• The synthesis and pharmacological activity of 3-arylecgonine methyl ester and 3-carbamoyloxyecgonine methyl ester analogues: Probes for the characterization of the dopaminergic cocaine receptor

      Kline, Richard Harry, Jr.; Wright, Jeremy, Ph.D. (1991)
      Several analogues of 3-arylecgonine methyl ester were designed, synthesized and characterized by {dollar}\sp1{dollar}H and {dollar}\sp{lcub}13{rcub}{dollar}C NMR, IR and MS. The compounds were synthesized as racemates from 2,4,6-cycloheptatriene carboxylic acid or as enantiomerically pure compounds from 1 R-cocaine hydrochloride. These analogues were assessed for their ability to inhibit ({dollar}\sp3{dollar}H) cocaine binding to bovine striatal tissue and ({dollar}\sp3{dollar}H) dopamine uptake into striatal synaptosomes. Methyl(1RS-2-exo-3-exo)-8-methyl-3-phenyl-8-azabicyclo (3.2.1) octane-2 carboxylate was the most potent showing IC{dollar}\sb{lcub}50{rcub}{dollar} values for inhibition of ({dollar}\sp3{dollar}H) cocaine binding and ({dollar}\sp3{dollar}H) dopamine uptake of 22 and 133 nM, respectively. The racemates and the 1R-isomers proved to be equally potent inhibitors of binding and uptake. The 1RS-{dollar}\alpha{dollar}-dinitrophenylecgonine methyl ester analogue had the lowest potency. IC{dollar}\sb{lcub}50{rcub}{dollar} values for inhibition of binding and uptake were 11 and 30 {dollar}\mu{dollar}M, respectively. One of these compounds, 1R-{dollar}\beta{dollar}-(p-aminophenyl)ecgonine methyl ester, was designed and synthesized as a potential ligand for affinity chromatography. Several (1{dollar}R{dollar}-2-{dollar}exo{dollar}-3-{dollar}exo{dollar})-3-({dollar}N\sp\prime{dollar}-phenylcarbamoyloxy)ecgonine methyl ester analogues were also designed, synthesized, and spectroscopically characterized. Many of these compounds were synthesized as 1{dollar}R{dollar}-stereoisomers from 1{dollar}R{dollar}-ecgonine methyl ester in good yields. These compounds were tested for their ability to inhibit ({dollar}\sp3{dollar}H) cocaine binding to rat striatal tissue and ({dollar}\sp3{dollar}H) dopamine uptake into synaptosomes prepared from the same tissue. The most potent of these analogues was (1{dollar}R{dollar}-2-{dollar}exo{dollar}-3-{dollar}exo{dollar})-3-({dollar}N\sp\prime{dollar}-3{dollar}\sp\prime {dollar}-nitrophenylcarbamoyloxy)-8-methyl-8-azabicyclo (3.2.1) octane-2-carboxylic acid methyl ester. IC{dollar}\sb{lcub}50{rcub}{dollar} values for inhibition binding and uptake were 37 and 178 nM, respectively. In general, compounds which contained meta substituents on the phenyl ring of the C{dollar}\sb3{dollar} phenylcarbamate side chain were more potent than those with para substituents. Also, analogues which contained electron withdrawing groups on the same phenyl ring had considerably higher potencies than those with electron donating groups. Meta and para substituted amino 3-carbamoyloxyecgonine methyl ester analogues were found to be potential affinity chromatography ligands. Two isothiocyanato derivatives proved to bind irreversibly to the receptor, and two azido compounds were shown to function as photoaffinity ligands. These probes may be instrumental in the eventual isolation and purification of the cocaine receptor/dopamine transporter.
    • Origins of infantile Tay-Sachs disease alleles in a Cajun population

      McDowell, Geraldine A. Edgell; Blitzer, Miriam G. (1991)
      The mechanisms responsible for the origin and maintenance of Tay-Sachs disease (TSD) alleles in the Ashkenazi Jewish population and others in which the occurrence of TSD is increased have yet to be delineated satisfactorily. As a model for studying these mechanisms, we evaluated five apparently unrelated Cajun families having members affected with TSD. The occurrence of TSD is elevated among Cajuns and preliminary studies suggest that the carrier frequency in the Cajun population may be increased over that of the general population. DNA sequence analysis revealed that, contrary to expectation, two different TSD alleles are present in the Cajun population: a {dollar}\beta{dollar}-hexosaminidase A {dollar}\alpha{dollar}-chain exon 11 insertion which is common among Jewish TSD carriers and an mRNA-negative allele which has not been described previously. Evaluation of pedigrees of 28 TSD carriers, identified by enzymatic and molecular screening of 95 relatives of the probands, revealed that the exon 11 insertion mutation has existed in the Cajun population since its establishment in the mid-1700's and most likely was introduced by members of a single family whose geographic origin is in France. Examination of these pedigrees for evidence of both increased fertility or survival among carriers of the insertion mutation compared to controls and preferential inheritance of this TSD allele produced no suggestion of heterozygote advantage. The presence of common ancestors among most of the exon 11 insertion carriers suggests that founder effect is responsible for the proliferation of this mutation among the Cajuns. The novel mutation has been in the Cajun population no more than four generations and appears confined to a single family. The Cajun population may well serve as a model for how founder effect and random genetic drift can result in the presence of two TSD alleles in a single population, one allele being found in the majority of carriers. This distribution of TSD alleles is similar to that seen in the Ashkenazi Jewish population. Of practical importance to the Cajun community is the likelihood that the exon 11 insertion mutation is widely distributed in southwest Louisiana and presents a significant public health problem within this community.
    • Partial purification and characterization of a C-methyltransferase from streptonigrin-producing Streptomyces flocculus

      Fox, Bonnie Marie; Speedie, Marilyn K. (1991)
      A C-methyltransferase which catalyzes the transfer of a methyl group from S-adenosyl-L-methionine to the C-3 position of the aliphatic side chain of L-tryptophan resulting in formation of {dollar}\beta{dollar}-methyl tryptophan, has been isolated from streptonigrin-producing Streptomyces flocculus. The enzyme catalyzes the first step in streptonigrin biosynthesis and is postulated to have a regulatory role in the pathway. The enzyme has been purified 217-fold by ammonium sulfate fractionation, followed by sequential gel filtration through Sephadex G-150 and Sephadex G-100 SF columns. Attempts at further purification have been hindered by very active proteases which co-purify with the enzyme. Protease inhibitors PMSF, pepstatin A, leupeptin, and trypsin inhibitor have failed to inactivate the protease activity. Based on comparison to reference proteins, the C-methyltransferase was estimated to have a molecular weight of 40,000 by Sephadex G-150 gel filtration. A narrow pH optimum of 7.5-8.0 was determined for the enzyme. The Sephadex G-100 SF fraction was highly unstable, losing 90 {dollar}\pm{dollar} 6% of its activity after 12 hours at 4{dollar}\sp\circ{dollar}C. S-Adenosyl-L-methionine and L-cysteine have been found to stabilize activity in the purified fractions. The enzyme is inhibited by sulfhydryl binding reagents, but no such inhibition is observed in the presence of substrate, suggesting an essential {dollar}-{dollar}SH group at or near the active site. Inhibition by carbonyl reagents was exhibited by the C-methyltransferase. Tritiated sodium cyanoborohydride treatment of the Sephadex G-100 SF fraction resulted in tritium incorporation and a concomitant 36 {dollar}\pm{dollar} 1% inactivation of the enzyme. These combined data led to the hypothesis that pyridoxal-5{dollar}\sp\prime{dollar}-phosphate may be involved as a cofactor in the C-methyltransferase. An enzymatic mechanism is proposed, and several studies related to this mechanism are presented.
    • A study of the association between social functioning and manic-depressive illness in family constellations with presumed genetic vulnerability for affective illness

      Scott, Alice Malone; Ephross, Paul H. (1991)
      This exploratory-descriptive study examines the association between social functioning and subclassifications of manic-depressive illness in family constellations with presumed genetic vulnerability for affective illness. The research question is: Are there differences in the social functioning of family members with bipolar I disorder, bipolar II disorder, and their biological relatives who are not affectively ill? A study sample of convenience was drawn from the Genetic Linkage Study of Affective illness conducted at the Johns Hopkins University School of Medicine. Twenty-one bipolar I's, 22 bipolar II's, and 20 unaffected participants were included. Instruments included interviewer administered scales (Hamilton Depression Rating Scale, Young Mania Rating Scale, and Health and Daily Living Assessment) and self administered scales (Social Adjustment Scale SR and Eysenck Personality Questionnaire). As hypothesized, findings reveal a statistically significant relationship between the diagnosis of bipolar illness and inadequate social functioning in the areas of close social relationships and overall social role adjustment. Both the bipolar I's and the bipolar II's had significantly fewer close relationships than their biological unaffected relatives. The two bipolar groups did not differ significantly from each other. When analyzed by Multiple Regression, personality as measured by the Eysenck Neuroticism Scale was found to be more highly predictive of overall social role adjustment than was the diagnosis of bipolar illness. Implications for social work and related practice in mental health settings and further research were drawn. Conceptual refinement of the global concepts social functioning within the interpersonal field and normality is greatly needed. Knowledge is lacking in the areas of nosology and the psychological and social environments which characterize individuals with subclassifications of bipolar illness. The impact of both ill and well intervals upon the family system needs to be explored.
    • The origin and meiotic behavior of Robertsonian translocations

      Wolff, Daynna Joanne; Schwartz, Stuart, 1951- (1991)
      Robertsonian translocations, whole arm chromosomal exchanges between acrocentric chromosomes, are the most common human structural chromosomal abnormality (1/1000 individuals). While these translocations have been thoroughly investigated in insects and some mammals, relatively little is known about the structure and formation of Robertsonian translocations in humans. In addition, the effect of this chromosomal aberration on the frequency and location of recombination during meiosis is poorly understood. Fluorescence in situ hybridization with five biotin-labeled probes was used to characterize 30 different Robertsonian translocations. Of 8 de novo homologous translocations, only one was interpreted as dicentric, while 19 of 22 nonhomologous Robertsonian translocations were dicentric. Two of 26 translocations studied using the beta satellite probe showed a positive signal, while ribosomal DNA was undetectable in ten cases studied. This analysis demonstrated that a majority of the breakpoints could be localized distal to the centromere and just proximal to the beta satellite and NOR regions of the short arm. The availability of appropriate DNA probes for restriction fragment length polymorphisms (RFLPs) permits the study of the origin of Robertsonian translocations, as well as the analysis of meiotic recombination frequencies for both the translocation chromosomes and their normal homologs. For the present study, 15 Robertsonian translocation families were studied using eight RFLPs from chromosome No. 21. Five families with de novo Robertsonian translocations were studied to determine the parental origin of the translocation. Two t(21;21) were found to originate from the father, while two out of three of the t(14;21) originated during maternal meioses (the last case was not informative). These results support previous findings of preferential formation of t(14;21) during maternal meiosis (20/21 of cases reported). Numerous studies in insects and in a few mammals have shown that chromosomal rearrangements, such as translocations, may cause intra- and interchromosomal effects. Studies in five familial Robertsonian translocations (one t(13;21) and four t(14;21)) revealed an increased frequency of crossing over in the long arm of chromosome 21, as well as a more proximal placement of the crossover events, in relation to the centromere. This finding demonstrates an intrachromosomal effect of the Robertsonian translocation on chromosomes 21 involved in the rearrangements. In contrast, no interchromosomal effect was revealed for chromosome 21 in five additional non-21 Robertsonian translocations (three t (13;14) and two t(14;22)). In these families, there was no significant alteration in either the frequency or location of crossing over.
    • A neuroendocrine investigation of the inhibitory effect of prolactin on LH release

      Park, Sung-Keun; Selmanoff, Michael K. (1991)
      Hyperprolactinemia (HP) is known to have an inhibitory effect on LH release. The aim of this research project was to further investigate the inhibitory mechanism(s) mediating hyperprolactinemic suppression of postcastration LH release in male rats. HP was induced by sc administration of purified ovine prolactin (oPRL) in a polyvinylpyrrolidone depot every 12 h, beginning at the time of orchidectomy (ORCH). oPRL suppressed postcastration LH release in graded, dose-dependent fashion. In contrast, shortloop autofeedback of oPRL on rat prolactin (rPRL) secreted from the in situ pituitary gland appeared regulated as a threshold response. The first significant suppression of LH secretion began in the high physiological range of plasma prolactin ({dollar}\sim{dollar}200 ng/ml) which is within the range encountered in pregnancy, pseudopregnancy and lactation in female rats. A striking finding was that this was the same dose which exerted PRL autofeedback. These data suggest that a common hypothalamic mechanism, stimulated by elevated PRL levels, may inhibit both LH and rPRL secretion. We next determined whether this dose-related suppression of mean LH levels resulted from differential, dose-related effects of oPRL on LH pulse amplitude versus pulse frequency. A smaller dose of oPRL significantly suppressed mean LH values by increasing the interpulse interval without changing pulse amplitude. Two higher doses of oPRL suppressed both LH pulse frequency and pulse amplitude. The mean maximal LH increments ({dollar}\Delta{dollar} LH) to two LHRH challenges were unaffected by oPRL indicating that the suppressive effect of oPRL was not by a direct action at the anterior pituitary gland. These data suggest that the neurons first affected by elevated PRL levels are the ones responsible for the coordinated firing of the LHRH neurons. Possible candidates for this neuronal mechanism would be the LHRH-LHRH collateral connections or other hypothalamic interneurons. We tried unsuccessfully to determine LHRH neuronal turnover by employing a recently published method using colchicine. In our hands, this method did not work for either LHRH or arginine vasopressinergic neurons. We characterized the responses of LHRH neurons to the neuroactive compounds norepinephrine (NE), epinephrine (E), bicuculline (BIC), neuropeptide Y (NPY) and N-methyl-D-aspartate (NMDA) to determine whether neurons utilizing these transmitters were involved in mediating HP suppression of LH. Third cerebroventricular administration of E and NPY and iv NMDA stimulated LH release. The mean maximal LH increments to these compounds in HP rats were suppressed. These data indicate that PRL either acts directly on the LHRH neurons to decrease their responsivity to all stimuli, and/or, that PRL acts indirectly to inhibit glutamatergic, NPYergic and adrenergic neurons that are stimulatory to LHRH release.
    • Rational design of controlled release matrix tablets using an ethylcellulose dispersion and fluid bed granulation technology

      Smith, Bruce Philip; Hollenbeck, R. Gary (1991)
      Application of an aqueous polymeric dispersion to control drug release from matrix tablets was investigated. The polymeric dispersion of ethylcellulose was applied as a binder in matrix tablet formulations prepared by fluid bed granulation. Examined were the effect of excipients (dicalcium phosphate, lactose, and microcrystalline cellulose), drug solubility (chlorpheniramine maleate and hydrochlorothiazide), level of water insoluble binder (5 to 30% w/w), and compression force (300 and 600 kg) on the granule and resulting tablet characteristics. These objectives were approached through the use of mixture experimental design. Examined also were the physico-chemical properties of the granules and tablets to further explain the in vitro release behavior of the different formulations. The mean particle size ranged from 85 to 700 {dollar}\mu{dollar}m and the majority of tablets possessed tensile strength values which ranged from 5 to 25 kg/cm{dollar}\sp2{dollar}. A critical assessment of the theoretical framework was tested by observing how the solubility of drug and excipient(s) changed the consolidation characteristics, porosity, and tortuosity of the matrix. Compression of the fluid bed granules into tablets was a requisite for sustained release of drug since the granules themselves rapidly released the drug. Excipients and level of binder had a major impact on the release characteristics of the tablets. In general, for tablets containing chlorpheniramine maleate higher binder levels and compression force decreased the release of drug from the matrix. In contrast, for tablets containing hydrochlorothiazide higher binder levels decreased the release of drug but the release was insensitive to compression force. Drug solubility played a disproportional role in the release rate with the more water soluble drug producing the fastest release in part due to the disruption of the matrix infrastructure. Addition of the polymeric dispersion to the raw materials, was found to decrease the mean yield value by 17 to 72% indicating increased plasticity of the granulations. The database generated from the mixture design was used to rationally fabricate matrix tablets which had a specified release rate least sensitive to compression force. This study demonstrates that a controlled release matrix system can be produced using an aqueous ethylcellulose dispersion and fluid bed granulation technology.
    • An ecological approach to reducing child maltreatment

      Mann, Linda Neunlist; Goldmeier, John (1991)
      This study was conducted to determine the effect of a parent training program on maltreating parents. The program was a brief intervention, based on the ecological model of child maltreatment, using both group and class sessions and was designed to provide parenting information and knowledge. The expectation was that at the end of the twelve-week program, the parents would increase in the parenting knowledge and skills, thereby increasing their parenting abilities and decreasing the likelihood that they would abuse or neglect their children. The study used three objective instruments in an attempt to measure changes in the parents' child abuse potential, levels of depression, and appraisal of social support. More than half of the subjects dropped out of the program prior to completion and a large number of the participants did not provide valid information on the three objective measures. However, in spite of these problems, the data analysis indicated that there were significant differences between the subjects' pre- and post-test scores, suggesting that participation in the parent training program had a positive benefit for a majority of the participants. The study findings indicate that, following the intervention, the parents had reduced levels of depression, reduced levels of child abuse potential, and increased appraisals of social support. In addition, there were significant differences between the participants who completed the program and those who dropped out and between the participants who provided valid information on the measures and those who provided invalid information. The study findings can be useful for social workers who are involved in planning and designing programs for maltreating parents and the findings suggest that parent training programs can be a beneficial intervention in efforts to reduce child maltreatment.
    • The relationships among psychological hardiness, faculty practice involvement, and perception of role stress of nurse educators

      Lambert, Clinton E., Jr.; Ruth, M. Virginia (1991)
      The primary purpose of this descriptive cross-sectional survey of nurse educators, employed full-time by a NLN accredited baccalaureate and higher degree granting school of nursing with both a graduate and an undergraduate program, was to identify if the variables, psychological hardiness, faculty practice involvement, and perceived role stress, were inter-related. An additional purpose was to determine whether there were differences between those involved in faculty practice and those not involved in faculty practice. It was predicted that: (1) the nurse educators perception of role stress would be negatively related to the level of psychological hardiness, (2) the perception of role stress by nurse educators involved in faculty practice would be greater than the perception of role stress by nurse educators not involved in faculty practice, and (3) the level of psychological hardiness of nurse educators involved in faculty practice would be greater than the level of psychological hardiness of nurse educators not involved in faculty practice. Each of 1345 identified nurse educators, whose name had been provided by the deans of 34 randomly selected schools of nursing, was requested to anonymously respond to a mailed, self-administered, pencil and paper questionnaire comprised of three instruments (Demographic Data Questionnaire, Personal Views Survey, and Role Conflict and Role Ambiguity Scale). A 66.5% subject response rate was obtained. Data from 871 of the respondents' questionnaires were analyzed using descriptive statistics and inferential statistics. The nurse educators perception of role stress was found to be significantly negatively correlated to the level of psychological hardiness. No significant difference was found between those involved in faculty practice and those not involved in faculty practice with respect to perception of role stress or to level of psychological hardiness. The results of this study suggest that faculty practice involvement of nurse educators in NLN accredited baccalaureate and higher degree granting schools of nursing with both a graduate and an undergraduate program is a self-selective process.
    • Acute sporadic non-A, non-B hepatitis and blood transfusion in Makkah province, Saudi Arabia

      Ghabrah, Tawfik Mohammed; Strickland, G. Thomas (1991)
      Non-A, non-B hepatitis (NANBH) is a disease of worldwide distribution. It represents an important public health problem in terms of morbidity and mortality throughout the world. The present study investigates the association between blood transfusion and acute NANBH and identifies other risk factors for NANBH using a hospital-based case-control study design. By using the first generation anti-HCV test, it also reports the frequency of hepatitis C virus (HCV) infection among acute NANBH cases and describes and compares the characteristics of HCV infection with those of other types of viral hepatitis. Risk factor information questionnaires concerning blood transfusion and other risk factors were completed for 97 acute NANBH cases and 194 controls, 13 years of age and older. After adjusting for both living or travelling into more endemic areas and contact with a jaundiced person, a history of blood transfusion was associated with about a 4-fold increase in risk for acute NANBH. Although this association was statistically nonsignificant, a significant dose-response effect was observed as a 3.5-fold increase in risk per transfusion. The two adjustment variables were the only ones among many risk factors which persisted in showing a significant association with the risk of acquiring acute NANBH throughout the analyses. Antibody to hepatitis C virus was found in 8 of the 97 acute NANBH cases for whom various characteristics including possible risk factors have been defined. In conclusion, commonly recognized risk factors (except parenteral drug abuse) for acquiring various types of viral hepatitis were present in 70% of acute NANBH cases. When compared to controls, NANBH cases did not significantly differ in frequency of parenteral exposures. On the contrary, the enterical route of transmission was more likely in NANBH cases who significantly differed from controls in reporting a history of both living or travelling into more endemic areas, especially the Indian subcontinent, and contact with a jaundiced person. Exposure to persons with subclinical infection or asymptomatic chronic carriers could account for the 30% of cases in which no specific risk factors could be identified. Early identification and implementation of preventive measures to reduce the disease incidence is recommended for high risk groups.
    • An evaluation of an intervention implemented to cause improved adjustment of prisoners

      Pugh, David N.; Varghese, Raju (1991)
      This dissertation is an evaluation of an intervention implemented to improve the adjustment of prisoners. The intervention, known as Decisions, is a structured educational model. It rests upon the assumption that criminals are poor problem-solvers and view themselves as victims of forces over which they have little or no control. Decisions attempts to cause enhanced prisoner adjustment by first causing improvement in problem-solving ability and a shift toward the internal dimension of personal control of prisoners. The literature review revealed three studies directly related to the underlying assumptions of Decisions. None of the studies lend any support to those assumptions. However, the literature does indicate that problem-solving ability and locus of control are related to prisoner adjustment. The literature reviewed also shows that the locus of control of prisoners is subject to at least short-term change via an intervention like Decisions. It was hypothesized that the experimental group would score significantly better on problem-solving scores and significantly more internal on locus of control scores at posttest and follow up. It was also hypothesized that the experimental group would score significantly better in adjustment at follow up. The analysis revealed a significant initial group difference on pretest locus of control scores, and on the scores of one adjustment scale. Attrition resulted in additional group differences on age and education. All analyses showed nonsignificant results. There were no significant differences between groups on posttest and follow up locus of control and problem-solving scores, nor on follow up prison adjustment scores. Only the variable age accounted for any significant variance of prison adjustment. Several interpretations of the results are offered. The first is that outcome is the cause of significant group differences both present at pretest and resulting from a high mortality rate. The second is that skewed distributions of two measures made it difficult for any change in the desired direction to be detected. A third and more plausible interpretation is that the intervention rests upon a weak theory. That is, neither the literature nor the data generated in this study support the assumption that prisoners are poor problem-solvers and externals. (Abstract shortened with permission of author.)
    • Characterization of endothelial barrier antigen of the blood-brain barrier

      Pulley, Michael Thomas; Sternberger, Ludwig A. (1991)
      The blood vessels of the brain have unique characteristics which regulate the passage of substances from the blood to the interstitial space of the brain. The special collection of selective permeability and specific transport of molecules through the vasculature of the brain is known as the blood-brain barrier (BBB). Our laboratory discovered a monoclonal antibody which reacted with blood vessels of the brain which possess a BBB but not the blood vessels of peripheral organs or areas of the brain without a BBB. Due to its spatial and functional relationship with the BBB, this antigen was named endothelial barrier antigen (EBA). These studies aimed to define some of the biological and biochemical characteristics of the EBA molecules. EBA was localized to the luminal plasma membrane of CNS endothelial cells (EC) possessing a BBB by electron microscopy. EBA expression is developmentally regulated and closely follows the maturation of the BBB. Injection of monoclonal anti-EBA results in specific, sustained binding to BBB EC. EBA is a BBB-specific antigen which probably has a role in the interaction of blood-borne cells or molecules with cerebral EC. Monoclonal anti-EBA may be useful for delivering drugs specifically to the BBB. The 25, 28 and 31KD EBA proteins were characterized by classical biochemical analyses. These analyses included amino acid composition, peptide mapping, investigation of posttranslational modifications, two-dimensional gel electrophoresis, and finally amino acid sequencing. Amino acid sequence data for two peptides from the 31KD EBA were identical to mouse histone H1 (Yang et al, 1987). The identity of the isolated proteins was also confirmed by Western blot analysis using monoclonal anti-H1 histone (Chemicon). The biochemical data generated were consistent with results one would anticipate for H1 histones. However, anti-EBA recognizes an antigen in the endothelial cell membrane distinct from histone H1. This antigen was solubilized by treating isolated cerebral microvessel membranes with the detergent sodium deoxycholate. The experiments performed will simplify the future isolation of the EBA protein(s).
    • Role of cytokines in Campylobacter jejuni infection and immunity in mice

      Baqar, Shahida; Rollwagen, Florence M.; Falkler, William A., Ph.D. (1991)
      Cytokines incorporated into agarose blocks and implanted subcutaneously into mice established an in vivo gradient which can be used to mimic a local inflammatory process. A model was developed in which cellular influx into cytokine impregnated blocks paralleled the normal cellular reaction to infections or wounds. Agarose blocks containing antigens of the intestinal pathogen, C. jejuni, were implanted in normal and infected mice. Kinetics of cellular influx into the blocks showed an early influx of lymphocytes in infected mice only. The two groups showed similar but discrete patterns of cytokine secretion within the blocks. Infected, but not normal mice, were actively synthesizing antibodies to C. jejuni at the local site (within the blocks), whereas the levels of total immunoglobulin were similar for the two groups. The results suggested that the agarose block model can be successfully applied to study local cytokine production and the role of various cells in infectious diseases. The primary infection with C. jejuni in mice resulted in the generation of sensitized B and T cells which could be boosted by rechallenge with homologous bacteria. These results indicated the presence of a functional immunity to C. jejuni challenge in mice. An antibody secreting cell assay allowed the early detection of specific antibody producing cells in circulation. In addition, data suggested a homing pattern of B cells to spleen as well as Peyer's patches. The profile of cytokine production at a local site (intestine) and in the circulation is suggestive of a role of local IL-1 and local as well as systemic IL-6 in immunity and in the pathogenesis of Campylobacter. Crude cytokine suspensions, when fed orally before challenging with C. jejuni, reduced the bacterial load from the gut and also augmented both humoral and cellular immune responses to the bacterial antigens. Oral treatment of mice with rIL-6 had an immediate effect in reducing the bacterial load whereas this effect was delayed in mice treated with rIL-5. Although IL-6 and IL-5 enhanced local as well as systemic antibodies to C. jejuni, these provided only a partial protection from rechallenge with homologous bacteria. On the other hand, mice treated with rIL-2 before challenge had better protection and a faster clearance of bacteria from their guts. In none of the groups could DTH to bacterial antigens be demonstrated, however, the lymphocytes responded to in vitro by stimulation with the same antigens. The data suggested that various cytokines have potential for use as immune modifiers to enhance the immune response to various enteric pathogens. (Abstract shortened with permission of author.)
    • Factors contributing to maternal protectiveness following the disclosure of intrafamilial child sexual abuse: A documentary study based on reports of Child Protective Service workers

      Heriot, Jessica K.; Ephross, Paul H. (1991)
      This study investigate maternal protectiveness following the disclosure of intrafamilial child sexual abuse. Two questions were posed: (1) What proportion of mothers act in a protective way following the disclosure of child sexual abuse, and (2) What factors are associated with maternal non protection? Maternal protectiveness was operationalized in two ways: (1) The mother takes action to physically separate herself and her abused child from the perpetrator, and (2) she feels and acts supportively toward her sexually abused child. The study investigated fourteen factors thought to be associated with maternal non protectiveness. They were grouped in three categories: individual maternal factors, child characteristics, and factors pertaining to the mother's relationship to the perpetrator. The study also investigated the relationship between maternal belief and maternal protectiveness. The study population was drawn from substantiated cases of child sexual abuse reported to Baltimore City and County Sexual Abuse Intake Units, Division of Child Protective Services. The sample consisted of 118 mothers whose children were abused by a family member or the mother's partner with whom the mother and the child were living when the abuse was reported to Child Protective Services. At the close of the intake period, data was collected on maternal protectiveness via a questionnaire given to intake workers. The majority of mothers took action to separate themselves and their children from the perpetrator (56.8%). Two-thirds of the mothers were supportive of their sexually abused children. Fifty-two percent of the mothers both separated from the perpetrator and were supportive of their children. Mothers whose feelings toward the perpetrator were warm and accepting were more likely to be non protective than mothers whose feelings were hostile and rejecting. Mothers of seriously abused children were more likely to be non protective than mothers of less seriously abused children. In addition, mothers who abused drug and/or alcohol and mothers of children abused by a husband or boyfriend were at risk for non protection. Finally, mothers of older children were less likely to be protective than mothers of younger children.
    • Characterization of the intrinsic protein kinase activity and the kinase catalytic motifs of the large subunit of the herpes simplex virus type 2 ribonucleotide reductase

      Luo, Jianhua; Aurelian, Laure (1991)
      The large subunit of Herpes Simplex Virus type 2 (HSV-2) ribonucleotide reductase (ICP10) is a chimera protein consisting of a serine/threonine protein kinase (PK) domain at the amino-terminus and a ribonucleotide reductase (RR) domain at the carboxyl-terminus. Like growth factor receptor PKs, ICP10 is myristylated, it has features of a signal peptide and putative transmembrane (TM) segment, and its PK activity is modulated by basic proteins and by antibodies to amino acid residues upstream of the TM. To further characterize this PK domain, we constructed a bacterial expression vector (pJL11) containing DNA sequences encoding ICP10 amino acid residues 1-445. Bacteria containing pJL11 were induced to express a 29 KDa protein (designated pp29{dollar}\sp{lcub}\rm la1{rcub}){dollar} that represents a truncated portion of the ICP10 PK domain as demonstrated by immunoprecipitation with antibodies that recognize different antigenic domains, competition studies with extracts of ICP10 positive eukaryotic cells, and peptide mapping. pp29{dollar}\sp{lcub}\rm la1{rcub}{dollar} has autophosphorylating and transphosphorylating activity for calmodulin. The enzyme is activated by Mn{dollar}\sp{lcub}2+{rcub}{dollar} but not by Mg{dollar}\sp{lcub}\rm 2+{rcub}{dollar} ions, and autophosphorylation is inhibited by histone. It differs from the authentic ICP10-PK in that phosphorylation is specific only for threonine. To determine the significance of ICP10 PK catalytic motifs, site-directed and deletion mutants in PK motifs I and II, the putative signal peptide and the TM segment were used to determine the role of these elements in ICP10-PK activity. PK activity was lost by deletion of the putative TM segment (amino acid residues 85-106). However, mutation of the central Gly in PK catalytic motif I (Gly{dollar}\sp{lcub}106{rcub}{dollar}) or of the invariant Lys in PK catalytic motif II (Lys{dollar}\sp{lcub}176{rcub}{dollar}) or deletion of both of these catalytic motifs (amino acid residues 106-178) did not abolish the kinase activity as determined both in auto- and transphosphorylation assays. PK activity of the mutant deleted in domains I and II was 4-fold lower than that of the wild type ICP10 and it was insensitive to Mn{dollar}\sp{lcub}2+{rcub}{dollar}, suggesting that these motifs are involved in Mn{dollar}\sp{lcub}2+{rcub}{dollar} activation of kinase activity. The result of immunoblotting demonstrated that ICP10 complexes with GTPase activating protein (GAP). Ras GTPase activity is significantly inhibited in ICP10 transformed (JHLa1) cells. These results suggested that ICP10 may constitutively activate ras activity by blocking its down-regulation process, implying a potential signal transduction mechanism for ICP10 induced transformation.
    • Transcriptional regulation and neoplastic transforming potential of the large subunit of ribonucleotide reductase from herpes simplex virus type 2

      Wymer, James Paul; Aurelian, Laure (1991)
      Herpes simplex virus (HSV) genes are regulated in a cascade of immediate early (IE), delayed early (DE), late (L). The large subunit of ribonucleotide reductase (RR) from HSV-2, designated ICP10, has been grouped with DE proteins. The amino-terminal domain of ICP10 has protein kinase (PK) activity and properties similar to growth factor receptor kinases that can be activated to transforming potential. The studies described in this dissertation sought to develop a better understanding of regulatory aspects of ICP10 regulation as well as the role of ICP10 expression in neoplastic transformation. Regulation of expression of the ICP10 gene was studied by immunofluorescence with the intact ICP10 gene or by chloramphenicol acetyltransferase (CAT) analysis with hybrid ICP10 promoter constructions containing the wild type ICP10 promoter or site-directed mutants deficit in specific cis-response motifs. Co-transfection of these constructions with DNA encoding an HSV nonspecific transactivator (IE110) or an IE gene-specific transactivator (Vmw65), enhanced expression at least 10-fold, regardless of the assay system. In contrast, expression was minimally enhanced by DNA encoding a DE gene transactivator (IE175) at low doses and slightly reduced at high doses. Sequence analysis of the ICP10 promoter revealed the presence of both herpesvirus IE gene-specific (TAATGARAT, GA-rich, and {dollar}\alpha{dollar}H2-{dollar}\alpha{dollar}H3 motifs), as well as cellular cis-response motifs (potential SP-1, consensus AP-1, and octamer transcription factor-1 (OTF-1) binding elements). Factors that bind to the ICP10 promoter were identified by gel retardation analysis with mixtures of uninfected cell nuclear extracts and virion lysates or in vitro synthesized OTF-1 and Vmw65. The Oct-1 motif (ATGCAAAT) was necessary for optimal Vmw65 binding to, but not for transactivation of the ICP10 promoter as evidenced by competition experiments with oligonucleotides overlapping the consensus IE110 promoter virion response element and by site-directed mutagenesis of the motif. The 3{dollar}\sp\prime{dollar} portion of the TAATGARAT motif (GARAT) was dispensible for binding but necessary for activation. These data suggest that ICP10 behaves as an IE gene and could therefore affect host gene regulation independent of lytic infection. ICP10-PK has neoplastic transforming potential in vitro. Anchorage independent growth was observed in cells transfected with the vectors that express the entire ICP10 protein or just the PK domain, but not a frameshift (not expressing ICP10) mutant or a carboxy-terminus (RR domain) expression vector.
    • Cytogenetic and molecular characterization of a human chromosomal region which renders in vitro phenotypic correction of xeroderma pigmentosum complementation group D

      Flejter, Wendy L.; Schultz, Roger Alan (1991)
      Xeroderma pigmentosum (XP) has been a focus of extensive investigation since its clinical description in 1874 and the subsequent demonstration of a DNA repair defect in 1968. However, identification of the mutant genes responsible for XP has been hindered by numerous experimental limitations and extensive genetic heterogeneity. The obvious in vitro phenotypes of UV sensitivity and defective DNA repair observed in XP fibroblasts can be complemented following transfer of chromosomes from normal cells. This strategy offers an excellent approach for gene mapping and cloning of the defective genes. Recently, a rearranged single human chromosome (Tneo) has been isolated which restores both UV light resistance and DNA repair activity in XP complementation group D (XP-D) cells. The cytogenetic and molecular characterization of Tneo was the focus of this dissertation. The results provided preliminary mapping of the XP-D locus and serve as a critical first step in the isolation of the gene(s) defective in XP-D cells. Classical cytogenetic analysis revealed that Tneo is a monocentric chromosome of undefined human origin. "Reverse in situ hybridization" by Alu-PCR indicated that this chromosome involves a complex rearrangement consisting of chromosome segments from 16q, 17p and 19. "Painting" with chromosome-specific DNA libraries was used to define the orientation of the chromosomal segments present in Tneo. Southern blot hybridizations using chromosome-specific probes further delineated the specific breakpoints involved in the Tneo rearrangement, as consistent with a der(16)(19pter{dollar}\to{dollar}19p13.2::17p?{dollar}\to{dollar}17p?::19p13.1{dollar}\to{dollar}19p13.1::16cen{dollar}\to{dollar}16q23q24?:: 19q13.2{dollar}\to{dollar}19q13.3::17p?{dollar}\to{dollar}17p?). Deletions of the complementing chromosome were generated and revealed that the restoration of UV resistance in XP-D cells resides in chromosomal material in the distal q-arm of Tneo. Molecular characterization demonstrated that the XP-D complementing region involves DNA derived from 19q13.2{dollar}\to{dollar}19q13.3 and 17p. Transfer of a normal human chromosome 17 into XP-D cells failed to correct the defective phenotypes, implying that the XP-D gene lies within or near 19q13.2{dollar}\to{dollar}19q13. It is of particular interest that this region includes a previously described human DNA repair gene cluster. The experiments described have generated materials which should prove valuable for the molecular cloning of the XP-D gene(s). Additionally, the data illustrate the power of combined cytogenetic and molecular mapping techniques for use in the isolation of genes defective in a variety of human genetic disorders which exhibit in vitro phenotypes.
    • A study of family life education experiences among Chewa grandmothers, mothers, and daughters in Malawi

      Banda, Eta Elizabeth; Ruth, M. Virginia; Kreider, Mildred Sherk, 1936- (1991)
      A significant problem in adolescent health care in Malawi is a lack of information about family life education. The purpose of this descriptive and correlational study was to describe family life education experiences of Chewa grandmothers, mothers and daughters as a means of identifying the nature of the organization and type of educational programme/learning experiences that traditionally have been offered for developmental task readiness for adult life. The subjects for preliminary interviews were limited to three sets of grandmothers, mothers and daughters, i.e. nine participants. This data was used to develop the tool utilized for data collection in the study. The sample size for this study was 300. Data analysis was carried out using descriptive statistics, chi-square, analysis of variance and content analysis. Family life education organization was primarily perceived to be either the responsibility of the family or a shared responsibility between the family, village and other social organizations. Family life education was mainly informally conducted within the family, although multiple resources were utilized for teaching. Music in combination with verbal communication played an important role in instruction and reading was observed to be almost absent as a method of teaching across all generations. Although mostly of the teaching was done didactically there was some practical experience in the sex education component where a female adolescent was given a male partner to learn and this male was called a 'fisi'. Significant differences were found in virtually all organizational variables, by generation and area of residence. Ten categories of family life education learning experiences were identified and tested for differences on the basis of generation and area of residence. The results revealed statistically significant intergenerational as well as geographic differences in learning experiences in sex education, menstruation and sanitary towel care, anatomy and physiology of the female reproductive system, socialization into adulthood, relationships with parents, elders, peers of the same and opposite sex, and disabled persons, traditional practices, psychological and spiritual issues. In addition analysis of variance to examine differences in traditional values between grandmothers, mothers and daughters revealed a statistically significant main effect for generation on traditional family values. (Abstract shortened with permission of author.)
    • Biochemical signalling responses of cultured neonatal rat heart myocytes to angiotensin II

      Abdellatif, Maha M.; Rogers, Terry Birkby (1991)
      Angiotensin II (AngII) has previously been shown to increase the beating frequency, decrease the force of contraction and induce phosphoinositide hydrolysis in cultured neonatal heart myocytes. These responses to AngII were transient, after which the cells became insensitive to any further application of the hormone. When the myocytes were incubated with 100 nM AngII there was a marked increase in total inositol phosphate (IPs) levels, that was maximal after 1 min, and returned to basal values after 5 min. This rapid desensitization process was concentration independent, and was completely reversible after 1 hr of hormone removal. The phenomenon was not due to an increase in the rate of degradation of inositol phosphates or depletion of the phospholipid pools. The homologous nature of the AngII-evoked desensitization, provided evidence that the receptor was the target of modification in this process. Binding studies with ({dollar}\sp{lcub}125{rcub}{dollar}I) AngII, revealed that the loss of surface receptors was not responsible for desensitization. Although activation of protein kinase C (PKC), desensitized the cells to AngII a role for the kinase in the hormone-evoked desensitization is not likely since the phenomenon was still observed in PKC-down regulated. The initial rapid burst of AngII evoked phosphoinositide turnover, was followed by a sustained (3 hr) low level of turnover. It was hypothesized that this long term signal may mediate a growth effect of the hormone on the heart cells. Consistent with this view, it was found that AngII increased the level of c-fos mRNA maximally after 0.5 hr. This response to the hormone was dose-dependent and mediated by the subtype-1 receptor through both PKC-dependent and independent pathways, but not through the initial 30s burst of inositol phosphates accumulation. The PKC-independent pathway was also independent of extracellular Ca{dollar}\sp{lcub}2+{rcub}{dollar}, arachidonic acid metabolites, calmodulin and the N{dollar}\sp{lcub}+{rcub}{dollar}/H{dollar}\sp{lcub}+{rcub}{dollar} exchanger. In conclusion, AngII induces a short lived, rapid hydrolysis of phosphoinositides, followed by a slower sustained effect in neonatal rat cardiac myocytes. The mechanism of desensitization of the cells to the former effect of the hormone is at the level of the receptor, but is independent of loss of surface receptors of PKC activation. (Abstract shortened with permission of author.)
    • Molecular cloning of GABA(A) receptor subunits from seizure prone (DBA/2J) and resistant (C57BL/6J) inbred mice

      Wang, Jia Bei; Burt, David R. (1991)
      Gamma-aminobutyric acid (GABA) is the major inhibitory transmitter in brain. The structure of the GABA{dollar}\sb{lcub}\rm A{rcub}{dollar} receptor is of particular interest because it contains several binding sites for clinically significant drugs such as benzodiazepines (BZ), barbiturates and alcohol. In addition, there is evidence which suggests the involvement of the GABA{dollar}\sb{lcub}\rm A{rcub}{dollar} receptor in several neurological afflictions. Molecular cloning of GABA{dollar}\sb{lcub}\rm A{rcub}{dollar} receptor subunits is the first step towards further understanding of this receptor through a molecular biological approach. The object of my thesis research is the use of molecular biological techniques to study the structure of subunits of the GABA{dollar}\sb{lcub}\rm A{rcub}{dollar} receptor at the cDNA and mRNA levels, extending this study to an animal model of epilepsy, in an attempt to establish the contribution of changes in the structure of the GABA{dollar}\sb{lcub}\rm A{rcub}{dollar} receptor to seizure susceptibility. Molecular cloning techniques have allowed determination of the nucleic acid and amino acid sequences of six subunits of GABA{dollar}\sb{lcub}\rm A{rcub}{dollar} receptors {dollar}(\alpha\sb{lcub}1-3{rcub},{dollar} {dollar}\gamma\sb{lcub}1-2{rcub}{dollar} and {dollar}\delta){dollar} from inbred DBA/2J and C57BL/6J mice. These subunits from mice have a high degree of sequence identity with those from rat, cattle and humans. GABA{dollar}\sb{lcub}\rm A{rcub}{dollar} receptor subunit proteins thus have structures which appear to be well conserved in mammals; presumably this reflects their key functional role. No amino acid sequence differences have been found between the two strains for these six subunits. Changes in regulation of gene expression of the receptor subunits could result in subunit under- or over-expression followed by dysfunction of the receptor. Unfortunately, our Northern blot data from the cerebellum, cortex and rest of the brain in both DBA/2 and C57BL/6 mouse brains did not indicate any major differences in these six subunits' expression. Our negative data should prove useful to others who are considering this obvious avenue of research to account for differential susceptibility to audiogenic seizures in DBA/2J and C57BL/6J mice. The discovery of {dollar}\gamma\sb{lcub}\rm 2L{rcub}{dollar} and {dollar}\gamma\sb{lcub}\rm 2S{rcub}{dollar} subunits supplies evidence that the generation of GABA{dollar}\sb{lcub}\rm A{rcub}{dollar} receptor heterogeneity occurs not only by the combinatorial association of subunit types and subunit isoforms, each of which are encoded by distinct genes, but also by alternative splicing of subunit gene transcripts. Our limited data about regional distribution and developmental profile of the subunit messenger mRNAs give another indication of GABA{dollar}\sb{lcub}\rm A{rcub}{dollar} receptor subtype diversity. The correlations existing among subunits in their regional distribution and developmental patterns may offer some hints as to the subunit combinations occurring in different native receptors and their possible functions in the brain.