[Molecular biological study on multifocal infection of HPV in uterine cervix, vagina and vulva]. Vaginal and vulvar scrapes from 30 patients which have HPV types 16 or 18 positive cervical neoplasia (13 of mild dysplasia, 5 of moderate dysplasia, 4 of severe dysplasia, 3 of carcinoma in situ, and 5 of invasive carcinoma) were examined by the polymerase chain reaction (PCR), to detect amplified E7 gene of HPV types 16 and 18 DNA sequences. In 24 vaginal and 15 vulvar scrapes, the same type of HPV as in cervical scrapes was detected. The frequencies of HPV DNA in vaginal and vulvar scrapes for histological diagnosis of cervical neoplasia were 84.6% (11/13) and 53.8% (7/13) in mild dysplasias, both were 100% (5/5) in moderate dysplasias, 75% (3/4) and 25% (1/4) in severe dysplasias, 66.7% (2/3) and 33.3% (1/3) in CISs, and 60% (3/5) and 20% (1/5) in invasive carcinomas, respectively. The results of vulvar scrapes showed that the frequency of HPV DNA in mild and moderate dysplasias was significantly higher than that in severe dysplasias, CISs and invasive carcinomas (p < 0.05). We suggest that, in addition to the investigation for the natural history of HPV infection, cell dynamics, local immunological status and any other factors in the HPV-infected lesion must be investigated to clarify the carcinogenesis of HPV in the uterine cervix. Furthermore, HPVs detected in the vagina and vulva were thought to have disappeared without reaching an integrated form during the follow-up of cervical neoplasia with HPV infection over a long period.


Cells transfected with human interleukin 6 cDNA acquire binding sites for the hepatitis B virus envelope protein. Earlier studies revealed that human interleukin 6 (IL-6) contains recognition sites for the hepatitis B virus (HBV) envelope (env) protein, and that IL-6 and anti-IL-6 antibodies, respectively, inhibited the interaction of cells expressing a receptor for HBV with the preS(21-47) segment of the HBV env protein, encompassing the complementary attachment site for IL-6. This suggested that IL-6 mediates HBV-cell interactions. We report that: (a) Chinese hamster ovary cells transfected with human IL-6 cDNA and Spodoptera frugiperda ovarian insect cells infected with recombinant baculovirus carrying human IL-6 cDNA expressed receptors for the preS(21-47) region of the HBV env protein, indicating that expression of IL-6 on the surface of cells is sufficient to endow them with receptors for HBV. (b) Among peptides covering the entire sequence of human IL-6 and the corresponding antipeptide antibodies, the peptide IL-6[35-66] and anti-IL-6[35-66] most effectively inhibited the interaction between human hepatoma HepG2 cells and the preS(21-47) ligand, suggesting that this region of the human IL-6 sequence encompasses a binding site for the HBV env protein. (c) Studies with replacement set peptides from the preS(21-47) sequence indicated that residues 21-25, 28, 31, 33-35, 39, and 43-45 can be replaced by alanine (serine) residues, while all the other residues are essential for maintaining the cell receptor/IL-6 binding activity. Further delineation of complementary sites on IL-6 and on the HBV env protein may contribute to the design of compounds inhibiting HBV replication.


Coexpression and functional cooperation of CTLA-4 and CD28 on activated T lymphocytes. T cell costimulation by molecules on the antigen presenting cell (APC) is required for optimal T cell proliferation. The B7 molecule on APC binds the T lymphocyte receptor CD28, triggering increased interleukin 2 (IL-2) production and subsequent T cell proliferation. CTLA-4 is a predicted T cell membrane receptor homologous to CD28, which also binds the B7 counter receptor, but whose distribution and function are unknown. Here we have developed monoclonal antibodies (mAbs) specific for CTLA-4 and have investigated these questions. mAbs were produced that bound CTLA-4 but not CD28, and that blocked binding of CTLA-4 to B7. CTLA-4 expression as measured by these mAbs was virtually undetectable on resting T cells, but was increased several hundred-fold during T cell activation. On activated lymphocytes, CTLA-4 was expressed equally on CD4+ and CD8+ T cell subsets and was coexpressed with CD25, CD28, and CD45RO. CTLA-4 expression was lower than that of CD28, reaching a maximum of approximately 1/30-50 the level of CD28. Despite its lower expression, CTLA-4 was responsible for much of the B7 binding by large activated T cells. Anti-CTLA-4 mAb 11D4 and anti-CD28 mAb 9.3 acted cooperatively to inhibit T cell adhesion to B7, and to block T cell proliferation in primary mixed lymphocyte culture. When coimmobilized with anti T cell receptor (TCR) mAb, anti-CTLA-4 mAbs were less effective than anti-CD28 mAb 9.3 at costimulating proliferation of resting or activated T cells. However, coimmobilized combinations of anti-CD28 and anti-CTLA-4 were synergistic in their ability to augment anti-TCR-induced proliferation of preactivated CD4+ T cells. These results indicate that CTLA-4 is coexpressed with CD28 on activated T lymphocytes and cooperatively regulates T cell adhesion and activation by B7.


Both intrathymic and peripheral selection modulate the differential expression of V beta 5 among CD4+ and CD8+ T cells. Murine T cells expressing V beta 5 are characterized by (a) intrathymic deletion in the presence of I-E and products of endogenous mouse mammary tumor viruses, and (b) a greater representation in CD8+ relative to CD4+ peripheral T cells, thought to be due to more efficient intrathymic positive selection on class I rather than class II major histocompatibility complex antigens. We have engineered mice that are transgenic for a rearranged gene encoding a V beta 5+ beta chain of the T cell receptor for antigen. Deletion is not predicted in I-E- V beta 5+ transgenic mice, and until the age of 2 wk, the CD4/CD8 ratio of peripheral T cells is > 3:1 and indistinguishable between transgenic and nontransgenic mice. Transgenic mice then show a rapid, age-dependent decline in the ratio of CD4+ to CD8+ T cells in the lymphoid periphery, reaching a low of 1:10 by 7 mo of age. Furthermore, the percent of peripheral CD4+ cells that express the transgene drops with age, reaching a low of about 60% at 7 mo, while the percent of CD8+ cells that express V beta 5 remains greater than 95% at all ages. The lymphoid periphery is implicated in this selection against CD4+ V beta 5+ T cells as it occurs more rapidly in thymectomized transgenic mice, and can be delayed in mice whose peripheral T cells are replaced by recent thymic emigrants after depletion by in vivo treatment with anti-Thy-1 antibodies. These results indicate that the relative expression of V beta 5 in T cell subsets can be influenced not only intrathymically in I-E+ V beta 5+ transgenic mice, but also by events in the periphery, in the absence of I-E expression.


Inhibition of mouse mammary tumor virus-induced T cell responses in vivo by antibodies to an open reading frame protein. Minor lymphocyte stimulating (Mls) antigens specifically stimulate T cell responses that are restricted to particular T cell receptor (TCR) beta chain variable domains. The Mls phenotype is genetically controlled by an open reading frame (orf) located in the 3' long terminal repeat of mouse mammary tumor virus (MMTV); however, the mechanism of action of the orf gene product is unknown. Whereas predicted orf amino acid sequences show strong overall homology, the 20-30 COOH-terminal residues are strikingly polymorphic. This polymorphic region correlates with TCR V beta specificity. We have generated monoclonal antibodies to a synthetic peptide encompassing the 19 COOH-terminal amino acid residues of Mtv-7 orf, which encodes the Mls-1a determinant. We show here that these antibodies block Mls responses in vitro and can interfere specifically with thymic clonal deletion of Mls-1a reactive V beta 6+ T cells in neonatal mice. Furthermore, the antibodies can inhibit V beta 6+ T cell responses in vivo to an infectious MMTV that shares orf sequence homology and TCR specificity with Mtv-7. These results confirm the predicted extracellular localization of the orf COOH terminus and imply that the orf proteins of both endogenous and exogenous MMTV interact directly with TCR V beta.


PAR village. Specialized intervention for cocaine abusing women and their children. Operation PAR in St. Petersburg received National Institute on Drug Abuse support in 1989 and state and local appropriations to establish PAR Village, a research demonstration program for treatment of cocaine abusing women with young children. Adjacent to PAR's residential therapeutic community, it includes 14 housing units and a day-care center for infants and children. Women live with their children while receiving long-term residential care. The program is being systematically evaluated by researchers from the University of South Florida Department of Psychiatry and Behavioral Medicine. Retention in treatment and post-discharge outcomes of the women are compared with those involved in the standard program where children remain in the community with relatives or are placed in foster care. Preliminary results suggest the demonstration program increases retention in treatment.


Actions of chiriquitoxin on frog skeletal muscle fibers and implications for the tetrodotoxin/saxitoxin receptor. Chiriquitoxin (CqTX) from the Costa Rican frog Atelopus chiriquensis differs from tetrodoxin (TTX) only in that a glycine residue replaces a methylene hydrogen of the C-11 hydroxymethyl function. On the voltage-clamped frog skeletal muscle fiber, in addition to blocking the sodium channel and unrelated to such an action, CqTX also slows the activation of the fast potassium current in approximately 40% of the muscle fiber population. At pH 7.25, CqTX is as potent as TTX in blocking the sodium channel, with an ED50 of 3.8 nM. Its ED50's at pH 6.50 and 8.25 are 6.8 and 2.3 nM, contrasted with 3.8 and 4.3 nM for TTX. These differences are attributable to changes in the chemical states in the glycine residue. The equipotency of CqTX with TTX at pH 7.25 is explainable by an intramolecular salt bridge between the amino and carboxyl groups of the glycine function, all other surface groups in TTX and CqTX being the same. From available information on these groups and those in saxitoxin (STX), the TTX/STX binding site is deduced to be in a pocket 9.5 A wide, 6 A high, and 5 A deep. The glycine residue of CqTX probably projects out of the entrance to this pocket. Such a view of the binding site could also account for the actions of STX analogues, including the C-11 sulfated gonyautoxins and the 21-sulfocarbamoyl analogues. In the gonyautoxins the sulfate groups are equivalently placed as the glycine in CqTX, whereas in the sulfocarbamoyl toxins the sulfate groups extend the carbamoyl side-chain, leading to steric hinderance to productive binding.


Grayanotoxin-I-modified eel electroplax sodium channels. Correlation with batrachotoxin and veratridine modifications. To probe the structure-function relationships of voltage-dependent sodium channels, we have been examining the mechanisms of channel modification by batrachotoxin (BTX), veratridine (VTD), and grayanotoxin-I (GTX), investigating the unifying mechanisms that underlie the diverse modifications of this class of neurotoxins. In this paper, highly purified sodium channel polypeptides from the electric organ of the electric eel were incorporated into planar lipid bilayers in the presence of GTX for comparison with our previous studies of BTX (Recio-Pinto, E., D. S. Duch, S. R. Levinson, and B. W. Urban. 1987. J. Gen. Physiol. 90:375-395) and VTD (Duch, D. S., E. Recio-Pinto, C. Frenkel, S. R. Levinson, and B. W. Urban. 1989. J. Gen. Physiol. 94:813-831) modifications. GTX-modified channels had a single channel conductance of 16 pS. An additional large GTX-modified open state (40-55 pS) was found which occurred in bursts correlated with channel openings and closings. Two voltage-dependent processes controlling the open time of these modified channels were characterized: (a) a concentration-dependent removal of inactivation analogous to VTD-modified channels, and (b) activation gating similar to BTX-modified channels, but occurring at more hyperpolarized potentials. The voltage dependence of removal of inactivation correlated with parallel voltage-dependent changes in the estimated K1/2 of VTD and GTX modifications. Ranking either the single channel conductances or the depolarization required for 50% activation, the same sequence is obtained: unmodified > BTX > GTX > VTD. The efficacy of the toxins as activators follows the same ranking (Catterall, W. A. 1977. J. Biol. Chem. 252:8669-8676).


Permeation and interaction of monovalent cations with the cGMP-gated channel of cone photoreceptors. We measured the ion selectivity of cGMP-dependent currents in detached membrane patches from the outer segment of cone photoreceptors isolated from the retina of striped bass. In inside-out patches excised from either single or twin cones the amplitude of these currents, under symmetric ionic solutions, changed with the concentration of cGMP with a dependence described by a Hill equation with average values, at +80 mV, of Km = 42.6 microM and n = 2.49. In the absence of divalent cations, and under symmetric ionic solutions, the I-V curves of the currents were linear over the range of -80 to +80 mV. The addition of Ca altered the form of the I-V curve to a new function well described by an empirical equation that also describes the I-V curve of the photocurrent measured in intact photoreceptors. The monovalent cation permeability sequence of the cGMP-gated channels in the absence of divalent ions was PK > PNa = PLi = PRb > PCs (1.11 > 1.0 = 0.99 = 0.96 > 0.82). The conductance selectivity sequence at +80 mV was GNa = GK > GRb > GCs > GLi (1.0 = 0.99 > 0.88 > 0.74 > 0.60). The organic cations tetramethylammonium (TMA) and arginine partially blocked the current, but the larger ion, arginine, was permeant, whereas the smaller ion, TMA, was not. The amplitude of the outward current through the channels increased with the concentration of monovalent cations on the cytoplasmic membrane surface, up to a saturating value. The increase was well described by the adsorption isotherm of a single ion binding site within the channel with average binding constants, at +80 mV, of 104 mM for Na and 37.6 mM for Li. By assuming that the ion channel contains a single ion binding site in an energy trough separated from each membrane surface by an energy barrier, and using Eyring rate theory, we simulated I-V curves that fit the experimental data measured under ionic concentration gradients. From this fit we conclude that the binding site interacts with one ion at a time and that the energy barriers are asymmetrically located within the membrane thickness. Comparison of the quantitative features of ion permeation and interaction between the cGMP-gated channels of rod and cone photoreceptors reveals that the ion binding sites are profoundly different in the two types of channels. This molecular difference may be particularly important in explaining the differences in the transduction signal of each receptor type.


A Gs protein couples P2-purinergic stimulation to cardiac Ca channels without cyclic AMP production. P2-purinergic stimulation of the L-type Ca current induced by the external application of 100 microM ATP gamma S was investigated in rat ventricular cardiomyocytes using the whole-cell patch-clamp technique. The purinergic-induced increase in ICa was slow and monophasic and reached a steady state within 3 min. In contrast to beta-adrenergic stimulation, after a brief agonist application the current did not continue to increase on washout; recovery started immediately after agonist removal. The P2-purinergic increase in ICa was significantly less in the presence of GDP beta S, but it occurred much faster and was twice as large when a low dose of GTP gamma S (100 microM) was added to a GTP-containing internal medium. This suggests that the ICa increase was mediated by a G protein. Based on electrophoretic mobility and susceptibility to cholera toxin and anti-G alpha s serum, it is proposed that the G protein involved during purinergic-induced ICa stimulation is an isoform of Gs not coupled to the adenylyl cyclase, since the cyclic AMP level was unaffected. High intracellular GTP gamma S (1 mM) maximally activated ICa so that neither beta-adrenergic nor P2-purinergic agonists further increased ICa. In the absence of GTP and an ATP-regenerating system, GTP gamma S was much more potent in increasing basal ICa and supporting purinergic stimulation. This indicates that a nucleoside diphosphate kinase activity might replenish endogenous GTP; GTP exchange with GTP gamma S on the G protein was promoted by the P2-purinergic stimulation and led to a reversible and reproducible increase in ICa. In the presence of 3 mM internal ATP gamma S, the P2-purinergic stimulation was also reversible and reproducible. Moreover, under these conditions (ATP gamma S or GTP gamma S) the increase in ICa was not maintained during prolonged agonist application. Such an inhibition occurred slowly and irreversibly; it might be related to the threefold increase in cyclic GMP. In conclusion, we propose that extracellular ATP induces both a stimulatory and an inhibitory effect on ICa, probably mediated by subtypes of P2-purinergic receptors. An isoform of the Gs protein is likely to mediate the stimulation.


New developments in the molecular epidemiology of adenovirus 8 keratoconjunctivitis. Four consecutive epidemics of keratoconjunctivitis caused by adenovirus 8 (Ad8) occurred over a 5-year period in Brest, France. A selection of 30 strains isolated during this period was studied by DNA restriction enzyme analysis using nine restriction enzymes. BglI and SacI were the most discriminative enzymes and allowed the recognition of four DNA variants, all different from the prototype strain Trim. Within each of the epidemics, the strains tested could not be distinguished in this analysis. Between strains from different epidemics differences in DNA structure could be detected however. Thus, the Ad8 epidemics of 1983/1984, 1984, 1987, and 1988 appear to have been due to DNA variants Ad8/D7, D8, D9, and D10, respectively. These results demonstrate that the DNA of Ad8 seems to display a considerable variability, comparable to that observed with Ad7 and Ad21. As has been described for Ad7, Ad21 and Ad41, successive DNA variants of Ad8 prevail during one or more years, and are then replaced by other, newly emerging variants sometimes associated with epidemics.


Case report: detection of varicella-zoster virus DNA in maternal breast milk. A 27-year-old woman developed chickenpox postpartum. Her 2-month-old son who was breast fed also acquired chickenpox 16 days after the onset of the mother's illness. Varicella-zoster virus (VZV) DNA was detected in maternal breast milk by the polymerase chain reaction. These results suggest that transmission of VZV may occur via maternal breast milk.


Passive immunizations of suckling mice and infants with bovine colostrum containing antibodies to human rotavirus. After immunizing 8-month pregnant Holstein cows with the human rotavirus MO strain, cow colostrum containing neutralizing antibody to four different serotypes of human rotavirus, designated Rota colostrum, was obtained. Oral inoculation of human rotavirus MO strain into 5-day-old BALB/c mice causes gastroenteritis characterized by diarrhea. Using this small animal model, passive protection of suckling mice against human rotavirus infection was achieved with the use of Rota colostrum. Rota colostrum completely protected against rotavirus infection, but purified IgG and IgA obtained from Rota colostrum were unable to protect against infection. After grouping randomly 20 infants from a baby care center, 10 infants received 20 ml of Rota colostrum per day for 2 weeks and 10 control infants did not. Rotavirus-associated diarrhea developed in 7 of 10 infants in the control group. None of the three infants in the every day recipient group of Rota colostrum had such symptoms, and one of three infants in the every other day recipient group developed rotavirus-induced diarrhea. All four infants who received Rota colostrum after symptoms appeared developed diarrhea. Oral administration of Rota colostrum seems to be an effective and safe means of preventing diarrhea caused by human rotavirus infection.


Coxsackievirus B4 infection alters thymic, splenic, and peripheral lymphocyte repertoire preceding onset of hyperglycemia in mice. Diabetogenic Coxsackievirus B4 infection may trigger autoimmune islet loss in diabetes-susceptible mice, resulting in hyperglycemia in nearly 90% of the animals at 6-8 weeks postinfection (p.i.). To ascertain whether changes in lymphocyte repertoire following infection could predispose these animals to diabetes, alterations in their thymic, splenic, and peripheral lymphocytes were analyzed. Additionally, lymphocyte changes were correlated with the virus load in these tissues and with lymphocyte migration to the inflammatory pancreas. Splenic B lymphocytes more than doubled at 72 hr p.i. and then continuously decreased by 16% of the noninfected controls at 8 weeks p.i. T lymphocytes (CD4+ + CD8+) decreased by about 50% at 72 hr and then increased to the control level by 8 weeks p.i.; CD8+ subset continuously decreased by 40% of the control at 8 weeks, resulting in a 67% increase in CD4+/CD8+ ratio. Macrophages and CD5+ B subset increased at 72 hr and then dipped by 93% and 84%, respectively, at 8 weeks. In contrast, peripheral B lymphocytes increased by 74% and T lymphocytes decreased by 11% at 8 weeks p.i. Macrophages increased by twofold at 72 hr and then dipped slightly (6%) at 8 weeks, whereas CD5+ B subset increased by 245%. Most prominent thymic T lymphocyte alteration was reflected by about 150% increase in CD4- CD8- cells at 8 weeks p.i. The peak viremia occurred at 72 hr p.i., with highest and lowest virus in the spleen and thymus, respectively. The thymus cleared virus by 3 days, the other tissues by 7 days. Insulitis and acinar necrosis followed infection; infiltrating lymphocytes were mostly CD4+. Virus-induced abnormal lymphocyte maturation may contribute to the development of insulitis and hyperglycemia.


Capsaicin-sensitive peptidergic neurons are involved in the zosteriform spread of herpes simplex virus infection. The intraneuronal transport of herpes simplex virus (HSV) is an essential component in disease pathogenesis. Capsaicin, a neuropharmacologic agent lacking direct antiviral activity, has been shown to protect animals against HSV-induced disease. It has been hypothesized that capsaicin acts by interfering with the intraneuronal transport of virus. Since animal models have been useful in studying the spread of virus, we used two guinea pig models of zosteriform herpes to examine the effect of capsaicin on HSV spread. Capsaicin was subcutaneously administered to Hartley guinea pigs prior to intravaginal or cutaneous HSV-2 inoculation. Treatment did not prevent the development of herpetic vesicles at the site of inoculation but significantly reduced the zosteriform spread of lesions in male and female animals. Further, after recovery from primary infection, capsaicin-treated male guinea pigs were observed to have fewer days with recurrent herpetic lesions. These results suggest that capsaicin-sensitive nerve fibers play a role in the pathogenesis of primary and recurrent HSV infections. Capsaicin appears to reduce the severity of cutaneous HSV infections by interfering with the spread of virus.


Rapid detection of respiratory viruses using mixtures of monoclonal antibodies on shell vial cultures. Eleven hundred and thirty-three clinical specimens submitted to the laboratory for diagnosis of respiratory virus infections were tested by direct immunofluorescence (DIF) for respiratory syncytial virus (RSV), by shell vial culture, and by conventional cell culture. The shell vial cultures were stained with 8 different monoclonal antibodies both 1 day and 3-7 days after inoculation. In order to limit the cost and the workload, mixtures of monoclonal antibodies were used. Coverslips with HEp-2 cells were incubated with a mixture of FITC-labeled monoclonal antibody to RSV and nonlabeled monoclonal antibody to adenovirus. When no RSV positive IF staining was observed after the first incubation step, the same coverslip was incubated once more with FITC-labeled anti-mouse antibody. A positive reaction at this stage indicated the presence of adenovirus. Similarly, cultures of tertiary monkey kidney cells were investigated with a mixture of two FITC-labeled monoclonals to the influenza viruses A and B and three nonlabeled monoclonals to the parainfluenza viruses 1, 2 and 3. If influenza virus or parainfluenza virus was detected, the exact type was determined by staining different parts of a duplicate coverslip. Shell vial cultures for cytomegalovirus (CMV) were always performed separately on human embryonic lung fibroblasts. Using this approach, we detected RSV (n = 248), CMV (n = 42), parainfluenza virus (n = 31), influenza virus (n = 28), and adenovirus (n = 6), in most cases after only one day of culture. For RSV, the sensitivity of the shell vial method was too low (74%) to allow omission of DIF (sensitivity 95%).(ABSTRACT TRUNCATED AT 250 WORDS)


Detection of hepatitis C viraemia in Caucasian patients with hepatocellular carcinoma. Potential risk factors for the development of hepatocellular carcinoma were analysed in 40 Caucasian patients with this malignancy. A higher proportion (14 of 40; 35%) had evidence of hepatitis C virus (HCV) infection than had evidence of either hepatitis B virus (HBV) carriage (17.5%) or alcohol abuse (30%). In all 14 patients whose sera were reactive by HCV ELISA (Ortho second generation test), the presence of antibodies to HCV were confirmed by recombinant immunoblot assay (Ortho RIBA-2). Furthermore, two independent laboratories detected HCV-RNA in 10 of the 14 (71%) anti-HCV positive sera. Two additional sera were shown to contain HCV-RNA when reanalysed by a modified PCR using oligonucleotide primers designed to amplify a shorter fragment of the 5' noncoding region of the genome. Seven of the anti-HCV positive patients also had evidence of prior HBV infection and 2 admitted to alcohol abuse. HCV infection was the only identifiable risk factor in 6 patients. These data confirm the association between HCV infection and hepatocellular carcinoma and suggest that persistent viral replication accompanies tumour development in the majority of patients whose serum contains anti-HCV.


A variant serotype G3 rotavirus isolated from an unusually severe outbreak of diarrhoea in piglets. About 80% of faecal samples from severe outbreak of porcine diarrhoea (scours) were positive for rotavirus. Rotavirus positive samples were analyzed for their antigenic properties and amino acid sequences of the glycoprotein genes. These viruses could not be assigned to any serotypes using serotyping monoclonal antibodies (MAbs) developed for porcine rotaviruses [Nagesha and Holmes: Journal of Medical Virology 35:206-211, 1991b]. When two such viruses were isolated in cell culture and analyzed by neutralization tests using hyperimmune sera they showed only one way antigenic relation with both human and porcine viruses belonging to serotype G3. In addition none of the serotyping MAbs neutralized these two virus isolates. There was no base variation between VP7 genes of faecal and cell culture isolates. Predicted amino acid sequences of the VP7 gene showed marked epitope variation from other porcine type G3 isolates with amino acid substitutions and an additional glycosylation site at residue 238. This antigenic variation seen in rotaviruses appears similar to that of influenza viruses undergoing antigenic drift.


Antibodies to a putative hepatitis C virus polyprotein in Japanese patients with chronic liver disease. To investigate the frequency of exposure to hepatitis C virus (HCV) in chronic liver disease, sera from Japanese patients were tested with the original anti-HCV assay (Ortho) and an anti-HCV assay based on synthetic peptides corresponding to a variety of regions in the HCV genome. Thirty-one (67%) of 46 patients with chronic non-A,non-B hepatitis were anti-HCV-positive by the Ortho ELISA, 20 of whom were also positive by ELISA based on synthetic HCV peptides. Eight (53%) of the 15 patients negative by the Ortho ELISA tested positive for anti-HCV by ELISA based on HCV peptides. Serum HCV RNA was detected in all cases positive for antibody to the HCV peptide and in 14 (78%) of 18 cases without antibody. Thirty-seven hepatitis B virus carriers were without anti-HCV by the Ortho ELISA and were negative for serum HCV RNA, six (16%) of whom were positive by ELISA based on HCV peptides. Antibody responses were directed against each synthetic HCV peptide used, with a considerable difference in incidence, indicating possible expression of the corresponding region in the course of HCV propagation. These findings indicate that exposure to HCV may be more common than expected based on the results of the Ortho ELISA.


Development and organization of the Drosophila olfactory system: an analysis using enhancer traps. Drosophila uses different olfactory organs at different developmental stages. The larval and adult olfactory organs are morphologically dissimilar and have different developmental origins: the antenno-maxillary complex (AMC), which houses the larval olfactory organ, is histolyzed during metamorphosis; the third antennal segment--the principal adult olfactory organ--derives from an imaginal disc. A screen for genes expressed in both larval and adult olfactory organs, but in relatively few other tissues, has been carried out. Seven enhancer trap lines showing reporter gene expression in both the larval AMC and in certain subsets of the adult antenna are described. The antennal staining pattern of one line shows a striking change over the first few days of adult life, with a time course comparable to that of the development of sexual maturity. A pronounced sexual dimorphism in antennal staining pattern is seen in another line. Some staining patterns resemble the patterns of certain classes of antennal sensilla; others show expression restricted to only a small number of cells. Some lines also show expression associated with other chemosensory organs at either the larval or adult stage, including the maxillary palps, labellum, and anterior wing margin. One line, which also shows staining in the male reproductive tract, is male sterile. The significance of these results is considered in terms of (1) the molecular organization of the olfactory system; (2) the recruitment of olfactory genes for use in two developmental contexts; (3) the sharing of genes among different sensory modalities; (4) the role of olfaction in sexual behavior; and (5) posteclosional changes in the olfactory system.


Can technetium-99m-mercaptoacetyltriglycine replace technetium-99m-dimercaptosuccinic acid in the exclusion of a focal renal defect? The presence of focal renal damage dictates different management of a child with urinary tract infection (UTI) compared with children who have normal kidneys. Technetium-99m-dimercaptosuccinic (DMSA) has a high sensitivity in the detection of a focal defect, and allows estimation of differential function. The introduction of 99mTc-MAG3 with high renal extraction suggests that this may be useful in children with UTI but its role remains speculative. Fifty-nine children with previous UTI underwent both 99mTc-DMSA and MAG3 within 4 wk of each other. Differential function and assessment of the images were undertaken. There is close correlation (R2 = 0.97) between the differential function. Analysis of the 99mTc-DMSA and functional MAG3 images showed that the functional image had a specificity of 88% and a sensitivity of 88% in the detection of a focal parenchymal defect. Technetium-99m-MAG3 in the clinical setting of a child with UTl allows accurate assessment of differential function and a high probability of detecting a focal renal abnormality.


Esophageal scintigraphy: reproducibility and normal ranges. Esophageal scintigraphy has been rather widely used, but much debated as a simple screening method of esophageal dysfunction. However, reports of normal ranges, age dependence and reproducibility are very limited. We studied 60 healthy subjects with a mean age of 43 yr (26, 19, 15 subjects aged 20-39, 40-59, and 60-79 yr) to establish normal ranges and variations of esophageal mean transit time and residual activity measured by a radionuclide method using [99mTc] pertechnetate labeled water. Mean transit time was calculated by Zierler's formula. The median values and 95% percentiles of single measurements of MTT and residual activity in the supine position were 6.1 (3.2-11.5) sec and 11.5 (3.0-50)%, respectively. The coefficients of variation (CV) were 20%-35% for mean transit time and 85%-120% for residual activity in the sitting and supine positions. When double measurements were used, the CVs were reduced to 10% for MTT and 40% for residual activity in the supine position. The values did not change with age except for a higher frequency of spikes in subjects over 40 yr. The study has demonstrated that mean transit time for radiolabeled water in the esophagus of healthy subjects, measured by double determinations, has rather low, age-independent, interobserver and intersubject variabilities. In contrast, measurements of residual activity shows unacceptably high variations.


Hypoxia in human gliomas: demonstration by PET with fluorine-18-fluoromisonidazole. Positron emission tomography (PET) with the hypoxic-cell tracer [18F]fluoromisonidazole presents a possible means of noninvasively demonstrating tumor hypoxia. PET studies using this tracer were performed in three patients with malignant glioma, and in all patients the tumor was clearly seen at 5 min postinjection and initial tumor activity exceeded cortical activity. In one patient, there was no tumor retention of [18F] fluoromisonidazole and tumor activity fell while cortical activity increased, with the two tissues reaching equality at 40-50 min. The tumor-to-plasma ratio was 0.71 at 3 hr. The other two patients showed variable tumor retention of [18F]fluoromisonidazole, with tumor-to-plasma ratios of 1.10 and 1.49 at 2 and 3 hr. These results demonstrate the feasibility of using [18F]fluoromisonidazole PET to detect hypoxia in human gliomas in vivo. Clinical trials are needed to determine whether a relationship exists between [18F]fluoromisonidazole uptake and tumor radiation response.


Identification and characterization of inebriated, a gene affecting neuronal excitability in Drosophila. On the basis of behavioral interactions with mutations in a potassium channel gene of Drosophila--Shaker (Sh)--we have isolated mutations in a new gene called inebriated (ine). In a wildtype background, ine mutants display no observable behavioral defects. However, in a Sh mutant background, ine mutations cause downturned wings and an indented thorax. This distinctive phenotype is also exhibited by flies of other genotypes that cause extreme neuronal hyperexcitability. We utilized the potassium channel blocking drugs quinidine and dideoxy forskolin (DDF) to test the effects of ine on synaptic transmission. DDF and ine mutations each potentiated the effects of quinidine on synaptic transmission, but neither had any observable effects in the absence of quinidine. Application of DDF to ine mutants had no effects either in the presence or absence of quinidine. We conclude that ine mutations increase neuronal membrane excitability and perhaps block a DDF-sensitive potassium channel.


Mapping the clock rhythm mutation to the period locus of Drosophila melanogaster by germline transformation. The Clock (Clk) mutation shortens circadian rhythms of locomotor activity and eclosion from ca. 24 h to 22.5-23 h. Clk was previously mapped, by meiotic recombination, very close to the period(per) locus on the X chromosome. To determine whether Clk is a mutation within the per gene or if the former is separate from the latter, two overlapping genomic fragments were cloned from Clk flies to produce a per-containing 13.2 kb construct, per01 flies (which by themselves are arrhythmic)--when transformed with this construct--expressed short-period rhythms. This indicates that the Clk mutation is contained within this 13.2 kb region and is almost certainly a new "fast-clock" allele of per.


Differential expression of the mitochondrial gene cytochrome oxidase II in benign and malignant breast tissue. Comparative analysis of expression levels of genes in benign and malignant tumours of the breast has been performed. Differential screening of cDNA libraries identified four genes of the mitochondrial genome as being expressed at different levels in the two tissues compared, but further investigations showed that only the gene encoding subunit 2 of cytochrome c oxidase (COII) is expressed at significantly higher levels in carcinomas compared with fibroadenomas. The mitochondrial genes encoding subunits 2 and 4 of NADH dehydrogenase, and subunit 6 of F0F1ATPase, were not found to be differentially expressed in carcinomas and fibroadenomas. All four genes were expressed in the epithelium of human breast carcinomas, as shown by in situ hybridization. The expression level of the COII gene is also correlated with carcinoma grade. No gross alterations to the mitochondrial DNA from these tumours could be detected. The possible implications of these results on the behavioural differences between fibroadenomas and carcinomas are discussed.


Immunoreactivity to ubiquitin-protein conjugates is present early in the disease process in the brains of scrapie-infected mice. Brains from mice infected with either the 87V or the ME7 strains of mouse-passaged sheep scrapie were taken at stages during the disease process and immunostained to show the localization of ubiquitin-protein conjugates. In both models, conjugates were seen as fine, dot-like structures; as coarser, granular lesions within or adjacent to neurones; and in areas surrounding plaques. The dot-like structures were visible at 28 days post-ME7 infection and at 55 days in 87V-infected mice. In both models, the extent of immunoreactive changes increased as the disease progressed and terminal infection was as described earlier by us (Lowe et al., J. Pathol 1990; 162: 61-66). The patterns of development of these features were distinctive in two ways: progression from region to region was observable and the density of the pathological lesions grew exponentially as the clinical symptoms appeared. The earliest pathological dot-like structures corresponded temporally with the earliest detection of PrPSC by Western blotting, and immunogold electron microscopic investigation of the dot-like lesions indicated that they were the multi-vesicular, lysosome-related, dense bodies that we have described previously in terminal disease (Laszlo et al., J Pathol 1992; 166: 333-341). Until now, ubiquitin-protein conjugates were seen mainly in inclusion bodies associated with the terminal stages of a range of human degenerative diseases. This study establishes that ubiquitin-protein conjugates accumulate in lysosome-related bodies very early and appear to be intimately related to the pathological processes in the animal disorders that we have studied.


Distribution and localization of Epstein-Barr virus subtypes A and B in AIDS-related lymphomas and lymphatic tissue of HIV-positive patients. Tissue samples of 21 HIV-positive patients have been studied for the presence and distribution of Epstein-Barr virus (EBV) subtypes A and B. This was done by PCR, EBER in situ hybridization, and immunohistochemical detection of EBV latent membrane protein (LMP) in AIDS-associated malignant lymphomas (16 cases) and lymphatic organs of patients without lymphoma (5 cases). Eleven cases were considered to be EBV-positive, with type A in four, and type B virus in four other cases. In patients with malignant lymphoma, EBV was localized in tumour tissue exclusively. One patient without lymphoma presented with multiple EBV genome type B-positive cells in all the lymphoid tissue samples examined. In HIV-positive patients, both subtypes of EBV, A and B, may play a role in the pathogenesis of lymphoproliferative lesions.


Aberrant expression of the tumour suppressor gene p53 is very frequent in Wilms' tumours. A series of 34 Wilms' tumours have been analysed for abnormal expression of the tumour suppressor gene p53 using frozen section immunohistochemistry. All tumours showed immunoreactivity with at least one of the specific antibodies used (monoclonal antibody PAb240, polyclonal antibodies CM1 and JG8). Abnormalities of p53 expression are very frequent in this type of childhood tumour.


Asbestos-stimulated tumour necrosis factor release from alveolar macrophages depends on fibre length and opsonization. Fibre length has been shown to be an important factor in the ability of respirable fibres to cause lung fibrosis and cancer. We have reported that a long sample of amosite asbestos is more carcinogenic and fibrogenic than a short sample of similar diameter. These amosite asbestos samples were studied with regard to their ability to stimulate the release of the pro-inflammatory cytokine tumour necrosis factor (TNF) from rat alveolar macrophages in vitro. The long fibre sample was found to stimulate substantially greater release of the cytokine than the short sample. Furthermore, on treatment of the fibres with rat immunoglobulin G (IgG), there was an increase in the ability of both the long and the short sample to stimulate TNF secretion, although the long sample retained by far the greatest activity. Coating of the fibres with a range of other proteins had no substantial effect on their ability to stimulate TNF secretion. Quartz and titanium dioxide (TiO2) were included as control particles and the TNF-stimulating activity of quartz was notably increased by opsonization with IgG. TiO2 showed a similar low activity to that of the short fibre sample of amosite but this again could be modestly increased by opsonization with IgG. The simulation of TNF release caused by treatment with immunoglobulin-opsonized long fibre amosite could be inhibited by treatment of the macrophages with the protein kinase C-inhibitor staurosporine. The study demonstrates a fibre length-related ability to stimulate cytokine secretion by alveolar macrophages, and its enhancement by opsonization with IgG.(ABSTRACT TRUNCATED AT 250 WORDS)


Correlation between tumor necrosis factor-alpha (TNF-alpha)-induced cytoskeletal changes and human collagenase gene induction. Tumor necrosis factor-alpha (TNF-alpha) has been shown not only to induce the biosynthesis and secretion of collagenase but also to change the organization of cytoskeletal components. In the present study we explore the correlation between the biosynthesis of collagenase (by mRNA hybridization, indirect immunofluorescence and collagenolytic activity), and cytoskeletal reorganization (by rhodamine-phalloidin staining of F-actin) induced in fibroblasts by recombinant TNF (rTNF). In the concentration range of 1-100 ng/ml, rTNF increased extracellular collagenase activity 8-fold and collagenase mRNA 4-fold. In addition, whereas the collagenase mRNA was detected as early as 24 h posttreatment, the appearance of extracellular collagenase activity required 48 h. Using phalloidin to follow the organization of the cytoskeleton we observed that rTNF disrupted the parallel array of stress fibers normally observed in the perinuclear region. In contrast to the time required to affect collagenase synthesis, the effect of rTNF on stress fiber organization occurred as early as 6 h post-treatment. Finally, while the number of cells exhibiting this change increased with increasing concentrations of rTNF, a maximum of about 30% of the cells showed this effect. Interestingly, double staining studies demonstrated that both stress fiber changes and procollagenase production occurred in the same cells. This finding, together with the observation that the cytoskeletal disorganization preceded collagenase gene induction by at least 18 h is consistent with the conclusion that the organizational status of the microfilaments may have a role as a regulator of procollagenase gene expression.


Periodontal bone loss in Porphyromonas gingivalis-infected specific pathogen-free rats after preinoculation with endogenous Streptococcus sanguis. Anaerobic Gram-negative bacteria dominate in periodontitis locations, while Gram-positive bacteria characterize healthy sites. A well-established Gram-positive flora might therefore inhibit the colonization of Gram-negative pathogens. The purpose of the present investigation was to examine whether endogenous S. sanguis could prevent, or reduce, periodontal bone loss in rats infected with a virulent P. gingivalis strain. Sixty specific pathogen-free Wistar rats were divided into 6 groups. Doxycycline was administered in the drinking water for 2 weeks to the groups A, B, C, and D to suppress the preexisting microflora in the mouth. Rats in groups A and C were subsequently inoculated with an S. sanguis strain, isolated from one of the rats, once a day for 5 d. Infection with P. gingivalis 381 was then carried out for 5 d in groups A, B, and E. Group F was not treated with doxycycline nor infected with bacteria and served as untreated control. Six weeks after the P. gingivalis inoculation, the rats were killed. Periodontal bone levels were assessed radiographically and morphometrically, and serum antibody against P. gingivalis 381 was determined by a fluorescence immunoassay. Periodontal bone support, determined radiographically, was reduced in group B (doxycycline-treated, P. gingivalis-inoculated) compared with the other groups. In contrast, the morphometric determination showed no differences between the groups. In group B antibody levels against two different P. gingivalis 381 cell surface antigens were significantly elevated.(ABSTRACT TRUNCATED AT 250 WORDS)


Binding of a cementum attachment protein to extracellular matrix components and to dental surfaces. Cementum proteins (CP) have been shown to mediate cell attachment. Among these, a 55 kDa protein was isolated. The purpose of the present study was to assess the capacity of CP to bind to non-demineralized and demineralized root surfaces and to support cell attachment to dentin. CP were prepared by sequential extraction of bovine cementum with 25 mM EDTA, 0.5 M acetic acid followed by 4 M guanidine HCl. The latter was subjected to ion exchange chromatography on a DEAE-3SW column and eluted stepwise with a 0-0.5 M NaCl gradient. CP were labelled with 125I and the capacity of 125I-CP to bind to mineralized and partially demineralized dentin, synthetic hydroxyapatite, collagen, fibronectin and fibrillar collagen-fibronectin complex was assessed. It was found that CP bind specifically to mineralized dentin and synthetic hydroxyapatite but not to demineralized dentin. The specific binding was 60% of the total binding. SDS-PAGE analysis of the proteins bound to dentin indicated that the main bound protein had a molecular weight of 55 kDa. CP exhibited high affinity for fibronectin (kD = 1.56 x 10(-10) M) and fibronectin-collagen complex, but their binding to either molecular or fibrillar collagen was negligible. It is suggested that CP may play an important role in the attachment of cells of the periodontium to cementum extracellular matrix during homeostasis and regeneration.


Southern blot hybridization and PCR in detection of oral human papillomavirus (HPV) infections in women with genital HPV infections. The presence of human papillomavirus (HPV) in biopsies taken from clinically normal buccal mucosa (n = 212) and clinical lesions (n = 60) was examined by Southern blot hybridization (SBH) using 32P-labelled HPV DNA probes. Furthermore, one hundred formalin-fixed, paraffin-embedded biopsies were analyzed by using polymerase chain reaction (PCR), combined with dot blot hybridization and biotinylated HPV DNA probes. With SBH and PCR, 15.4% and 29.4% of the biopsies, respectively, contained HPV DNA. In clinically normal epithelium, 15.6% and 23.1% of the samples were HPV-positive with SBH and PCR, respectively. The HPV types detected in the genital and oral mucosa of index patients differed in all except two cases. Histology could not be relied on distinguishing HPV DNA positive and HPV DNA negative samples. Hand warts were encountered significantly more frequently in patients with a concomitant oral HPV infection. To conclude, oral HPV infections as detected by SBH and PCR are surprisingly common, but similar to the genital tract, the virus seems to exist in a latent form in the vast majority of cases. The frequent concomitant finding of skin warts and oral HPV infection may suggest some kind of HPV-specific immunosuppression.


Detection of human papilloma virus type 16 DNA in oral squames from normal young adults. We have employed the polymerase chain reaction (PCR) to detect Human Papillomavirus (HPV) type 16 in oral squames and mononuclear cells from 62 healthy young adult volunteers. Two groups were screened for the presence of this virus, but in not all cases was DNA obtained from the scrapes. In the first (n = 30), the results show that 43% of normal individuals harbour HPV 16 (a genital type) in their buccal mucosa, epithelium of dorsum of tongue and hard palate. In the second group (n = 18), 44% of individuals were positive for HPV 16 in their oral epithelial scrapes, while only 6% were positive for the same virus in mononuclear cells. Interestingly, in 2 cases, peripheral blood lymphocyte DNA gave a positive reaction with the HPV 16 primers. To investigate possible HPV infection of lymphocytes, a further 42 lymphocyte samples, taken from the same age group as the epithelial study group, were analysed. None of these lymphocytes were positive for the presence of HPV 16 DNA.


Ontogenesis of peripheral benzodiazepine receptors: demonstration of selective up-regulation in rat testis as a function of maturation. Peripheral benzodiazepine receptors (PBR) have been localized to the outer mitochondrial membrane in a variety of organs, where they apparently play a role in steroidogenesis, oxidative processes, and/or growth and development. Previous studies have demonstrated ontogenetic changes in heart and lung PBR, with maximal PBR density at 31 days, as opposed to negligible changes in brain PBR during the prenatal through postnatal periods. The present study was designed to examine the influence of maturation and aging upon PBR binding characteristics. Rats aged 1, 2, 12, 18, and 24 months were sacrificed, and the following organs were removed according to standard protocol: heart, lungs, kidneys, adrenal gland, and testes. Binding studies were performed using [3H]PK 11195 as a radioligand. A 3-fold increase in PBR density was demonstrated in testis during maturation, with maximal values appearing at 18 months, followed by a decline at 24 months. None of the other organs examined showed significant changes in PBR density. No alterations were observed in affinity values for the various organs and ages. Since testicular PBR are putatively involved in testosterone production, these results might reflect critical interactions between PBR and gonadal hormone activity during development.


Invasive cervical cancer in young women. Clinicopathologic correlation and demonstration of human papillomavirus by in situ hybridization. It has been suggested that invasive cervical carcinoma is biologically more aggressive in young patients than in older patients. The clinicopathologic characteristics and human papillomavirus (HPV) status of women < or = 35 years of age with invasive cervical carcinoma treated at Emory University Hospital from 1985-1989, were evaluated in a retrospective study. The group consisted of 23 patients, 35% of all patients with invasive cervical carcinoma. Age at diagnosis, clinical stage, histologic classification, lymph node status, treatment and clinical follow-up were obtained from medical records. HPV status was evaluated by colorimetric in situ hybridization for HPV types 6, 11, 16, 18, 31, 33 and 35 in archival formalin-fixed paraffin-embedded tumor material available from 16 of the patients. This study found that the young patients had lower survival and greater extent of disease than predicted by clinical staging. The rate of detection of HPV (69% positive) and HPV typing (type 16 predominated) in these young patients showed no difference from other series of cervical carcinomas from all age groups. Although a higher proportion of patients who died of disease had HPV-negative tumors (3/7, 43%) than those patients who were alive and free of disease (2/9, 22%), this difference was not statistically significant (P = .73, Fisher's Exact T-Test).


Culicoides boydi (Diptera: Ceratopogonidae): a potential vector of hemorrhagic disease viruses to desert bighorn sheep in southern California. Culicoides boydi new species is described from Riverside County, CA. A taxonomic key, table of numerical characters, and female wing photographs are presented to distinguish it from the three other species of the Culicoides pusillus species group of the subgenus Avaritia. Brief notes are presented on its biology and distribution and suspected vector potential in the transmission of bluetongue virus to desert bighorn sheep in California.


Dietary intake of fiber and decreased risk of cancers of the colon and rectum: evidence from the combined analysis of 13 case-control studies. Colorectal cancer is a major public health problem in both North America and western Europe, and incidence and mortality rates are rapidly increasing in many previously low-risk countries. It has been hypothesized that increased intakes of fiber, vitamin C, and beta carotene could decrease the risk of colorectal cancer. The objective of this study was to examine the effects of fiber, vitamin C, and beta-carotene intakes on colorectal cancer risk in a combined analysis of data from 13 case-control studies previously conducted in populations with differing colorectal cancer rates and dietary practices. The study was designed to estimate risks in the pooled data, to test the consistency of the associations across the studies, and to examine interactions of the effects of the nutrients with cancer site, sex, and age. Original data records for 5287 case subjects with colorectal cancer and 10,470 control subjects without disease were combined. Logistic regression analysis was used to estimate relative risks and confidence intervals for intakes of fiber, vitamin C, and beta carotene, with the effects of study, sex, and age group being adjusted by stratification. Risk decreased as fiber intake increased; relative risks were 0.79, 0.69, 0.63, and 0.53 for the four highest quintiles of intake compared with the lowest quintile (trend, P < .0001). The inverse association with fiber is seen in 12 of the 13 studies and is similar in magnitude for left- and right-sided colon and rectal cancers, for men and for women, and for different age groups. In contrast, after adjustment for fiber intake, only weak inverse associations are seen for the intakes of vitamin C and beta carotene. This analysis provides substantive evidence that intake of fiber-rich foods is inversely related to risk of cancers of both the colon and rectum. If causality is assumed, we estimate that risk of colorectal cancer in the U.S. population could be reduced about 31% (50,000 cases annually) by an average increase in fiber intake from food sources of about 13 g/d, corresponding to an average increase of about 70%.


Enhancement of nitrosourea activity in medulloblastoma and glioblastoma multiforme. Although chemotherapy offers promise of increased survival for children with medulloblastoma and glioblastoma multiforme, drug resistance occurs frequently, resulting in tumor progression and death. Resistance to nitrosoureas and methylating agents, which damage DNA, can be mediated by a DNA repair protein, O6-alkylguanine-DNA alkyltransferase (AGAT). Depletion of this protein with alkylguanines or methylating agents, however, restores tumor cell sensitivity to the cytotoxicity of chloroethylnitrosoureas (e.g., carmustine [BCNU]). This study was designed to determine whether resistance to the activity of nitrosourea (the drug BCNU) in BCNU-resistant human medulloblastoma (D341 Med) and human glioblastoma multiforme (D-456 MG) can be reversed by the methylating agent streptozocin and the O6-substituted guanines O6-methylguanine and O6-benzylguanine. Xenografts were grown subcutaneously in athymic BALB/c mice. BCNU was administered as a single intraperitoneal injection at doses of 100 mg/m2, 75 mg/m2, or 38 mg/m2--i.e., 1.0, 0.75, or 0.38, respectively, of the dose lethal to 10% of treated animals (LD10). Mice were treated intraperitoneally with a single dose of O6-benzylguanine or O6-methylguanine (240 mg/m2) or with streptozocin (600 mg/m2) daily for 4 days. Response was assessed by tumor growth delay and tumor regression. AGAT activity in the xenografts was measured at 1 and 6 hours after pretreatment, at the time tumors were excised. Pretreatment with O6-benzylguanine, O6-methylguanine, or streptozocin reduced AGAT activity to 4%, 25%, and 95% of control values, respectively, in D341 Med and 0%, 0%, and 25% of control values, respectively, in D-456 MG 1 hour after injection. After 6 hours, levels changed to 7%, 61%, and 116% of control values in D341 Med and 0%, 79%, and 21% of control values in D-456 MG, respectively. Both D341 Med and D-456 MG xenografts were completely resistant to BCNU at its LD10. Pretreatment with O6-benzylguanine increased BCNU sensitivity in both types of xenograft. In contrast, treatment with BCNU plus O6-methylguanine or streptozocin did not produce growth delays substantially different from those produced by BCNU alone, reflecting the more efficient depletion of AGAT by O6-benzylguanine. Following therapy with BCNU plus O6-benzylguanine at 0.38 LD10, tumor regressions were seen in eight of 10 D341 Med and in all 10 D-456 MG xenografts. We recommend comprehensive clinical toxicologic evaluation of combination therapy with O6-benzylguanine plus BCNU, which would allow subsequent design of phase I clinical trials.


Diffuse hyperpigmentation associated with acquired immunodeficiency syndrome. This article reports a case of diffuse hyperpigmentation in a 56-year-old Latino male after he was diagnosed with acquired immunodeficiency syndrome (AIDS). The possible causes of this hyperpigmentation are discussed.


The Fifth Edition of the Addiction Severity Index. The Addiction Severity Index (ASI) is 12 years old and has been revised to include a new section on family history of alcohol, drug, and psychiatric problems. New items were added in existing sections to assess route of drug administration; additional illegal activities; emotional, physical, and sexual abuse; quality of the recovery environment; and history of close personal relationships. No changes were made in the composite scoring to maintain comparability with previous editions. This article discusses the clinical and research uses of the ASI over the past 12 years, emphasizing some special circumstances that affect its administration. The article then describes the rationale for and description of the changes made in the ASI. The final section provides "normative data" on the composite scores and severity ratings for samples of opiate, alcohol, and cocaine abusers as well as drug abusing inmates, pregnant women, homeless men, and psychiatrically ill substance abusers.


A comparison of male and female clients in substance abuse treatment. To assess the extent to which women appear to have special treatment needs, this paper compares male and female patients receiving inpatient substance abuse treatment. The author analyzed completed intake interview forms from the files of all clients entering two private, nonprofit inpatient substance abuse treatment facilities during an 8-month period in 1989 (a total of 181 men and 48 women). The women were similar to the men with respect to sociodemographic characteristics, family history, alcohol/drug history, and treatment completion. However, they were more likely to report a sexual abuse history and indicated more emotional distress than the men. Implications for treatment are discussed.


Organizational change required for paradigmatic shift in addiction treatment. The requirement for major organizational change to accomplish a paradigmatic shift for a staff at a residential addiction treatment center in Central Texas necessitates a number of strategies. The paradigmatic shift involves moving a staff accustomed to conceptualizing addiction treatment as a fixed-length program with similar components for all and levels of care as discrete programs to the concept of treatment as a flexible process that involves fluid movement of a patient through an individualized set of program components and multiple levels of care within one treatment episode. Conceptualization of the programmatic changes, necessary language changes, and the use of new criteria for patient placement and flexible movement between levels of care results not only in improved patient care, but also in improved staff morale if handled with cognizance for the principles of organizational change and adult education.


[Low reactive-level laser therapy near the stellate ganglion for postherpetic facial neuralgia]. Low reactive-level laser therapy near the stellate ganglion was given for a 68-year-old female with postherpetic neuralgia, suffering from burning pain in the right forehead for 11 years. Stellate ganglion block and supraorbital nerve block with oral medication were not effective to relieve this pain. The laser irradiation induced warm sensation in her face followed by an excellent pain relief. Thermograms illustrated a remarkable increase from 30.6 degrees C to 31.5 degrees C in temperature of her right face. The irradiation near the right carotid artery also had the similar effect. The results imply that the irradiation with low reactive-level laser of the stellate ganglion and/or the carotid artery increases a facial blood flow and relieves facial neuralgia.


[Diagnostic significance of angiography for small hepatocellular carcinoma--comparison with other examinations]. The diagnostic value of angiography for small hepatocellular carcinoma was evaluated in comparison with histology, contrast-enhanced computed tomography, and ultrasonography. A total of 120 patients with small hepatocellular carcinoma (less than 3 cm in size) were examined. The definitive detection rate for primary tumors less than 2 cm in size was 44.9% by angiography, while it was 68.6% for primary tumors between 2 cm and 3 cm in size. When the primary tumor was less than 2 cm in size and without tumor vessels on angiography, it tended to be of Edmondson's grade 1 and to show fatty change. When the primary tumor was less than 2 cm in size and without tumor stain while non-cancerous parenchyma showed irregular stain, it tended to be of Edmondson's grade 1 and normotrabecular type. Angiography was found to be of particular value in detecting satellite tumors with a nodular parenchymal echo pattern in non-cancerous areas, because ultrasonography often fails to differentiate these satellite tumors from non-affected parenchyma.


[Multivariate analysis on the relationship between subjective symptoms and risk factors for the development of symptoms including working conditions, life habits and physical status in forestry workers using chain saw]. To determine the risk factors of the subjective symptoms experienced by chain saw workers, we surveyed 206 forestry workers using this device in their work. The strength of the relationship between their symptoms and such potential risk factors as occupational, behavioral, and physical factors was evaluated by a multiple logistic regression model. It was found that many years of chain saw operation, and numerous hours of chain saw operation per day, increased the risk of white finger, numbness of hands and arms, chillness of hands and arms and difficulty in joint movement, indicating that these symptoms were closely related to vibration exposure. Many years of forestry work before chain saw work also increased the risk of joint pain, suggesting that bone and joint disorders are related to heavy manual work not using a chain saw. A second job involving manual labor increased the risk of weakness in the arms, stiffness of the shoulders, and lumbago. Infrequent bathing was associated with increased risk of pain in hands and arms and lumbago. Older workers had a higher prevalence of stomach discomfort. No risk factor was found to significantly increase the risk of easy fatigability, forgetfulness and sleep disorder. It was found that not only exposure to vibration but also other factors contributed to the prevalence of pain in hands and arms, joint pain, weakness in arms, stiffness of shoulder, and lumbago. Second job and infrequent bathing appeared to be related to the occurrence of certain subjective symptoms. These contributory factors should be taken into account when evaluating subjective symptoms encountered in field studies of forestry workers.


An attempt to eradicate Herpesvirus simiae from a rhesus monkey breeding colony. In the fall of 1987 an attempt to establish a Herpesvirus simiae (B-virus)-negative rhesus monkey (Macaca mulatta) breeding colony was initiated at the Armstrong Laboratory. A serologic testing program was used to identify all monkeys into groups that were either positive or negative to B-virus based on serologic tests. Segregation of the groups allowed the creation of breeding harems that were exclusively seropositive or -negative to B-virus. Animals that were serologically positive were kept in breeding to maintain infant production levels not unlike those previous to segregation. Decreasing numbers of animals converted to a positive status during the first three serum tests for B-virus in the program. During 1990, an increase in the number of monkeys converting to positive status and the discovery of an indeterminate status demonstrated that latency of B-virus in the rhesus may have the potential to defeat an eradication attempt not conscientiously pursued.


[Neurologic syndromes associated with anti-Hu antibody. Study of 24 patients]. Twenty-four patients with neurologic involvement and anti-Hu antibodies were studied with the aim of defining the type of tumor associated, evaluating whether the clinico-pathologic picture agreed with the concept of paraneoplastic encephalomyelitis (PEM) and evaluating the treatments used. The study was retrospective with the clinical histories being reviewed to define the neurologic syndromes, their evolution and response to the different treatments, time of appearance and type of tumor as well as the neuropathologic changes in the patients undergoing autopsy. In 18 patients a neoplasm was diagnosed as small cell pulmonary carcinoma (SCPC) in 89% of the cases. The neurologic picture preceded the tumor by an average of five months. The clinical pictures included: sensitive neuropathy (20 patients), cerebellous and truncus encephalicus involvement (8 patients), motor neuropathy (6 patients), cortical involvement (5 patients) and neurovegetative dysfunction (4 patients). In 55% of the patients more than one area was altered. Post mortem studies carried out on 5 patients demonstrated inflammatory infiltrates and neuronal loss in multiple areas of the nervous system. None of the patients improved with treatment. The 9 patients who only received immunodepressants evolved in a way similar to those who were not treated. In 7 of the 11 patients who received antitumoral therapy, the neurologic syndrome stabilized for at least 6 months. The clinicopathological picture and the associated tumor seen in patients with anti-Hu antibodies are identical to those seen in PEM. Antitumoral treatment seems to be more effective than immunodepressant treatment.


[Embolization of the hepatic artery in diffuse malignant tumors of the liver]. The paper presented the results of therapeutic embolization of the hepatic artery (EHA) in 20 patients with malignant tumors that involved more than 70% of the liver. Simultaneous occlusion of all the arteries running to the tumor caused a severe hepatic failure and the death of 2 out of 4 patients. The blood flow reduction in 4 patients appeared to be safe but effective partially. Successive lobular EHA resulted in the improvement of the condition in 3 patients and failed in 2 cases. Two-staged EHA (first reduction and then occlusion) turned to be safe and in 6 out of 7 patients had led to significant improvement. Advantages and faults of the embolization technique considered are discussed.


[The use of molecular epidemiologic markers in the study of an epidemic outbreak of legionnaires' disease of nosocomial origin]. Epidemiological investigations of Legionella infections are based, since recently, on molecular techniques that are more sensitive and specific than phenotypic traits. We were interested in these methods for subtyping isolates of L. pneumophila serogroup 1 and confirm the epidemic spread of an outbreak of legionnaires' disease at the Universitary Hospital Germans Trias i Pujol (HUGTiP) in Badalona. Environmental samples taken from domestic water, heating and cooling water systems and oxygen humidifiers were examined. Clinical and environmental isolates of L. pneumophila serogroup 1 were compared by analysis of genomic DNA by restriction endonucleases. We could found L. pneumophila serogroup 1 and 9 in domestic hot water and heating systems and L. micdadei in cooling water system. Cleavage of genomic DNA showed that all restriction fragment patterns coming from clinical and environmental isolates of L. pneumophila serogroup 1 were identical and different from isolates belonging to the same species and serogroup but coming from community area. Molecular analysis of clinical and environmental isolates of L. pneumophila serogroup 1 has allowed to identify a reservoir related to a nosocomial outbreak of legionnaires' disease at the HUGTiP, and a clonal population of L. pneumophila serogroup 1 in environmental samples genotypically identical to the clinical ones.


[Subtraction scintigraphy with thallium-201 chloride and technetium-99m pertechnetate versus high resolution ultrasonography in the localization of the parathyroid glands in primary hyperparathyroidism]. The aim of this study was to evaluate the use of scintigraphy with thallium-201 chloride (201 Tl) and technetium-99m pertechnetate (99mTc) in the diagnosis of the localization of the pathological parathyroid glands in primary hyperparathyroidism and compare the results with those of high resolution ultrasonography. Twelve patients of 56.1 +/- 7.8 years of age diagnosed with primary hyperparathyroidism were studied between March 1987 and June 1990. High resolution ultrasonography with a 7.5 MHz transducer and scintigraphy of digital subtraction with 201Tl-99mTc were carried out preoperatively in all the patients. None of the patients had had previous cervical surgery and diagnosis was proven following surgery by histopathologic study. Ultrasonography detected 9 out of 11 adenomas and 3 out of 4 hyperplastic glands. Scintigraphy identified 9 adenomas and only 2 of the hyperplastic glands. With this latter technique there was one false positive. The sensitivity of ultrasonography was 80% and that of scintigraphy was 73% with specificity being 100% vs 96.9%, respectively. Subtraction 201 Tl-99mTc scintigraphy is useful in the diagnosis of the localization of pathologic parathyroid glands in patients with no previous cervical surgery although in this series this technique did not surpass that of high resolution ultrasonography.


[BK and JC viruses--2 polyomaviruses causing disease in immunosuppressed patients]. BK and JC viruses are two polyoma viruses designated by the initials of the patients from whom they were first isolated. After the primary infection, usually occurring in childhood or early school age, the viruses become latent. Reactivation occurs during immunosuppression, and the BK virus has been shown to be the main cause of viral hemorrhagic cystitis in bone marrow transplanted patients, while the JC virus has been found to cause progressive multifocal leukoencephalopathy, PML. The paper consists in a report of results obtained with an established method for the amplification of BK and JC virus DNA. Of 20 urine specimens from patients with hemorrhagic cystitis, 13 were found to be BK virus-positive. Post-transplantation follow-up shows that the virus continues to remain detectable for long periods. JC virus positivity was found in central nervous system material (a cerebrospinal spinal fluid specimen in one case) from two PML patients.


Proton relaxation enhancement by means of serum albumin and poly-L-lysine labeled with DTPA-Gd3+: relaxivities as a function of molecular weight and conjugation efficiency. A series of poly-L-lysine chains, with molecular weight ranging from 3300 up to 102,000 Da, were labeled with DTPA-Gd3+. No significant differences in longitudinal and transversal relaxivity, could be demonstrated as a function of the chain length. The R1 and R2 relaxivities were respectively 2.5 and 5 times superior to those of plain DTPA-Gd3+ (at 2.4 T). Bovine serum albumin was also labeled in a way that a wide (DTPA-Gd3+)/BSA range (3-39) was obtained. The longitudinal relaxivity, of these paramagnetically labeled albumins, increased with increasing (DTPA-Gd3+)/BSA ratios. This effect was most pronounced at very low (DTPA-Gd3+)/BSA ratios.


Temporal lobe epilepsy of the rat: differential expression of mRNAs of chromogranin B, secretogranin II, synaptin/synaptophysin and p65 in subfield of the hippocampus. We have investigated by in situ hybridization changes in the content of mRNAs encoding for chromogranin B, secretogranin II, synaptin/synaptophysin and p65 after kainic acid-induced seizures and pentylenetetrazol kindling. Kainic acid seizures resulted in marked but transient increases in secretogranin II mRNA concentrations in the granule cell layer and throughout the pyramidal cell layers of the hippocampus (by 100-500%) as well as in various areas of the cerebral cortex (by up to 900%) and the thalamus (up to 300%) 12 h after injection of the toxin. Chromogranin B mRNA concentrations were persistently increased in granule cells (but not in pyramidal cells) of the hippocampus (suprapyramidal blade, 450%) and in cortical areas (250%) at all time intervals after kainic acid injection (12 h to 60 days). Accordingly chromogranin B immunoreactivity was enhanced in the terminal field of mossy fibers and in the inner part of the molecular layer 30 days after kainic acid. Secretogranin II immunoreactivity was also markedly increased in CA1, the paraventricular thalamic nucleus and in the central amygdala. In rats kindled with pentylenetetrazol only chromogranin B (by 200%) but not secretogranin II mRNA was increased in dentate granule cells. In contrast to the mRNAs of these secretory proteins concentrations of mRNAs encoding synaptin/synaptophysin and p65, two membrane proteins of synaptic vesicles, were not altered in any of these brain structures. These data demonstrate that in brain the biosynthesis of chromogranin B and secretogranin II is regulated like that of neuropeptides which is consistent with a role of these secretory polypeptides as precursors of functional peptides. Activation of neurons induces an increased synthesis of neuropeptides but not a concomitant synthesis of membrane proteins of synaptic vesicle. This might lead to an increased quantal content available for transmission.


Neurotransmitter-stimulated immediate-early gene responses are organized through differential post-synaptic receptor mechanisms. The products of the cellular immediate-early genes (IEGs) are thought to act as messengers in the coupling of trans-synaptic stimuli with altered neuronal gene expression. However, the manner in which neurotransmission specifies particular responses through the IEGs is undefined. In this report, mRNA and transcription analysis of a precisely-timed, physiological IEG response illustrates how an IEG signal may be organized through differential neurotransmitter receptor activation. The nocturnal pattern of IEG expression in the rat pineal gland has been shown to be differentially regulated through post-synaptic adrenergic receptors. Induction of the c-fos gene is primarily mediated through alpha 1-receptors, whereas the coordinately regulated jun-B gene exhibits dual regulation through alpha 1- and beta-receptors. A simultaneous repression of c-jun expression is partly mediated through a beta-receptor mechanism. In vitro analysis of IEGs in cultured pineal glands has confirmed the receptor-specific link between adrenergic neurotransmission and IEG induction. The pineal is a unique neuroendocrine model in which the characteristics and function of the IEG third messenger system may be defined.


Glucocorticoid regulation of preproenkephalin gene expression in the rat forebrain. The effects of glucocorticoids on the levels of preproenkephalin (PPE) mRNA in the rat forebrain were analyzed with in situ hybridization and dot blots. In adrenally-intact rats, high levels of PPE mRNA, as assessed by in situ hybridization, were localized in the caudate-putamen, nucleus accumbens, central amygdala, and ventrolateral ventromedial hypothalamus (VMHVL), and low levels in the hippocampus. After adrenalectomy, the density of PPE mRNA-positive cells and the level of PPE mRNA/cell were decreased in all regions except the hippocampus. Acute treatment with corticosterone (CORT) in adrenalectomized rats increased the level of PPE mRNA/cell in the caudate-putamen and VMHVL. In intact rats, chronic treatment with CORT increased the density of PPE mRNA-positive cells in the caudate-putamen and hippocampus, and the level of PPE mRNA/cell in the caudate-putamen and nucleus accumbens. The effect of chronic CORT treatment on PPE mRNA in the striatum, amygdala, hippocampus and mediobasal hypothalamus was assessed with dot blots. Chronic CORT treatment increased PPE mRNA levels in the caudate-putamen and hippocampus. There was a good correlation between results on the effect of chronic CORT treatment on PPE mRNA levels in intact rats, obtained from dot blots and in situ hybridization. Results from this study suggest that glucocorticoids are required for the maintenance of basal PPE mRNA levels in most regions of the rat forebrain. There is, however, considerable regional heterogeneity in the effect of glucocorticoid treatment on PPE mRNA levels in adrenalectomized and intact rats. Increased PPE mRNA levels in response to high circulating levels of glucocorticoids, e.g. in stress, may have important pathophysiological consequences.


Expression of amyloid precursor protein mRNAs in endothelial, neuronal and glial cells: modulation by interleukin-1. The origin of beta-amyloid deposited in senile plaques in Alzheimer's disease (AD) is not known. We compared the expression of protein precursor of beta-amyloid (APP) in the cell types involved in plaque formation. The levels of APP mRNA were determined in primary rat neurons and glial cells in culture, human endothelial cells and in a murine brain-derived endothelial cell line. Northern blot analysis was performed using an APP cDNA probe to detect the general APP sequence and an oligonucleotide (40 mer) complementary to the sequence of the Kunitz protease inhibitor (APP-KPI). The APP mRNA transcripts were abundant in all three cell types. The highest level of APP, normalized to beta-actin mRNA content, was expressed in neurons, followed by glial cells, where the APP expression was similar (94%) while in endothelial cells was lower (53%). The proportion between APP-KPI mRNA and total APP mRNA was high in endothelial, intermediate in glial and low in neuronal cells. We compared the effects of exposure to interleukin-1 (IL-1), a cytokine involved in several biological processes and elevated in AD, on APP mRNA expression in neuronal, glial and endothelial cells. In human endothelial and in brain-derived murine endothelial cells we observed a similar increase (50%) of total APP mRNA or APP-KPI mRNA after treatment with human recombinant IL-1 beta. In neuronal cells, IL-1 (200 ng/ml) substantially increased APP mRNA (175%), detected with both probes. In glial cells, the expression of APP mRNA did not appear to be altered by IL-1 (50-400 ng/ml). The results suggest a role of IL-1 in the neuronal mechanisms related to beta-amyloid protein deposition in AD.


Expression of the tissue specific splicing protein SmN in neuronal cell lines and in regions of the brain with different splicing capacities. The SmN protein is closely related to the ubiquitously expressed SmB and B' RNA splicing proteins but is expressed in only a limited range of tissues and cell types. The expression of SmN in a range of neuronal and non-neuronal cell lines correlates with their ability to splice the calcitonin/CGRP transcript to produce the mRNA encoding CGRP rather than that encoding calcitonin. Moreover, the SmN mRNA shows a widespread distribution within the brain and spinal ganglia being present in neuronal cells in all regions which naturally produce CGRP as well as in those areas which do not naturally express the calcitonin/CGRP gene but which can correctly splice the CGRP mRNA in transgenic mice expressing the calcitonin/CGRP gene in all cell types. Interestingly however the mRNA encoding SmN is also found in a few areas of the brain which can only carry out calcitonin-specific splicing in transgenic mice, such as the Purkinje layer of the cerebellum and the inferior colliculus. The possible role of SmN in the regulation of splicing in neuronal cells is discussed in the light of these results.


Expression of the pro-opiomelanocortin gene in dorsal root ganglia, spinal cord and sciatic nerve after sciatic nerve crush in the rat. Neuropeptides related to alpha-melanocyte-stimulating hormone (alpha-MSH) stimulate nerve outgrowth following peripheral nerve injury and may play an important physiological role in peripheral nerve regeneration. The mechanism of action underlying the neurotrophic effect of pharmacologically administered alpha-MSH is unknown. Here we investigate the hypothesis that reexpression of the proopiomelanocortin (POMC) gene, the prohormone of alpha-MSH/adrenocorticotropic hormone (ACTH)-like peptides, is part of the endogenous repertoire of peripheral nerve responses following injury. The effect of sciatic nerve crush on the expression of POMC mRNA between 0.5 h and 14 days after crush was investigated using polymerase chain reaction (PCR) and Northern blot analysis. The presence of a POMC transcript in dorsal root ganglia (DRG), spinal cord and in the sciatic nerve at the crush site could be demonstrated in both control and lesioned animals by PCR using primers located in exon 1 and 3 of the POMC gene. Minute quantities of two POMC transcripts (1200 nt and 800 nt) could be detected by Northern blot analysis of total RNA prepared from DRG, spinal cord and the sciatic nerve of control animals and of animals subjected to nerve crush. POMC mRNA expression was, however, not increased following nerve crush. Probes specific for exons 1 and 2 or specific for exon 3 of the POMC gene were employed to demonstrate that the 800 nt transcript represents the truncated POMC mRNA previously shown to be present in extra-pituitary tissue. The larger 1200 nt transcript comigrates with the full length POMC mRNA expressed in the pituitary gland. The present results demonstrate the expression of small amounts of POMC mRNA in all compartments of the sciatic nerve. The absence of an induction of POMC expression in response to nerve crush suggests that the stimulating effect of exogenously applied alpha-MSH does not mimic a POMC derived neurotrophic peptide induced in the nerve following nerve injury.


Regulation of prodynorphin gene expression in the hippocampus by glucocorticoids. The regulation of prodynorphin gene expression by glucocorticoids in the hippocampus was examined in rats that were adrenalectomized (ADX) either 7, 30, 60 and 90 days prior to sacrifice. Peptide levels in the hippocampus of ADX rats were determined by radioimmunoassay and immunocytochemistry. Prodynorphin (PDYN) mRNA was measured by Northern blot analysis and in situ hybridization. A time-dependent decrease in dynorphin A(1-8)(DYN) levels in the hippocampus (18% at 7 days; 44% at 30 days; 58% at 60 days) of ADX rats was found, which was accompanied by a comparable decrease in the abundance of PDYN mRNA. An in situ hybridization analysis revealed that both the number of positively hybridized cells and the number of silver grains per cell were decreased in the dentate gyrus after ADX. The administration of dexamethasone after surgery reversed the peptide and mRNA attenuation induced by ADX. ADX had no effect on the expression of proenkephalin mRNA or [Met5]-enkephalin immunoreactivity in the hippocampus. Examination of thionin-counterstained tissue showed that the dentate granule cell layer was intact. The decrement of DYN expression in this system is proposed to have resulted from the removal of glucocorticoid input and not dentate granule cell loss. This study provides the strong evidence for a differential susceptibility of these two opioid peptides in the hippocampus to the removal of glucocorticoids. In addition, these data provide support for a potentially selective, glucocorticoid-permissive component in PDYN gene expression.


Temporal and spatial expression of a fos-lacZ transgene in the developing nervous system. A Fos-lacZ transgenic mouse has been described that accurately recapitulates both constitutive and inducible patterns of c-fos expression in adult mice. Here we describe the developmental expression of the transgene in the brain during the early postnatal period. On the day of birth, expression of the transgene is observed in several discrete regions of the CNS; including the olfactory bulb, hippocampus, retrosplenial cortex, parafascicular nucleus of the thalamus, and several cranial nerve nuclei. In these regions, expression declines to adult levels by three weeks. In other regions of the CNS, expression appeared transiently after P0.


Cellular alterations produced by the experimental increase in intracellular calcium and the nature of protective effects from pretreatment with nimodipine. The immortalized septal cell line, SN56 B5 G4, generated by the fusion of mouse septal area cells and neuroblastoma cells, was used to determine if nimodipine, an antagonist of voltage sensitive calcium 'L' channels, might act in a neuroprotective fashion when intracellular calcium levels were raised by incubation in ouabain and monensin. Fluorescent indicator dyes and the automated spectrofluorometer, the CytoFluor 2300, were used to analyze specific cellular targets and functions affected by ouabain and monensin and possible protection by prior incubation with nimodipine. Ouabain and monensin were used together to create a time- and dose-dependent toxic episode. Increases in the emission intensity of Fluo3-AM demonstrated that the concentration of intracellular calcium was monotonically increased by increasing levels of ouabain-monensin. The calcein-AM fluorescent probe indicated that there were no changes in plasma membrane permeability during the toxic episode. Lysosomal integrity decreased as indicated by decreases in neutral red retention. The concentration of free radicals increased as shown by the increase in emission intensity of 2',7'-dichlorfluorescein. Nimodipine pretreatment of the cells incubated with ouabain and monensin resulted in apparent protection of lysosomes and a reduction in the level of free radicals. While nimodipine, by itself, produced a small decrease in intracellular calcium, it actually augmented the ouabain-monensin induced increase in intracellular calcium. The data suggest that in immortalized septal cells, (a) nimodipine offers protection to certain of the responses induced by ouabain-monensin, (b) the protection offered by nimodipine may be independent of antagonism of voltage sensitive calcium channels, and (c) that the protective changes can occur at the same time that intracellular calcium is increasing. These latter observations question the hypothesis that the protection against cell death and dysfunction offered by nimodipine is due solely to maintaining calcium homeostasis.


Gene expression of Ca2+/calmodulin-dependent protein kinase of the cerebellar granule cell type or type IV in the mature and developing rat brain. The localization and ontogenic changes of expression of the mRNA for Ca2+/calmodulin-dependent protein kinase of the cerebellar granule cell type or type IV (CaM kinase Gr or IV) in the rat brain were examined by in situ hybridization histochemistry. At the young adult stage, intense expression signals for this kinase mRNA were detected in the cerebellar granule cells, the hippocampal pyramidal cells, the dentate granule cells, and the piriform cortex. Moderate levels of the mRNA were expressed in the thalamic nuclei and the cerebral cortex. No distinct expression signals were detected in the Purkinje cells and most brainstem nuclei except for the pontine nuclei, locus ceruleus and inferior olive which showed weak expression. During development, two chronological patterns of changes in the gene expression for this kinase were discerned. The first was a high and persistent expression from the developing stages till the adult stage, which was observed in the cerebellar granule cells, the hippocampal pyramidal cells and the dentate granule cells. The other was a transiently high expression during limited developmental periods, which was observed in the Purkinje cells, neurons in the inferior olive, various brain stem nuclei, and the subventricular neuronal cells. These findings suggest that this Ca2+/calmodulin-dependent protein kinase is involved differentially in multiple Ca2+ signaling pathways in different developing and mature neurons.


Chronic GABA exposure down-regulates GABA-benzodiazepine receptor-ionophore complex in cultured cerebral cortical neurons. Cerebral cortical cultured neurons were characterized for GABA-benzodiazepine (BZ) receptor complex, and the effect of chronic exposure of cortical neurons to GABA on GABA-BZ receptor system was investigated. In the intact cells, the [3H]flunitrazepam binding was rapid and saturable, with an apparent Kd of 4.2 +/- 1.5 nM and Bmax of 776 +/- 54 fmol/mg protein. Specifically bound [3H]flunitrazepam was displaced in a concentration-dependent manner by various BZ receptor ligands such as Ro15-1788, DMCM, Ro15-4513, clonazepam, alprazolam, diazepam and zolpidem, and enhanced by GABA, muscimol and pentobarbital. GABA induced enhancement of 36Cl-influx in a concentration-dependent manner (EC50 = 9 +/- 2 microM). Chronic exposure of the cultured neurons to GABA resulted in a reduced [3H]flunitrazepam, [3H]GABA, [3H]Ro15-1788, [3H]Ro15-4513 and [35S]TBPS binding, a reduced enhancement of [3H]flunitrazepam binding by GABA, and a reduced GABA-induced 36Cl-influx susceptible to reversal by concomitant exposure of the cultures to R 5135, a GABAA-receptor antagonist. These findings indicate that cerebral cortical cultured neurons provide an ideal model to study GABA-BZ receptor complex using binding and 36Cl-influx assays, and chronic exposure of cortical cultures to GABA leads to a down-regulation of GABA-BZ receptor system. It is a GABAA receptor-mediated slow process.


Expression of a carboxy-terminal region of the beta-amyloid precursor protein in a heterogeneous culture of neuroblastoma cells: evidence for altered processing and selective neurotoxicity. Six independent clonal isolates from a morphologically heterogeneous human neuroblastoma cell line stably expressed several products of the human amyloid precursor protein (APP) from an introduced DNA construct; the "substrate-adherent" phenotype (fibroblast-like cells) predominated in all 6; these displayed immunoreactivity of vimentin, but little to no reactivity of neuron-specific enolase. A stably transfected isolate which did not show any expression from the identical construct (presumably because of a position effect) exhibited the predominantly neuronal phenotype of the parental cells (neuron-specific enolase positive). These results suggest selective neurotoxicity of the expressed products. Two of the 6 stably expressing cell lines showed a decrease of native mRNA for APP to levels that were 1/4-1/3 that of the parental cells and a decrease of their growth rates to half that of the parental cells; these decreased growth rates were improved by conditioned medium from the parental cell line. Western blot analysis revealed at least four distinct fragments of the COOH-terminus of APP in the isolate which expressed protein and mRNA in greatest abundance, suggesting that overexpression of APP in a human neural cell line leads to aberrant cleavage of APP.


Melatonin influences Fos expression in the rat suprachiasmatic. Administration of the pineal hormone melatonin to rats induces expression of Fos, the protein product of the c-fos proto-oncogene, in the suprachiasmatic nucleus (SCN), the putative biological clock of mammals. Expression of the Fos protein is dependent on circadian phase: injections in the late subjective night (circadian time (CT) 22) induce Fos expression in cells within the ventral SCN whereas injections during the subjective day are ineffective. Since melatonin injections in the late subjective day have previously been shown to phase advance circadian rhythms, these results indicate that phase-advances of the circadian system can occur without increased expression of Fos protein in the SCN, at least at levels detectable by immunohistochemistry. In support of in situ hybridization histochemical evidence obtained previously, immunocytochemical data from vehicle-injected control rats suggest that the Fos protein undergoes an endogenous fluctuation with peak levels in the SCN occurring during the subjective night. These observations indicate that melatonin can affect immediate early gene expression within the SCN.


Differential expression of two alpha 2-adrenergic receptor subtype mRNAs in human tissues. Genetic subtypes of alpha 2-adrenergic receptors (AR) may mediate distinct physiological functions, and undergo differential cell type-specific regulation. Thus, these distinct receptor subtypes are possible targets for the development of subtype-selective drugs. We have analyzed the tissue distribution of two human alpha 2-adrenoceptor subtype gene mRNAs, alpha 2-C4 and alpha 2-C10, in normal human fetal and adult tissues. Both receptor subtype mRNAs were abundantly expressed in fetal brain and choroid plexus. In non-neural fetal tissues, alpha 2-C10 mRNA was detected in spleen, kidney, adrenal gland, and skin, while alpha 2-C4 transcripts were observed only in kidney and skin. Most regions of the adult brain also expressed both subtypes, but with marked quantitative differences. For example, cerebral cortex contained predominantly alpha 2-C10 mRNA, whereas the caudate nucleus expressed mostly alpha 2-C4 mRNA. In adult peripheral tissues, alpha 2-C10 mRNA expression was most abundant in spleen and renal cortex, and expression of alpha 2-C4 mRNA was strongest in renal cortex and medulla. These different expression patterns provide evidence for the differential regulation of the two alpha 2-adrenergic receptor genes and warrant further investigation with techniques capable of improved anatomical resolution. Regional differences in receptor subtype expression may be valuable for the development of new, subtype-selective pharmacological agents with more targeted actions compared to currently used alpha 2-adrenoceptor agonists and antagonists.


Comparison of the concentration of messenger RNA encoding four muscarinic receptor subtypes in control and Alzheimer brains. We determined the concentration of the messenger RNA species which encode four (m1-m4) of the five cloned muscarinic receptors in brains of Alzheimer's disease patients as compared to age-matched controls. Assays were performed using the quantitative method of DNA-excess solution hybridization in the cerebral cortex (frontal, temporal and occipital), hippocampus, nucleus basalis of Meynert and brainstem. The results suggest a statistically significant decrease in the m1 muscarinic receptor message in the temporal and occipital cortex, with no change in other regions. There was no change in the level of mRNA encoding the m2, m3 or m4 receptors in any of the brain regions studied.


Chemical characterization of a novel peptide from the neuroendocrine light yellow cells of Lymnaea stagnalis. The neuroendocrine light yellow cells of Lymnaea stagnalis form two clusters of cells in the visceral and right parietal ganglion, respectively. These cells are endogenously bursting neurons whose activities are modified during egg-laying and feeding. Using gel permeation chromatography and reverse phase HPLC we have purified two highly related novel peptides from the light yellow cells. These peptides differ only in length, due to truncation of the amino-terminal aspartic acid residue, which causes a major shift in the charge of the molecule. We conclude that the longer peptide is the immediate precursor of the shorter form. Using whole mount immunocytochemistry, it was confirmed that the light yellow cells produce these peptides.


A noninvasive assessment of myocardial oxygen tension: 19F NMR spectroscopy of sequestered perfluorocarbon emulsion. Fluorine NMR spectroscopy of sequestered perfluorocarbon emulsion has been used to measure myocardial oxygen tension. This novel application provides a rapid noninvasive assessment of changes in oxygen tension in response to ischemia and reperfusion. Rats were predosed with Oxypherol-ET (emulsion of perfluorotributylamine). Following vascular clearance of the emulsion the heart was excised and perfused using the Langendorff retrograde technique. 19F spin-lattice relaxation time measurements provided an accurate estimate of myocardial pO2. Using a two-point determination with a time resolution of 1 s, the loss of oxygen was found to be complete within 40 s of the onset of global ischemia. The fall in oxygen tension correlated closely with an observed loss of ventricular pressure. Magnetic resonance imaging showed that perfluorocarbon was distributed throughout the heart; thus, this reporter molecule provides a global measurement of oxygen tension.