Heat-stable antigen is a costimulatory molecule for CD4 T cell growth. Optimal induction of clonal expansion by normal CD4 T cells requires a ligand that can engage the T cell receptor as well as functionally defined costimulatory activity on the same antigen-presenting cell surface. While the presence of effective costimulation induces proliferation, T cell receptor ligation in its absence renders T cells inactive or anergic. The molecular basis of this costimulatory activity remains to be defined. Here we describe a monoclonal antibody that can block the costimulatory activity of splenic accessory cells. Treatment with this antibody not only blocks the proliferation of CD4 T cells to a T cell receptor ligand, but also induces T cell nonresponsiveness to subsequent stimulation. Sequence analysis of the antigen recognized by this antibody indicates that it recognizes a protein that is identical to heat-stable antigen. Gene transfer experiments directly demonstrate that this protein has costimulatory activity. Thus, heat-stable antigen meets the criteria for a costimulator of T cell clonal expansion.


Acquired Mls-1a-like clonal deletion in Mls-1b mice. BALB/c mice (H-2d, Mls-1b) from one colony progressively modify their T cell repertoire during aging, by deleting T cells that express products of the V beta 6 and V beta 8.1 genes of the T cell receptor. Clonal deletion occurs only in 50% of mice between 27 and 43 wk of age, affecting thymus, spleen, and lymph node T cells. The phenomenon is progressive and seems to affect nearly all thymuses between 14 and 19 wk of age. CD4+CD8- mature T cells are more affected than CD4-CD8+ cells. In the thymus, deletion occurs at the stage of immature J11d+ cells expressing a high level of V beta 6, while J11d+V beta 6low-expressing cells are not modified. Clonal deletion is thus an early phenomenon that deletes cells of the immature generative compartment in the thymus. This Mls-1a-like clonal deletion is associated neither with the expression of an Mls-1a-like antigen that could be identified in mixed lymphocyte reaction in vitro, nor with the presence of Mtv-7, the endogenous mouse mammary tumor virus (MMTV) proviral loci. Spleen cells, bone marrow cells, and total thymocytes injected into newborn thymuses cannot induce V beta 6+ cell deletion. However, newborn thymuses grafted into old BALB/c mice behave like their recipients, suggesting that a new antigen, present in these old BALB/c mice, is indeed able to induce an Mls-1a-like clonal deletion. As other BALB/c colonies tested do not behave in same way, the hypothesis of a new exogenous deleting factor rather than a genetic transmission is likely. This may suggest that acquired clonal deletion is a more common phenomenon than expected, and may be the spontaneous reaction of the immune system to the introduction of a new retrovirus or other superantigen.


Human mature T cells that are anergic in vivo prevail in SCID mice reconstituted with human peripheral blood. In these studies we have characterized the human cells that repopulate severe combined immunodeficient (SCID) mice after injection of adult peripheral blood or cord blood (hu-PBL-SCID mice). In all organs of the chimeras, and at any time point tested, single-positive (CD4+ or CD8+) T cells that expressed the alpha/beta T cell receptor (TCR) prevailed. All T cells were CD45RO+ and the majority were also HLA-DR+. Thus, the human T cells in the chimeras exhibited the phenotype of mature, memory cells that showed signs of recent activation. Cell cycle studies revealed a mitotically active human T cell population in the murine host. However, when freshly isolated from the chimeras, the human T cells were refractory to stimulation by anti-CD3 antibody but proliferated in response to exogenous interleukin 2. Chimera-derived human T cell lines retained this state of unresponsiveness to TCR-triggered proliferation for 4-6 wk in vitro. Subsequently, the T cell lines developed responses to anti-CD3 stimulation and 9 of 11 of the lines also proliferated in response to splenic stimulator cells of SCID mice. These data demonstrate that the human T cells are in a state of reversible anergy in the murine host and that xenoreactivity might play a critical role in hu-PBL-SCID mice. Mechanisms that may determine repopulation of SCID mice with human peripheral blood mononuclear cells are discussed.


The OX-44 molecule couples to signaling pathways and is associated with CD2 on rat T lymphocytes and a natural killer cell line. The MRC OX-44 molecule, which is expressed on all peripheral leukocytes, identifies the subset of thymocytes capable of proliferating in response to alloantigens and lectins (Paterson, D.J., J.R. Green, W.A. Jefferies, M. Puklavec, and A.F. Williams. 1987. J. Exp. Med. 165:1). When we isolated monoclonal antibodies (mAbs) on the basis of their ability to activate the phosphatidylinositol signaling pathway in RNK-16 cells (a rat leukemia line with natural killer activity), three of the resulting mAbs recognized the OX-44 molecule. Addition of these mAbs to RNK-16 elicits protein tyrosine phosphorylation, generates inositol phosphates, and increases the concentration of cytoplasmic free calcium. These responses require the addition of intact mAb and are not observed with F(ab')2 fragments. One of these mAbs (7D2) is mitogenic for freshly isolated rat splenic T cells and synergizes with a mAb to the T cell antigen receptor in this activation. A 50-60-kD glycoprotein coprecipitates with the OX-44 molecule from RNK-16 cells and rat splenic T cells. Peptide mapping and reprecipitation studies indicate that the coprecipitating molecule is CD2. Thus, the OX-44 molecule can couple to multiple signaling pathways and associates with CD2 on both RNK-16 and rat T cells.


The cumulative risk of AIDS as the CD4 lymphocyte count declines. A method is proposed for assessing the cumulative risk of various AIDS-defining conditions as the CD4 lymphocyte count declines in HIV-infected individuals. The method is analogous to survival analysis but is based on the CD4 lymphocyte count rather than on time. Thus, the level to which the CD4 lymphocyte count has declined, rather than the length of time since seroconversion, is considered as an individual's survival interval. The survival interval may be censored (due to lack of follow-up) or treated as an interval to failure (if the individual develops AIDS). The Kaplan-Meier (product-limit) estimates, of the proportion of individuals developing AIDS before reaching a given low CD4 lymphocyte count, may be useful for determining when prophylactic treatment should begin.


Synthesis and evaluation of some water-soluble prodrugs and derivatives of taxol with antitumor activity. The synthesis and evaluation of some 2'- and 7-amino acid derivatives of taxol (1) are reported. Reaction of taxol with N-protected amino acids gave 2'-N-protected amino acid esters of taxol. However, deprotection of the amino group and subsequent isolation of products were complex and only successful when formic acid was used to deprotect a t-BOC protecting group. Esterification of taxol using N,N-dialkylated amino acids gave 2'-amino acid esters of taxol, 2'-(N,N-dimethylglycyl)taxol (4) and 2'-[3-(N,N-diethylamino)propionyl]taxol as its methanesulfonic acid salt (5b), in good yield. The 7-derivatives, 7-(N,N-dimethylglycyl)taxol (9) and 7-L-alanyltaxol (12), were prepared by two alternate methods. In the first approach, the 2'-hydroxyl group was protected using the [(2,2,2-trichloro-ethyl)oxy]carbonyl, or troc, protecting group followed by the esterification of the 7-hydroxyl and subsequent deprotection of the amino and troc groups. In the second approach, taxol was allowed to react with more than 2 molar equiv of the N-protected amino acids or N,N-dialkylated amino acids to give 2',7-diamino acid esters of taxol. For the protected amino acids, the deprotection of the amino group followed by removal of the 2'-substituent gave the 7-amino acid esters of taxol. The methanesulfonic acid salts of both 2'- and 7-amino acid esters showed improved solubility ranging from 2 to greater than 10 mg/mL. The 7-derivatives were effective in promoting microtubule assembly in vitro while 2'-derivatives showed little in vitro activity. The derivatives 2'-(N,N-dimethylglycyl)taxol (4) and 2'-[3-(N,N-diethylamino)propionyl]taxol (5) inhibited proliferation of B16 melanoma cells to an extent similar to that of taxol, while the other derivatives were about 50% as cytotoxic. In a mammary tumor screen, 2'-[3-(N,N-diethylamino)propionyl]taxol showed the greatest antitumor activity compared to the other analogues. The lower activities of the 7-derivatives in inhibiting tumor growth and melanoma cell proliferation (although they were almost as active as taxol in inducing microtubule assembly in vitro) may be due to differences in drug uptake by the cells. The similar cytotoxic and antitumor activities of the 2'-analogues and taxol can be explained by their conversion to taxol or an active taxol metabolite. Therefore, the 2'-analogues appear to behave as prodrugs and have the potential to be developed as chemotherapeutic agents.


Preparation and opioid activity of analogues of the analgesic dipeptide 2,6-dimethyl-L-tyrosyl-N-(3-phenylpropyl)-D-alaninamide. A number of analogues of the recently disclosed analgesic dipeptide 2,6-dimethyl-L-tyrosyl-D-alanine-phenylpropylamide (SC-39566, 2) were prepared. These analogues contained oxymethylene, aminomethylene, ketomethylene, bismethylene, and trans double bond (including vinyl fluoride) isosteric replacements for the amide bond between the D-alanine and phenylpropylamine units in 2. These compounds were tested in opioid binding assays and in the mouse writhing assay for analgesic activity. Though not as potent as 2, the oxymethylene, and trans double bond isosteres showed analgesic activity. The aminomethylene analogues also showed binding activity in subnanomolar concentrations at the mu receptor. The amide bond between 2,6-dimethyl-L-tyrosine and D-alanine units seems to be critical for opioid activity.


Risk of strut fracture of Björk-Shiley valves. The incidence of and factors that predispose to outlet strut fracture of Björk-Shiley heart valves are still not known. To obtain such information a retrospective cohort study was conducted on all 2303 patients in the Netherlands with a 60 degrees convexo-concave (60 degrees CC) or a 70 degrees convexo-concave (70 degrees CC) Björk-Shiley heart valve. Patients have been followed-up for a mean of 6.6 years (range 1-4271 days). 42 cases of mechanical failure due to outlet strut fracture have been recorded-6 of the 7 patients with fracture of the aortic valve died, as did 18 of the 35 patients with fracture of the mitral valve. Multivariate analysis identified wide opening angle (70 degrees), large valve size (greater than or equal to 29 mm diameter), and young age (less than 50 years) as risk factors for outlet strut fracture. For large 70 degrees CC mitral valves the cumulative risk of outlet strut fracture after 8 years was 17.4% (95% CI 9.1-31.6). Unlike previous findings, this excessive risk applied to late as well as to early batches of valves. In patients with a large 60 degrees CC mitral valve the cumulative risk after 8 years was 4.2% (95% CI 2.7-6.5). The incidence rate of outlet strut fracture in 60 degrees CC and 70 degrees CC valves (aortic and mitral) was constant over time. Overall survival since implantation was better for patients with 60 degrees CC prostheses than for those with 70 degrees CC prostheses; the adjusted hazard ratio for mortality for patients receiving a 70 degrees CC prosthesis was 1.5 (95% CI 1.1-2.0). Together with the low (24%) necropsy rate, this ratio suggests that the reported incidence of strut fracture for the 70 degrees CC valves is an underestimate. The data indicate that prophylactic replacement of 60 degrees CC and 70 degrees CC valves is advisable for selected groups of patients. Since the case-fatality rate is 50% for emergency replacement of faulty valves, patients suspected of Björk-Shiley heart-valve failure should be referred without delay to a cardiothoracic centre.


Breast milk and subsequent intelligence quotient in children born preterm. There is considerable controversy over whether nutrition in early life has a long-term influence on neurodevelopment. We have shown previously that, in preterm infants, mother's choice to provide breast milk was associated with higher developmental scores at 18 months. We now report data on intelligence quotient (IQ) in the same children seen at 7 1/2-8 years. IQ was assessed in 300 children with an abbreviated version of the Weschler Intelligence Scale for Children (revised Anglicised). Children who had consumed mother's milk in the early weeks of life had a significantly higher IQ at 7 1/2-8 years than did those who received no maternal milk. An 8.3 point advantage (over half a standard deviation) in IQ remained even after adjustment for differences between groups in mother's education and social class (p less than 0.0001). This advantage was associated with being fed mother's milk by tube rather than with the process of breastfeeding. There was a dose-response relation between the proportion of mother's milk in the diet and subsequent IQ. Children whose mothers chose to provide milk but failed to do so had the same IQ as those whose mothers elected not to provide breast milk. Although these results could be explained by differences between groups in parenting skills or genetic potential (even after adjustment for social and educational factors), our data point to a beneficial effect of human milk on neurodevelopment.


High-frequency diaphragmatic flutter: symptoms and treatment by carbamazepine. Classic diaphragmatic flutter, a rare disorder associated with dyspnoea, thoracic or abdominal wall pain, and epigastric pulsations, is caused by involuntary contractions of the diaphragm with a frequency of 0.5-8.0 Hz. We have seen three patients with diaphragmatic flutter of higher frequency not associated with respiratory disease. The patients presented with longstanding oesophageal belching, hiccups, and retching, respectively. The diagnosis was established by the presence on electromyography of the diaphragm and scalene and parasternal intercostal muscles of repetitive discharges of 9-15 Hz. Spirographic tracings, especially those of volume or flow vs time, showed similar high-frequency oscillations superimposed on tidal respiratory movements. Treatment with carbamazepine 200-400 mg three times daily led to disappearance or great improvement of flutter and clinical symptoms in all three patients. The phenomenon was not seen in other patients with chronic hiccups or oesophageal belching or in patients without these symptoms who had undergone electromyography or spirography for other reasons. Thus, high-frequency diaphragmatic flutter seems to be a new disease entity. The response to carbamazepine, which suggests that the flutter causes the symptoms, requires further study.


Anti-GOR and hepatitis C virus in autoimmune liver diseases. Anti-GOR is an autoantibody found in hepatitis C virus (HCV) infection. We have studied the specificity of this antibody for HCV infection in various groups of autoimmune liver diseases. Anti-HCV was detected by a second generation HCV enzyme-linked immunosorbent assay in 14 of 29 patients with liver-kidney-microsomal (LKM-1) -antibody-positive autoimmune hepatitis type 2 and in all 6 control patients with HCV-RNA-positive chronic hepatitis C. Anti-HCV was not found in those with antinuclear-antibody-positive autoimmune hepatitis type 1 (10 patients), with soluble-liver-protein-antibody-positive autoimmune hepatitis type 3 (8), with primary biliary cirrhosis (9), with systemic lupus erythematosus (SLE) (10), or in healthy controls (13). Anti-GOR was detected in 11 of 14 patients with autoimmune hepatitis type 2 who were all positive for anti-HCV but only in 1 of 15 LKM-1 patients who were negative for anti-HCV. We did not find anti-GOR in any other group of autoimmune liver disease, SLE, or control sera, but this antibody was detected in 3 of 6 patients with chronic hepatitis C. Autoimmune hepatitis type 2 patients who were anti-GOR positive and anti-HCV positive were less likely to be female, were older (p less than 0.001), and had lower LKM-1 antibody titres (p less than 0.001), lower disease activity, and responded less effectively to immuno- suppression than did those who were anti-HCV negative/anti-GOR negative. The findings show that anti-GOR reflects HCV-specific autoimmunity. HCV seems to induce autoimmunity to both GOR (an HCV-specific autoepitope) and LKM-1 (an epitope that is also recognised by autoimmune hepatitis sera of a different cause). Anti-GOR and LKM-1 antibodies contribute to a better differentiation of chronic hepatitis, a finding that has therapeutic implications.


Indomethacin and postprandial gallbladder emptying. Patients with gallstone disease commonly have impaired gallbladder emptying. To see whether non-steroidal anti-inflammatory drugs (NSAIDs) prevent gallstone formation by improving gallbladder emptying, we assessed the effect of indomethacin on postprandial emptying in healthy subjects and in patients with gallstone disease. Subjects received indomethacin 25 mg three times a day for a week and matching placebo for another week. Compared with placebo, indomethacin improved postprandial gallbladder emptying in all 7 patients with gallstone disease. This finding was not recorded in healthy subjects with normal gallbladders. The prevention of gallstone formation associated with ingestion of NSAIDs may be due mainly to a prokinetic effect on the gallbladder since there is no evidence to suggest that these drugs affect cholesterol crystal nucleation at ordinary therapeutic doses in man or animals.


Detection of full fragile X mutation.

Detection of full fragile X mutation. In fragile X syndrome, the most common inherited cause of mental deficiency, the underlying mutation is a large increase in the number of CGG repeats in a gene on chromosome X. We have developed a polymerase chain reaction (PCR) method to amplify across the full mutation in affected individuals. In this report, a fragile X family including a positive prenatally diagnosed fetus was analysed by PCR, and the results are consistent with direct genomic Southern blot analysis. Genetic screening of at-risk populations for fragile X can now be achieved by PCR rapidly, inexpensively, and on small samples.


Intrarectal challenge of macaques vaccinated with formalin-inactivated simian immunodeficiency virus. Macaques can be protected from intravenous infection with simian immunodeficiency virus (SIV) by vaccination with chemically inactivated virus. However, protection against infection via a mucosal surface has not been demonstrated. We vaccinated four rhesus macaques with formalin-inactivated SIV given intramuscularly. These monkeys, which had remained virus free for 10 months after intravenous challenge with SIV, were given a further dose of vaccine and together with four unvaccinated controls were challenged intrarectally with SIV. Subsequently, virus was isolated from all control animals on five successive occasions, but the vaccinated animals remained free of virus. Proviral DNA could not be detected in peripheral blood mononuclear cells from the vaccinated animals. Preliminary data indicate that vaccinated animals make a local antibody response.


Long-term follow-up of childhood Henoch-Schönlein nephritis. A study of long-term outcome of 78 subjects who had had Henoch-Schönlein nephritis during childhood (at a mean of 23.4 years after onset) shows that severity of clinical presentation and initial findings on renal biopsy correlate well with outcome but have poor predictive value in individuals. 44% of patients who had nephritic, nephrotic, or nephritic/nephrotic syndromes at onset have hypertension or impaired renal function, whereas 82% of those who presented with haematuria (with or without proteinuria) are normal. 17 patients deteriorated clinically from an initial assessment in 1971; 7 of these had apparently completely recovered in 1976. 16 of 44 full-term pregnancies were complicated by proteinuria and/or hypertension, even in the absence of active renal disease. These findings indicate that childhood Henoch-Schönlein nephritis requires long-term follow-up, especially during pregnancy.


Customised antenatal growth charts.

Customised antenatal growth charts. Charts for fetal growth do not take physiological variables into account. We have therefore designed a computer-generated antenatal chart that can be easily "customised" for each individual pregnancy, taking the mother's characteristics and birthweights from previous pregnancies into consideration. The adjusted birthweight range expected at 40 weeks' gestation is combined with a standard, longitudinal ultrasound-derived curve for intrauterine weight gain. Review at the Queen's Medical Centre, Nottingham, UK, of 4179 pregnancies with ultrasound-confirmed dates showed that, in addition to gestation and sex, maternal weight at first antenatal-clinic visit, height, ethnic group, and parity were significant determinants of birthweight in our population. Correction factors were calculated for each of these variables and entered into a computer program to adjust the normal birthweight centile limits. With adjusted centiles we found that 28% of babies conventionally designated small for gestational age (less than 10th centile) and 22% of those designated large (greater than 90th centile) were in fact within normal limits for the pregnancy. Conversely, 24% and 26% of babies identified as small or large, respectively, with adjusted centiles were "missed" by conventional unadjusted centile assessment. Adjustment for physiological variables will make assessment of fetal growth more precise and reduce unnecessary investigations, interventions, and parental anxiety.


Sudden cardiac tamponade after chemotherapy for marrow transplantation in thalassaemia. Published work suggests that cardiac tamponade occurs only occasionally after bone-marrow transplantation (BMT) but the worrying number of cases encountered in the transplant programme in Pesaro, Italy, has led to an analysis of this complication. Cardiac tamponade occurred in 8 (2%) of 400 consecutive thalassaemic patients during conditioning for or within a month of BMT. 6 cases were fatal; these represented 9% of all causes of death and 29% of those occurring between start of conditioning regimen and 30 days post transplant. The syndrome was characterised by sudden onset of circulatory shock and cardiac arrest. The only effective treatment was immediate fluid removal. The absence of myocardial lesions and the complete resolution of the syndrome after pericardiocentesis suggest that the pericardial membranes played the main part in the pathogenesis of the syndrome. Since irradiation was not part of the conditioning regimen and since 3 of the affected patients had bacteraemia, the triggering factor for the syndrome could have been the drugs used for conditioning, acting alone or together with bacteraemia and trauma. The frequency with which we encountered the syndrome, and the similarity among our patients in clinical picture, and in characteristics of the effusion, indicate that cardiac tamponade occurring in thalassaemic patients after start of chemotherapy as conditioning for BMT is a specific syndrome requiring rapid treatment.


Drug dependence with oestrogen replacement therapy. Dependence on some drugs can be hard to recognize. Hormone replacement therapy (HRT) has been widely prescribed only in the past two decades, and the indications for treatment and the risk/benefit ratio are still disputed. Oestrogens are psychoactive: they lift mood, can be given by injection, and their use has powerful psychological effects. Reports of women with supraphysiological oestradiol concentrations may represent tolerance and withdrawal. Dependence on substances occurring naturally in the body has been reported before. We propose that HRT dependence occurs.


Overexpression of glutathione S-transferase and elevation of thiol pools in a multidrug-resistant human colon cancer cell line. A human colon cancer cell line with acquired multidrug resistance (MDR) was assayed for the intracellular GSH level and the activity of GSH-S-transferase (GST), which catalyzes the conjugation reaction of electrophilic drugs with GSH. The GSH level and GST activity (as measured with 1-chloro-2,4-dinitrobenzene) were elevated in the resistant cells by 1.7-fold and 2-fold, respectively. This elevated catalytic activity of the resistant cells was reflected in a 2-fold increase in GST-pi mRNA, which was not the result of gene amplification. In addition, buthionine sulfoximine, a specific inhibitor of GSH synthesis, significantly increased Adriamycin sensitivity in both the MDR and the parental cells, affecting the former more than the latter. The effects seen with buthionine sulfoximine were not seen with puromycin and actinomycin D. A dramatic overexpression of mdr1, a P-glycoprotein gene responsible for the MDR phenotype, was also observed in the MDR cells. In contrast, none of these products (i.e., mdr P-glycoprotein, GSH level, total GST activity, GST-pi gene copy, and GST-pi mRNA level) was elevated in HeLa cells resistant to cisplatin and some alkylating agents, supporting the notion that the acquisition of cisplatin resistance differs from the mechanism of MDR. These results indicate that the intrinsic GSH level and GST-pi activity affect anthracycline resistance per se and not MDR in the human colon cancer cells.


Anti-oncogenic activity of signalling-defective epidermal growth factor receptor mutants. Overexpression and autocrine activation of the epidermal growth factor receptor (EGF-R) cause transformation of cultured cells and correlate with tumor progression in cancer patients. Dimerization and transphosphorylation are crucial events in the process by which receptors with tyrosine kinase activity generate normal and transforming cellular signals. Interruption of this process by inactive receptor mutants offers the potential to inhibit ligand-induced cellular responses. Using recombinant retroviruses, we have examined the effects of signalling-incompetent EGF-R mutants on the growth-promoting and transforming potential of ligand-activated, overexpressed wild-type EGF-R and the v-erbB oncogene product. Expression of a soluble extracellular EGF-R domain had little if any effect on the growth and transformation of NIH 3T3 cells by either tyrosine kinase. However, both a kinase-negative EGF-R point mutant (HERK721A) and an EGF-R lacking 533 C-terminal amino acids efficiently inhibited wild-type EGF-R-mediated, de novo DNA synthesis and cell transformation in a dose-dependent manner. Furthermore, coexpression with the v-erbBES4 oncogene product in NIH 3T3 cells resulted in transphosphorylation of the HERK721A mutant receptor and reduced soft-agar colony growth but had no effect in a focus formation assay. These results demonstrate that signalling-defective receptor tyrosine kinase mutants differentially interfere with oncogenic signals generated by either overexpressed EGF-R or the retroviral v-erbBES4 oncogene product.


Fetal liver pro-B and pre-B lymphocyte clones: expression of lymphoid-specific genes, surface markers, growth requirements, colonization of the bone marrow, and generation of B lymphocytes in vivo and in vitro. We describe here the development and characterization of the FLS4.1 stromal line derived from 15-day fetal liver of BALB/c embryos and defined culture conditions that efficiently support the cloning and long-term growth of nontransformed B-220+ 14-day fetal liver cells at two stages of B-cell development, namely, pro-B lymphocytes (immunoglobulin [Ig] genes in germ line configuration) and pre-B cells (JH-rearranged genes with both light-chain Ig genes in the germ line state). All B-cell precursor clones require recombinant interleukin-7 (rIL-7) and FLS4.1 stromal cells for continuous growth in culture, but pro-B lymphocyte clones can also proliferate in rIL-3. None proliferate in rIL-1, rIL-2, rIL-4, rIL-5, rIL-6, or leukemia inhibitory factor. FLS4.1 stromal cells synthesize mRNA for Steel factor but not for IL-1 to IL-7; all pro-B and pre-B clones express c-Kit, the receptor for Steel factor, and a c-Kit-specific antibody inhibits the enhanced proliferative response of fetal liver B-220+ B-cell precursors supported by FLS4.1 stromal cells and exogenous rIL-7 but does not affect that promoted by rIL-7 alone. Northern (RNA) blot analysis of the expression of the MB-1, lambda 5, Vpre-B, c mu, RAG-1, and RAG-2 genes in pro-B and pre-B clones show that transcription of the MB-1 gene precedes IgH gene rearrangement and RNA synthesis from c mu, RAG-1, RAG-2, lambda 5, and Vpre-B genes. All clones at the pre-B-cell stage synthesize mRNA for c mu, RAG-1, and RAG-2 genes; transcription of the lambda 5 and Vpre-B genes seems to start after D-to-JH rearrangement in B-cell precursors, indicating that the proteins encoded by either gene are not required for B-cell progenitors to undergo D-to-JH gene rearrangement. These findings mark transcription of the MB-1 gene as one of the earliest molecular events in commitment to develop along the B-lymphocyte pathway. Indeed, both pro-B and pre-B clones can generate in vitro and in vivo B lymphocytes but not T lymphocytes; moreover, these clones do not express the CD3-gamma T-cell-specific gene, nor do they have rearranged gamma, delta, or beta T-cell antigen receptor genes.


The Oct-1 POU domain mediates interactions between Oct-1 and other POU proteins. The POU domain is the conserved DNA binding domain of a family of gene regulatory proteins. It consists of a POU-specific domain and a POU homeodomain, connected by a variable linker region. Oct-1 is a ubiquitously expressed POU domain transcription factor. It binds to the canonical octamer sequence (ATGCAAAT) as a monomer. Here we show by chemical cross-linking and protein affinity chromatography that the Oct-1 POU domain monomers can interact in solution. This association requires both the POU homeodomain and the POU-specific domain. The interaction is transient in solution and can be stabilized by binding to the heptamer-octamer sequence in the immunoglobulin heavy-chain promoter. This correlates with cooperative DNA binding to this site. POU proteins from different subclasses, including Oct-1, Oct-2A, Oct-6, and a chimeric Oct-1 protein containing the Pit-1 POU domain, can bind cooperatively to a double binding site and form a heteromeric complex.


A controlled trial of early versus late treatment with zidovudine in symptomatic human immunodeficiency virus infection. Results of the Veterans Affairs Cooperative Study. Zidovudine is recommended for asymptomatic and early symptomatic human immunodeficiency virus (HIV) infection. The best time to initiate zidovudine treatment remains uncertain, however, and whether early treatment improves survival has not been established. We conducted a multicenter, randomized, double-blind trial that compared early zidovudine therapy (beginning at 1500 mg per day) with late therapy in HIV-infected patients who were symptomatic and had CD4+ counts between 0.2 x 10(9) and 0.5 x 10(9) cells per liter (200 to 500 per cubic millimeter) at entry. Those assigned to late therapy initially received placebo and began zidovudine when their CD4+ counts fell below 0.2 x 10(9) per liter (200 per cubic millimeter) or when the acquired immunodeficiency syndrome (AIDS) developed. During a mean follow-up period of more than two years, there were 23 deaths in the early-therapy group (n = 170) and 20 deaths in the late-therapy group (n = 168) (P = 0.48; relative risk [late vs. early], 0.81; 95 percent confidence interval, 0.44 to 1.59). In the early-therapy group, 28 patients progressed to AIDS, as compared with 48 in the late-therapy group (P = 0.02; relative risk, 1.76; 95 percent confidence interval, 1.1 to 2.8). Early therapy increased the time until CD4+ counts fell below 0.2 x 10(9) per liter (200 per cubic millimeter), and it produced more conversions from positive to negative for serum p24 antigen. Early therapy was associated with more anemia, leukopenia, nausea, vomiting, and diarrhea, whereas late therapy was associated with more skin rash. In symptomatic patients with HIV infection, early treatment with zidovudine delays progression to AIDS, but in this controlled study it did not improve survival, and it was associated with more side effects.


Fatal familial insomnia, a prion disease with a mutation at codon 178 of the prion protein gene. We previously described two members of a family affected by an apparently genetically determined fatal disease characterized clinically by progressive insomnia, dysautonomia, and motor signs and characterized pathologically by severe atrophy of the anterior ventral and mediodorsal thalamic nuclei. Five other family members who died of this disease, which we termed "fatal familial insomnia," had broader neuropathologic changes suggesting that fatal familial insomnia could be a prion disease. We used antibodies to prion protein (PrP) to perform dot and Western blot analyses, with and without proteinase K, on brain tissue obtained at autopsy from two patients with fatal familial insomnia, three patients with sporadic Creutzfeldt-Jakob disease, and six control subjects. The coding region of the PrP gene was amplified and sequenced in the samples from the two patients with fatal familial insomnia. Restriction-enzyme analysis was carried out with amplified PrP DNA from 33 members of the kindred. Protease-resistant PrP was found in both patients with fatal familial insomnia, but the size and number of protease-resistant fragments differed from those in Creutzfeldt-Jakob disease. In the family with fatal familial insomnia, all 4 affected members and 11 of the 29 unaffected members had a point mutation in PrP codon 178 that results in the substitution of asparagine for aspartic acid and elimination of the Tth111 I restriction site. Linkage analysis showed a close relation between the point mutation and the disease (maximal lod score, 3.4 when theta was zero). Fatal familial insomnia is a prion disease with a mutation in codon 178 of the PrP gene, but the disease phenotype seems to differ from that of previously described kindreds with the same point mutation.


The use of beta-agonists and the risk of death and near death from asthma. Morbidity and mortality from asthma appear to be increasing, and it has been suggested that medications used to treat asthma are contributing to this trend. We investigated a possible association between death or near death from asthma and the regular use of beta 2-agonist bronchodilators. Using linked health insurance data bases from Saskatchewan, Canada, we conducted a matched case-control study of subjects drawn from a cohort of 12,301 patients for whom asthma medications had been prescribed between 1978 and 1987. We matched 129 case patients who had fatal or near-fatal asthma with 655 controls (who had received medications for asthma but had not had fatal or near-fatal events) with respect to region of residence, age, receipt of social assistance, and previous hospitalization for asthma. The use of beta-agonists administered by a metered-dose inhaler was associated with an increased risk of death from asthma (odds ratio, 2.6 per canister per month; 95 percent confidence interval, 1.7 to 3.9) and of death or near death from asthma, considered together (odds ratio, 1.9; 95 percent confidence interval, 1.6 to 2.4). For death from asthma, use of the beta-agonist fenoterol was associated with an odds ratio of 5.4 per canister, as compared with 2.4 for the beta-agonist albuterol. On a microgram-equivalent basis, the odds ratio for this outcome with fenoterol was 2.3, as compared with 2.4 with albuterol. An increased risk of death or near death from asthma was associated with the regular use of inhaled beta 2-agonist bronchodilators, especially fenoterol. Regardless of whether beta-agonists are directly responsible for these adverse effects or are simply a marker for more severe asthma, heavy use of these agents should alert clinicians that it is necessary to reevaluate the patient's condition.


[Anti-angina effect of amiodarone in therapy-resistant angina pectoris]. In a double-blind randomized trial 63 patients on the waiting list for coronary artery bypass grafting, with stable angina NYHA III and a positive stress test on triple (nitrates, beta- and calcium entry blockers) combination therapy, were studied for an additional antianginal effect of amiodarone over a period of 2 months. In the treatment group an increase in exercise duration and a decrease of the double product and of ST-depression were noted. Thus, amiodarone is an effective antianginal agent in patients with limiting angina pectoris on conventional triple therapy.


Role of testosterone in gamma-glutamyltranspeptidase-dependent renal methylmercury uptake in mice. To elucidate the mechanisms responsible for sex and age differences in renal methylmercury uptake, effects of castration and testosterone treatment on mercury content and activity of renal gamma-glutamyltranspeptidase (gamma-GTP), which supposedly plays an important role in renal mercury uptake, were investigated in mice. Between 2 and 8 weeks of age, renal methylmercury uptake in male mice determined 4 hr after injection of a nontoxic dose of methylmercuric chloride (MMC, 1 mumol/kg, sc) increased about fivefold. At 4 weeks of age, a significant sex difference in renal mercury uptake first appeared. Renal mercury content in 4-week-old male mice was twofold higher than that of females and increased with age, but remained constant in females. Small but significant (p less than 0.05) differences in mercury content in other tissues were observed, which could not account for the marked sex- and age-related differences in renal mercury concentrations. Renal gamma-GTP activity gradually increased in males with maturation, and a sexual dimorphism of renal gamma-GTP was apparent after the fourth week. Seven days after castration of 4-week-old male mice, both renal mercury content and gamma-GTP activity were decreased to the levels in females. Activity of gamma-GTP was subsequently elevated to control male levels by sc injection of testosterone (5 mg/kg/day x 7 days). In female mice, both renal mercury content and gamma-GTP activity were increased to the level of males by testosterone treatment (5 mg/kg/day x 14 days). Thus, the renal mercury content was closely correlated with changes in renal gamma-GTP activity. These results suggest that sex and age differences in renal methylmercury accumulation may be due to a difference in renal gamma-GTP activity controlled at least in part by testosterone.


Inhibition of interleukin 2 receptor expression in normal human T cells by cyclosporine. Demonstration at the mRNA, protein, and functional levels. In view of the importance of the IL-2 receptors in the expression of antiallograft immunity and the currently existing controversy regarding the effect of CsA on the induction of IL-2 receptors, we explored the effect of cyclosporine on the induction of interleukin-2 receptor alpha and beta in normal human T cells. The effect of CsA on the induction of IL-2 receptors was examined at the levels of mRNA expression (with the aid of the polymerase chain reaction), protein (by SDS-PAGE analysis of chemically crosslinked 125I-IL-2 membrane protein complexes and by FACS), and function (by Scatchard analysis of 125I-IL-2 binding to T cells). The T cells were signaled with sn-1,2-dioctanoylglycerol and ionomycin or with crosslinked anti-CD3 and anti-CD2 mAbs. Our experimental design revealed that (A) CsA inhibits the induction of IL-2 receptor alpha and beta in normal human T cells, (B) the inhibitory activity is realized by a direct effect on T cells, and (C) the inhibitory activity is detectable at the pretranslational level--CsA significantly reduced the induction of mRNA encoding IL-2 receptor alpha and IL-2 receptor beta. These observations together persuasively demonstrate the ability of CsA to interrupt the emergence of IL-2 receptors on the surface of normal human T cells.


Evidence that multiple defects in cell-surface molecule interactions across species differences are responsible for diminished xenogeneic T cell responses. The purpose of the present study was to identify which of the several possible defects in cell-surface-molecule interactions are responsible for diminished mouse helper T cell responses to xenoantigens. We measured primary mouse anti-monkey, anti-pig, and anti-human proliferation in vitro in experimental systems in which potential defects were partially corrected by lymphokine supplementation and/or the use of transgenic or hybridoma cell populations. We found that the diminished mouse helper T cell responses to xenoantigens result from at least two defects in cell-surface-molecule interactions between T cells and xenogeneic APCs, specifically TCR and/or CD8 interactions with xenogeneic class I MHC molecules and accessory molecule interactions with their ligands (probably LFA-1 with ICAM-1/ICAM-2 and/or LFA-2 with LFA-3). Other investigators have identified additional defects, such as in lymphokine function across species differences. Thus, there appear to be multiple defects responsible for the diminished cellular immune response to xenoantigens.


Mitochondrial DNA mutation and heteroplasmy in type I Leber hereditary optic neuropathy. Leber hereditary optic neuropathy (LHON) is a maternally inherited disorder characterized by bilateral acute or subacute loss of central vision, primarily in young males. A G----A single base mutation at 11778nt of the mitochondrial genome which eliminates a SfaNI restriction site [Wallace et al., 1988; Holt et al., 1989; Hotta et al., 1989; Singh et al., 1989; Vilkki et al., 1989; Yoneda et al., 1989; Stone et al., 1990; Lott et al., 1990.] has been found in more than 60% of the families with LHON studied. We studied 25 persons from 4 families with LHON using SfaNI and Mae III digestion of a 201 base pair polymerase chain reaction (PCR) product encompassing the 11778nt mutation. The loss of the SfaNI site and the acquisition of a Mae III site at 11778nt were identified in all maternal relatives of the LHON families studied. The mutation was heteroplasmic in all affected individuals, female carriers, and males at-risk. The heteroplasmy of mitochondrial DNA (mtDNA) was also identified by direct DNA sequencing of PCR amplified by direct DNA sequencing of PCR amplified mtDNA digested by SfaNI or Mae III. It appears that the proportion of the mutant mtDNA correlates with the severity of the disease.


Five missense mutations at the adenosine deaminase locus (ADA) detected by altered restriction fragments and their frequency in ADA--patients with severe combined immunodeficiency (ADA-SCID). Severe combined immunodeficiency (SCID) is a heterogeneous syndrome, due to X-linked and autosomal recessive defects. A significant proportion of the autosomal recessive forms of SCID are due to mutations at the adenosine deaminase (ADA) locus. Nine different mutations at the ADA locus, including 7 missense point mutations, have been reported in children with ADA-SCID. We could detect 5 of the 7 missense mutations associated with ADA-SCID by alterations in restriction fragments utilizing standard restriction digestion of genomic DNA and hybridization of radiolabelled ADA genomic probes to Southern transfers. We additionally developed more rapid nonradioactive methods employing digestion of genomic DNA amplified by PCR that also detected all 5 mutations. Using these methods, we have examined a sample of 45 ADA-SCID chromosomes and report that these 5 missense mutations account for one third of the ADA--chromosomes studied, with 2 mutations being relatively common.


Rapid identification of deoxyribonucleic acid sequence differences in cytochrome P-450 21-hydroxylase (CYP21) genes with denaturing gradient gel blots. Denaturing gradient gel electrophoresis was used to identify single-base differences in the cytochrome P-450 21-hydroxylase (CYP21) genes of 132 unrelated control individuals and family members of three unrelated patients with 21-hydroxylase deficiency. The salt-wasting variety was caused by gene deletion and gene conversion/deletion mutations in affected members of two families studied. The simple virilizing form, present in the third family, was caused by an apparent point mutation not detectable by routine Southern blots. We have detected many restriction fragment melting polymorphisms in the CYP21 genes of the members of both salt-wasting families and normal individuals with denaturing gradient gel electrophoresis. We also identified a restriction fragment melting polymorphism specific for the simple virilizing patient in the third family. The data demonstrate that the CYP21 genes are highly polymorphic and that denaturing gradient gel electrophoresis is useful for genomic deoxyribonucleic acid analysis of patients with 21-hydroxylase deficiency.


Exercise inhibits glucocorticoid-induced glutamine synthetase expression in red skeletal muscles. One purpose of this study was to determine whether the suppression of glucocorticoid-induced glutamine synthetase (GS) gene expression by exercise is localized to fiber types that are known to be primarily recruited during endurance running. A second purpose examined whether denervation, which is associated with a reduction in contractile activity, would upregulate GS expression. Exercise consisted of treadmill running at 31 m/min for 12-16 wk. Glucocorticoid treatment (100 mg/kg body wt hydrocortisone 21-acetate) was administered during the last 11 days of the exercise program. Basal GS expression was lowest (GS enzyme activity, 43 +/- 3 nmol.h-1.mg protein-1; GS mRNA, 1.0 arbitrary units) in the slow-twitch red soleus, a muscle type that is known to resist glucocorticoid-induced muscle wasting, intermediate (74 +/- 10 and 1.7 +/- 0.2) in fast-twitch red quadriceps, a muscle type susceptible to atrophy, and highest (106 +/- 16 and 5.4 +/- 1.3) in fast-twitch white quadriceps, a muscle type known to be most susceptible to atrophy. Hormone treatment increased GS enzyme activity and mRNA by two- to fourfold in all muscle types. Exercise diminished GS enzyme activity and mRNA in the fast-twitch red fibers to 35-70% of sedentary control values in both basal and glucocorticoid-stimulated muscles. The running also reduced GS enzyme activity in hormone-treated slow-twitch fibers but did not alter basal or glucocorticoid-induced GS expression in fast-twitch white fibers. These results indicate that glucocorticoids induce similar relative GS expression across all muscle types, but the low absolute levels of expression in slow-twitch muscles are not related to any atrophy.(ABSTRACT TRUNCATED AT 250 WORDS)


Mechanisms of cAMP-mediated relaxation of distal circular muscle in rabbit colon. Photolytic release of free adenosine 3',5'-cyclic monophosphate (cAMP) from its caged form was used to evaluate the physiological role of several proposed mechanisms of cAMP-mediated relaxation of circular smooth muscle in the distal rabbit colon. Photolysis of caged cAMP produced a rapid relaxation of bethanechol-contracted distal circular muscle strips that was dependent on ultraviolet exposure time. An increase in release of free cAMP, associated with increased ultraviolet exposure, was confirmed with high-performance liquid chromatography. Vanadate (an ATPase inhibitor) (3 mM) caused a 48% decrease in cAMP-mediated relaxation, while ouabain and a zero K+ bath solution failed to affect relaxation. cAMP-mediated relaxation of KCl-contracted strips was significantly less effective than that of bethanechol-contracted strips. Although this finding suggested that cAMP-mediated relaxation may involve K+ channel modulation, specific (glibenclamide, charybdotoxin) and nonspecific (TEA) K+ channel blockade failed to affect cAMP-mediated relaxation of bethanechol-contracted strips. The photolytic release of cAMP failed to relax Ca(2+)-contracted saponin skinned muscle strips. These studies suggest 1) modulation of Ca2+ pumps plays an important role in this model of relaxation of distal colonic circular muscle in the rabbit colon, 2) modulation of the Na+ pump or sarcolemmal K+ channels may not play an important physiological role in relaxation induced by a rapid rise in intracellular cAMP, and 3) cAMP does not seem to have a significant physiological effect on the Ca2+ sensitivity contractile apparatus.


Intestinal acid inhibits gastric acid secretion by neural and hormonal mechanisms in rats. To determine the relative contributions of neural reflexes and intestinal hormones to the inhibition of gastric acid secretion by intestinal acidification, rats with an extrinsically denervated, transplanted segment of jejunum, and those with an innervated segment of jejunum, were studied. Postoperatively, meal-stimulated gastric acid secretion was measured. When the acid secretory response to intragastric liver extract reached a plateau, graded concentrations of hydrochloric acid or saline were instilled into the jejunal segments. Gastric acid secretion was inhibited by intrajejunal acid (pH 2.5) by 79% in the innervated rats and by 64% in the transplanted group. Thus at a pH of 2.5 there was a 15% greater maximum inhibition of plateau acid response in the innervated rats than in the transplanted rats, presumably because of the extrinsic neural contribution. To examine the hormonal mediators, the effects of a somatostatin monoclonal antibody and a CCK-A receptor antagonist (L 364718) on acid-induced inhibition of gastric acid secretion were studied in transplanted rats. Treatment with a somatostatin monoclonal antibody or with L 364718 reduced the acid-induced (pH 2.5) inhibition of gastric acid secretion by 93 and 27%, respectively. Jejunal acidification inhibits gastric acid secretion in the rat by both neural and hormonal mechanisms. The hormonal mechanism is mediated by somatostatin and CCK.


Hemodynamic responses evoked by endothelin from central cardiovascular neural substrates. Endothelin-1 (ET-1, 3-10 pmol) applied to the fourth cerebral ventricle of anesthetized ventilated rats decreased mean arterial pressure (MAP, 37 +/- 5 to 55 +/- 5%), heart rate (13 +/- 7 to 21 +/- 3%), and renal blood flow (RBF, 41 +/- 7 to 45 +/- 8%; all values are means +/- SE) for 30-90 min. At a 30-pmol dose of ET-1, the decrease in MAP was preceded by an increase (58 +/- 16%). Micropneumophoresis of ET-1 (100-300 fmol) into discrete glutamate-responsive cardiovascular loci within the nucleus tractus solitarii (NTS), viz., the dorsal strip and the commissural subnucleus, produced depressor and bradycardic responses. However, central ET-1 was ineffective in evoking swallowing responses when microinjected into glutamate-responsive deglutitive sites in the NTS. These data suggest that, at low doses, ET-1 evokes hypotension and bradycardia by a specific neuronal action in the central nervous system; one site of action appears to be the cardiovascular neural substrates within the NTS; decreases in RBF may be secondary to the hypotension, since renal vascular resistance also decreased. In anesthetized nonventilated rats, ET-1 (3 and 10 pmol) applied to the fourth ventricle produced profound respiratory depression accompanied by a transient pressor effect. Thus centrally administered ET-1 can elicit complex cardiovascular responses by a direct action on cardiovascular substrates and/or indirectly via respiratory depression.


Halothane anesthesia abolishes pulmonary vascular responses to neural antagonists. We investigated the effects of the inhalational anesthetic halothane on autonomic nervous system (ANS) regulation of the baseline pulmonary vascular pressure-flow (P/Q) relationship compared with that measured in the conscious state. Multipoint pulmonary vascular P/Q plots were constructed by stepwise constriction of the thoracic inferior vena cava to decrease venous return and Q. P/Q plots were generated in the same dogs in the conscious state and during halothane anesthesia (approximately 1.2% end tidal) in the intact (no drug) condition and after administration of selective ANS antagonists. In conscious dogs, sympathetic alpha 1-adrenoreceptor block with prazosin decreased (P less than 0.01) the pulmonary vascular pressure gradient [pulmonary arterial pressure-pulmonary arterial wedge pressure (PAP-PAWP)] over the entire range of Q studied; i.e., inhibition of endogenous alpha 1-adrenoreceptor activity caused pulmonary vasodilation. In contrast, alpha 1-adrenoreceptor block had no effect on PAP-PAWP at any value of Q during halothane anesthesia. In conscious dogs, sympathetic beta-adrenoreceptor block with propranolol increased (P less than 0.01) PAP-PAWP over the entire range of Q studied; i.e., inhibition of endogenous beta-adrenoreceptor activity resulted in pulmonary vasoconstriction. However, beta-adrenoreceptor block had no effect on PAP-PAWP at any value of Q during halothane anesthesia. Finally, cholinergic receptor block with atropine decreased (P less than 0.05) PAP-PAWP at values of Q greater than 100 ml.min-1.kg-1 in conscious dogs but had no effect on PAP-PAWP at any value of Q during halothane anesthesia. These results indicate that endogenous ANS regulation of the baseline pulmonary vascular P/Q relationship observed in conscious dogs is abolished during halothane anesthesia.


Beta 2-adrenoceptor-mediated positive dromotropic effects on atrioventricular node of dogs. The functional significance of beta 2-adrenoceptors in atrioventricular (AV) nodal conduction was investigated by using canine isolated blood-perfused AV node preparations. Dose-dependent shortening of the atrio-His bundle (A-H) interval by dl-procaterol hydrochloride hemihydrate (0.03-1 nmol) injected intra-arterially into the AV node artery was affected little by an infusion of dl-atenolol (10 nmol/min) into the same artery, whereas the dose-response curve for the positive dromotropic effect of procaterol was shifted markedly to the right by approximately 1.5 and 2.5 log units with ICI 118551 [erythro-dl-1-(7-methylindan-4-yloxy)-3-isopropylamino-bu tan-2-ol]- hydrochloride (1 and 10 nmol/min). The positive dromotropic effect of dl-T-1583 [alpha-(3,4,5-trimethoxyphenethylaminomethyl)-3,4-dihydroxybenzyl- alcohol] hydrochloride (30-300 pmol), a selective beta 1-adrenoceptor agonist, was shifted to the right by approximately 1.2 log units with dl-atenolol (10 nmol/min) but was affected little by ICI 118551 (10 nmol/min). The dose-response curve for l-isoproterenol hydrochloride (3-100 pmol) was shifted to the right by 0.7 log unit with ICI 118551 (10 nmol/min) and by 1.7 log units with atenolol (10 nmol/min). The dose-response curve for dl-norepinephrine (0.03-1 nmol) was shifted to the right by 1.0 log unit with atenolol only, whereas the curve for l-epinephrine (0.03-1 nmol) was shifted by 0.45 log unit with ICI 118551 (10 nmol/min). These results suggest that both beta 1- and beta 2-adrenoceptors, which coexist on the AV node, play a functional role in AV nodal conduction.


Fluid restitution and shift of blood volume in anesthetized rabbits subject to cyclic hemorrhage. We investigated the effect of a 10% cyclic blood volume change with a period of 2 or 4 min to study the short-term control of blood volume. In experiments with pentobarbital-anesthetized rabbits, the blood density variation over a 2-min cycle is 0.94 +/- 0.04 (SE) g/l, and the plasma density variation is 0.17 +/- 0.04 g/l. The plasma density variation could result from a fluid restitution from the extravascular space (with a density 1,005 g/l), with a volume equal to 14% of the withdrawn blood volume. This restitution cannot account, however, for the entire observed density change in arterial blood. Because of the Fahraeus effect in microvascular flow, a shift in blood volume from the microvasculature is another mechanism that could lead to a decrease in the density of arterial blood. An analysis of the blood and plasma density variations indicates that a blood volume (49% of the shed volume) is shifted from the micro- to macrocirculation. This volume compensation by fluid restitution and volume shift acts to minimize the effect of hemorrhage on the filling of the venous system. We found that the blood density waveform parallels the change in blood volume. When the blood volume change reverses its direction, the density change also reverses direction with a time delay less than 8 s. The blood density variations are not altered by bilateral vagotomy or its combination with hexamethonium (a sympathetic ganglionic blocker). These observations of anesthetized rabbits indicate that the short-term compensation is primarily due to the volume shift from the microcirculation and is not regulated by humoral or neural mechanisms but by local mechanisms such as autoregulation and the passive response due to changes in microvascular pressure.


Myocardial oxygenation in the isolated working rabbit heart as a function of work. Myocardial O2 consumption (MVO2) was stimulated up to two-fold by either increasing afterload or beta-receptor stimulation in working normothermic isolated rabbit hearts while noninvasively monitoring the O2 delivery or phosphate compounds (total n = 48). Intracellular O2 delivery was estimated with the use of myocardial optical absorbance changes centered at 603.5 and 582 nm that correlate with cytochrome aa3 redox and myoglobin oxygenation states. Phosphate-containing metabolites (ATP, phosphocreatine, free ADP) were assessed using 31P nuclear magnetic resonance spectroscopy. Measurements were made both with intact autoregulation and after maximal vasodilation by 1 microM nitroprusside (NP). When afterload was used to increase MVO2, absorbance decreased at 603.5 nm and increased at 582 nm, consistent with a 10-15% increase in myocardial oxygenation, without an associated change in cardiac phosphate compounds. NP caused a further increase in myocardial oxygenation and venous PO2 consistent with an increase in the O2 supply-to-demand ratio. Increases in MVO2 due to beta-stimulation alone were not associated with changes in 603.5-nm absorbance or phosphate compounds, but in combination with NP were accompanied by increased oxygenation, venous PO2, and cardiac phosphocreatine. KCl arrest caused maximal increases in oxygenation and phosphocreatine. These findings suggest that neither cytochrome aa3 nor myoglobin in the isolated working rabbit heart is fully oxidized or oxygenated, respectively. Furthermore, the oxygenation state of the tissue varied both with afterload-induced changes in cardiac work and with changes in O2 supply/demand.


True histiocytic malignancy associated with a malignant teratoma in a patient with 46XY gonadal dysgenesis. The relatively frequent association of hematologic neoplasia and primary mediastinal germ cell tumors has been reported. Of these hematologic malignancies, nine were classified as malignant histiocytosis or acute monoblastic leukemia, and all occurred in males. We now report on a patient who was phenotypically female, with 46XY gonadal dysgenesis, and who developed a true histiocytic malignancy that presented as a large hepatic tumor and also involved the spleen, right kidney, and lymph nodes. Twenty-six months before the development of the histiocytic malignancy, an ovarian malignant teratoma with yolk sac elements was removed; the patient subsequently received chemotherapy. The neoplasm was composed of large pleomorphic cells and the histiocytic nature was established by cytologic, cytochemical, immunologic, and ultrastructural studies. In the course of her illness, the patient developed classic acute monoblastic leukemia 8 months after the diagnosis of histiocytic malignancy. Karyotypic analysis of the hepatic tumor, bone marrow, and blood showed 46XY gonadal dysgenesis. We believe that this is the first reported case of a phenotypically female patient who developed these two rare malignancies. It suggests that the association between germ cell tumors and histiocytic malignancy in genotypically male individuals may not be coincidental or secondary to therapy, but may be a phenomenon related to dysgenetic gonads in the presence of a Y chromosome.


Inhibition of DNA synthesis by somatostatin in rat hepatocytes stimulated by hepatocyte growth factor or epidermal growth factor. The antiproliferative effects of somatostatin on hepatocytes stimulated by hepatocyte growth factor (HGF) or epidermal growth factor (EGF) were investigated using primary cultures of adult rat hepatocytes. Somatostatin inhibits HGF-induced (at a dose of 10 ng/mL) or EGF-induced (at a dose of 100 ng/mL) 3H-thymidine incorporation into hepatocytes in a dose-dependent manner (10(-10) to 10(-8) M). This inhibition was confirmed by autoradiography. The effect of somatostatin was nontoxic as judged by preserved albumin synthesis, a marker for differentiated hepatocyte function. In the presence or absence of somatostatin, neither HGF nor EGF significantly altered intracellular cyclic adenosine monophosphate (cAMP). We conclude that somatostatin is a potent inhibitor of HGF- or EGF-induced deoxyribonucleic acid synthesis in adult rat hepatocytes. The mechanism of this inhibition appears to be independent of cAMP. The significance of somatostatin in liver regeneration has yet to be assessed.


Platelet activating factor-induced changes in gastric motility and vascular resistance. Our study evaluated the hypothesis that gastric contractions may contribute to ischemia by increasing vascular resistance. Using an ex vivo segment of the dog's stomach as the experimental model, contractions were induced with platelet activating factor (PAF) and bethanechol, a cholinergic vasodilator. Spontaneous contractions produced slight increases in luminal pressure and corresponding increases in vascular resistance. PAF caused statistically significant, dose-dependent increases in the force of gastric contractions that were highly correlated with phasic changes in vascular resistance. To ensure that the relationship between contractions and vascular resistance was independent of vascular tone, we next examined responses to bethanechol. Bethanechol stimulated contractions that also transiently increased both luminal pressure and vascular resistance. Our results demonstrate that gastric contractions markedly increase vascular resistance and support the hypothesis that hypercontractility may contribute to the development of mucosal ischemia during ulceration.


Somatostatin stimulation of the normal esophagus. The inhibitory effects of somatostatin on gastric and small bowel motor function are well documented. However, the effects of somatostatin on esophageal body motility and lower esophageal sphincter tone are not completely defined. We investigated the effects of octreotide, a long-acting somatostatin analogue, on the esophageal body and the lower esophageal sphincter in 15 healthy volunteers. Lower esophageal sphincter tone was increased by octreotide infusion. Esophageal body contraction amplitude and velocity were also increased by octreotide infusion. Our data show that somatostatin stimulates the normal human esophagus, an action mediated either by a direct effect, a central nervous system action, or the inhibition of the secretion of gastrointestinal hormones that influence esophageal motor activity.


Ovarian strumal carcinoid in association with multiple endocrine neoplasia, type IIA. Strumal carcinoid is an unusual form of monodermal ovarian teratoma with thyroid-like follicles admixed with typical carcinoid tumor patterns. We encountered a case of this neoplasm in a patient with multiple endocrine neoplasia, type IIA (Sipple's syndrome), including a medullary thyroid carcinoma diagnosed 24 years previously. During evaluation of bilateral adrenal pheochromocytomas, a unilateral left ovarian strumal carcinoid was discovered. Subsequently, the patient had a parathyroid adenoma excised. The ovarian tumor was immunohistochemically reactive for neuron-specific enolase, chromogranin, synaptophysin, and serotonin, but did not stain for calcitonin. The follicular structures stained for thyroglobulin. This unusual case shows that ovarian strumal carcinoid, like carcinoid tumors at other sites, may arise in association with multiple endocrine neoplasia.


SDZ 280-446, a novel semi-synthetic cyclopeptolide: in vitro and in vivo circumvention of the P-glycoprotein-mediated tumour cell multidrug resistance. SDZ 280-446 is a semi-synthetic derivative of a natural cyclic peptolide. Its ability to sensitise in vitro tumour cells whose resistance is due to P-glycoprotein-mediated anticancer-drug efflux was shown using four different pairs of parental drug-sensitive (Par-) and multidrug-resistant (MDR-) cell lines, from three different species (mouse, human, Chinese hamster) representing four different cell lineages (monocytic leukaemia, nasopharyngeal epithelial carcinoma, colon epithelial carcinoma, ovary fibroblastoid carcinoma), and using four different drug classes (colchicine, vincristine, daunomycin/doxorubicin and etoposide). By measuring its capacity to restore normal drug sensitivity of MDR-cells in culture in vitro, it appeared that SDZ 280-446 belongs to the same class of very potent chemosensitisers as the cyclosporin derivative SDZ PSC 833: both are about one order of magnitude more active than cyclosporin A (CsA), which is itself about one order of magnitude more active than other known chemosensitisers (including verapamil, quinidine and amiodarone which have already entered clinical trials in MDR reversal). Low concentrations of SDZ 280-446 could also restore cellular daunomycin retention in MDR-P388 cells to the levels found in the Par-P388 cells. SDZ 280-446 was also effective as a chemosensitiser when given orally in vivo. In a syngeneic mouse model, combined therapy with vinca alkaloids given i.p. and SDZ 280-446 given per os for 5 consecutive days significantly prolonged the survival of MDR-P388 tumour-bearing mice, when compared with mice receiving vinca alkaloids alone. Another protocol, using three cycles of i.p. doxorubicin at 4 day intervals, could also not increase MDR-P388 tumour-bearing mouse survival unless the mice received SDZ 280-446 orally 4 h before each doxorubicin injection. Though only very few combined therapy treatment protocols have been tested so far, clear increases in survival time of MDR-tumour-bearing mice were regularly obtained, leaving hope for major improvement of the therapy using other dosing schedules.


Prenatal ethanol exposure during the last third of gestation in rat reduces hippocampal NMDA agonist binding site density in 45-day-old offspring. The effect of ethanol exposure during different periods of prenatal or postnatal development on hippocampal N-methyl-D-aspartate (NMDA) receptor binding was studied in rat. Fetal rat pups were exposed to ethanol for different periods of time during gestation via maternal consumption of a 3.35% ethanol liquid diet. In a separate experiment, neonatal pups were fed 2.51 g ethanol/kg body weight/day from Postnatal Day (PD) 4 to PD 10 via intragastric feeding tube. These two ethanol administration paradigms produced average peak maternal and pup blood ethanol concentrations of 39 mg/dl and 57 mg/dl, respectively. At 45 days of age, offspring from each treatment group were sacrificed for measurements of hippocampal NMDA-sensitive [3H]-glutamate binding site density using in vitro radiohistochemical techniques. As observed previously, prenatal ethanol exposure throughout gestation resulted in NMDA-sensitive [3H]-glutamate binding site reductions in the apical dendritic field regions of dentate gyrus, hippocampal CA1 and subiculum of dorsal hippocampal formation compared to the ad lib or pair-fed control groups. NMDA-sensitive [3H]-glutamate binding was not different than control in rats exposed to ethanol during the first half of gestation only. Prenatal ethanol exposure during the last half or the last third of gestation resulted in NMDA-sensitive [3H]-glutamate binding site reductions comparable to the binding site reductions observed in rats exposed to ethanol throughout gestation. Hippocampal NMDA-sensitive [3H]-glutamate binding site density in postnatal ethanol-exposed rats was not different than the suckling or gastrostomy control groups.(ABSTRACT TRUNCATED AT 250 WORDS)


Relationship between c-erbB-2 protein product expression and response to endocrine therapy in advanced breast cancer. Of 221 patients with breast cancer of known epidermal growth factor receptor (EGFR) and oestrogen receptor (ER) status, 99 had developed recurrences during the period of follow-up (range 3-60 months, median 24 months). Of these, 72 received endocrine therapy as first-line treatment for relapse. Immunohistochemical assessment of c-erbB-2 protein product expression was made using paraffin-embedded tumour tissue from 65 of these 72 patients. Including patients whose disease remained stable for more than 6 months with those showing an objective response (CR or PR for more than 3 months), only one (7%) of 14 c-erbB-2 positive tumours responded to endocrine manipulation compared with 19 (37%) of 51 c-erbB-2 negative tumours (P less than 0.05). Coexpression of c-erbB-2 reduced the response rate of ER positive patients from 48% to 20% and of ER negative cases from 27% to 0% (P less than 0.01). EGFR and c-erbB-2 protein appeared to have additive effects in reducing the likelihood of response, and none of eight patients with EGFR positive, c-erbB-2 positive tumours derived benefit from endocrine therapy. The results of this study suggest that c-erbB-2 protein overexpression, a marker of poor prognosis in breast cancer, is associated with a lack of response to endocrine therapy on relapse, and particularly in combination with EGFR may be useful in directing therapeutic choices.


Effects of interferon-alpha on human B cell responsiveness: biphasic effects in cultures stimulated with Staphylococcus aureus. Although interferon-alpha (IFN-alpha) has been found to be involved in the immune regulation in vivo, the effects of IFN-alpha on human B cells have not yet been clarified because of conflicting results in the literature. The present study therefore examined the effects of several subtypes of IFN-alpha (natural, alpha 1, alpha 2a, alpha 2b) on B cell responsiveness in detail by comparing different experimental conditions. Highly purified B cells from normal human individuals were cultured with Staphylococcus aureus (SA) + IL-2 or with immobilized anti-CD3-activated T4 cells in the presence or absence of IFN-alpha. IFN-alpha enhanced the immunoglobulin (Ig) production induced by immobilized anti-CD3-activated T4 cells. By contrast, IFN-alpha (5-50,000 IU/ml) suppressed the Ig production induced by SA + IL-2. The suppression by IFN-alpha was dependent on the concentration of SA. The inhibitory effects of IFN-alpha in SA-stimulated cultures were exerted in the first 72 hr of cultures and required the presence of IL-2, whereas IFN-alpha enhanced the maturation of B cells when it was added after 72 hr of cultures. The suppressive effects of IFN-alpha were overcome by addition of immobilized anti-CD3-preactivated T cells that had been treated with mitomycin C, but not by the addition of fresh T cells or soluble factors produced by activated T cells. Of interest, IFN-alpha did not inhibit the expression of IL-2R, but inhibited that of intercellular adhesion molecule-1 (ICAM-1) on B cells after stimulation with SA + IL-2, suggesting that the suppressive effects of IFN-alpha might be related to the regulation of B cell-B cell contacts through ICAM-1. There was no significant difference in effects on B cells among various subtypes of IFN-alpha. These results suggest that the effects of IFN-alpha on human B cell responsiveness may be different depending on the nature of stimulation. Moreover, the data indicate that IFN-alpha enhances the differentiation of activated B cells irrespective of the activation signals.


Modification by ketamine on the neuromuscular actions of magnesium, vecuronium, pancuronium and alpha-bungarotoxin in the primate. The neuromuscular effects of ketamine, at cumulative doses of 2.5 and 10 mg.kg-1 iv, were studied by electromyographically quantifying the thumb response evoked by ulnar nerve stimulation in 25 monkeys anaesthetized with pentobarbital-N2O-O2. Ketamine alone at these doses had no neuromuscular effects. When the EMG response was maintained at 50% of control by a continuous infusion of magnesium, vecuronium, or pancuronium, ketamine depressed the responses by an additional 13 +/- 3%, 34 +/- 7% and 32.5 +/- 3.3% (mean +/- SEM), respectively, at the highest dose, P less than 0.05. In contrast, ketamine had no effect on the neuromuscular block produced by incremental doses of alpha-bungarotoxin. These results indicate that ketamine does not act on the postjunctional acetylcholine receptor. It plays a secondary role in neuromuscular block, possibly by prejunctional or postjunctional effects independent of receptor occupation.


Esmolol prevents and suppresses arrhythmias during halothane anaesthesia in dogs. The antiarrhythmic effect of esmolol, a selective beta 1 adrenoreceptor blocker, was evaluated in the presence of epinephrine induced arrhythmias in dogs (n = 6). The arrhythmogenic dose of epinephrine (ADE) during 1.2 MAC halothane in dogs was increased from 3.23 +/- 0.25 (mean +/- SD) to 30.90 +/- 3.56 micrograms.kg-1.min-1 (P less than 0.001) by the prior administration of esmolol 0.5 microgram.kg-1 bolus followed by an infusion at the rate of 150 micrograms.kg-1.min-1. Higher esmolol infusion doses of 200 micrograms.kg-1.min-1 further increased ADE to 99.0 +/- 2.92 micrograms.kg-1.min-1 (P less than 0.001). After discontinuation of esmolol and during continued halothane anaesthesia, ventricular tachycardia was induced by increasing the infusion rate of the 100 micrograms.ml-1 solution of epinephrine. In all dogs ventricular tachycardia was restored to sinus rhythm by a bolus dose of esmolol (1 microgram.kg-1). We conclude that esmolol pretreatment increases the ADE during halothane anaesthesia in dogs. Our data suggest that esmolol may be useful as an antiarrhythmic agent in the management of epinephrine-related ventricular arrhythmias during anaesthesia in man.


Expression of P-glycoprotein gene in marine sponges. Identification and characterization of the 125 kDa drug-binding glycoprotein. In the present paper it is shown that the marine sponges Geodia cydonium and Verongia aerophoba contain the gene coding for P-glycoprotein P170, also known as a multidrug-resistance gene. Western blot studies revealed that polyclonal antibodies raised against hamster P170 cross-react with the sponge polypeptide of Mr 125,000. After endoglycosidase F treatment, the sponge P125 is converted to a polypeptide of Mr 105,000. Northern blot studies, using the human P170 cDNA probe, revealed a size of 4.2 kb for the sponge P125 transcript. The level of this transcript does not change in response to incubation with the aggregation factor. Confocal laser scanning microscopy showed that P125 is a cell membrane bound protein. In addition, sponge membrane vesicles possess a potential to bind in vitro 2-acetylamino-fluorene, vincristine and daunomycin. This process is Verapamil-sensitive, a characteristic known also for the mammalian vesicle associated P170. The data reported demonstrate that the classical multidrug resistance mechanism, described in drug-resistant tumor cell lines, functions also in sponges and may explain the relative resistance of these animals to pollution.


Potent inhibition of aflatoxin-induced hepatic tumorigenesis by the monofunctional enzyme inducer 1,2-dithiole-3-thione. 1,2-Dithiole-3-thiones are five-membered cyclic sulfur-containing compounds with antioxidant, chemotherapeutic, radioprotective and chemoprotective properties. Several substituted 1,2-dithiole-3-thiones are used medicinally and one of these, oltipraz [5-(2-pyrazinyl)-4-methyl-1,2-dithiole-3-thione], has been recently shown to be an inhibitor of aflatoxin B1 (AFB1) hepatocarcinogenesis in the rat. Structure-activity studies have been undertaken to probe the mechanisms by which dithiolethiones inhibit carcinogenesis. Such studies revealed that unsubstituted 1,2-dithiole-3-thione was more effective than oltipraz at inhibiting aflatoxin-DNA adduct formation in vivo and at inducing electrophile detoxication enzymes in cell culture. In the present studies the effects of dietary administration of 1,2-dithiole-3-thione on the induction of xenobiotic metabolizing enzymes and inhibition of aflatoxin-induced hepatic tumorigenesis were examined. Male F344 rats were fed graded doses of 1,2-dithiole-3-thione (0.001-0.03%) for 4 weeks. During the second and third weeks of 1,2-dithiole-3-thione feeding, rats were dosed by gavage with 250 micrograms of AFB1/kg five times a week. Rats were then restored to control AIN-76A diet 1 week after cessation of AFB1 dosing. At 4 months, focal areas of hepatocellular alteration were identified and quantified by staining sections of liver for gamma-glutamyltranspeptidase (GGT) activity and glutathione S-transferase P (GST-P) expression. Treatment with 1,2-dithiole-3-thione at the lowest dose (0.001%) reduced by greater than 80% the volume of liver occupied by GGT or GST-P foci; higher dietary concentrations provided greater than 98% reductions in the volume per cent of these markers for presumptive preneoplastic lesions. All dietary concentrations of 1,2-dithiole-3-thione resulted in significant elevations in hepatic GST activities. In accord with the protective effects against tumorigenesis, 4- to 6-fold increases in the specific activities of aflatoxin-glutathione conjugation were observed in cytosols prepared from livers of animals fed 1,2-dithiole-3-thione. By contrast, 1,2-dithiole-3-thione did not have any detectable inductive effects on hepatic microsomal cytochrome P450 levels or activities. Dietary administration of 1,2-dithiole-3-thione also elevated activities of GSTs and other phase II enzymes in several extrahepatic organs. This broad pattern of induction of detoxication enzymes by 1,2-dithiole-3-thione supports the potential widespread use of this compound as a protective agent against chemical carcinogenesis and other forms of electrophile toxicity.


Induction of intercellular adhesion molecule-1 (CD54) on human hepatoma cell line HepG2: influence of cytokines and hepatitis B virus-DNA transfection. Human hepatocyte expression of intercellular adhesion molecule-1 (ICAM-1) (CD54) was studied in vitro by exposing the well differentiated human hepatoblastoma cell line HepG2 to various cytokines. In addition, hepatitis B virus (HBV)-DNA transfected HepG2 cells were also analysed. Expression of ICAM-1 on HepG2 cells was then revealed with an immunohistochemical procedure. Untreated HepG2 cells were unreactive, but showed strong cytoplasmic ICAM-1 immunoreactivity after treatment with interferon-gamma (IFN-gamma). This induction was completely inhibited by addition of a neutralizing antibody directed to IFN-gamma. IL-1, IL-6, tumour necrosis factor-alpha (TNF-alpha) and IFN-alpha, used alone or in combination, did not induce ICAM-1 expression, neither did they inhibit the IFN-gamma-induced expression of this adhesion molecule on HepG2 cells. Untreated hepatitis B virus-DNA transfected HepG2 cells expressed membranous ICAM-1. These results indicate that IFN-gamma is the main cytokine trigger for ICAM-1 expression on HepG2 cells, suggesting that in areas of liver inflammation this adhesion molecule is up-regulated on hepatocytes by locally released IFN-gamma. In addition, expression of ICAM-1 by hepatitis B virus-DNA transfected HepG2 cells suggests other, still unknown, triggering mechanisms in the induction of such adhesion molecules, for instance gene activation by viral genome, or autocrine virus-induced hepatocellular cytokine production.


Therapeutic response to somatostatin analogue, BIM 23014, in metastatic prostatic cancer. Metastatic prostate cancer is well known to respond to hormonal manipulations, but once progression occurs new treatment modalities are required. Specific and systemic antitumour therapy is preferable to local treatments such as radiotherapy in such patients. The finding that somatostatin analogue, BIM 23014, inhibits prostatic tumour growth in animal models is of great interest. We treated 25 poor risk patients with progressive metastatic prostate cancer. Sixteen had also failed to respond to 'total androgen blockade'. Two patients have achieved a partial remission, one of which is maintained at over 30 months, and three had stable disease for over 6 months. Side effects have consisted of mild diarrhoea and abdominal cramp in the first few days of treatment in a minority of the patients. These results are encouraging and further randomized studies are in progress.


Localization of the rat insulin I gene (INS1) to chromosome 1q55 by fluorescence in situ hybridization. The rat insulin I gene (INS1) was assigned to chromosome 1q55 using fluorescent in situ hybridization. In addition, several RFLPs were detected among 11 inbred rat strains.


Gamma-aminobutyric acid-glutamate interaction in the control of somatostatin release from hypothalamic neurons in primary culture: in vivo corroboration. Recent studies have provided new data on the neuroendocrine role of glutamate (the major excitatory neurotransmitter) on somatostatin release. The neuroendocrine role of gamma-aminobutyric acid (GABA) (the major inhibitory neurotransmitter) on this same secretion, is also well established. Our objective was thus to investigate whether GABA and glutamate, which have opposite neurotransmission signals, could interact in the control of hypothalamic somatostatin release. Pharmacological manipulations of the two types of receptors were performed in vitro on primary cultures of hypothalamic neurons secreting somatostatin. We found that tonic release of somatostatin was reduced by 76% in the presence of tetrodotoxin (TTX) and was regulated by endogenous secretion of glutamate and GABA. CGS 19755, a highly selective N-methyl-D-aspartate (NMDA) receptor antagonist, significantly reduced tonic somatostatin secretion whereas it was strongly increased by picrotoxin and bicuculline, two GABAA antagonists. When CGS 19755 was applied with picrotoxin, somatostatin release was the same as levels obtained in the control group with TTX. GABA reduced tonic somatostatin release (in the presence or absence of TTX), and glutamate-stimulated secretion in a dose-dependent manner. Picrotoxin stimulation of tonic somatostatin release was additive with that obtained after glutamate stimulation and was also dose-dependent. This interaction was also studied in vivo in unanesthetized rats bearing a push-pull cannula stereotaxically implanted into the median eminence. Ip injected CGS 19755 (an antagonist that can freely permeate the blood-brain barrier) completely blocked the peak secretion of somatostatin observed after ip picrotoxin administration, whereas there was no significant effect when it was injected alone. These findings corroborated our in vitro data and allow us to postulate that GABA and glutamate interact in the control of somatostatin.


Time course and mechanism of growth hormone's negative feedback effect on its own spontaneous release. Endogenous pulsatile GH secretion is blunted by the administration of exogenous GH; however, few data are available on the time course of GH negative feedback, and the mechanism by which this occurs still remains unclear. In the present study, we examined the temporal pattern of the inhibitory effect induced by an acute (single) and chronic (5 days) sc recombinant human (rh) GH injection regimen on spontaneous GH release in the rat and assessed the possible involvement of the hypothalamic GH-inhibitory peptide, somatostatin (SRIF), in this response. Eight-hour (0800-1600 h) GH secretory profiles, obtained from free-moving adult male rats administered a single sc injection of 200 micrograms rhGH at 0800 h, revealed a marked suppression of spontaneous GH pulses (GH peak amplitude: 45.7 +/- 10.9 vs. 207.8 +/- 31.7 ng/ml in H2O-injected control rats; P less than 0.001) lasting for up to 4.1 +/- 0.1 h after the injection (mean 4-h plasma GH level: 13.6 +/- 3.6 vs. 49.4 +/- 7.0 ng/ml in H2O-injected controls; P less than 0.01). During the subsequent 4- to 8-h period, recovery of spontaneous GH secretory bursts was evident, and neither the GH peak amplitude nor mean 4-h plasma GH level of rhGH-treated rats was significantly different from that of H2O-injected controls. The magnitude, time course, and recovery of the rhGH-induced inhibitory effect on pulsatile GH release after chronic rhGH treatment was similar to that after a single injection. Passive immunization of rhGH-treated rats with SRIF antiserum reversed the rhGH-induced inhibition of spontaneous GH pulses (peak amplitude: 131.7 +/- 53.7 vs. 7.1 +/- 3.4 ng/ml in rhGH-treated control rats given normal sheep serum; P less than 0.05) and restored both the GH peak amplitude and mean plasma GH level to values similar to those in H2O-injected controls. Taken together, these results demonstrate that: 1) the inhibitory effect of rhGH on endogenous pulsatile GH release is of short duration (approximately 4 h); 2) the time course of this response does not change after 5-day repeated rhGH administration; and 3) the feedback effect of GH on its own spontaneous release is exerted, at least in part, by increasing hypothalamic SRIF secretion. Such a mechanism of GH feedback may be important in the physiological control of pulsatile GH secretion.


Developmentally regulated polyadenylation of two discrete messenger ribonucleic acids for müllerian inhibiting substance. Mullerian inhibiting substance (MIS) is a 140-kilodalton homodimeric glycoprotein that causes regression of the Mullerian ducts in male embryos, and may also have a role in both males and females in the regulation of germ cell maturation. We examined the ontogeny of MIS messenger RNA (mRNA) in rat testes from midgestation through adulthood and found two discrete MIS mRNA species that are developmentally regulated. The larger 2.0-kilobase species is abundant at embryonic day 14, then decreases in late gestation, and is barely detectable after birth. The smaller 1.8-kilobase species is first noted at embryonic day 18 and is the major species detected postnatally. Both species are abundant just prior to birth, at embryonic day 21, then decrease markedly after birth. This variation in MIS mRNA levels correlates with the developmental expression of MIS protein. A series of oligonucleotide-directed ribonuclease H mapping experiments determined that the two mRNA species differ at their 3' ends in the extent of polyadenylation. Thus, differential polyadenylation of MIS mRNA may be an additional mechanism for regulating MIS expression during fetal and postnatal development.


Pituitary adenylate cyclase activating polypeptide, growth hormone (GH)-releasing peptide and GH-releasing hormone stimulate GH release through distinct pituitary receptors. GH secretion has been thought traditionally to be regulated by the two hypothalamic hormones, GH-releasing hormone (GHRH) and somatostatin (SRIF). Recent evidence has suggested that other factors may be involved. These factors include the natural ligand for the synthetic hexapeptide GH-releasing peptide (GHRP) and the putative hypophysiotropic factor pituitary adenylate cyclase-activating polypeptide (PA-CAP). Accordingly, we examined the effects of GHRP and PACAP on GH secretion at the single cell level using the reverse hemolytic plaque assay which allows distinction of effects on the number of secreting cells and the amount of hormone each cell secretes. Both factors stimulated GH secretion in a dose-dependent fashion, with PACAP being more effective. PACAP increased both the number of cells secreting and the mean amount of hormone secreted per cell. In contrast, GHRP increased the number of secreting cells, although it had no effect on the amount of secretion per cell. GH secretion induced by GHRH, GHRP, and PACAP was inhibited by SRIF, but the effect was predominantly on the number of cells secreting rather than the amount secreted per cell. Specific antagonists to GHRP and GHRH inhibited GH secretion induced by the respective agonist but not that induced by the other factor nor by PACAP. These findings confirm the complex nature of the regulation of GH secretion at the level of the somatotrope. At least three factors, operating via distinct receptors, are able to increase GH secretion. In addition, they ascribe a potential physiological role for the hitherto putative hypophysiotropic factor PACAP.


Synthesis and binding of peptide T and aminoacyl derivatives to CD4+ lymphocytes. 1. Peptide T and four other aminoacyl derivatives of this octapeptide were synthesized on solid phase support using the Boc and Fmoc procedures. 2. The octapeptides were modified by chloroacetylation and radiolabelled by halogen exchange with 125I. 3. Purified and crude extracts of lymphocytes were used to determine the binding of the octapeptides at different concentrations.


Reinnervation of syngeneic mouse pancreatic islets transplanted into renal subcapsular space. A syngeneic transplantation of 150 islets into the subcapsular renal space was performed on normoglycemic or alloxan-induced diabetic male C57BL/6 mice. Six, 8, 14, or 20-21 wk after transplantation, the graft-bearing kidney was removed and processed for microscopical examinations with indirect immunofluorescence for neuropeptides and tyrosine hydroxylase, and with acetylcholinesterase staining to visualize nerve fibers within the graft. Six weeks after implantation, only a few scattered nerve fibers were observed within the grafts. A progressive increase in the number of nerves was observed until 14 wk after transplantation, after which, a stable level was reached. Alloxan-induced diabetic mice showed quantitatively and qualitatively similar reinnervation to normoglycemic mice 20 wk after transplantation. The findings demonstrate the presence of sympathetic nerve fibers (containing tyrosine hydroxylase and neuropeptide Y), mainly accompanying ingrowing blood vessels; parasympathetic nerve fibers (containing acetylcholinesterase and vasoactive intestinal peptide), possibly reaching the graft from the adjacent renal capsule; and afferent nerve fibers (containing substance P and calcitonin gene-related peptide), which were less numerous. The data suggest that transplanted islets become reinnervated by ingrowth of nerve fibers from the implantation organ and that several types of nerves are present.


Preferential mutation of the neurofibromatosis type 1 gene in paternally derived chromosomes. An interesting feature of neurofibromatosis type 1 (NF1) is its high mutation rate of 1 x 10(-4) per gamete per generation. The molecular basis for frequent NF1 mutation in unknown; the gene is not deletion prone. We have found that in all ten families examined, the apparent new NF1 mutation occurred on the paternally-derived chromosome. The probability of observing this result by chance is less than 0.001 assuming an equal frequency of mutation of paternal and maternal NF1 genes. We hypothesize a role for genomic imprinting that may either enhance mutation of the paternal NF1 gene or confer protection from mutation to the maternal NF1 gene.


The effect of chronic phenytoin treatment on serum lipid profile in adult epileptic patients. Total cholesterol, high-density lipoprotein (HDL), low-density lipoprotein (LDL), and very-low-density lipoprotein cholesterol, apolipoproteins A, A1, and B and gamma-glutamyltransferase (ggt) serum concentrations were measured in 100 adult epileptic patients receiving chronic phenytoin (PHT) treatment and in 100 control subjects. In relation to controls, patients showed higher HDL cholesterol, apolipoproteins A and A1, and ggt levels and lower LDL cholesterol and apolipoprotein B values; the significance of the results was greater in women than in men. Among patients, ggt levels were positively correlated with PHT plasma concentrations; likewise, a negative correlation was found between the apolipoprotein A/A1 ratio and the PHT and ggt plasma levels, and a positive correlation between the apolipoprotein A/A1 ratio and the LDL/HDL cholesterol ratio. These data indicate that PHT exerts a beneficial effect on the serum lipids profile.


Sib pair linkage analysis of renin gene haplotypes in human essential hypertension. Although essential arterial hypertension is believed to have a strong genetic predisposition, the gene(s) responsible are unknown. The mechanisms underlying the regulation of blood pressure and experimental studies place the renin gene among the main candidate genes that need to be tested in humans. We tested the hypothesis of a linkage between the renin gene and essential hypertension using the affected sib pair method. Siblings (133 subjects, 52.1 +/- 10.9 years) from 57 families were selected for sustained hypertension (160.7 +/- 22.9/99.5 +/- 12.8 mmHg with 80% of patients under antihypertensive treatment), of early onset (40.7 +/- 12.0 years), in the absence of obesity, diabetes mellitus, and secondary hypertension. Eight renin haplotypes were generated from three diallelic renin restriction fragment length polymorphisms (RFLPs) (TaqI, HinfI, HindIII) located throughout the renin gene. The allelic concordance between the sib pairs was analyzed by identity by state relationships for 98 sib pairs (41 for 41 couples, 39 for 13 trios, 18 for 3 quartets). Allelic frequencies in the 57 hypertensive probands were similar to those observed among 102 hypertensive subjects studied previously. Six of eight possible haplotypes were observed, the informativity of the marker corresponded to 70% of heterozygosity. Allelic concordance for all sib pairs according to sibship size was not significantly different from that expected under the hypothesis of no linkage (t = 0.52, P = 0.15) reflecting only a small excess of renin alleles shared by the hypertensive sibs (1.44 +/- 0.6 vs 1.36 +/- 0.6). Likewise the linkage hypothesis was unsupported by weighted estimates to correct for possible bias due to large sibship size. Thus, the sib pair analysis suggests that the renin gene does not have a frequent role in the pathogenesis of essential hypertension; further more powerful linkage studies or other approaches will be needed to detect contributions at the renin locus to the heritability of essential hypertension.


Estimating the stability of the proposed imprinted state of the fragile-X mutation when transmitted by females. Fragile-X syndrome is a major cause of mental retardation in humans. The X-inactivation imprinting model accounts for the unusual pattern of inheritance and expression of this syndrome. According to this model, the fragile-X mutation creates a local block to the attempted reactivation of the mutant X chromosome prior to oogenesis. This local block results in an "imprinted" fragile-X chromosome that is deleterious in males and in females for whom this chromosome is predominantly the active X chromosome. The imprinted state of the fragile-X mutation is inferred to be stable when transmitted by an imprinted female because the penetrance of the syndrome in sons of affected females is estimated to be 1.0. To provide a more precise estimate of the stability of the proposed fragile-X imprint, we have analyzed published pedigrees that include restriction fragment length polymorphism and cytogenetic data from sibships with mothers who are interpreted as having an imprinted fragile-X allele. We conclude that the fragile-X imprint was stable in 46 out of 48 female meioses. This analysis leads to a preliminary estimate of about 96% for the stability of the imprint through female meiosis. Two imprinted females had progeny who appeared to be carriers of a nonimprinted fragile-X allele. If this interpretation is correct, then reversion from the imprinted to the nonimprinted state, or "erasure," can occasionally occur when the mutant fragile-X allele is transmitted by an imprinted female. We discuss the genetic and epigenetic significance of possible female erasure. We request DNA and cytogenetic information from unpublished pedigrees to quantify further the stability, during female meiosis, of the proposed imprinted state of the mutant fragile-X allele.


Localization of a gene for the human low-voltage EEG on 20q and genetic heterogeneity. The localization of a gene responsible for a normal variant of the human electroencephalogram to the distal part of chromosome 20q is reported. A linkage analysis, including 17 families with 191 individuals, tested with 73 RFLPs and 22 blood and serological markers, was performed for the low-voltage electroencephalogram. This is a normal variant of the human electroencephalogram with an autosomal dominant mode of inheritance. The results present strong evidence for close linkage with the highly polymorphic marker CMM6 (D20S19) and for genetic heterogeneity.


The human heat-shock genes HSPA6 and HSPA7 are both expressed and localize to chromosome 1. HSPA6 is a member of the human heat-shock protein gene family, encoding a basic 70-kDa protein, with unique induction characteristics (Leung et al., 1990, Biochem. J. 267: 125-132). Hybridization analyses with a somatic cell hybrid DNA panel localized the gene to chromosome 1q. The highly related HSPA7 DNA sequence (Voellmy et al., 1985, Proc. Natl. Acad. Sci. USA 82: 4949-4953) colocalized. Both HSPA6 and HSPA7 represent functional genes, as determined by analyses of mRNA from heat-shocked human cells using specific oligonucleotides, although their pattern of expression differed. Neither mRNA was detected in the absence of heat stress. A BamHI polymorphism in the HSPA7 gene was present in a predominantly Asian population.