Effects of quinolinic acid on messenger RNAs encoding somatostatin and glutamic acid decarboxylases in the striatum of adult rats. The level of expression of mRNAs encoding somatostatin and two isoforms of glutamic acid decarboxylase (Mr 65,000, GAD65 and 67,000, GAD67) was examined by quantitative in situ hybridization histochemistry in the striatum of adult rats after local injections of quinolinic acid. After a 2-week survival period, Nissl strains showed a profound loss of neurons in the injected striata. With a dose of 120 nmol quinolinic acid, the lesioned area was completely devoid of somatostatin mRNA-positive neurons but contained cells expressing nicotinamide adenine dinucleotide-diaphorase activity (a marker of somatostatinergic interneurons in striatum). After 60 nmol of quinolinic acid, the number of neurons expressing somatostatin mRNA in the lesioned area was similar to controls but the level of labeling per neuron was increased. In the lesioned area, labeling for GAD65 mRNA was abolished and labeling for GAD67 mRNA markedly reduced. However, scattered neurons expressing GAD67 mRNA could still be detected. The majority of surviving GABA-ergic neurons expressed immunoreactivity to parvalbumin, a marker for striatal GABA-ergic interneurons. The results show that quinolinic acid induces dose-dependent alterations in the expression of striatal somatostatin mRNA and reveal a relative sparing of GABA-ergic interneurons in the quinolinic acid-lesioned rat striatum.


Molecular cloning and sequence analysis of bovine T-cell receptor gamma and delta chain genes. Nine bovine T-cell receptor (Tcr) gamma chain (Tcrg) and three Tcr delta chain (Tcrd) cDNA clones were isolated from the cDNA libraries constructed from peripheral blood lymphocytes and thymocytes. Of nine Tcrg cDNA clones, only four were rearranged and contained specific V, J, and C gene segments, but the remaining five contained specific J and C or only C gene segments without the V gene segment. Three kinds of Tcrg-C, which were highly related at the nucleotide and amino acid levels, were found and designated as Tcrg-C1, Tcrg-C2, and Tcrg-C3. Compared with human and mouse Tcrg-C, bovine Tcrg-C sequences are much longer, with about 27-55 amino acids corresponding to the hinge and connector regions, where the characteristic repetitive 5-amino acid motif (TTEPP or TTKPP) exists in sheep Tcrg-C as previously reported. From three Tcrd cDNA clones, two Tcrd-V and three Tcrd-J segments were isolated. The nucleotide and deduced amino acid sequences of bovine Tcrd-C, especially the transmembrane and cytoplasmic domains, are well conserved among species. As in bovine Tcrg-C, diversity of amino acid residues in the Tcrd-C region is concentrated in the hinge regions. Southern blot analysis showed that there are at least three Tcrg-C genes and one Tcrd-C gene in the bovine genome. The analysis also revealed the presence of Tcrg-C- and Tcrd-C-associated restriction fragment length polymorphisms among bovine breeds.


Differential actions of angiotensin II and angiotensin-(1-7) on transmitter release. The central cardiovascular and dipsogenic effects of angiotensin II involve interactions with norepinephrine, dopamine, and serotonin. Our findings that angiotensin II receptors and substance P immunoreactivity show a parallel distribution in the dorsal medulla and that angiotensin II releases substance P from perfused rat medulla slices revealed the potential for a functional relation between these peptidergic systems as well. Additional evidence suggests that the heptapeptide angiotensin-(1-7) exerts its biological activities via selective angiotensin receptor subtypes. Thus, we compared the effects of these two peptides on release of substance P and monoamines in perfused slices of medulla and hypothalamus from 77 male Sprague-Dawley rats. Transmitter levels were determined in 6-minute collections of perfusate before (basal), during (experimental), and after (recovery) perfusion with either angiotensin-(1-7), angiotensin II, or Krebs' solution alone (control). Substance P was measured by radioimmunoassay and monoamines and their metabolites by high-performance liquid chromatography with electrochemical detection. In the medulla, 2 microM angiotensin II but not angiotensin-(1-7) significantly increased efflux of substance P (221 +/- 87% of basal) and norepinephrine (130 +/- 17% of basal) during the experimental period. The effect of angiotensin II on substance P was sustained into the recovery period. Dopamine and its metabolite 3,4-dihydroxyphenylacetic acid were not detected in this brain region. In the hypothalamus, both angiotensin-(1-7) and angiotensin II increased substance P (169 +/- 30% and 141 +/- 35% of basal, respectively); the effect of angiotensin II was sustained throughout the recovery period.(ABSTRACT TRUNCATED AT 250 WORDS)


Allelic loss on chromosome 22 correlates with histopathological predictors of recurrence of meningiomas. Meningiomas are common tumors of the nervous system. Although usually benign, they may exhibit variable degrees of aggressiveness. Their probability of recurrence after subtotal resection has been correlated with several histological parameters. Independently, a loss of chromosome 22, as evidenced either by cytogenetics or by somatic loss of alleles, has been observed in about half of the cases studied. In 34 meningiomas we have examined the relationship between loss of chromosome 22 alleles and 6 histological predictors of recurrence. Significant correlations were found for 3 of these, i.e. prominent nucleoli (p less than 0.002), microscope count of mitoses (p less than 0.05) and nuclear pleomorphism (p less than 0.02). Correlation with the other 3, i.e. sheeting of cells, vascularity and micronecrosis, did not reach significance. Total tumor score, defined by the sum of the individual scores for these 6 parameters, was strongly correlated to allelic loss (p less than 0.0001). Thus, the loss of chromosome 22 alleles, which possibly contribute to the inactivation of tumor-suppressor gene(s), might be a potent genetic marker of the aggressiveness of meningiomas.


The alcohol dehydrogenase polymorphism in natural populations of Drosophila melanogaster: restriction map variation in the region of the Adh locus in populations from two hemispheres. Restriction endonuclease variation in the 12 kb region surrounding the Adh locus was measured in seven Australian and six Chinese populations of Drosophila melanogaster. There is a higher level of nucleotide-substitution variation in the Australian populations than in the Chinese, which is possibly a reflection of their origins. None of the restriction site polymorphisms, nor any of the insertions, showed a significant association with latitude. A 0.2 kb deletion varied with latitude in the Chinese populations. In accordance with previous studies, a majority of the insertions were located in a region 1.5-3.5 kb 3' from the Adh coding region, and a majority of the deletions were at a site 3 kb 5' to the Adh coding region. Two of the insertions shared homologies with known mobile elements. Overall, the data suggest that restriction endonuclease variation in the Adh region is not related to the cline in Adhs frequencies.


Population structure and mitochondrial DNA gene flow in Old World populations of Drosophila subobscura. An extensive survey of mitochondrial DNA (mtDNA) restriction polymorphism in 156 isofemale lines from 29 different geographic populations of Drosophila subobscura distributed throughout the Old World was carried out. Ten restriction enzymes were used, five of which revealed restriction site polymorphism. Of the 31 restriction sites detected, 13 were found to be polymorphic. Comparisons with the mtDNA map of Drosophila yakuba indicate that the variable sites are mainly concentrated in protein genes, especially those corresponding to the NADH complex. A total of 13 different haplotypes were observed, two of which (haplotypes I and II) are quite frequent and widely distributed throughout the populations, whereas the other 11 with the exception of VIII, which deserves special attention, are each restricted to one population only and occur at low frequencies. The observed distribution of haplotypes, corroborated by a parsimonious unrooted tree, suggests an ancient origin of haplotypes I and II in the continent. In order to compare genetic structure according to mtDNA and allozymes, the 10 populations with higher population sizes were studied for 10 polymorphic allozymes also. One striking result is the high degree of population structure of the mtDNA when compared to that obtained for allozymes. If an island model is assumed, estimates of gene flow give values of 0.013 and 1.89 migrants per generation for mtDNA and allozymes, respectively. What is apparent from these estimates is that Drosophila subobscura populations are effectively subdivided for mtDNA genes at migration rates at which nuclear genes (allozymes) are almost panmictic.


Effect of BSO on the radiation response at low (0-4 Gy) doses. Depletion of thiols by various agents has been shown to radiosensitize hypoxic cells. The agent BSO is used to specifically inhibit GSH synthesis, and the effect of this treatment on radiation response, particularly in the region of clinical interest, was studied using automated microscopic identification of cells for measurement of high survival levels. In both CHO and V79 cell lines, the enhancement rations were greater at low doses (80% S) than at high doses (2% S) in cells irradiated in hypoxia. GSH depletion also affected the aerobic response in both dose regions, with again higher ER's observed at low doses. These results support previous studies which suggest that GSH levels affect the shoulder portion of the survival curve. However, as with untreated cells, the OER's remain higher at high doses than at low doses, in BSO treated cells of both lines. These studies complement an earlier low-dose study on diamide by Skarsgard and co-workers, and were carried out to increase our understanding of the factors which affect radiation sensitivity in the range of doses used in fractionated regimens in the clinic.


Taxol: a novel radiation sensitizer.

Taxol: a novel radiation sensitizer. The investigational antineoplastic agent, taxol, a natural product from the yew, Taxus sp. L., is currently being evaluated in a series of Phase II clinical trials. To date, the drug has shown activity against ovarian cancer, lung cancer, and melanoma. Taxol is a potent microtubule stabilizing agent that selectively blocks cells in the G2 and M phases of the cell cycle and is cytotoxic in a time-concentration dependent manner. It is well known from radiobiological principles that G2 and M are the most radiosensitive phases of the cell cycle. On the rationale that taxol could function as a cell-cycle selective radiosensitizer, we examined the consequences of combined drug-radiation exposures on the human grade 3 astrocytoma cell line, G18. Survival curve analysis shows a dramatic interaction between taxol and ionizing radiation with the degree of enhanced cell killing dependent on taxol concentration and on the fraction of cells in the G2 or M phases of the cell cycle. The sensitizer enhancement ratio (SER) for 10 nM taxol at 10% survival is approximately 1.8. These results obtained with cycling aerated radioresistant brain tumor cells indicate that significant advantage may derive from appropriate time-concentration dependent interactions in combined modality protocols.


Characterization of the regulon controlled by the leucine-responsive regulatory protein in Escherichia coli. The leucine-responsive regulatory protein (Lrp) has been shown to regulate, either positively or negatively, the transcription of several Escherichia coli genes in response to leucine. We have used two-dimensional gel electrophoresis to analyze the patterns of polypeptide expression in isogenic lrp+ and lrp mutant strains in the presence or absence of leucine. The absence of a functional Lrp protein alters the expression of at least 30 polypeptides. The expression of the majority of these polypeptides is not affected by the presence or absence of 10 mM exogenous leucine. Outer membrane porins OmpC and OmpF, glutamine synthetase (GlnA), the small subunit of glutamate synthase (GltD), lysyl-tRNA synthetase form II (LysU), a high-affinity periplasmic binding protein specific for branched-chain amino acids (LivJ), W protein, and the enzymes of the pathway converting threonine to glycine, namely, threonine dehydrogenase (Tdh) and 2-amino-3-ketobutyrate coenzyme A ligase (Kbl), were identified as members of the Lrp regulon by electrophoretic analysis. We have shown that Lrp is a positive regulator of glutamate synthase and glutamine synthetase and that exogenous leucine has little or no effect on the expression of these proteins. In strains carrying a glnL deletion and in strains carrying the glnL2302 allele, which directs the synthesis of a GlnL protein that is constitutively active, expression of glutamine synthetase is no longer regulated by Lrp, demonstrating that the effect of Lrp on glutamine synthetase levels is indirect and requires an intact glnL gene. lrp::Tn10 strains grow poorly when arginine or ornithine is present as the sole nitrogen source in the medium. On the bases of present studies and previous research, we propose that Lrp is involved in the adaptation of E. coli cells to major shifts in environment, such as those which occur when E. coli leaves the intestinal tract of its animal host. Several genes required for amino acid and peptide transport and catabolism are negatively regulated by Lrp, and other genes required for amino acid biosynthesis and ammonia assimilation in a nitrogen-poor environment are positively regulated by Lrp.


Adhesion molecules on murine brain microvascular endothelial cells: expression and regulation of ICAM-1 and Lgp 55. The mechanisms for the initiation of immune reactions in the central nervous system are poorly understood. In this report, we describe the presence of intercellular adhesion molecule-1 (ICAM-1) and Lgp 55 (suggested mouse homologue of human intercellular adhesion molecule-2, ICAM-2) on the surface of brain microvessel endothelium (EN) cells and show in vitro induction of ICAM-1 molecules on EN cells with pro-inflammatory cytokines. ICAM-1 expression was detected using flow cytometry analysis with biotinylated anti-ICAM-1 antibody (YN1/1.7.4). Lgp 55 expression was characterized using PA3 monoclonal antibody. According to our results, 30-40% of the non-activated brain EN cells expressed ICAM-1 and 15-20% expressed Lgp 55 molecules. The ICAM-1 molecule expression was increased after the activation of the cells with recombinant murine gamma interferon (IFN-gamma), tumor necrosis factor (TNF-alpha), and interleukin-1 alpha (IL1-alpha) in a dose-dependent manner. The increased ICAM-1 expression was detected as early as 2 h following the cytokine treatment and reached its maximum after 24 h. Transforming growth factor-beta (TGF-beta) did not influence the expression of ICAM-1 molecule. Lgp 55 molecule does not seem to be regulated by pro-inflammatory cytokines. ICAM-1 and Lgp 55 expression was found to be polarized on the luminal surface of EN by confocal laser microscopy suggesting accessibility for leukocytes. Inducible ICAM-1 expression may play a critical role in formation of inflammatory reactions inside the central nervous system.


The stem cells of the liver--a selective review. The current status of the much-debated question of the still-hypothetical stem cells of the liver is reviewed, with an emphasis on their role in hepatocarcinogenesis. The widely held view of the primacy of the hepatocyte, notably of the mononuclear diploid type, in this process--the "hepatocytic theory"--has been compared with variants of the "stem cell hypothesis" based on the "non-parenchymal epithelial cells" of the liver--the "oval" or biliary ductular cells, the "nondescript periductular" cells and the "primitive" bipotential epithelial cells. An attempt has been made to concentrate mainly on the more recent publications, in an effort to balance the conflicting opinions expressed by comparing results obtained by the newer procedures currently in use. Despite some interesting and relevant findings it appears that the evidence in favour of the stem-cell hypothesis is still circumstantial and that the hepatocytic theory has not been invalidated. Presumably the question of the hepatic stem cells will be answered when the riddle of hepatocarcinogenesis has been solved.


Diagnostic criteria for polyarteritis nodosa in childhood. Clinical and laboratory features of 31 children with a diagnosis of polyarteritis nodosa were evaluated retrospectively. All the patients had musculoskeletal involvement, renal involvement, or both during the course of the disease. We have defined involvement of these two systems as the major diagnostic criteria in polyarteritis nodosa. Ten additional minor criteria were defined: (1) cutaneous findings, (2) gastrointestinal involvement, (3) peripheral neuropathy, (4) central nervous system involvement, (5) hypertension, (6) cardiac involvement, (7) lung involvement, (8) constitutional symptoms, (9) presence of acute-phase reactants, and (10) presence of hepatitis B surface antigen. We propose that the presence of five of these criteria, including at least one major criterion, is highly suggestive of polyarteritis nodosa; such a combination was present in 97% of our patients. Fourteen of the patients were treated with corticosteroids alone and 14 were treated with a combination of steroids plus cyclophosphamide or azathioprine. At the last follow-up examination six patients in the steroid group and nine in the combination group were considered to have complete remission of disease or inactive disease with persisting symptoms in an organ system. The overall mortality rate was 16%; renal involvement had the greatest adverse effect on outcome. We suggest that in patients with five of the 12 diagnostic criteria, especially those with renal involvement, therapy should be initiated promptly while diagnostic procedures are being carried out.


Comparison of the postoperative intraocular pressure with Betagan, Betoptic, Timoptic, Iopidine, Diamox, Pilopine Gel, and Miostat. A randomized, masked study measuring postoperative intraocular pressure at 4, 8, and 24 hours, two to seven days, and one month after planned extracapsular cataract extraction with posterior chamber lens implantation was conducted. Seven commonly used ocular hypotensive agents and a control, given at the completion of surgery, were compared: timolol maleate (Timoptic), levobunolol hydrochloride (Betagan), betaxolol hydrochloride (Betoptic), pilocarpine hydrochloride (Pilopine Gel), carbachol (Miostat), apraclonidine hydrochloride (Iopidine), acetazolamide (Diamox). There were significant differences between agents. Miostat was the most effective in controlling postoperative IOP, followed by Timoptic. Diamox, Pilopine Gel, and Betagan were equally effective. Betoptic was somewhat less effective and Iopidine was not significantly better than the control.


Modulation and function of intercellular adhesion molecule-1 (CD54) on human retinal pigment epithelial cells. As part of the blood-retina barrier, the neuroectodermally-derived retinal pigment epithelial (RPE) monolayer is strategically positioned to interact with circulating leukocytes and regulate their access to the retina. We, therefore, studied whether human RPE cells express intercellular adhesion molecule-1 (ICAM-1), a specialized cell surface glycoprotein that binds the leukocyte function antigen-1 receptor present on all leukocytes. Using specific monoclonal antibody to ICAM-1, immunohistochemical staining of freshly-isolated primary and fourth passaged human RPE cells resulted in delicate reaction product that increased dramatically upon exposure to human recombinant (r) interferon-gamma (rIFN-gamma), interleukin-1-beta (rIL-1 beta), or tumor necrosis factor-alpha (rTNF-alpha). Fluorescence-activated cell sorting analysis demonstrated 2-fold increases in constitutive RPE ICAM-1 expression within 6 hours of exposure to physiologic concentrations of rIFN-gamma, rIL-1 beta, or rTNF-alpha. In standardized leukocyte adherence assays, cultured RPE cells showed avid binding of neutrophils that increased significantly after stimulation with rIFN-gamma, rIL-1 beta, or rTNF-alpha (p less than 0.001). In parallel assays, monoclonal antibody to either ICAM-1 on RPE cells, or subunits of leukocyte function antigen-1 receptors on leukocytes significantly blocked leukocyte binding to unstimulated (p less than 0.001) or rIFN-gamma-stimulated RPE cells (p less than 0.001). To demonstrate RPE ICAM-1 expression in intact human tissue, fresh uveoretinal explants were exposed to rIFN-gamma, rIL-1 beta, or rTNF-alpha and stained using mAb to ICAM-1. Tissue sections of cytokine-stimulated explants revealed dramatic increases in RPE ICAM-1 immunoreactivity over the low levels observed in unstimulated uveoretinal tissue. Our results indicate that: (a) ICAM-1 is expressed at low levels on unstimulated RPE cells, (b) RPE ICAM-1 may be augmented by inflammatory cytokines, and (c) RPE ICAM-1 is a functional receptor mediating leukocyte binding. ICAM-1 on RPE cells at the blood-retina barrier may regulate leukocytic infiltration in ocular diseases in which leukocytes are important pathogenetically and may be important to the generation of ocular immune responses.


Methylation status of the major breakpoint cluster region in Philadelphia chromosome negative leukemias. It has been shown that a 600 bp long cluster of cell lineage specific hypomethylated sites in the major breakpoint cluster region (M-bcr) on chromosome 22 exists in hematopoietic cells. To determine possible relationships between methylation patterns within the M-bcr and the stage of hematopoietic cell development, the M-bcr methylation status of 39 patients with leukemia and lymphoma and two patients with myelodysplastic syndrome with non-rearranged M-bcrs was examined by BgIII-HpaII digestion. In the myeloid malignancies, the presence of a hypermethylated 4.8 kb BgIII-BgIII M-bcr allele was directly proportional to the combined myeloblast and promyelocyte percentage of the specimen, whereas the presence of a 2.5 kb BgIII-HpaII allele was directly proportional to the combined percentage of monocytic cells and neutrophils. All five acute monoblastic leukemias showed a methylation pattern that closely resembled neutrophils. All of thirteen surface immunoglobulin positive B-cell malignancies showed a distinct methylation pattern consisting of three or more BgIII-HpaII restriction fragments of 2.5 kb or less in length. The B-cell precursor leukemias showed heterogeneous M-bcr methylation patterns, with four of seven showing a B-cell pattern and three showing a hypermethylated pattern with 4.8, 3.1/3.0 and/or 2.5 kb BgIII-HpaII M-bcr alleles. It is concluded that the M-bcr methylation status is related to the maturation of the neutrophil series; the surface immunoglobulin positive B-cell malignancies are characterized by a distinct, extreme hypomethylation pattern of the M-bcr; and the B-cell precursor malignancies appear to have a heterogeneous M-bcr methylation pattern.


Potentiation of opioid analgesia by cocaine: the role of spinal and supraspinal receptors. These studies examined the effect of cocaine on the analgesia produced by systemically and centrally administered opioid agonists. Cocaine (50 mg/kg, s.c.) increased the analgesic potency of systemic, ICV and IT morphine; and the ICV and IT analgesic effects of the delta selective peptide, [D-Pen2,D-Pen5]enkephalin (DPDPE). Cocaine also increased the analgesic potency of the mu selective ligand [D-Ala2,NMePhe4,Gly-ol5]enkephalin (DAGO) administered ICV. However, cocaine did not alter the ED50 for IT DAGO. GC-MS studies indicated that brain cocaine concentration was approximately 3.0 micrograms/g wet weight 45 min following s.c. administration. These results suggest that cocaine-induced increases in opioid analgesic potency are mediated at brain mu and delta receptors and spinal mu receptors. Furthermore, there might be functional differences between spinal and supraspinal sites at which DAGO produces analgesia.


Hypoglycemic effects of a beta-agonist, Ro 16-8714, in streptozotocin-diabetic rats: decreased hepatic glucose production and increased glucose utilization in oxidative muscles. Streptozotocin (STZ)-induced diabetic rats are glycosuric, hyperglycemic, hyperketonemic, overproduce glucose, and have a decreased glucose utilization in oxidative muscles. Treatment with a beta-agonist, Ro 16-8714, decreases the glycosuria, hyperglycemia, hyperketonemia, and hepatic glucose production. Tissue glucose utilization was unchanged, except in oxidative muscles, where it was increased.


Correlation of genetic and physical structure in the region surrounding the I2 Fusarium oxysporum resistance locus in tomato. The dominant gene I2 confers on tomato (Lycopersicon esculentum) resistance against the fungus Fusarium oxysporum f. sp. lycopersici race 2. A restriction fragment length polymorphism (RFLP) marker, TG105, has recently been found to be tightly linked to I2. The potential for cloning this gene by a reverse genetics approach prompted us to describe in both genetic and physical detail the region surrounding the I2 locus on chromosome 11. We have analyzed patterns of segregation of RFLP markers on chromosome 11 and Fusarium resistance in 140 F2 plants from a cross between Fusarium-resistant and susceptible parental lines. Marker TG105 mapped 0.4 centiMorgan (CM) from I2. Physical analysis of TG105 and its flanking RFLP markers, TG26 and TG36, by pulsed field gradient gel electrophoresis (PFGE) yielded a restriction map for this region encompassing at least 620 kb of the tomato genome. TG105 and TG26 hybridized to the same 175 kb MluI-NruI restriction fragment. We have therefore linked two genetically distinct RFLP markers. Based on the 4.1 cM distance between them, we have assigned a mean value of 43 kb for each cM recombination distance in the vicinity of I2. This local ratio between physical and genetic distances is more than 10-fold below the average for the tomato genome. It should therefore be possible to clone I2 by chromosome walking from TG105.


Tuberculous meningitis in patients infected with the human immunodeficiency virus. Tuberculosis is a frequent complication of human immunodeficiency virus (HIV) infection. We describe the clinical manifestations and outcomes of tuberculous meningitis in patients with HIV infection, and compare them with those in non-HIV-infected patients. We reviewed the records from 1985 through 1990 at two large referral hospitals in Madrid for patients who had Mycobacterium tuberculosis isolated from cerebrospinal fluid. Of 2205 patients with tuberculosis, 455 (21 percent) also had HIV infection, of whom 45 had M. tuberculosis isolated from the cerebrospinal fluid. Of the 37 HIV-infected patients with tuberculous meningitis for whom records were available, 24 (65 percent) had clinical or radiologic evidence of extrameningeal tuberculosis at the time of admission. In 18 of 26 patients (69 percent), a CT scan of the head was abnormal. In most patients, analysis of cerebrospinal fluid showed pleocytosis (median white-cell count, 0.234 x 10(9) per liter) and hypoglycorrhachia (median glucose level, 1.3 mmol per liter), but in 43 percent (15 of 35), the level of protein in cerebrospinal fluid was normal. In four patients with HIV infection, tuberculosis was only discovered after their deaths. Of the 33 patients who received antituberculous treatment, 7 died (in-hospital mortality, 21 percent). Illness lasting more than 14 days before admission and a CD4+ cell count of less than 0.2 x 10(9) per liter (200 per cubic millimeter) were associated with a poor prognosis. Comparison with tuberculous meningitis in patients without HIV infection showed that the presentation, clinical manifestations, cerebrospinal fluid findings, and mortality were generally similar in the two groups. However, of the 1750 patients without HIV infection, only 2 percent (38 patients) had tuberculous meningitis, as compared with 10 percent of the HIV-infected patients (P less than 0.001). HIV-infected patients with tuberculosis are at increased risk for meningitis, but infection with HIV does not appear to change the clinical manifestations or the outcome of tuberculous meningitis.


Glutamate receptors in the substantia nigra of Parkinson's disease brains. Glutamic acid and its analogs are excitotoxins that might contribute to the pathogenesis of Parkinson's disease (PD). We measured four subtypes of glutamate binding sites autoradiographically in 20-microns sections from control and PD midbrains. N-Methyl-D-aspartate (NMDA) binding sites (eight control, eight PD) were very low in control (20 +/- 7 [SEM] fmol/mg protein) and were reduced in the PD pars compacta (2.6 +/- 1.1 fmol/mg protein; p less than 0.02). NMDA binding was also reduced in the red nucleus but not in periaqueductal gray (PAG). We measured alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA), metabotropic, and non-NMDA, nonkainate, non-quisqualate (NNKQ) sites in 10 PD and 12 control midbrains. AMPA binding sites were reduced from 175 +/- 20 to 99 +/- 16 (p less than 0.05) fmol/mg protein in PD pars compacta, NNKQ sites from 86 +/- 10 to 50 +/- 12 (p less than 0.05) fmol/mg protein in total nigra, and metabotropic sites (15 +/- 5 fmol/mg protein) were unchanged. AMPA, metabotropic, and NNKQ binding were unchanged in red nucleus and PAG. The very low number of NMDA binding sites suggests that factors other than excitotoxicity mediated via NMDA receptors on nigral cell bodies play roles in the pathogenesis of PD. There may be a generalized loss of NMDA receptors in PD brains. AMPA and NNKQ binding sites appear to be located on dopamine neurons, although the role of NNKQ sites in normal nervous system function and human disease is unknown.


Clinical and pathologic significance of the c-erbB-2 (HER-2/neu) oncogene. Activation of the c-erbB-2 oncogene can occur by amplification of c-erbB-2 DNA and by overproduction of c-erbB-2 mRNA and c-erbB-2 protein. Approximately 20 percent of breast carcinomas show evidence of c-erbB-2 activation, which correlates with a poor prognosis primarily in patients with metastasis to axillary lymph nodes. Studies that have attempted to correlate c-erbB-2 activation with other prognostic factors in breast carcinoma have reported conflicting conclusions. The pathologic and clinical significance of c-erbB-2 activation in other neoplasms is unclear and should be assessed by additional studies.


Nonconservative segregation of parental nucleosomes during simian virus 40 chromosome replication in vitro. Simian virus 40 chromosomes can be replicated in vitro with the same set of purified proteins required for the replication of naked DNA containing the viral origin. With these reconstituted systems, the fate of parental histones during replication was examined in vitro. The assembly of nucleosomes on replicating chromosomes was hardly affected by the presence of simultaneously replicating naked DNA competitor, suggesting that replication forks can traverse nucleosomes without the displacement of histones. Moreover, we demonstrate that the nascent nucleosomes were distributed almost equally between the leading and lagging strands. This distributive mode of nucleosome segregation favors the propagation of parental chromatin structures to both daughter cells, which can maintain cellular functions dictated by these structures during cell proliferation.


[Sudden death in heart failure. Analysis and prevention]. Sudden death is a frequent complication of heart failure occurring in 35 to 45 per cent of the cases. This multifactorial event may be of haemodynamic origin (acute heart failure, electro-mechanical dissociation) or, more often, of rhythmic origin (torsade de pointe, sustained ventricular tachycardia, ventricular fibrillation, bradycardia, asystole). Numerous structural, haemodynamic, metabolic, ionic, neurohormonal and iatrogenic factors facilitate ventricular hyperexcitability. The main predictive factors of sudden death in heart failure are the presence of coronary heart disease and of reduced left ventricular ejection fraction; the prognostic value of ventricular rhythm disorders is controverted. Prevention of sudden death begins with correcting those factors which facilitate disturbances in rhythm and conduction. Beta-blockers are effective in the post-infarction period, but there is no evidence that other drugs are useful. Identifying patients at high risk and determining the therapeutic approach that reduces this risk are still incompletely resolved problems.


Trophic doses of E2 prostaglandins do not influence the exocrine and endocrine pancreas in the presence of high levels of plasma somatostatin. The aim of the present investigation was to study the effect of a long-term and a short-term treatment regimen with 15-R-15-methyl prostaglandin E2 and natural prostaglandin E2 (PGE2) on the endocrine cell populations of the rat pancreas. Graded oral doses of the analogue (5 and 50 micrograms/kg) and PGE2 (5000 micrograms/kg) were given twice daily for 4 weeks. The pancreas was carefully excised and weighed. Sections from randomly taken pancreatic biopsy specimens were processed for immunohistochemistry or hematoxylin and eosin staining before quantitative estimations were made, using stereologic methods. The total pancreatic volumes of insulin-, glucagon-, polypeptide P-, somatostatin-, and chromogranin A-immunoreactive cells were not affected by E2 prostaglandins. Neither the total volume of the islets of Langerhans nor that of the pancreatic cell nuclei was affected. The size of pancreatic cell nuclei was the same in the groups. The plasma levels of the antitrophic peptide somatostatin were significantly increased in rats treated with doses of both the analogue and PGE2 (p less than 0.05). In an additional short-term study rats were given oral placebo or 5000 micrograms/kg PGE2 twice daily for 5 days. The total endocrine pancreatic volume was not affected by PGE2. As in the long-term study, natural PGE2 did not affect the total pancreas volume or the total volume of pancreatic cell nuclei. These findings indicate that E2 prostaglandins produce no changes in the exocrine or endocrine pancreas in a dose range known to induce hyperplasia in the gastrointestinal epithelium.(ABSTRACT TRUNCATED AT 250 WORDS)


Guanylin: an endogenous activator of intestinal guanylate cyclase. Intestinal guanylate cyclase mediates the action of the heat-stable enterotoxin to cause a decrease in intestinal fluid absorption and to increase chloride secretion, ultimately causing diarrhea. An endogenous ligand that acts on this guanylate cyclase has not previously been found. To search for a potential endogenous ligand, we utilized T84 cells, a human colon carcinoma-derived cell line, in culture as a bioassay. This cell line selectively responds to the toxin in a very sensitive manner with an increase in intracellular cyclic GMP. In the present study, we describe the purification and structure of a peptide from rat jejunum that activates this enzyme. This peptide, which we have termed guanylin, is composed of 15 amino acids and has the following amino acid sequence, PNTCEICAYAACTGC, as determined by automated Edman degradation sequence analysis and electrospray mass spectrometry. Analysis of the amino acid sequence of this peptide reveals a high degree of homology with heat-stable enterotoxins. Solid-phase synthesis of this peptide confirmed that it stimulates increases in T84 cyclic GMP levels. Guanylin required oxidation for expression of bioactivity and subsequent reduction of the oxidized peptide eliminated the effect on cyclic GMP, indicating a requirement for cysteine disulfide bond formation. Synthetic guanylin also displaces heat-stable enterotoxin binding to cultured T84 cells. Based on these data, we propose that guanylin is an activator of intestinal guanylate cyclase and that it stimulates this enzyme through the same receptor binding region as the heat-stable enterotoxins.


Safety of pyridostigmine in hypertensive patients receiving beta blockers. In the last decade, pyridostigmine, a quaternary carbamate that reversibly inhibits the enzyme acetylcholinesterase, was proposed for pretreatment of nerve gas (organophosphate) poisoning. The objective of this study was to assess the cardiovascular effects of pyridostigmine in patients treated with beta blockers. Eight hypertensive patients receiving regular treatment with beta blockers were randomized in a double-blind crossover study to receive pyridostigmine (30 mg 3 times daily) or placebo for 2 days. Heart rate and blood pressure in the supine and standing positions were recorded every 2 hours during the day, and 24-hour Holter monitoring was performed. In addition, a symptom-limited exercise test was performed, and plasma catecholamine levels were determined at rest and at peak exercise. Pyridostigmine, as compared with placebo, did not induce any significant effect on heart rate, plasma catecholamine levels or resting blood pressure. Both systolic and diastolic blood pressures increased in accordance with exercise intensity (p less than 0.01), although a significantly lower diastolic blood pressure was observed when pyridostigmine was used (average decrease 5 mm Hg compared with placebo; p less than 0.01). No clinical adverse reactions were observed, confirming the relative safety of the combination of low-dose pyridostigmine with beta-adrenergic blocking agents.


The relation between nitrite inhalants, unprotected receptive anal intercourse, and the risk of human immunodeficiency virus infection. The role of nitrite was evaluated between 1985 and 1988 in a study of sexual transmission of the human immunodeficiency virus (HIV) among homosexual male couples in Boston, Massachusetts. Initial enrollment data suggested that a history of unprotected receptive anal intercourse (odds ratio (OR) = 2.3, 95% confidence interval (CI) 1.4-3.6) and a history of nitrite use (OR = 1.7, 95% CI 1.1-2.5) were independent risk factors for HIV infection. In addition, interaction between nitrite use and unprotected receptive anal intercourse was observed (OR = 5.5, 95% CI 2.8-11.1) after controlling for number of unprotected receptive anal sex partners and history of sexually transmitted diseases. Since it was felt that nitrite use might be a marker for unprotected receptive anal sexual activity, a supplemental questionnaire was administered to obtain information on simultaneous nitrite use and unprotected receptive anal intercourse. The supplemental data suggested a strong interaction between nitrite use and unprotected receptive anal intercourse in increasing the risk of HIV infection. In the adjusted analyses, the odds ratio for HIV infection was considerably greater among men who always used nitrites during unprotected receptive anal intercourse (OR = 31.8, 95% CI 12.9-76.7) compared with men who sometimes (OR = 7.1, 95% CI 2.1-23.6) or never (OR = 9.0, 95% CI 2.5-32.1) used them. These findings have preventive public health implications and may add insight into our understanding of the mechanism by which HIV infection spread rapidly among homosexual men in the early 1980s.


Enhanced cerebral blood flow autoregulation in the newborn piglet by d-tubocurarine and pancuronium but not by vecuronium. Neuromuscular blockers may affect cerebral blood flow (CBF) regulation in the newborn. We studied the effects of d-tubocurarine (0.1 mg.kg-1, n = 8), pancuronium (0.1 and 0.4 mg.kg-1, n = 6 and 7), and vecuronium (0.1 and 0.4 mg.kg-1, n = 6 and 7) on CBF measured over the same range of mean systemic blood pressure ([BP] 15-122 mmHg) in each group of newborn pigs; controls received normal saline (n = 7). The levels of BP during hypotension and hypertension were scaled at intervals of 5 +/- 1.6 mmHg and adjusted by inflating balloon-tipped catheters placed in the aorta. After saline, the low dose of pancuronium (0.1 mg.kg-1), and the two doses of vecuronium, CBF was constant over the BP range of 50-90 mmHg (r = -0.07-0.35, P greater than 0.20) but varied directly with BP beyond this range (tau = 0.38 - 0.60, P less than 0.05). In contrast, in pigs treated with d-tubocurarine and high-dose pancuronium, CBF remained constant from 35 to 122 mmHg of BP (r = 0.14 - 0.37, P greater than 0.10) and changed minimally (4-12%) with BP greater than 105 mmHg compared to the other groups (41-59%, P less than 0.01). When BP was reduced below 30 mmHg, CBF also decreased less (20-38%) in animals treated with d-tubocurarine and high dose-pancuronium than after the other treatments (58-67%, P less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)


Effect of increasing dosage of an inhaled beta-2 specific agonist on pulmonary function, tremor, and cardiovascular indices. In this investigation we evaluated the effect of increasing dosage, using an inhaled beta-2 specific agonist, pirbuterol, administered by a metered dose inhaler on pulmonary function, tremor, and cardiovascular parameters in nonacute adult asthmatic patients. This study was conducted with a randomized crossover study design in which each individual was administered a single dose of pirbuterol (0.4, 0.8 and 1.2 mg) [corrected] on separate days. Measurements of pulmonary function, tremor (by accelerometer readings), electrocardiogram, blood pressure and pulse were obtained at 0, 15, 30, 60, 90, 120, and 180 minutes following test drug administration where appropriate. These measurements were performed until there had been no change from baseline in tremor by 60 minutes or return of tremor measurements to within 15% of baseline for up to six hours. The data demonstrated that the onset of bronchodilator effectiveness had occurred by 15 minutes after test drug administration (the first testing time). The peak percent change from baseline for the FEV1 occurred at 60 minutes after administration for all three test doses. The duration of activity was never truly established as there was no significant difference between any of the potential side effects for any of the three test drugs at any testing time period. This study demonstrated that with the beta-2 specific agent pirbuterol, administered as a metered dose inhaler, there is little risk of development of skeletal muscle or cardiovascular toxicity when as much as three times the recommended dose is used in a single usage, and that there is no direct correlation between the onset, peak, and/or duration of tremor with the onset and peak of bronchodilator efficacy with this agent.


Suppression of epicutaneous reactivity by terfenadine and loratadine. We compared the relative antihistaminic effects of loratadine (10 mg), terfenadine (120 mg), and placebo in patients with tree pollen-induced allergic rhinitis. Wheals were produced by epicutaneous tests with serial dilutions of histamine phosphate, tree and grass pollen extracts before ingestion of medication. Repeat tests were performed after seven days of treatment. Terfenadine was more effective than both loratadine (P less than .01) and placebo (P less than .001) at suppressing histamine induced wheals. Terfenadine was more effective than placebo (P less than .01), but not loratadine at inhibiting both tree-induced and grass-induced epicutaneous reactions. In this study, terfenadine was more potent than loratadine in inhibiting both histamine-induced and allergen-induced epicutaneous wheals.


Clonidine for patients with rapid atrial fibrillation. To determine whether clonidine can slow ventricular rate in patients with rapid atrial fibrillation. Randomized, controlled trial, with a 4-hour follow-up period. Emergency room of a university hospital. A consecutive sample of 18 hemodynamically stable patients who were evaluated or treated for rapid atrial fibrillation. Exclusion criteria included acute or terminal illness; current use of antiarrhythmic agents, calcium-channel blockers, or beta-blockers; excessive hypertension; pulmonary, valvular, or pericardial disease; and electrolyte imbalance. Patients were randomly assigned to receive either "no treatment" (control group) or clonidine, 0.075 mg orally, at baseline and after 2 hours if heart rate did not decrease by at least 20%. Blood pressure was measured by the same nurse in the same arm for 4 consecutive hours, and a full 12-lead electrocardiographic evaluation was done. Heart rate decreased to below 100 beats/min in eight of nine patients receiving clonidine compared with two of nine patients in the control group. The difference in the mean decreases in heart rate was 38 beats/min (95% CI, 20 to 56 beats/min). Six patients who were treated with clonidine and one patient in the control group reverted to normal sinus rhythm. Systolic blood pressure decreased slightly in both groups, without significant differences. Clinical follow-up was uneventful. Low-dose clonidine was an easy, efficient, and effective treatment for patients with rapid atrial fibrillation who were hemodynamically stable.


[Postoperative infection control in patients with hepatic, biliary tract, and pancreatic cancers]. Postoperative infection occurs more frequently in patients with malignant disease than in patients with benign disease. Postoperative infection control in patients with hepatic cancer, biliary tract cancer and pancreatic cancer is studied. Although in patients with jaundice due to malignancy the rate of positive bacterial culture of the bile collected at the time of PTCD was low, the rate of positive bile culture increased after 10 to 14 days of PTCD. The predominant strain was Enterococcus spp., followed by Klebsiella spp., Enterobacter spp. and E. coli in that order. These bacteria isolated from the bile were considered to be causative organisms of postoperative infection. Prophylactic antibiotics after the operation for jaundice due to malignancy should be chosen based on the results of bile culture. In patients undergoing hepatectomy, which is considered to be an aseptic operation, gram positive cocci such as S. aureus was the most frequently encountered organism. On the other hand, in patients undergoing hepatectomy and intestinal anastomosis, enteric bacteria were frequently isolated from the infectious foci. In this study there were 6 cases of methicillin-resistant S. aureus (MRSA) postoperative infection, 3 cases after pancreatoduodenectomy, and 3 cases after hepatectomy. Even after an aseptic operation, postoperative MRSA infection is likely to occur in patients undergoing a more invasive operation, so hospital infection control should be again emphasized.


Investigations into the mechanism by which sulfated polysaccharides inhibit HIV infection in vitro. Sulfated polysaccharides have been shown to inhibit human immunodeficiency virus (HIV) infection in vitro. Dextrin sulfate, fucoidan, and dextran sulfate fail to neutralize virions directly, but interact with target cells to inhibit virus entry. Ionic interactions of sulfated polyanions with oppositely charged cell surface components, including CD4, have been assumed to be the inhibitory mechanism. It is shown that the sulfated polysaccharides inhibit infection of both CD4+ and CD4- cell lines by HIV and also that they inhibit HTLV-1 and, to a lesser extent, the simian retrovirus, MPMV, which use receptors other than CD4. One binding site for radiolabeled fucoidan on the surface of human T cells is an 18 kD protein, but its significance is not yet clear.


Dual tropism of HIV-1 IIIB for chimpanzee lymphocytes and monocytes. In humans, macrophages serve as a major reservoir of human immunodeficiency virus (HIV-1) in the infected host and may play a role in the pathogenesis of the disease. In HIV-1-infected chimpanzees, however, virus could not be recovered from cells of the monocyte/macrophage lineage, leaving the question of macrophage tropism of HIV-1 in this species unresolved. The data reported that HIV-1 IIIB shows dual tropism and is infectious for both chimpanzee monocytes and lymphocytes in vitro. Viral replication in chimpanzee monocytes was clearly demonstrated by infection of allogeneic phytohemagglutinin (PHA) blasts in vitro and by electron microscopy (EM). EM revealed HIV particles associated with 10-15% of the HIV-1 IIIB-infected chimpanzee monocytes. Viral particles budding from the monocyte surface in the typical crescent form were noted as well. This is in contrast to the human situation, where monocytotropic HIV strains preferentially bud into and accumulate in cytoplasmic vacuoles. These results indicate that both lymphocytes and cells of the monocyte/macrophage lineage replicate virus in the chimpanzee; the cell tropism of viral strains, however, is different in chimpanzees and humans.


Nature of the intermediate in the 3-oxo-delta 5-steroid isomerase reaction. The role of Tyr-14 of 3-oxo-delta 5-steroid isomerase (KSI) was probed by analysis of the spectra of 3-amino-1,3,5(10)-estratrien-17 beta-ol (4) and equilenin (5) bound to the active site of KSI. The ultraviolet spectrum of 4 bound to KSI is identical to that for 4 in neutral solution. This observation indicates that Tyr-14 does not protonate the amine group of 4 at the active site. By analogy, it is argued that the 3-oxo group of steroid substrates for KSI is not protonated during the reaction. In contrast, the fluorescence excitation spectra of 5 bound to KSI show characteristics of an ionized phenol, even at pH values as low as 3.8. It is concluded that the pKa of equilenin is perturbed from its value in solution of 9 to less than or equal to 3.5 at the active site of KSI. Similarly, the pKa of the intermediate dienol in the KSI reaction should be lowered to less than or equal to 4.5 when it is bound to KSI. Thus, the function of Tyr-14 as an electrophilic catalyst is likely the stabilization of the anion of the dienol by hydrogen bonding rather than by proton transfer.


Role of Glu318 at the putative distal site in the catalytic function of cytochrome P450d. Most microsomal P450s have a conserved "threonine cluster" composed of three Thrs (Thr319, Thr321, Thr322 for P450d) at a putative distal site. An ionic amino acid at 318 is also well conserved as Glu or Asp for most P450s. To understand the role of these conserved polar amino acids at the putative distal site in the catalytic function of microsomal P450, we studied how mutations at this site of P450d influence the activation of molecular oxygen in the reconstituted system. Catalytic activity (0.02 min-1) toward 7-ethoxycoumarin of the Glu318Ala mutant of P450d was just 6% of that (0.33 min-1) of the wild type, while those of Glu318Asp, Thr319Ala, and Thr322Ala were comparable to or even higher than that of the wild type. Consumption rates of O2 and formation rates of H2O2 of those mutants varied in accord with the catalytic activities. Especially, the efficiency (0.5%) of incorporated oxygen atom to the substrate versus produced H2O2 for the Glu318Ala mutant was much lower than that (3.7%) of the wild type, while that (58.8%) for the mutant Glu318Asp was 16-fold higher than that of the wild type. In addition, the autoxidation [Fe(II)---- Fe(III)] rate (0.074 s-1) of the Glu318Ala mutant was much lower than those (0.374-0.803 s-1) of the wild type and other mutants. Thus, we strongly suggest that Glu318 plays an important role in the catalytic function toward 7-ethoxycoumarin of microsomal P450d.


Fe3+ binding to ovotransferrin in the presence of alpha-amino acids. The ability of L-alpha-amino acids as synergistic anions for iron binding to ovotransferrin was investigated through electronic spectroscopy. Glycine and glutamic acid were found to form by far the most stable ternary Fe(3+)-ovotransferrin-amino acid complexes. Less stable adducts were formed by amino acids with a hydroxy, amide or sulphur-containing group in the side chain, while the complexes with leucine, isoleucine, valine, lysine, arginine, tyrosine and tryptophan failed to form. Evidence is obtained that the synergistic effectiveness of the H2N-CH-COO- moiety is determined not only by the isoelectric point of the amino acid and the steric hindrance of its side chain, but a significant role is also played by interactions of the side chain itself with residues in the metal binding domains. Zn2+, Cd2+ and Co2+ are found to bind to ovotransferrin in the presence of glycine. 113Cd-NMR spectra on the Cd-derivative indicate that, according to the interlocking-sites model, the amino group of glycine directly binds to the metal ion.


Altered glucose metabolism in adriamycin-induced heart failure. Spontaneously hypertensive rats received 1 mg/kg of Adriamycin intravenously once a week for up to 12 weeks; their hearts were excised and perfused with buffer containing 5 mM [1-13C]glucose. Histological evidence of Adriamycin cardiotoxicity was evident after 8 and 12 weeks of treatment and was accompanied by a significant decrease in cardiac function. There were only minor changes in the 31P-NMR spectra in hearts following treatment; however, 13C-NMR spectra revealed decreased incorporation of label into the lactate, alanine and glutamate pools in hearts with severe tissue damage compared to hearts from untreated animals.


Factor XIII-dependent generation of 5th complement component(C5)-derived monocyte chemotactic factor coinciding with plasma clotting. Blood coagulation or plasma clotting caused generation of a monocyte chemotactic factor(s) in vitro. The chemotactic factor, of which the apparent molecular mass was 75 kDa, shared antigenicity with complement C5 and possessed the affinity to monocytes, but not to polymorphonuclear leukocytes. The generation of the chemotactic factor was hindered in the presence of a thiol enzyme inhibitor, p-chloromercuriphenyl sulfonic acid, at the concentration of 1 mmol/l, although the gelation of plasma was apparently completed. Furthermore, the generation of chemotactic factor was not observed when a plasma deficient in blood coagulation factor XIII, which is a precursor of a thiol enzyme, plasma transglutaminase, was used; and the activity normally appeared when the deficient plasma was reconstituted with purified factor XIII or with a tissue transglutaminase prior to clotting. When the human sera were injected into guinea pig skin, the serum derived from normal plasma or from the reconstituted factor XIII deficient one caused mononuclear cell infiltration, however, the serum from the deficient plasma without reconstitution infiltrated to a significantly smaller extent. These results indicated that the complement system was initiated somehow during the clotting process resulting in the generation of the C5-derived monocyte chemotactic factor in cooperation with factor XIIIa (activated factor XIII).


Characterization of the myeloid-specific CD11b promoter. The CD11b/CD18 heterodimeric surface antigen is expressed exclusively on human monocytes, macrophages, granulocytes, and natural killer cells. During differentiation of myeloid cell lines, CD11b steady state messenger RNA levels increase significantly; we show here that CD11b transcription rates increase commensurately. A 1.7-kb fragment of CD11b 5' flanking sequence directs expression of a reporter gene specifically in myeloid cell lines. Deletion analysis localizes elements directing high levels of tissue-specific reporter gene expression to the 412 bp proximal to the transcriptional start site. This sequence contains two consensus binding sites for Sp1, a GATA motif, and a purine-rich sequence that presents potential binding sites for members of the ets family of genes. Analysis of this promoter should result in the isolation of myeloid-specific transcription factors and the development of methods to direct the myeloid-specific expression of heterologous genes.


Treatment of hairy cell leukemia with 2-chlorodeoxyadenosine (2-CdA). We administered one course of 2-chlorodeoxyadenosine (2CdA) at 4 mg/m2 daily for 7 days by continuous intravenous infusion to 46 patients with hairy cell leukemia. Complete remissions occurred in 36 patients (78%; 95% confidence limits, 63% to 89%), partial remissions in five (11%), and a minor response in one. One patient died of candida sepsis 3 weeks after beginning treatment and three patients were clearly resistant to therapy. These three either had morphologically atypical hairy cells, less than 20% of which expressed Ig light chain on the cell surface, or had failed prior treatment with deoxycoformycin and interferon-alpha. At a median of 37 weeks since discontinuation of therapy, recurrent thrombocytopenia has developed in one patient, whose marrow remains normal, while a bone marrow relapse has occurred in another patient, whose blood counts remain normal. Treatment produced a greater than 50% decrease in neutrophil count in 26 patients, which lasted 3 to 4 weeks and was associated with an increased incidence of febrile episodes. These episodes occurred in 21 patients but were associated with documented infection in only four patients. Decreases in the number of CD4+ lymphocytes appeared to occur regularly after treatment and have persisted for a median of 18 weeks without obvious clinical significance. Although years of follow-up will be needed, our results confirm Piro et al's observation (N Engl J Med 322: 1117, 1990) that 2CdA appears to be highly effective in the treatment of hairy cell leukemia.


Rapid recovery from cytopenia in hairy cell leukemia after treatment with 2-chloro-2'-deoxyadenosine (CdA): relation to opportunistic infections. Sixteen patients with symptomatic hairy cell leukemia were treated with a single course of 2-chloro-2'-deoxyadenosine (CdA), 0.7 mg/kg total dose. Twelve patients achieved complete remission (CR). One patient with a CD19+/CD5+/CD25- phenotype and one with a pentostatin-treated CD19+/CD25- variant form had minor responses. Two patients with advanced disease and poor performance status died early from invasive mycosis. Three patients recovered from infections caused by cytomegalovirus and by candida. No patient had infections caused by bacteria or by unknown organisms. The median time to full recovery from anemia and thrombocytopenia was 6 and 2 weeks from start of therapy, respectively. Patients with infections, however, recovered at 13 and 5 weeks, respectively. Neutrophil, monocyte, and lymphocyte counts returned to normal at a median of 5, 5, and 10 weeks, respectively. Infections developed more frequently in pancytopenic patients than in those with one or more blood cell count within the normal range (P less than .01). All patients with one or no previous therapy had a CR, whereas those with more than one previous regimen had a lower CR rate (P less than .01). Thus, 1 week of CdA therapy frequently induced CR also in patients resistant to interferon. Toxicity was limited, and recovery from cytopenia was faster than what is reported during interferon therapy.


'Natural history' of hepatectomy.

'Natural history' of hepatectomy. The aim of this study was to describe biochemical and liver function test changes after hepatectomy in 33 patients with the following characteristics: absence of underlying liver disease, no blood or plasma transfusion during the perioperative period, uneventful postoperative course. Resection with a temporary pedicle inflow occlusion (10-45 min) consisted of unisegmentectomy or less in 15 patients and bisegmentectomy or more in 18. Blood tests showed: a correlation between aminotransferase rise and duration of ischaemia, and a fall in prothrombin time and factor V levels correlating with the weight of resected specimen at day 1; a moderate gamma-glutamyl transpeptidase and alkaline phosphatase elevation and a rise in fibrinogen level correlating with the extent of resection at day 7. Changes in haemoglobin level, white cell count, platelet count, prothrombin time, factor V level and serum bilirubin level tended to return to preoperative levels by day 7. For gamma-glutamyl transpeptidase and alkaline phosphatase, increased levels persisted for 8-12 weeks after resection. These results, in this selected group of patients, allow a description of the 'natural history' of hepatectomy. The knowledge of these 'natural' changes may contribute to the early detection of postoperative complications.


Interferon-induced increase in sensitivity of ovarian cancer targets to lysis by lymphokine-activated killer cells: selective effects on HER2/neu-overexpressing cells. Overexpression of the HER2/neu oncogene in ovarian tumor cells is associated with relative resistance to lymphokine-activated killer (LAK) cell cytotoxicity. Treatment with gamma-interferon (IFN-gamma) (200-2000 units/ml) for 3 days markedly enhanced the sensitivity of HER2/neu-overexpressing ovarian tumor cells to LAK cells but had no effect on the sensitivity of nonexpressing ovarian targets. Increased sensitivity to lysis was associated with an increase in effector-target conjugate formation, the induction of target cell intercellular adhesion molecule 1 (ICAM-1) expression, and the down-regulation of HER2/neu expression. Anti-ICAM-1 antibody blocked the enhanced lysis, indicating that ICAM-1 is important in the increased sensitivity to LAK cells. However, induction of ICAM-1 expression did not correlate well with enhanced sensitivity to lysis; it was maximal after 24 h of exposure to IFN-gamma and still present 24 h after removing IFN-gamma. In contrast, enhanced lysis required 3 days of exposure to IFN-gamma and was reversed within 24 h after removal of IFN-gamma. These data indicate that, although ICAM-1 is necessary, it is not sufficient for the IFN-gamma-induced enhancement of sensitivity to LAK lysis.


A general theory for host seeking decisions in mosquitoes. We develop a theory for host seeking decisions in mosquitoes that explicitly considers the tradeoffs mosquitoes face in allocation to somatic and gametic function. Specifically, we consider conditions under which mosquitoes should seek out nectar and blood hosts upon encountering host odours. Results from development of a dynamic model that considers free and crop energy states suggest that mosquitoes should seek out blood hosts under a wide variety of conditions but that decisions to seek nectar depends upon crop volume, concentration and free energy. This pattern arises because mosquitoes carrying large crop loads are constrained in their ability to obtain large blood meals due to space limitations in the abdomen. The predicted patterns of behaviour are supported by published observations of mosquito behaviour.


Consistent association of 1p loss of heterozygosity with pheochromocytomas from patients with multiple endocrine neoplasia type 2 syndromes. Pheochromocytomas and medullary thyroid cancers (MTCs) are neuroendocrine tumors which arise sporadically or as part of the multiple endocrine neoplasia type 2 (MEN-2) hereditary syndromes. The most consistent molecular genetic abnormality which has been described in these tumors is loss of heterozygosity (LOH) of the short arm of chromosome 1 (1p). This finding is particularly interesting because the predisposition gene for the hereditary form of these tumors has been mapped to chromosome 10, but LOH on chromosome 10 in MEN-2 tumors is found rarely. We have used a battery of 1p DNA probes to elucidate the region of loss of 1p in 18 pheochromocytomas and 27 MTCs. Using restriction fragment length polymorphism analysis, we identified loss of all or a portion of 1p in 12 of 18 pheochromocytomas. 1p LOH was identified in nine of nine pheochromocytomas in MEN-2A and -2B patients, compared with only two of seven sporadic pheochromocytomas. We also found 1p LOH in one of two von Hippel-Lindau patients. LOH on 1p was noted in only three of 24 informative MTCs, and these were from patients with MEN-2A. In most of the pheochromocytomas, the entire short arm of chromosome 1p appears to have been lost; however, in three of the non-MEN pheochromocytomas and in three MEN-2A MTCs, the region of loss is smaller, allowing estimation of the smallest region of overlap. The combined data for MTCs and pheochromocytomas suggest that the smallest region of overlap of LOH is bounded by D1S15 (1pter-p22) and D1Z2 (1P36.3), excluding a region around MYCL (1p32). Although other regions of 1p should not be completely ruled out, the data suggest that this region may harbor a tumor suppressor gene or genes whose inactivation is important in the development of these tumors. Furthermore, the strong association between 1p LOH and the MEN-2 syndromes, especially in pheochromocytomas, suggests a relationship between the predisposition gene on chromosome 10 and the loss of the suppressor gene on 1p. Alternatively, other loci may be more important in sporadic disease.


Molecular heterogeneity of somatostatin analogue BIM-23014C receptors in human breast carcinoma cells using the chemical cross-linking assay. Distinct proteins complexed with somatostatin and the somatostatin analogue BIM-23014C were revealed in human breast cancer cells using the cross-linking assay. One BIM-23014C-specific complex (Mr 57,000) was observed in MCF-7 (monolayer, nodule, and tumor) and T47D. Growth inhibition of MCF-7 tumor xenografts by BIM-23014C was dose related in the 6-day subrenal capsule assay. Three complexes (Mr 27,000, 42,000, and 57,000) were detected in MDA-MB-231, and no complex was visible in HBL-100. No correlation was found between receptors for BIM-23014C and epidermal growth factor in these lines. Twenty-seven of 30 human breast tumors (90%) had at least one BIM-23014C receptor. Sixteen had three complexes (Mr 27,000, 42,000, and 57,000). Six had the two complexes (Mr 27,000 and 57,000), two had Mr 42,000 and 57,000 complexes, two had just the Mr 27,000 complex, and one had just the Mr 42,000 complex. The presence of the three BIM-23014C receptors was positively correlated (P less than 0.05) to the low amount of sex steroid receptors (less than 20 fmol/mg) [seven of eight (estrogen receptor negative, progesterone receptor negative) versus four of 14 (estrogen receptor positive, progesterone receptor positive)]. Another positive correlation was established between the absence of progesterone receptors and the presence of these three complexes [12 of 16 (progesterone receptor negative) versus four of 14 (progesterone receptor positive)]. This high percentage of BIM-23014C receptor-positive biopsies and its inhibitory activity would support its clinical potential for the treatment of breast cancer.


Deletion mapping on the short arm of chromosome 3 in squamous cell carcinoma and adenocarcinoma of the lung. We examined loss of heterozygosity in 49 adenocarcinomas and 18 squamous cell carcinomas of the lung with 19 RFLP markers on the short arm of chromosome 3. Although no interstitial deletions were observed in any squamous cell carcinomas, interstitial or partial deletions were detected in 23 adenocarcinomas. Identification of two common regions of deletion in adenocarcinomas, at 3p21.3 and 3p14.1-21.1, suggested the presence of at least two tumor suppressor genes on 3p within the same regions commonly deleted in renal cell carcinomas. Correlation between the frequency of loss of heterozygosity on 3p and histopathological grade of adenocarcinoma also was observed. These results imply an etiological difference between two major types of non-small cell lung cancers, adenocarcinoma and squamous cell carcinoma.


Glutaminase and glutamine synthetase activities in human cirrhotic liver and hepatocellular carcinoma. Glutamine synthetase and glutaminase activities in human cirrhotic liver tissues and hepatocellular carcinomas were determined for comparison with normal liver tissues. In hepatocellular carcinoma, glutamine synthetase activity was approximately one-third of that in normal liver, whereas no detectable change in the enzyme activity was observed in cirrhotic liver. Phosphate-dependent and phosphate-independent glutaminase activities were increased approximately 20-fold and 6-fold, respectively, both in the carcinoma and cirrhotic liver compared with those from normal liver, Oxypolarographic tests showed that the rate of glutamine oxidation in the tumor and cirrhotic liver mitochondria was about 5-fold higher than that in the liver mitochondria. The rate of glutamate oxidation in the liver mitochondria was comparable to that in the cirrhotic liver and tumor mitochondria. Glutamine oxidation was inhibited by prior incubation of the mitochondria with 6-diazo-5-oxo-L-norleucine, which inhibited mitochondrial glutaminase. These results indicate that the product of glutamine hydrolysis, glutamate, is catabolized in the tumor and cirrhotic liver mitochondria to supply ATP. In the liver and cirrhotic liver mitochondria, glutamate was oxidized via the routes of transamination and deamination. On the other hand, glutamate oxidation was initiated preferentially via a transamination pathway in the tumor mitochondria.


Effect of tumor size on the enhancement by gamma interferon of the localization of radiolabeled F(ab')2 fragments of anti-intercellular adhesion molecule-1 monoclonal antibodies in human colon carcinoma cells grafted in nude mice. Because tumor size has been shown to influence the specific accumulation of radiolabeled anti-tumor-associated antigen monoclonal antibodies (mAb), the present study has investigated the effect of the tumor size on the enhancement by gamma interferon (IFN-gamma) of the accumulation of radiolabeled mAb in malignant lesions. Intercellular adhesion molecule-1 (ICAM-1) has been used as a marker because of its high susceptibility to modulation by IFN-gamma. F(ab')2 fragments of anti-ICAM-1 mAb CL207.14 have been selected to visualize malignant lesions, because they had been shown to be more sensitive probes for our experiments than whole IgG. Administration of IFN-gamma to human colon carcinoma-bearing nude mice increased the expression of ICAM-1 in the xenografts and the specific accumulation of 125I-F(ab')2 fragments of anti-ICAM-1 mAb CL207.14. The latter effect is influenced by the size of the lesions, because it was observed only in tumors with an approximate diameter of 8 mm and an approximate weight of 250 mg. If these results obtained in an animal model system are applicable to patients with malignant diseases, the present investigation suggests that administration of IFN-gamma enhances the sensitivity of immunoscintigraphy and the efficacy of immunotherapy with radiolabeled mAb which recognize tumor-associated antigens that are susceptible to modulation by IFN-gamma. However, the effect of IFN-gamma is not a general phenomenon but is influenced by the size of the malignant lesions.


Chromosome alterations in human small cell lung cancer: frequent involvement of 5q. Deletions of the 3p chromosome region and molecular alterations of the tumor suppressor genes RB1 and TP53, located, respectively, at 13q14 and 17p13, are well-documented in small cell lung cancer (SCLC). Because of technical difficulties, karyotypes of primary SCLC specimens are rarely reported. In this study, detailed cytogenetic analysis was performed on 13 early passage SCLC cell lines and fresh specimens, including 4 lung primaries. Numerous chromosome alterations were found, even in newly diagnosed primary tumors. Consistent with previous molecular studies, chromosomal losses of 3p (13 cases) and 17p13 (12 cases) were frequently observed. Numerical losses of chromosome 13 and structural rearrangements affecting 13q14 were identified in 10 specimens. In addition, losses of chromosome 5 and structural alterations of 5q occurred in 12 tumors; among these, 9 displayed losses of region 5q13-q21. Double minutes were found in 4 cases (3 of 5 specimens from patients who received prior cytotoxic therapy but only 1 of 8 from untreated patients). DNA analysis revealed amplification of either MYC1 or MYCN in cells from each of these 4 tumors. Overall, the cytogenetic findings underscore that progression of SCLC involves multiple genetic changes and suggest further that a tumor suppressor gene(s) on 5q may contribute to SCLC tumorigenesis.


Inhibitory effect of circulating insulin on glucagon secretion during hypoglycemia in type I diabetic patients. To clarify whether the circulating insulin level influences hormonal responses, glucagon secretion in particular, during hypoglycemia in patients with insulin-dependent (type I) diabetes. Nine type I diabetic patients were studied. During two separate experiments, hypoglycemia was induced by low-dose (244 pmol.kg-1.h-1) and high-dose (1034 pmol.kg-1.h-1) intravenous insulin infusions for 180 min in each case. The arterial blood glucose level was directly monitored every 1.5 min, and glucose was infused in the high-dose test to clamp the arterial blood glucose level to be identical as in the low-dose test. Despite the fact that the plasma insulin level was four times higher in the high-dose than in the low-dose test (740 +/- 50 vs. 180 +/- 14 pM), a close to identical arterial hypoglycemia of approximately 3.3 mM was obtained in the two experiments. During hypoglycemia, a significant rise of the plasma glucagon level was found only in the low-dose test (188 +/- 29 vs. 237 +/- 37 ng/L, P less than 0.05), and the incremental area under the glucagon curve was significantly greater in the low-dose than in the high-dose test (140 +/- 19 vs. -22.7 +/- 34 ng/L.h-1, P less than 0.005). The responses of plasma epinephrine, norepinephrine, growth hormone, pancreatic polypeptide, and somatostatin were similar in both tests and, consequently, were not significantly modified by the circulating insulin level. This study demonstrates that, in type I diabetic patients, the glucagon response to hypoglycemia is suppressed by a high level of circulating insulin within the physiological range. Our findings may help to explain the impairment of glucagon secretion during hypoglycemia frequently seen in these patients.


Somatostatin receptors in human colorectal cancer. We have previously reported that somatostatin may reduce tumour cellular proliferation in patients with colorectal carcinoma. However, it is not known what proportion of primary colorectal cancers express somatostatin receptors. We have therefore evaluated somatostatin receptor status in 50 primary colorectal cancers. Twelve (24%) of the cancers were shown to be somatostatin receptor positive. There was no correlation between receptor status and tumour stage or grade.


Detection of human papillomavirus types 16 and 18 in the exfoliated cervical cells using the polymerase chain reaction. We applied the polymerase chain reaction (PCR) to detect HPV 16 and 18 in cytological samples obtained from the uterine cervices of Japanese women. HPV infection was detected in 17 (25%) of 67 with CIN and 11 (37%) of 30 with cervical carcinoma. It is notable that 11 (16%) of 69 women with normal cervices were infected with either HPV 16 or 18. The polymerase chain reaction is sensitive and useful for epidemiological studies.


Hormonal and ultrasound characteristics of menstrual function during chronic hemodialysis and after successful renal transplantation. The cycles of 11 renal transplant recipients (RTR), at least 24 months after stabilization of graft function and four hemodialyzed (HD) patients, menstruating regularly, were evaluated by concurrent and systematic determinations throughout the cycle of LH, FSH, estradiol, progesterone, testosterone, prolactin and SHBG and in the case of RTR also by ultrasound follow-up. Biphasic estradiol secretion, midcycle LH and FSH surge, duration of luteal phase, midluteal progesterone values and in the case of RTR, ultrasonic parameters were consistent with: (1) normal ovulatory cycles in five RTR; (2) ovulatory cycles with luteal phase deficiency in five RTR and two HD patients; (3) anovulatory cycles in one RTR and two HD patients. Thus, in HD patients only abnormal cycles of central etiology were found, while in RTR, luteal phase deficiency was a very common syndrome, in equal percentage with normal ovulatory cycles.


Macrophage- and lymphocyte-subtypes in the endometrium during different phases of the ovarian cycle. The presence of immunocompetetive cells in the endometrium during the proliferative and secretory phase of the ovarian cycle is demonstrated on the light and electron microscopic level using monoclonal antibodies (MoAb). Subtypes of monocytes, macrophages and T-lymphocytes appear during the different phases in variable extent and different localization. Some subpopulations of the monocyte/macrophage system and T-helper lymphocytes increase in number on day 21/22. Our observations indicate that cells with bone marrow origin take part in functional events of the endometrium during the ovarian cycle.


Effect of the vibratory acoustic stimulation on fetal heart rate patterns of premature fetuses. The purpose of this study was to examine the heart rate patterns before and after a standardized external vibratory acoustic stimulation in a group of 24 healthy premature fetuses at 32-35 weeks gestational age. FHR was analysed on line by Sonicaid Computer System 8000. A significant increase in the number of accelerations and an increase of variation after stimulus were observed. All other FHR patterns such as baseline, high and low episodes did not change significantly.


Role of thioridazine in unexplained infertility. A total of 452 women with unexplained infertility were selected for the present study. From them 310 women were put on thioridazine tablet (5 mg), 1 h after dinner from the 8th day of the menstrual cycle to the 18th day in each cycle. Coitus was advised about 1-2 h after the drug intake and proper posture was advised to the patients. The other 142 patients were given placebo therapy. Patients were followed up for pregnancy for 1 year which was confirmed by ultrasonographic examination. Ninety-four patients (30.2%) in the study group conceived in contrast to 22 (15.42%) in the control group (P less than 0.001). Incidence of abortions, congenital malformations and perinatal mortality and mode of delivery were not significantly different in the two groups. Thioridazine due to anxiolytic effect in low dosage appears to be promising in the treatment of unexplained infertility.


Ogilvie's syndrome.

Ogilvie's syndrome. Four cases of Ogilvie's syndrome (acute colonic pseudo-obstruction) are reported. All occurred in the early puerperium following cesarean section and cesarean hysterectomy. In three of the patients, the diameter of the distended cecum was less than 9.0 cm and so management was conservative while in the fourth patient it was more than 9.0 cm, and so surgical intervention was carried out. A cecal diameter of 9.0 cm or above is an indication for surgical intervention to prevent possible colonic perforation. Other indications for surgery include established cecal perforation and failed conservative management. It is important that an early diagnosis is made and management instituted in order to prevent complications and associated high mortality.


Management of isoimmunization in pregnancy. ACOG Technical Bulletin number 148--October 1990. Isoimmunization is diagnosed by a positive antibody screen and requires identification of the specific antigen responsible, titration of the level of antibody response, and classification of the antigen into either a clinically significant or benign group. The paternal antigen status and zygosity should be determined whenever possible. A significant antibody response to an antigen associated with erythroblastosis should be monitored at regular intervals by obtaining serial titers, and repetitive amniocentesis or fetal blood sampling may be required to adequately monitor the fetal condition. Early and continued consultation with a perinatologist and a neonatologist who are experts in the management of this condition is critical in developing an appropriate therapeutic plan that includes proper management at delivery and optimal neonatal support. New technologies and expertise now allow better outcome for severely affected fetuses.


Deception. ACOG Committee opinion: Committee on Ethics number 87--November 1990. Deception is the deliberate misrepresentation of facts through words or actions in order to make a person believe that which is not true. The forms deception can take include explicit lying, deception by implication, and deception by omission of information that patients need to make decisions in their own regard. Deception intended to advantage the physician economically or otherwise at the expense of the patient is unethical.


Uterine motility in patients with bicornuate uterus. This study analyzes uterine motility in 12 women with a bicornuate uterus using the results of the recordings of endo-uterine pressure, obtained with two balloon-closed catheters. Seven patients had symmetric uterine cavities, while the rest (5 patient) had very dissimilar ones. The registration of the uterine motility was carried out during various phases of the cycle and after the administration of two drugs (oxitocin and methylergobasine), with the following results: the bicornuate uterus has a spontaneous activity similar to that of a normal uterus. A similar contractile response was observed in the uteri with two anatomically symmetric horns, whereas a dissimilar response was typical of the uteri with marked anatomic differences between the two horns.


Developmental potential.

Developmental potential. In summary (and probably to no one's genuine surprise), it seems clear that some of the key themes in the mechanisms employed during development reiterate themselves throughout the animal kingdom. Yet, as our understanding becomes more refined, new and beguiling observations point to unique aspects of each developmental program. The concentration and absolute position of a variety of positional signaling molecules is likely to be very important in determinative events (establishment of the anteroposterior positioning in a field as in retinal development, establishment or enactment of a hox code, and selector gene regulation through gradients in Drosophila). Appropriate signalling responses are virtually certain to depend critically on the appropriate expression of each component of cellular signal transduction pathways (initiated by the activation of cell-surface receptor protein kinases to finally eliciting gene expression changes through the differential activity of specific transcription factors). The important biochemical details of transcription factor activation of specific respondent genes may be either simpler (as indicated from the murine/Drosophila domain swap experiments) or more complicated (from the responses of mim-1 to cellular versus viral myb proteins) than we had heretofore anticipated.


The polyhomeotic gene of Drosophila encodes a chromatin protein that shares polytene chromosome-binding sites with Polycomb. The Polycomb group (PcG) genes in Drosophila melanogaster are required for maintenance of correct spatial expression of homeotic genes, and their products are thought to form either a regulatory network or act as a multimeric complex. Recently, it has been suggested that because of homology between Polycomb (Pc) and Su(var)205, PcG genes encode chromatin proteins required for the maintenance of a determined state in chromatin. The polyhomeotic (ph) gene is a member of the PcG of genes. We present DNA sequence of a ph cDNA, which encodes a 169-kD protein with a single putative zinc finger, a serine/threonine-rich region, and has glutamine repeats, suggesting that ph is a DNA-binding protein. Polyclonal antisera directed against ph protein bind to approximately 80 sites on polytene chromosomes. Most of these sites appear to be the same as those recognized by antibodies to Pc protein. ph protein binds to insertion sites of constructs containing DNA from the bithoraxoid (bxd) region of the Bithorax complex, showing that ph binding to chromatin is DNA dependent. The same bxd constructs are recognized by Pc protein, strongly supporting the hypothesis that ph and Pc interact directly.


Effect of overproduction of heat shock chaperones GroESL and DnaK on human procollagenase production in Escherichia coli. The effect of overexpression of the heat shock chaperone genes dnaK and groESL on heterologous protein production in Escherichia coli was examined, using a set of related human procollagenase proteins. A diverse range of effects on protein solubility, secretion, and accumulation was observed, and these effects were highly dependent on the particular chaperone/procollagenase pairing involved. Both chaperones caused a large increase in the apparent solubility of a fusion of the LamB signal peptide to procollagenase. GroESL had no effect on the accumulation of mature (secreted) procollagenase, while DnaK suppressed secretion considerably. In the absence of a signal peptide, overexpression of either chaperone resulted in a dramatic increase in both solubility and accumulation of procollagenase. The 10-fold increase in accumulation was associated with an increase in in vivo protein half-life.


Insulin activation of acetyl-CoA carboxylase accompanied by inhibition of the 5'-AMP-activated protein kinase. The activity of acetyl-CoA carboxylase (ACC), a rate-limiting enzyme of fatty acid biosynthesis and malonyl-CoA production, can be regulated by several mechanisms, including multisite covalent phosphorylation, both in vitro and in intact cells. Evidence has been presented by others to indicate that a 5'-AMP-activated protein kinase (AMPK) is likely the major regulatory kinase active on ACC. While insulin is known to activate ACC in several cell types, accompanied by changes in ACC phosphorylation, the mechanism underlying this activation has been obscure. In the present study, we have examined, in Fao hepatoma cells, the effects of insulin on ACC and AMPK activity, the latter measured with a synthetic peptide corresponding to one of the phosphorylation sites on ACC for AMPK. Our results show that insulin leads to inhibition of kinase activity prior to the onset of ACC activation; the peak of maximal kinase inhibition (approximately 35% at 10 min) is seen to precede the onset of ACC activation (20 min). The inhibition of kinase activity due to insulin is observed both in the absence and presence of varying stimulating concentrations of added 5'-AMP. Both kinase inhibition and ACC activation display similar insulin sensitivity (A50 0.3 nM). Preservation of this insulin-induced kinase inhibition requires the presence of protein phosphatase inhibitors in the cell lysis buffer, suggesting that AMPK itself might be regulated by insulin-stimulated changes in kinase phosphorylation. Taken together, these data are consistent with the hypothesis that the 5'-AMP-activated protein kinase is a regulated component of the insulin signal transduction pathway and may be the major target for insulin regulation of ACC.


Brain somatostatin receptor-G protein interaction. G alpha C-terminal antibodies demonstrate coupling of the soluble receptor with Gi(1-3) but not with Go. Somatostatin (SST) receptors activate potassium channels, stimulate protein phosphatases, inhibit adenylate cyclase and close calcium channels. These multiple effects are controlled by guanine nucleotide binding (G) proteins of the pertussis toxin-sensitive Gi and Go types. In the present study we have identified the G proteins coupling with brain SST receptors. To this end, brain SST receptors were solubilized in G-protein coupled form. Binding of the SST analogue MK 678 to the solubilized receptor was completely inhibited by guanosine 5'-O-thiotriphosphate (IC50 = 100 nM), reflecting decreased receptor affinity for agonist following uncoupling of the receptor and G protein(s). Antibodies raised against specific COOH-terminal peptides of the G proteins Gi(1-3), Go, and Gz were used to probe for SST receptor-G protein coupling in this system. Antibodies binding to the COOH-terminal regions of Gi1 and Gi2 (antibody AS) and Gi3 (antibody EC) inhibited binding of 125I-MK 678 (75 pM) by 57 +/- 4% and 48 +/- 5%, respectively. The effects of these antibodies were concentration-dependent and additive, such that in combination AS and EC completely inhibited binding. Antibodies binding to the COOH-terminal region of Go (GO) and Gz (QN) did not affect binding of 125I-MK 678, indicating that neither Go nor Gz are associated with the brain SST receptor. Prelabeling of the receptor with 125I-MK 678 prior to addition of antibody induced the formation of a "locked conformation" of the agonist-bound receptor-G protein complex which was insensitive to antibody. In conclusion, Gi1 and/or Gi2 and Gi3 are coupled in approximately equal proportions to the brain 125I-MK 678-binding SST receptor, accounting for all of the G protein coupling of this receptor.


Genetic cause of leukocyte adhesion molecule deficiency. Abnormal splicing and a missense mutation in a conserved region of CD18 impair cell surface expression of beta 2 integrins. Patients with leukocyte adhesion molecule (CD11/CD18, beta 2 integrins) deficiency have structural defects in the common beta subunit (CD18), which prevent heterodimer formation and normal cell surface expression of these receptors, leading to life-threatening bacterial infections. To elucidate the nature of these defects in a patient with partial (type II) deficiency, abnormal CD18 cDNA clones were isolated, using the polymerase chain reaction to amplify the patient's B cell-derived cDNAs. Sequence analysis revealed two mutant alleles. cDNA clones, representing a maternal allele, contained both a 12-base pair insertion resulting in an in-frame addition of four amino acids between P247 and E248 and a C1756----T nucleotide transition, resulting in an R586----W substitution in the normal CD18 protein. The inframe insertion arose by a single nucleotide C----A transversion in the 3' terminus of an intron, generating aberrant splice acceptor site. Other cDNA clones contained an A1052----G nucleotide transition not present in either parent which resulted in an N351----S substitution. To determine the functional importance of these changes, cDNA encoding a normal alpha chain (CD11b) was cotransfected into COS with CD18 cDNAs encoding for wild-type, maternal mutant allele, or CD18 containing N351----S substitution. Immunostaining of transfectants with anti-CD18 monoclonal antibodies revealed no cell surface expression of the maternal mutant CD18, and 22% surface expression of N351----S CD18. Both the insertion and the N351----S mutations occurred in a 250 amino acid extracellular region of CD18 that is highly conserved among beta integrins supporting a role for this region in heterodimer formation.


NH2-terminal globular domain of human platelet glycoprotein Ib alpha has a methionine 145/threonine145 amino acid polymorphism, which is associated with the HPA-2 (Ko) alloantigens. The glycoprotein (GP) Ib/IX complex, a prominent platelet GP complex, is the primary receptor for vWF. Previously, we have established that an antigenic polymorphism of platelets, the HPA-2 or Ko alloantigen system, is located on the 45-kD amino-terminal globular domain of GPIb alpha. With the polymerase chain reaction, we have amplified two segments of the GPIb alpha gene coding for the first 382 amino acids of two HPA-2a and two HPA-2b homozygous individuals. Nucleotide sequence analysis revealed as the only difference a C-T polymorphism at position 434 of the coding region for the mature protein. This base change results in a substitution of threonine (ACG) in HPA-2a (Kob) to methionine (ATG) in HPA-2b (Koa) at amino acid position 145. The C-T polymorphism is reflected in a difference in restriction enzyme recognition, resulting in an Aha 2-site in the HPA-2b allele and a SfaN1 site in the HPA-2a allele. Restriction fragment length polymorphism analysis of the amplified DNA of 3 HPA-2(a-,b+), 2 HPA-2(a+,b+), and 11 HPA-2(a+,b-) donors showed that these restriction sites were associated with the HPA-2 alleles. DNA-typing for the HPA-2 alloantigen system on genomic DNA obtained from a small number of cells may be applied for determining the genotype of a fetus from an immunized mother or of severely thrombocytopenic patients.


Cloning and expression of a mutant methylmalonyl coenzyme A mutase with altered cobalamin affinity that causes mut- methylmalonic aciduria. Distinct genotypic and phenotypic forms of methylmalonyl CoA mutase (MCM) apoenzyme deficiency can be delineated by biochemical analysis of mutant fibroblasts. One form, designated mut-, expresses a phenotype in which residual enzyme activity is evident in cultured cells exposed to high concentrations of hydroxycobalamin. We describe cloning of an MCM cDNA from cells exhibiting a mut- phenotype and characterization of the mutant gene product overexpressed in primary muto human fibroblasts and Saccharomyces cerevisiae. Three novel base changes were observed. Recombinant clones containing one of these base changes (G717V) express four characteristics of the mut- phenotype: failure to constitute [14C]propionate incorporation activity in fibroblasts assayed under basal cell culture conditions, constitution of [14C]propionate incorporation activity in fibroblasts stimulated with 0.1-1.0 micrograms/ml hydroxycobalamin, interallelic complementation with alleles bearing an R93H mutation, and an apparent Km (adenosylcobalamin) 1,000-fold higher than normal. These results demonstrate that the G717V mutation produces the mut- phenotype and localizes determinants for adenosylcobalamin binding near the carboxyl terminus of MCM.


Identification of three mutant alleles of the gene for mitochondrial acetoacetyl-coenzyme A thiolase. A complete analysis of two generations of a family with 3-ketothiolase deficiency. 3-Ketothiolase deficiency (3KTD) stems from a deficiency of mitochondrial acetoacetyl-coenzyme A thiolase (T2). We analyzed the molecular basis of 3KTD in two generations of a family. A boy (patient 2, GK04), his father (patient 1, GK05), his mother, and his brother were studied; three mutant alleles of T2 gene were identified. Patient 1 is a compound heterozygote: one allele has a point mutation of G to A at position 547 on his T2 cDNA, causing Gly150 to Arg substitution of the mature T2 subunit, and the other allele has GT to TT transition at the 5' splice site of intron 8, causing exon 8's skipping of the T2 cDNA. Patient 2 is also a compound heterozygote: one allele inherited from his mother has AG to CG transition at the 3' splice site of intron 10, causing exon 11's skipping of the T2 cDNA, and the other allele derived from patient 1 has the G to A mutation (Gly to Arg). The brother of patient 2 is an obligatory carrier with the mutant allele causing the exon 8 skipping. This report seems to be the first complete molecular definition of 3KTD at the gene level.


Neutrophil adherence to isolated adult cardiac myocytes. Induction by cardiac lymph collected during ischemia and reperfusion. Canine neutrophils can be induced to adhere in vitro to isolated adult cardiac myocytes by stimulation of the neutrophils with chemotactic factors such as zymosan-activated serum (ZAS) only if the myocytes have been previously exposed to cytokines such as interleukin 1 (IL-1) or tumor necrosis factor-alpha. These cytokines induce synthesis and surface expression of intercellular adhesion molecule-1 (ICAM-1) on the myocyte, and neutrophil adhesion is almost entirely CD18 and ICAM-1 dependent. The present study examines cardiac-specific lymph collected from awake dogs during 1-h coronary occlusion and 3 d of reperfusion for its ability to induce both ICAM-1 expression in cardiac myocytes, and neutrophil-myocyte adherence. Reperfusion lymph induced ICAM-1 expression in isolated myocytes, and myocyte adherence to ZAS-stimulated neutrophils that was completely inhibited by anti-CD18 and anti-ICAM-1 monoclonal antibodies. This activity peaked at 90 min of reperfusion and persisted for up to 72 h. Preischemic lymph was not stimulatory. IL-1 appeared not to be a stimulating factor in lymph in that dilutions of lymph were found to inhibit the stimulatory effects of recombinant IL-1 beta. However, investigation of interleukin 6 (IL-6) revealed that recombinant IL-6 stimulated myocyte adhesiveness for ZAS-stimulated neutrophils (ED50 = 0.002 U/ml) and expression of ICAM-1 by isolated myocytes. IL-6 neutralizing antibody markedly reduced the ability of reperfusion lymph to stimulate adhesion and ICAM-1 expression, and estimates of levels of IL-6 in reperfusion lymph ranged from 0.035 to 0.14 U/ml. These results indicate that cytokines capable of promoting neutrophil-myocyte adhesion occur in extracellular fluid during reperfusion of ischemic myocardium, and that one of these cytokines is IL-6. Neutrophil-myocyte adhesion may be of pathogenic significance because it may enhance the cytotoxic activity of the neutrophil.


A common precursor for CD4+ T cells producing IL-2 or IL-4. To investigate whether CD4+ T cells are predetermined to produce a given pattern of lymphokines, we have used a culture system that allows the controlled induction of either IL-2- or IL-4-producing CD4+ T cells. Single, freshly isolated murine CD4+ T cells were activated with Con A, rIL-2, and APC; the developing clones were split and then cultured for an additional 14 days with either rIL-2 alone or with rIL-2 and anti-CD3 stimulation. Subclones expanded in the presence of rIL-2 alone produced predominantly IL-2, although subclones derived from the same precursor and expanded in the presence of rIL-2 and a mitogenic antibody to CD3 released predominantly IL-4. Subclones expanded for 2 wk in the presence of rIL-2 plus a mitogenic mAb to CD3 released up to 60 times more IL-4 but only 1/90 the amount of IL-2 released by subclones derived from the same precursor cell and expanded with rIL-2. Both phenotypes can be derived from IL-2-producing precursor cells. These results demonstrate that IL-2-producing clones can be derived from the same cells as IL-4-producing clones and are most consistent with the view that the IL-2-producing Th1 or the IL-4-producing Th2 phenotype of a T cell clone is acquired during T cell differentiation and is not secondary to the expansion of distinct subpopulations that are predetermined to produce a specific cytokine pattern.