Sequence of the nucleocapsid protein gene of Machupo virus: close relationship with another South American pathogenic arenavirus, Junín. The sequence of the nucleocapsid (N) protein of Machupo virus (causative agent of Bolivian haemorrhagic fever) has been determined, and used to infer a phylogenetic relationship to other arenaviruses. The relationship of the virus to Junín and Tacaribe viruses, together with previous demonstrations of antigenic similarity and cross-protection by heterologous viruses, suggest that vaccines developed against Argentine haemorrhagic fever might also be effective against the Bolivian disease.


Cloning and restriction endonuclease mapping of the genome of an equine herpesvirus 4 (equine rhinopneumonitis virus), strain 405/76. Purified virion DNA of an Australian isolate of equine herpesvirus 4(EHV 4.405/76) was digested with restriction enzymes and the DNA fragments were cloned into pUC19. The resulting recombinant plasmid library, representing 92% of the virus genome, was used in hybridization analyses to construct restriction maps for BamHI, EcoRI, and SalI for the EHV4 genome. The results show that the genome of EHV 4.405/76 was approximately 145 kb and comprised a unique long (UL) region of 112 kb and a unique short (US) region of 12.4 kb. US is flanked by an internal and terminal repetitive sequence (IRS and TRS) of about 10.3 kb. The BamHI and EcoRI restriction maps are similar to those previously published for an English isolate EHV 4.1942 strain although some differences such as location of an additional fragment and changes in positions of two other small fragments were found.


Chronic fatigue syndrome. A review from the general practice perspective. There is no doubt that the chronic fatigue syndrome exists. It is a condition that is debilitating and of unknown cause. Research into chronic fatigue syndrome demonstrates possible psychiatric or organic causes. The truth may be somewhere in between. Evidence for the existence of an ongoing chronic infection is now not convincing. Treatment should be based on supportive counselling, explanation, psychiatric help (both pharmacological and non pharmacological) and a graded programme of increased activity with the eventual aim of resumption of full functioning.


Platelet-derived growth factor in human malignancy. Platelet-derived growth factor (PDGF) was first implicated in the process of transformation when one of its peptide chains was found to be homologous to the viral sis oncogene (v-sis). Since that time, there have been multiple demonstrations of the transforming activity of v-sis in fibroblasts. Because of the near identity of the v-sis protein with the PDGF B chain, v-sis is thought to transform through an autocrine stimulatory mechanism of cell growth. Consistent with this view are studies which demonstrate inhibition of v-sis-mediated transformation by anti-PDGF antibodies. Expression of the cellular sis gene (c-sis) and its receptors, and secretion of PDGF-like factors have been demonstrated in many types of human malignant cells. Nevertheless, a causative role for c-sis in inducing or maintaining the transformed phenotype in human malignancies remains to be established. There are significant differences in structure between v-sis and c-sis. Studies of transforming ability have yielded conflicting results in transfection models, depending on the transfected vector and target cell type utilized. While there is compelling evidence for the involvement of PDGF in an autocrine growth mechanism in transformed fibroblasts, the evidence in human epithelial tumor types is less convincing because PDGF receptors are usually not detectable on the cell surface. The recent demonstration of intracellular co-localization of active PDGF precursors and PDGF receptors, however, supports the existence of an internal autocrine pathway independent of PDGF secretion. Further investigation of such a mechanism in de novo human malignancies is warranted to establish the role of PDGF in the development of these neoplasms.


Pyrroloquinoline quinone (coenzyme PQQ) and the oxidation of SH residues in proteins. Pyrroloquinoline quinone (PQQ) catalyzes the oxidation of cysteamine at neutral pH with a second order rate constant K2 = 0.45 M-1 s-1. The reduction of PQQ was monitored by absorption and fluorescence spectroscopy, whereas the oxidation of cysteamine to cystamine was followed by titration with 5,5'-dithiobis(2-nitrobenzoic acid). PQQ also catalyzes the oxidation of thiol groups critically connected with the function of two proteins, i.e. thioredoxin and phosphoribulose kinase. The reaction of PQQ with reduced thioredoxin brings about the oxidation of two thiol groups of the oxireductase, whereas the enzyme phosphoribulose kinase is inactivated at 25 degrees C. The oxidized disulfide bond of phosphoribulose kinase is reduced by dithiothreitol and the enzyme recovers catalytic activity. The ability of PQQ to catalyze the oxidation of vicinal cysteinyl residues to generate disulfide bonds under mild experimental conditions can be exploited to define the precise role of modified thiol residues in either catalysis or stabilization of protein structure.


Molecular cloning of a new variant of human papillomavirus type 6 isolated from a Chinese woman and expression of its L1 gene in E. coli--molecular cloning & expression of HPV6 gene. A human papillomavirus genome DNA of 7.9 kb from a Chinese woman with genital condyloma acuminata was cloned in BamHI site of pAT153. According to the results obtained from Southern blotting, restriction mapping as well as partial DNA sequencing, the isolated genome (HPV6BV) had obvious variance and was referred to as a new variant of HPV6 found in China the first time. HPV6BV L1 gene was successfully expressed in E. coli as a fusion protein with pUR288. The beta-galactosidase/L1 fusion protein reacted with both beta-galactosidase antiserum and HPV antibody using Western blot technique. The E. coli-produced fusion protein, possessing HPV antigenicity, may provide a reagent for clinical diagnosis and epidemiological survey.


Evaluation of a putative vitamin D response element in the avian calcium binding protein gene. A primary response of the avian intestine to 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] is increased synthesis of a 28-kD calcium-binding protein, calbindin-D28k (CaBP). This study examined whether 1,25-(OH)2D3 regulates CaBP gene transcription by an interaction of the vitamin D receptor (VDR) with a vitamin D-responsive element (VDRE) in the CaBP promoter. A genomic clone of CaBP containing about 1 kb of 5'-flanking DNA and 13 kb of the structural gene was isolated. 5'-Flanking DNA from -320 to -306 had considerable sequence similarity to VDREs identified in other genes. Indeed, a portion of the CaBP gene containing this region (-743 to +47) linked to a growth hormone reporter construct elicited a 1,25-(OH)2D3-dependent, VDR-dependent increase in reporter expression in transiently transfected chicken embryo fibroblasts. However, deletion analysis demonstrated that the sequences responsible for this induction reside 3' to -133 and the putative VDRE at -320 to -306 was not involved in the response. Furthermore, transfection of heterologous promoter constructs consisting of a Ban I fragment (-354 to -252) linked to the Herpes simplex thymidine kinase promoter revealed no effect of this region on reporter expression. Gel mobility shift analysis confirmed that this putative VDRE in the CaBP promoter was not a high-affinity binding site for VDR. Consequently, functional significance with respect to the primary induction of CaBP by 1,25-(OH)2D3 cannot be ascribed to this region of the CaBP promoter.


Molecular and phylogenetic analysis of calmodulin-dependent protein phosphatase (calcineurin) catalytic subunit genes. In the mammalian brain, there are multiple catalytic subunits for the Ca(2+)- and calmodulin-dependent protein phosphatase [also called protein phosphatase 2B (PP-2B) and calcineurin] that are derived from two structural genes. The coding sequences of these two genes are distinguished by the absence (PP2B alpha 1) or the presence (PP2B alpha 2) of an amino terminus containing polyproline. Both of these genes can produce intragenic isoforms through alternative splicing. In the present study, a potential phylogenetic relationship of these genes was inferred from analysis of genomic DNA and from studies of mRNA and protein expression. Southern blot analysis showed unique restriction fragments for both genes in seven mammalian species; however, in organisms from two nonmammalian vertebrates (chicken and lizard), hybridization was observed only for PP2B alpha 1. In agreement with these results, Northern blots of mammalian brain RNA showed transcripts for both genes, with about two to three times more of the PP2B alpha 1 mRNAs, whereas in chicken and lizard, only PP2B alpha 1 transcripts were detected. An analysis of protein expression by two-dimensional electrophoresis was also consistent with these findings. For the purified mammalian brain protein, eight to ten variants were observed with isoelectric points of 5.2-5.8; immunoblot analysis using anti-peptide antibodies indicated that the majority of these were PP2B alpha 1 forms. In chicken brain, multiple isoforms were recognized by antibodies against the PP2B alpha 1 forms, but no reactivity was seen with those against the PP2B alpha 2 forms. Taken together, these findings suggest that: (i) in mammals, the predominant catalytic subunit isoforms in brain are PP2B alpha 1 products and (ii) the gene for the polyproline-containing catalytic subunit of calmodulin-dependent phosphatase (PP2B alpha 2) may have evolved after the avian/reptilian branching point, perhaps to carry out a role(s) of particular significance in mammals.


Ab initio phase determination for viruses with high symmetry: a feasibility study. Conditions that would permit the complete structure determination of spherical viruses that have high internal symmetry were examined starting only from an initial spherical shell model. Problems were considered that might arise due to the following. 1. Creation of centric phases due to the simple shell model and its position in the unit cell. The centric symmetry can generally be broken on averaging an initial electron density map based on observed structure amplitudes, provided that the internal molecular symmetry is sufficiently non-parallel to the crystallographic symmetry. 2. Choice of the average model shell radius. Some incorrect radii led to the Babinet opposite solution (electron density is negative instead of positive). Phases derived from other models with incorrect radii failed to converge to the correct solution. 3. Error in structure amplitude measurements. 4. Lack of a complete data set. 5. Error in positioning the initial spherical-shell model within the crystal unit cell. It was found that an error of 1.6 A caused noticeable phasing error at a resolution greater than 20 A.


Acetaldehyde decreases the antitryptic activity of alpha 1-proteinase inhibitor. The trypsin-inhibiting activity of human serum is lowered upon addition of formaldehyde or acetaldehyde thereto. Acetaldehyde reacts with alpha-1-proteinase inhibitor (alpha 1-PI) to decrease its trypsin-inhibiting ability. Acetaldehyde has only a slight effect on the tryptic hydrolysis of benzoyl-DL-arginine-p-nitroanilide. It did not decrease the inhibitory activity of the Kunitz inhibitor (Aprotinin) or soybean trypsin inhibitor. Since aldehydes form covalent products with primary amines, primary amides, arginine, tyrosine, and tryptophan in protein, as well as methylene bridges thereby crosslinking functional groups, it is proposed that one or more such interactions affect alpha 1-PI activity. It is further suggested that chronically high levels of acetaldehyde, as a metabolic produce of ethanol, may be a contributory factor to the generation of pancreatitis in alcoholics by possibly lowering the effective alpha 1-PI level which is a natural protective element from proteolysis by trypsin.


Cerebellar GABAA receptors in two rat lines selected for high and low sensitivity to moderate alcohol doses: pharmacological and genetic studies. Alcohol-sensitive (ANT) rat line produced by selective outbreeding for high acute sensitivity to the motor-impairing effects of ethanol, displays unusual cerebellar GABAA receptor pharmacology. The ANT rats have enhanced benzodiazepine agonist affinity at their binding sites for an imidazobenzodiazepine, [3H]Ro 15-4513, normally not affected by agonists at all, and reduced GABAA agonist, [3H]muscimol, binding, when compared to the alcohol-insensitive (AT) rat line. In the present study, the benzodiazepine receptor difference was localized to the cerebellar granule cell layer. This receptor difference was not found in ex vivo binding studies after lorazepam administration, although brain lorazepam concentrations in both rat lines similarly exceeded 1 microM. An indication for differential binding in vivo between the lines was, however, observed, as pretreatment with lorazepam accentuated the relative accumulation of radioactivity only in the cerebellum of the AT rat line after an intravenous injection of a trace amount of [3H]Ro 15-4513, thus revealing benzodiazepine insensitivity for a portion of the cerebellar [3H]Ro 15-4513 binding in the AT but not in the ANT rats. In the second generation of AT/ANT cross-breeding, there was no clear association of alcohol sensitivity and cerebellar receptor binding. There was, however, a significant positive correlation between the [3H]muscimol binding and the diazepam-insensitive [3H]Ro 15-4513 binding in the cerebellum. In conclusion, the receptor defect in the cerebellar granular cell layer of the alcohol-sensitive ANT rats was also detectable in vivo, but it may not explain the enhanced alcohol sensitivity of these rats.


Alzheimer's disease and alcoholism: possible interactions. The purpose of this investigation was to test the hypothesis that chronic exposure to alcohol may accelerate Alzheimer's disease (AD), either by independently adding receptor losses or by accelerating the AD disease process itself. Muscarinic [3H]quinuclidinyl benzilate and benzodiazepine [3H]-flunitrazepam receptor binding in homogenates of human autopsy brains were determined in four nonalcoholic and seven alcoholic AD brains and in histologically normal brains from 20 alcoholics and 20 nonalcoholics. Muscarinic binding was decreased in alcoholic AD compared with nonalcoholic AD in the parahippocampal region of frontal cortex, premotor temporal cortex, and amygdala, but not in the hippocampus. Benzodiazepine receptors were lost from the temporal cortex and amygdala, but the difference in the amygdala was not statistically significant. Plaque counts considered a marker of the severity of AD were not increased in the brains of alcoholics compared with nonalcoholics. Larger receptor losses in some alcoholic AD were associated with low plaque counts. Since all of these patients were severely demented, it is tentatively suggested that the receptor losses resulting from alcoholism may have contributed to the dementia in these AD patients.


Functional properties of GABAA receptors in two rat lines selected for high and low alcohol sensitivity. The effects of lorazepam and sodium barbital on GABAA receptor function were evaluated in rat lines selected for differential sensitivity to the motor-impairing effects of ethanol [alcohol-insensitive (AT) and alcohol-sensitive (ANT) lines]. The effect of GABA on [3H]flunitrazepam and [3H]Ro 15-4513 binding and the effects of lorazepam and sodium barbital on [3H]muscimol binding were measured in cerebellar, cerebrocortical, and hippocampal membrane preparations. The effects of lorazepam and sodium barbital on muscimol-stimulated 36Cl- influx were measured using membrane vesicle suspensions from the same brain areas. No differences were found between the rat lines in the GABA-induced stimulation of [3H]flunitrazepam binding or in the lorazepam and sodium barbital-induced enhancement of either [3]muscimol binding or muscimol-stimulated 36Cl- flux. Neither was desensitization of the 36Cl- flux affected differently by ethanol, lorazepam, and barbital in vitro between the lines. The affinity of cerebellar diazepam-insensitive (DZ-IS) [3H]Ro 15-4513-binding sites for benzodiazepine agonists has been shown to be much greater in the ANT than the AT rats. In the present study, at 0 degrees C, GABA decreased [3H]Ro 15-4513 binding in the presence of diazepam only in ANT rats. Similarly, GABA decreased this binding at 37 degrees C in ANT rats having a high affinity for diazepam, whereas it enhanced the binding in all AT samples in those ANT samples where diazepam had a poor AT-like affinity. The decrease in binding in ANT samples is apparently caused by the enhancing effect of GABA on diazepam binding to DZ-IS [3H]Ro 15-4513-binding sites.(ABSTRACT TRUNCATED AT 250 WORDS)


Localization of mRNAs coding for CMD1, myogenin and the alpha-subunit of the acetylcholine receptor during skeletal muscle development in the chicken. Myogenin and CMD1, the chicken homologue of MyoD, transactivate the promoter of the alpha-subunit of the acetylcholine receptor (AChR) in chicken fibroblasts. The expression of these three genes was followed by in situ hybridization. In two-day-old embryos the CMD1 gene is expressed shortly before the AChR alpha-subunit and the myogenin genes. At day 19 extrajunctional AChR mRNA clusters have disappeared and myogenin mRNAs are no longer detected in PLD muscle. Moreover, both myogenin and CMD1 mRNA levels increase after muscle denervation in chicks. These data are compatible with a role for myogenic factors in the induction and maintenance of extra-junctional expression of the AChR genes during early muscle development. Using digoxygenin labelled RNA probes, we also show that the mRNAs for the AChR alpha-subunit display a punctated, probably perinuclear distribution, whereas mRNAs for myogenic genes accumulate in the sarcoplasm around subsets of nuclei in the muscle fiber.


Diurnal variation in peak expiratory flow rate among workers exposed to toluene diisocyanate in the polyurethane foam manufacturing industry. The diurnal variation in peak expiratory flow rate (PEFR) was studied in 26 mixers from eight factories making polyurethane foam, who were exposed to toluene diisocyanate (TDI), and 26 unexposed controls matched for age, race, and smoking. They were all men. The mean diurnal variation in PEFR of the mixers was 6.2%, which was significantly higher than the 4.3% for controls. Six mixers had a diurnal variation of greater than 15% on at least one day compared with none among the controls. There was, however, no overt cause of occupational asthma. All but one of the 24 environmental samples taken exceeded the short term exposure limit of 0.02 ppm for TDI. This accounted for the high prevalence (50%) of irritative symptoms such as cough and eye irritation. Forced expiratory volume in one second (FEV1) and FEV1/forced vital capacity (FVC) (%) was negatively correlated with duration of exposure to TDI. Foam workers may still have high exposure to TDI, high prevalence of irritative symptoms, increased diurnal variation in PEFR and evidence of chronic airway obstruction, particularly in those with greater than or equal to 10 years of exposure.


[Primary tumors of the parotid. An anatomicoclinical review]. It was studied 54 cases of tumors of parotid treated in the Hospital Universitario of Granada from 1978 to 1989. It was also studied the patient age and sex, the year and month of disease arise, the tumors stages at diagnosis, the different treatments followed and the histological classification. Finally the results are discussed.


[Calcium phosphates in otological surgery. II. The bony integration of calcium phosphates. An experimental study]. Calcium phosphates have been proposed for obliteration of the mastoid cavity as well as TORP and PORP. Their use is based on a very good biocompatibility and host acceptance. The osseous integration of a dense and a porous hydroxyapatite is studied experimentally in artificially created epitympanic holes of rabbits. Osteoneogenesis was observed from the third week after surgery starting from the walls od the defects themselves. This had been evidenced using intravital fluorescent dyes. Porous granules showed more "osteoconductivity" in the beginning, probably due to their greater surface area. Bonding osteogenesis with both materials did not show differences after six months. No resorption was observed, excepting remodelation processes.


Estimation by an electrophysiological method of the expression of oxytocin receptor mRNA in human myometrium during pregnancy. In order to evaluate the changes in uterine oxytocin receptor-specific mRNA during pregnancy, receptor expression in Xenopus oocytes are examined electrophysiologically following microinjection of mRNA from human uterus. In voltage-clamped oocytes injected with term myometrial mRNA, oxytocin elicited an inward current response. The amplitude of the oxytocin-induced current increased with increasing dose of oxytocin, but no current was elicited following stimulation with vasopressin. The oxytocin-induced current was completely eliminated as a result of pretreatment with a specific oxytocin antagonist. 21 of 27 oocytes injected with term myometrial mRNA showed a large amplitude (77.0 +/- 16.1 nA) reaction to oxytocin. In comparison, only 3 of 13 oocytes injected with early gestational myometrial mRNA exhibited a small amplitude (4.6 +/- 1.4 nA) reaction to oxytocin. No oxytocin response was observed in oocytes injected with non-pregnant myometrial mRNA. These results indicate that the striking increment in oxytocin sensitivity in term uterus depends on the increase in mRNA encoding oxytocin receptors.


[The presence of the Epstein-Barr virus in Waldeyer's lymphatic ring during the acute phase of infectious mononucleosis]. The aim of our research was to detect the existence of virus Epstein-Barr genome in the palatine- and the nasopharyngeal tonsil obtained from six patients during the acute phase of the infectious mononucleosis. The presence of EBV-DNA was demonstrated with the help of nucleic acid hybridization techniques (Southern-Blot and in situ hybridization) in the two components of the lymphoepithelial ring of the throat. The pathogenesis and the therapeutic implications of these findings are discussed.


The plasma membrane of epididymal epithelial cells has a specific receptor which binds to androgen-binding protein and sex steroid-binding protein. The binding of [3H] delta 6-testosterone photoaffinity-labelled rat androgen-binding protein (rABP) has been studied in an enriched fraction of plasma membranes of epithelial epididymal cells in immature (15 days) and adult rats (40 days). The binding was maximal in less than 30 min and more rapid at 4 degrees C than at 34 degrees C. It was calcium and pH dependent. Scatchard plots of the binding data gave curvilinear plots with two types of binding sites corresponding to a K(ass1) of 18.2 nM-1 and K(ass2) of 1.6 nM-1 (2.2 x 10(11) sites/mg protein and 5.4 x 10(11) sites/mg protein, respectively). In adult rats, only one type of binding site was found, with a K(ass) of 3.7 nM-1 (4.5 x 10(11) sites/mg protein). The number of receptors was 5-fold lower in the cauda than in the caput of the epididymis. The pretreatment of the isolated intact cells with streptozotocin induced a 45% reduction of the binding. Only unlabelled rABP and hSBP (human sex steroid-binding protein) but not other proteins (lactotransferrin, serotransferrin, asialofetuine, fetuine and bovine serum albumin) competed with the labelled ligand to bind plasma membranes. The membrane fraction was solubilized by triton X-100. Its incubation with labelled rABP and hSBP provoked the elution of the tracer as an aggregate into the void volume fraction of superose 6B mini-gel filtration columns. Structural homology between hSBP and rABP could be responsible for the common behaviour of the steroid-carrier molecules for the ABP receptor of rat epididymal epithelial cells.


Differential effects of adrenocorticotropic hormone on steroid hydroxylase activities in the inner and outer zones of the guinea pig adrenal cortex. We have studied the effects of ACTH treatment on steroid hydroxylase activities in the inner (zona reticularis) and outer (zona fasciculata plus zona glomerulosa) zones of the guinea pig adrenal cortex. Animals received 5 or 10 U of ACTH daily for 6 days and enzyme activities were then assessed in isolated microsomal or mitochondrial preparations. In control animals, microsomal cytochrome P-450 concentrations were greater in the inner than outer zone, but mitochondrial P-450 levels were similar in the two zones. Microsomal 17 alpha-hydroxylase and mitochondrial 11 beta-hydroxylase activities were greater in the outer than inner zone, but microsomal 21-hydroxylase activity was greater in the inner zone. ACTH treatment decreased cytochrome P-450 concentrations in inner but not outer zone microsomes; mitochondrial P-450 levels were unaffected in both zones. ACTH caused a dose-dependent increase in inner zone 17 alpha-hydroxylase activity and decrease in 21-hydroxylase activity without affecting the activity of either enzyme in outer zone microsomes. ACTH also decreased 11 beta-hydroxylase activity in outer but not inner zone mitochondrial preparations. The net effect of ACTH treatment was to diminish the differences in steroid metabolism between the two zones. The results indicate that the effects of ACTH on steroid hydroxylase activities are both zone- and enzyme-dependent, suggesting the existence of multiple and independent regulatory mechanisms.


Alternating chemotherapy for small-cell lung cancer. A twelve-week schedule of six drugs. We have treated sixty-two patients (21 with limited disease, 41 with extensive disease), on an outpatient-basis schedule of six drugs administered weekly for twelve weeks. Cyclophosphamide, 400 mg/m2, adriamycin, 20 mg/m2 and vincristine, 2 mg, full dose, were administered during weeks 1, 5 and 9; cisplatin, 50 mg/m2 and etoposide, 100 mg/m2 during weeks 2, 6 and 10; adriamycin and vincristine at the same doses during weeks 3, 7 and 11; methotrexate 30 mg/m2, during weeks 4, 8 and 12. After the first 28 patients vincristine was replaced by teniposide (VM-26) due to neurotoxicity. The overall response rate was 64.5% (complete remission 13 p., partial remission 27 p.). Toxicity grade 3-4, mainly nausea and vomiting or neutropenia, was recorded in 17 patients. Alopecia grade 1-2 was universal. One toxic death occurred from sepsis. The overall survival was 8 months (range 1-40), (95% CL: 53-77%); 8 months in limited disease (range 1-40), and 7 months in extensive disease (range 1-23). Time to treatment failure was 6 months (7 limited disease, 5 extensive disease). In conclusion, the results of this alternating schedule are poorer than those attained with standard, high-dose treatments, mainly in limited disease, but could be a less toxic option for patients with extensive disease.


Therapy-related acute myeloid leukemia secondary to inhibitors of topoisomerase II: from the bedside to the target genes. In the past five years, several groups have reported acute myeloid leukemia (AML) often monoblastic, as a complication of chemotherapy regimens including the epipodophyllotoxins, etoposide and teniposide. This syndrome is distinct clinically, pathologically and cytogenetically from classical therapy-related myelodysplasia and AML. There is also evidence that other topoisomerase II inhibitors, such as the intercalating agents (including doxorubicin, mitoxantrone, and actinomycin D) may be leukemogenic. Furthermore, there may be further interactions from concomitant topoisomerase II inhibitors and alkylating agents. Topoisomerase II inhibitors induce DNA cleavage and other chromosomal aberrations, including sister chromatid exchanges. These clastogenic abnormalities are not fully understood, and may be specific for each cytotoxic agent. Work is in progress to clone breakpoints such as the t(9;11) and t(8;21) and the use of the resultant DNA probes will enhance our understanding of the leukemogenic process. Given the potential diversity in patients with secondary leukemia, cytogenetic studies should be mandatory for both enhancing our knowledge base and guiding treatment in individual patients. Clinicians must also be wary of the leukemogenic potential of 'dose-intense' regimens including agents such as etoposide and doxorubicin.


A randomized prospective study of cisplatin and vinblastine versus cisplatin, vinblastine and mitomycin in advanced non-small cell lung cancer. From June 1986 to February 1989, 103 patients with advanced non-small cell lung cancer, with no previous chemotherapy, were randomized to receive either a combination of cisplatin and vinblastine (group A) or the same combination with the addition of mitomycin (group B). In group A, 15/48 evaluable patients had objective responses, as did 8/45 in group B. The median survivals were 35 and 32 weeks, respectively. The median survival of patients with response or stable disease was 43 weeks. Response and survival did not differ significantly between treatment groups. The addition of mitomycin to the two-drug combination showed no major therapeutic benefit, while bone marrow toxicity was increased. Three patients in group B died of sepsis. Among the different patient characteristics, disease stage, performance status and response had influence on survival.


Site-directed conversion of a cysteine to aspartate leads to the assembly of a [3Fe-4S] cluster in PsaC of photosystem I. The photoreduction of FA is independent of FB. The terminal electron acceptors FA and FB exist as two [4Fe-4S] clusters located on the 8.9-kDa PsaC protein in photosystem I. We have used site-directed mutagenesis to produce a complementary pair of mutant PsaC proteins in which specific cysteine ligands to the [4Fe-4S] clusters were changed to aspartic acid residues. The mutant proteins, denoted C14D and C51D, were overproduced in Escherichia coli; the iron-sulfur clusters were inserted in vitro; and the reconstituted proteins were rebound to the P700-FX core of Synechococcus sp. PCC 6301 in the presence of the PsaD protein. In complexes reconstituted with C51D a rhombic ESR spectrum with g-values of 2.063, 1.934, and 1.879 in the reduced state identifies the intact [4Fe-4S] cluster as FB, while an intense axial spectrum with g-values of 2.020 and 1.997 in the oxidized state identifies the altered cluster in the aspartate site as a [3Fe-4S] cluster. The [3Fe-4S] cluster corresponding to FA can be reduced chemically with dithionite and photochemically by illumination at room temperature but is not reduced by illumination at 15 K. With reconstituted C14D a rhombic ESR spectrum with g-values of 2.043, 1.942, and 1.853 in the reduced state identified the unaltered [4Fe-4S] cluster as FA, while a complex spectrum with a gz-value of 2.194 and an asymmetric gx,y set of resonances between 2.092 and 1.999 indicates an altered cluster of unknown identity in the site containing the aspartate ligand. The ESR signals arising from the altered cluster corresponding to FB are not diminished by illumination at either room temperature or 15 K.(ABSTRACT TRUNCATED AT 250 WORDS)


Kinetics and mechanism of the folding of cytochrome c. The reversible folding of cytochrome c in urea at pH 4.0 was investigated by repetitive pressure perturbation kinetics and by equilibrium spectroscopic methods. Two folding reactions were observed in the 1 ms to 10 s time range. The rates and amplitudes of these reactions depend on urea concentration in a complex manner, which is different for each process. The absorbance spectra of the kinetic amplitudes of the two reactions also differ from each other. A model with a three-state mechanism can quantitatively account for all of the kinetic and equilibrium data, and it enables us to determine the rate constants and volume changes of the two steps. If a rapid protonation step is added to the mechanism, the analysis can be extended to calculate the pH dependence of the rate and amplitude of the faster folding step. This pH dependence is in excellent agreement with previously published data [Tsong, T. Y. (1977) J. Biol. Chem. 252, 8778-8780]. Kinetic experiments in the 695-nm band show clearly that the axial ligand methionine-80 is involved in the slow folding process and the other axial ligand, histidine-18, is involved in the fast process. Additional experiments with a cyanogen bromide fragment of the protein, and fluorescence detection of the folding kinetics of the intact protein, support an interpretation of the model in terms of known structural elements of cytochrome c. This work provides new information about the mechanism of the folding of cytochrome c, resolves conflicts in earlier interpretations, and demonstrates the applicability of the repetitive pressure perturbation kinetics method to protein folding.


Phospholipids chiral at phosphorus. Stereochemical mechanism for the formation of inositol 1-phosphate catalyzed by phosphatidylinositol-specific phospholipase C. The phosphatidylinositol-specific phospholipase C (PI-PLC) from mammalian sources catalyzes the simultaneous formation of both inositol 1,2-cyclic phosphate (IcP) and inositol 1-phosphate (IP). It has not been established whether the two products are formed in sequential or parallel reactions, even though the latter has been favored in previous reports. This problem was investigated by using a stereochemical approach. Diastereomers of 1,2-dipalmitoyl-sn-glycero-3-(1D- [16O,17O]phosphoinositol) ([16O,17O]DPPI) and 1,2-dipalmitoyl-sn-glycero-3-(1D-thiophosphoinositol) (DPPsI) were synthesized, the latter with known configuration. Desulfurization of the DPPsI isomers of known configurations in H2(18)O gave [16O,18O]DPPI with known configurations, which allowed assignment of the configurations of [16O,17O]DPPI on the basis of 31P NMR analyses of silylated [16O,18O]DPPI and [16O,17O]DPPI (the inositol moiety was fully protected in this operation). (Rp)- and (Sp)-[16O,17O]DPPI were then converted into trans- and cis-[16O,17O]IcP, respectively, by PI-PLC from Bacillus cereus, which had been shown to proceed with inversion of configuration at phosphorus [Lin, G., Bennett, F. C., & Tsai, M.-D. (1990) Biochemistry 29, 2747-2757]. 31P NMR analysis was again used to differentiate the silylated products of the two isomers of IcP, which then permitted assignments of IcP with unknown configuration derived from transesterification of (Rp)- and (Sp)-[16O,17O]DPPI by bovine brain PI-PLC-beta 1. The results indicated inversion of configuration, in agreement with the steric course of the same reaction catalyzed by PI-PLCs from B. cereus and guinea pig uterus reported previously. For the steric course of the formation of inositol 1-phosphate catalyzed by PI-PLC, (Rp)- and (Sp)-[16O,17O]DPPI were hydrolyzed in H2(18)O to afford 1-[16O,17O,18O]IP, which was then converted to IcP chemically and analyzed by 31P NMR. The results indicated that both B. cereus PI-PLC and the PI-PLC-beta 1 from bovine brain catalyze conversion of DPPI to IP with overall retention of configuration at phosphorus. These results suggest that both bacterial and mammalian PI-PLCs catalyze the formation of IcP and IP by a sequential mechanism. However, the conversion of IcP to IP was detectable by 31P NMR only for the bacterial enzyme. Thus an alternative mechanism in which IcP and IP are formed by totally independent pathways, with formation of IP involving a covalent enzyme-phosphoinositol intermediate, cannot be ruled out for the mammalian enzyme. It was also found that both PI-PLCs displayed lack of stereo-specifically toward the 1,2-diacylglycerol moiety, which suggests that the hydrophobic part of phosphatidylinositol is not recognized by PI-PLC.


Subcellular localization of squalene synthase in human hepatoma cell line Hep G2. Using the Hep G2 cell line as a model for the human hepatocyte the question was studied whether Hep G2-peroxisomes could be able to synthesize cholesterol. Hep G2 cell homogenates were applied to density gradient centrifugation on Nycodenz, resulting in good separation between the organelles. The different organelle fractions were characterized by assaying the following marker enzymes: catalase for peroxisomes, glutamate dehydrogenase for mitochondria and esterase for endoplasmic reticulum. Squalene synthase activity was not detectable in the peroxisomal fraction. Incubation of Hep G2 cells with U18666A, an inhibitor of the cholesterol synthesis at the site of oxidosqualene cyclase, together with heavy high density lipoprotein, which stimulates the efflux of cholesterol, led to a marked increase in the activity of squalene synthase as well as HMG-CoA reductase, whereas no significant effect on the marker enzymes was observed. Neither enzyme activity was detectable in the peroxisomal density gradient fraction, suggesting that in Hep G2-peroxisomes cholesterol synthesis from the water-soluble early intermediates of the pathway cannot take place. Both stimulated and non-stimulated cells gave rise to preparations where squalene synthase activity was comigrating with the reductase activity at the lower density side of the microsomal fraction; however, it was also present at the high density side of the microsomal peak, where reductase activity was not detected.


Epizootiological aspects of type 1 and type 4 equine herpesvirus infections among horse populations. The dissemination of equine herpesvirus types 1 (EHV-1) and 4 (EHV-4) among various horse populations in Japan was investigated through the isolation and typing of virus strains from horses with respiratory diseases. Type specific monoclonal antibody pools were used for the typing of isolates. The 42 strains of EHV-1 and 64 strains of EHV-4 were isolated from 4593 nasal swabs and/or blood plasma samples collected from 3326 horses during a period from 1979 to 1990. All the strains of EHV-1 were isolated from racehorses only and during the winter season exclusively, when the epizootic of respiratory diseases occurred among racehorse populations at two Training Centers of the Japan Racing Association. In contrast, the strains of EHV-4 were isolated from horses irrespective of the season, facility, or horse population; foals and yearlings in breeding farms and our institute, rearing horses in rearing farms, and racehorses. Especially, 4 strains of EHV-4 were isolated from plasma samples containing buffy coat cells. We believe this is the first reported case of EHV-4 cell-associated viremia in the world. All 87 strains isolated from aborted fetuses were identified as EHV-1. The results suggest that EHV-1 is responsible for epizootic respiratory diseases in racehorses in the winter and abortion among mares at the late stage of gestation, and that EHV-4 causes respiratory diseases throughout the year among all horse populations.


Antibody responses of swine to type A influenza viruses in the most recent several years. A serological survey was conducted on 4,080 swine sera collected for the years 1985-90. The swine sera positive to A/New Jersey/8/76 (swine type H1N1) strain were observed in annual (10-20%) and monthly (20-40%) incidences during the observation period except for occasional months. Antibodies to recent human H1N1 viruses in swine were recognized in relation to the human H1N1 influenza epidemics. Antibody responses of swine to human H3N2 strains appeared irrespective of human epidemics with the virus in the years 1985-87. However, in 1988 almost no antibodies to three human H3N2 isolates of 1983-88 were observed for this year except a few months though the human epidemic occurred in the area. Although in 1989-90 many swine had antibodies to the three strains in the percentage of 3 to 35, no antibody to the latest isolate, A/Hokkaido/20/89 (H3N2), was found for almost all the months of both years. These findings differed markedly from the possible relationship between the prevalence of H3N2 virus-antibodies in swine and the human influenza epidemics, which were described previously in many reports including our studies.


Isolation and serial propagation of porcine epidemic diarrhea virus in cell cultures and partial characterization of the isolate. Porcine epidemic diarrhea virus (PEDV) was isolated in Vero cell cultures from the small intestine of a piglet experimentally infected with porcine coronavirus 83P-5, that had been isolated during outbreaks of porcine acute diarrhea and passaged in piglets. The isolation of the PEDV was successful only in Vero cells maintained in the maintenance medium (MM) containing trypsin. Infected Vero cell cultures exhibited CPE characterized by cell-fusion and syncytial formation, as well as cytoplasmic fluorescence when examined by the indirect immunofluorescent test using rabbit anti-83P-5 virus serum. The isolate was adapted to serial propagation in Vero cell cultures by adding trypsin to MM. Vero cell-adapted PEDV was successfully propagated in the MA104, CPK and ESK cell lines in the presence of trypsin in MM. Vero cell-adapted PEDV had morphologic and physicochemical characteristics similar to those of other members of the coronaviridae. The isolate differed serologically from porcine transmissible gastroenteritis (TGE) and porcine hemagglutinating encephalomyelitis viruses, and no antigenic relationship between the isolate and TGE virus could be detected by the indirect immunofluorescent test. Attempts to isolate PEDV in 6 types of primary fetal pig cell cultures and 6 of 10 established cell lines resulted in the failure, probably because these cells were damaged by the action of trypsin.


The effect of experimental infection of mouse preimplantation embryos with paramyxovirus Sendai. Zona-intact and zona-free mouse embryos at the morula stage were exposed to Sendai virus in vitro, and then transferred to the uteri of recipient foster mothers. Both embryos developed into expanded blastocyst stage after 48 hr of culture. Zona-intact embryos were resistant to infection and subsequent transfer resulted in the development of fetuses, indicating that the zona pellucida plays a role of a barrier to virus infection. On the other hand, zona-free embryos were susceptible to infection and only one fetus out of 64 transfers developed to term. Implantation sites were scarcely observed in the uteri of the foster mothers that received zona-free embryos, suggesting that most of the embryos did not develop after embryo transfer. Sendai virus was shed in the culture fluid of the zona-free embryos indicating viral replication in the embryonic cells. By immunofluorescence assay, viral antigens were detected in the embryos, tissues of the fetus and implantation site derived from the zona-free embryos. These findings indicate that replication of Sendai virus in the embryonic cells interfere with early embryonic development and fetal growth of the embryo.


Role of protein conformation changes and transphosphorylations in the function of Na+/K(+)-transporting adenosine triphosphatase: an attempt at an integration into the Na+/K+ pump mechanism. The particular aim of the review on some basic facets of the mechanism of Na+/K(+)-transporting ATPase (Na/K-ATPase) has been to integrate the experimental findings concerning the Na(+)- and K(+)-elicited protein conformation changes and transphosphorylations into the perspective of an allosterically regulated, phosphoryl energy transferring enzyme. This has led the authors to the following summarizing evaluations. 1. The currently dominating hypothesis on a link between protein conformation changes ('E1 in equilibrium with E2') and Na+/K+ transport (the 'Albers-Post scheme') has been constructed from a variety of partial reactions and elementary steps, which, however, do not all unequivocally support the hypothesis. 2. The Na(+)- and K(+)-elicited protein conformation changes are inducible by a variety of other ligands and modulatory factors and therefore cannot be accepted as evidence for their direct participation in effecting cation translocation. 3. There is no evidence that the 'E1 in equilibrium with E2' protein conformation changes are moving Na+ and K+ across the plasma membrane. 4. The allosterically caused ER in equilibrium with ET ('E1 in equilibrium with E2') conformer transitions and the associated cation 'occlusion' in equilibrium with 'de-occlusion' processes regulate the actual catalytic power of an enzyme ensemble. 5. A host of experimental variables determines the proportion of functionally competent ER enzyme conformers and incompetent ET conformers so that any enzyme population, even at the start of a reaction, consists of an unknown mixture of these conformers. These circumstances account for the occurrence of contradictory observations and apparent failures in their comparability. 6. The modelling of the mechanism of the Na/K-ATPase and Na+/K+ pump from the results of reductionistically designed experiments requires the careful consideration of the physiological boundary conditions. 7. Na+ and K+ ligandation of Na/K-ATPase controls the geometry and chemical reactivity of the catalytic centre in the cycle of E1 in equilibrium with E2 state conversions. This is possibly effected by hinge-bending, concerted motions of three adjacent, intracellularly exposed peptide sequences, which shape open and closed forms of the catalytic centre in lock-and-key responses. 8. The Na(+)-dependent enzyme phosphorylation with ATP and the K(+)-dependent hydrolysis of the phosphoenzyme formed are integral steps in the transport mechanism of Na/K-ATPase, but the translocations of Na+ and K+ do not occur via a phosphate-cation symport mechanism.(ABSTRACT TRUNCATED AT 400 WORDS)


The new macrolide antibiotics: azithromycin, clarithromycin, dirithromycin, and roxithromycin. To review the chemistry, antimicrobial spectrum, pharmacokinetics, clinical trials, adverse effects, and drug interactions of four new macrolide antibiotics: azithromycin, clarithromycin, dirithromycin, and roxithromycin. Information was obtained from comparative clinical trials, abstracts, conference proceedings, and review articles. Indexing terms included azithromycin, clarithromycin, dirithromycin, erythromycin, roxithromycin, and macrolide antibiotics. Emphasis was placed on comparative clinical trials involving the new macrolide antibiotics. Data from human studies published in the English language were evaluated. Trials were assessed by sample size, macrolide dosage regimen, and therapeutic response. The erythromycins have gained widespread use in treating a variety of infections. Although they are effective, limitations include the need to administer four times a day and the intolerable adverse gastrointestinal effects. Four of the more extensively studied agents, azithromycin, clarithromycin, dirithromycin, and roxithromycin, are currently being studied in patients. Based on the studies to date, the newer macrolides may offer several advantages over erythromycin, including: (1) greater antimicrobial activity against certain organisms; (2) longer elimination half-life, thus allowing less frequent administration; and (3) lower incidence of adverse gastrointestinal effects. The new macrolide antibiotics appear to offer an improvement over erythromycin. Definitive conclusions about the role of these drugs should await completion of ongoing clinical studies.


Purification and characterization of human lymphoblast N-acetylglucosamine-1-phosphotransferase. N-Acetylglucosamine-1-phosphotransferase (GlcNAcPTase) was solubilized with 2% Tergitol NP-10 from cultured human lymphoblast cells and purified 3840-fold with 14% recovery using lentil lectin-Sepharose 4B, DEAE-Sephacel and Sephacryl S-400 chromatographies. The partially purified enzyme requires the non-ionic detergent Tergitol NP-10 and a divalent cation, Mn2+ or Mg2+, for its activity and exhibits an optimal pH at 7.2-7.5 in Tris-maleate buffer. Kinetic studies demonstrated an apparent Km of 24 microM for the donor UDP-N-acetylglucosamine and of 117 mM for the artificial acceptor alpha-methylmannoside. The GlcNAcPTase is inhibited by UDP and UDP-glucose, and by negatively charged phospholipids including phosphatidylserine, phosphatidylglycerol and phosphatidic acid. The apparent mol. wt of the human lymphoblast GlcNAcPTase is approximately 1000 kDa, which is analogous to that reported for the partially purified enzyme from rat liver (Waheed et al., 1982).


Homonuclear three-dimensional NMR methods for the complete assignment of proton NMR spectra of oligosaccharides--application to Gal beta 1-4(Fuc alpha 1-3)GlcNAc beta 1-3Gal beta 1-4Glc. Two new homonuclear three-dimensional NMR techniques are described for the simplification of proton resonance assignment in oligosaccharides, namely HOHAHA-COSY and ROESY-COSY. The former technique is of value in the resonance assignment of gluco-configuration monosaccharide residues, whereas the latter is more suited to resonance assignment of galacto-configuration monosaccharide residues. The value of these techniques is illustrated by application to the proton resonance assignment of the pentasaccharide Gal beta 1-4(Fuc alpha 1-3)GlcNAc beta 1-3 Gal beta 1-4Glc, a compound which exhibits a variety of assignment problems due to severe cross-peak overlap in conventional COSY or HOHAHA spectra.


Clinical trial with inactivated hepatitis A vaccine and recommendations for its use. To compare the reactogenicity and immunogenicity of an inactivated hepatitis A vaccine in two different immunisation schedules. Randomised trial. One London teaching hospital. 104 healthy adult volunteers (71 men, 33 women aged 19-60). Hepatitis A vaccine to group 1 (54 volunteers) at 0, 1, and 2 months and to group 2 (50) at 0, 1, and 6 months. Symptoms at and after each dose; liver function, hepatitis A virus specific serum immune response; and responses in saliva and parotid fluid in immunised volunteers and subjects with natural immunity. The vaccine was well tolerated; 97% (96/99) and 100% of those immunised developed serum antibody after one and two doses of vaccine respectively. Geometric mean titres increased progressively after each dose and were significantly higher in men but not women in group 2 after the third dose (ratio between geometric mean titres 0.265, 95% confidence interval 0.18 to 0.39; p less than 0.001). At one year this group-sex interaction was absent; geometric mean titres for both sexes were significantly higher in group 2 (ratio 0.330, 0.227 to 0.478; p less than 0.0001). Antibody responses were not significantly different between the groups at two years. Compared with naturally infected subjects immunised volunteers developed poor or undetectable virus specific IgG and IgA responses in saliva and parotid fluid. The vaccine was safe and highly immunogenic, and the differences in the immune responses in saliva and parotid fluid are unlikely to affect its efficacy.


Presence, distribution and spread of productive varicella zoster virus infection in nervous tissues. Nervous tissue lesions were retrospectively studied for detection of productive varicella zoster virus (VZV) infection in 33 autopsied cases, including 19 herpes zoster (HZ) (10 trigeminal, nine spinal) and 14 cases of nodular brainstem encephalitis without HZ. Immunocytochemistry for VZV antigens and in situ hybridization with a biotinylated VZV DNA probe were used on formol-fixed paraffin sections. Peripheral and central nervous system, skin and striated muscle were investigated in serial sections; available tissue blocks, however, varied between cases. Varicella zoster virus production (both antigen and DNA) in nervous tissue was found in HZ cases but only of short survival after a rash of up to 7 wks (eight out of 12 patients). Varicella zoster virus was visualized in nerve cells, glial cells, Schwann cells and blood vessels. In the central nervous system (CNS), VZV was detected in trigeminal nuclei (one out of 10 brains) or disseminated nodular brainstem lesions (one out of 10 brains), in subependymal microvessels (one out of 10 brains) or vasculitic arteries (two out of 19 brains or spinal cords). In the peripheral nervous system (PNS), VZV (DNA and antigen) was found in neurons and satellite cells of sensory ganglia (four out of seven cases with sampling of ganglia), and in damaged nerve fibres including a muscle nerve in one case; myositis with VZV in affected muscle fibres was found in the latter case. In nodular brainstem encephalitis, one case contained VZV within nodular lesions. We conclude that (i) VZV neural spread is suggested by detectable virus in ganglia, nerve fibres and CNS target nuclei; (ii) haematogenous spread of VZV is suggested by detection of virus in CNS microvessels and in disseminated brainstem encephalitis; (iii) VZV myositis may occur in zosteric myotomes; and (iv) VZV is a possible agent in nodular brainstem encephalitis.


Neuron-specific enolase reflects metabolic activity in mesencephalic neurons of the rat. Numerous studies on the local rate of energy metabolism of various brain regions during development and following experimental manipulation have been conducted using 2-deoxyglucose uptake and cytochrome oxidase (CO) histochemistry, both considered to be reliable indicators of long-term and short-term alterations in neuronal activity, respectively. Another method which has been related to neuronal activity is neuron-specific enolase (NSE) immunohistochemistry. An isoenzyme of enolase, a key element in the glycolytic pathway, NSE is present in neurons and neural-related cells e.g. neuroendocrine cells, pituicytes, and many tumor cells, but not in glia. The distribution on adjacent tissue sections of immunoreactive NSE and histochemically determined CO were mapped in the rat mesencephalon and adrenal medulla. Both methods showed highly restricted localization of staining which coincided with few exceptions in the most reactive areas, namely the superior colliculus, medial and lateral geniculate nuclei, red nucleus, lateral mammillary nucleus, interpeduncular nucleus and substantia nigra pars lateralis and pars reticulata. Immunoreactivity of varying intensity for NSE was also observed in perikarya and in processes of numerous scattered neurons throughout the mesencephalon, including the substantia nigra pars compacta, and reticular formation. The general correspondence in staining patterns between CO and NSE in the midbrain, supports the utility of NSE as a useful index of metabolic activity in neurons.


Rat brain hypothalamic and hippocampal monoamine and hippocampal beta-adrenergic receptor changes during pregnancy. The concentration of noradrenaline (NA), dopamine (DA), serotonin (5-HT), and their metabolites was measured in hypothalamic and hippocampal brain tissue obtained from non-pregnant, 15- or 20-day pregnant and 4-day postpartum rats. At 20 days of pregnancy, hypothalamic NA and DA concentrations were significantly decreased and their turnover increased relative to postpartum and estrous values, respectively. Hippocampal 3-methoxy-4-hydroxy-phenylglycol (MHPG) levels were significantly decreased at 15 days of pregnancy and 4 days postpartum compared to estrous and 20-day pregnant levels and the MHPG/NA ratio was significantly reduced at 4 days postpartum relative to the estrous value. Hippocampal 5-HT and 5-hydroxyindole-3-acetic acid (5-HIAA) levels were significantly decreased at 15 days of pregnancy while 5-HIAA levels and the 5-HIAA/5-HT ratio were significantly decreased at 20 days of pregnancy. Hippocampal beta-adrenergic receptor density was significantly lower at 4 days postpartum than at 15 days of pregnancy. A positive correlation was observed between plasma progesterone and hippocampal beta-adrenoceptor Kd values, suggesting a possible causal relationship between these two variables. The monoamine and beta-adrenoceptor changes which occur during pregnancy may be an important contributing factor in determining the mood changes which occur during pregnancy and postpartum.


Cyclic GMP modulators and excitotoxic injury in cerebral cortical cultures. N-Methyl-D-aspartate (NMDA) receptor activation generates nitric oxide (NO) and cyclic GMP (cGMP) and produces 'excitotoxic' neuronal injury. To examine the possible role of cGMP in excitotoxicity, we evaluated the effects of agents that stimulate or inhibit cGMP activity on the release of lactate dehydrogenase from neuron-enriched cortical cultures. cGMP analogs exhibited no toxicity, and inhibitors of guanylate cyclase or of cGMP-dependent enzymes failed to protect cultures from the toxic effects of NMDA or the NO donor sodium nitroprusside. These findings argue against a role for cGMP in the pathogenesis of excitotoxic neuronal injury.


Clinical cases in AIDS. 2.

Clinical cases in AIDS. 2. As the human immunodeficiency virus (HIV) epidemic continues, there are increasing numbers of patients with HIV-related disease. Those doctors studying for the MRCP exam will need to be familiar with the common manifestations of HIV infection and acquired immunodeficiency syndrome.


[Demonstration of delta sleep inducing peptide in a strain of human small cell lung cancer by immunocytology]. The "delta sleep inducing peptide" (DSIP) is a regulatory peptide localized in the brain, the hypophysis and some endocrine cells of the gut. The present immunological study, performed with a monoclonal antibody to DSIP, provides evidence for the presence of DSIP-like immunoreactivity (DSIP-LI) in a strain of small cell carcinoma. The specificity of the immunoreaction was assessed by the tests using heterologous antigen known to be secreted by these cells. The DSIP could play a role in the course of this disease.


High prevalence of human papillomavirus transcripts in all grades of cervical intraepithelial glandular neoplasia. Cervical biopsy specimens containing cervical intraepithelial glandular neoplasia (CIGN) were examined for the presence of human papillomavirus (HPV) RNA transcripts by in situ hybridization with iodine 125-labeled riboprobes. HPV RNA was detectable in 95.2% of biopsy specimens. HPV 16 RNA was present in 12, HPV 18 in 27, and both in 1 of the 42 cases examined. Among HPV-positive cases, HPV RNA was detectable in all grades of CIGN and, in three cases, in glands displaying only minimal nuclear abnormality insufficient for a diagnosis of CIGN. Patients with HPV RNA-positive CIGN were younger than those with negative findings for HPV, and patients with less severe grades of CIGN showed a trend toward a younger age of presentation than patients with severe glandular lesions. Increasing grades of CIGN may reflect progressive stages in the development of cervical adenocarcinoma, and this progression may closely involve HPV gene expression from its earliest stages.


Malignant cell-specific gelatinase activity in human endometrial carcinoma. The protease activity leading to degradation of the extracellular matrix was compared between human endometrial cancer and normal uterine endometrium. Conditioned medium from tumor cells and normal endometrial cells was subjected to electrophoresis on sodium dodecyl sulfate (SDS)-polyacrylamide gel containing gelatin as a substrate. After electrophoresis, the gel was stained with Coomassie blue, and then the enzyme activity, expressed as the zone of dye clearing, was analyzed by densitometry. Densitometric analysis showed that all the endometrial cancers expressed a very high molecular weight enzyme activity (Mr 220,000), which was not detected in medium from normal endometrial cells. The analysis also showed that in endometrial cancer the activity of a Mr 92,000 enzyme was always superior to that of a Mr 64,000 enzyme, which was in contrast to the situation for normal endometrium. These results indicate that the expression of Mr 220,000 enzyme activity and the higher activity of the Mr 92,000 enzyme than the Mr 64,000 enzyme are involved in the malignant phenotype of native endometrial cancer.


Epstein-Barr virus integration in human lymphomas and lymphoid cell lines. Epstein-Barr virus (EBV) is maintained as an episome in most infected cells. The presence of fused terminal restriction enzyme fragments distinguishes the circular DNA form from the linear virion form. EBV genomic structure was analyzed in 8 lymphoid cell lines and 21 human lymphoma specimens by the Southern blot technique. Evidence of viral integration into host chromosomal DNA was identified in four cell lines. In the Namalwa and BL30-B95.8 cell lines, integration occurred through the terminal repeat (TR) sequences. In the BL41-P3HR1 and BL41-B95.8 cell lines, there was loss of left-end viral genomic sequences, including ori-P sequences required for episome maintenance, implying that integration was required for viral genome persistence. Integration was not detected in four other cell lines (Raji, Daudi, B95.8, and BL30-P3HR1). In 21 EBV-containing human lymphomas, including 18 immunodeficiency-related lymphomas, fused TR sequences were identified without evidence of viral genomic integration. These findings suggest that, although viral integration is common in Burkitt lymphoma cell lines infected in vitro, integration is not common in human lymphomas that develop in vivo in normal or immunodeficient people.


Does hepatitis C virus infection contribute to hepatocellular carcinoma in Hong Kong? Hong Kong has a high incidence of hepatocellular carcinoma (HCC) and, because it is an endemic area for hepatitis B virus (HBV) infection, the etiologic association between HCC and HBV infection is reported to be as high as 80%. Hepatitis C virus (HCV) recently was shown to be a possible pathogenetic agent for HCC in a number of countries. To assess the relative importance of these two viruses in HCC in Hong Kong, a retrospective study of 424 Chinese patients with HCC was performed. Three hundred forty-one (80.3%) patients were found to be carriers of hepatitis B surface antigen (HBsAg). Hepatitis C antibodies (anti-HCV) were detected in 31 patients (7.3%). Fifteen patients with positive findings for anti-HCV had concurrent HBV infection, 11 had serologic evidence of previous HBV infection, and only 5 patients had anti-HCV marker alone. Patients with positive findings for anti-HCV were older than those with HBsAg (mean ages, 60 and 53 years, respectively). A higher preponderance of male patients was found in the HBsAg-positive group; the male to female ratio was 11:1, compared with 7:1 among patients with anti-HCV. Anti-HCV was detected in 0.64% of 175 age-matched and sex-matched controls. These data indicate a possible causal role of HCV infection in HCC, but it is of relatively minor epidemiologic significance in Hong Kong, where HBV infection is overwhelming.


Tumor encapsulation in hepatocellular carcinoma. A pathologic study of 189 cases. One hundred eighty-nine surgically resected hepatocellular carcinomas (HCC) were analyzed to study tumor encapsulation and the pathologic features that might account for the better prognosis in relation to it, and to examine the prognostic and pathobiologic significance of capsular thickness. Tumor encapsulation was found in 72 (46.8%) of the 154 cases with adequate histologic sections of the tumor-nontumor junctions. Encapsulated tumors showed a much lower incidence of direct liver invasion (P less than 0.0001), tumor microsatellites (P less than 0.0001), and venous permeation (P = 0.02) when compared with nonencapsulated ones. Significantly better disease-free and actuarial survival times were observed in patients with encapsulated tumors (medians, 9.9 and 18.3 months, respectively), compared with those with nonencapsulated ones (medians, 4.0 and 5.9 months, respectively; P = 0.0001 and 0.001, respectively). The incidence of tumor encapsulation did not increase or decrease with tumor size. Tumor encapsulation did not correlate with the presence of cirrhosis or the abundance of tumor stroma, suggesting that formation of the tumor capsule was independent of the degree of fibrosis within and outside the tumor. Among the 72 cases of encapsulated HCC, the capsular thickness ranged from 0.13 to 3.09 mm (mean +/- standard deviation = 0.87 +/- 0.59 mm), and it was unrelated to tumor size or presence of cirrhosis. Although it was apparent that a lower extensive tumor invasiveness contributed significantly to the better prognosis in encapsulated HCC, there was no correlation between capsular thickness and liver invasion, microsatellites, venous permeation, or survivals. Therefore, the thickness of tumor capsules was not helpful in prognostication.


The use of IVIG in neurological disease. The studies cited herein highlight the potential benefits of IVIG therapy in a group of neurological disorders that are associated with aberrant immune responses. Indeed, all of the disorders discussed, except epilepsy, are associated with autoreactivity. The trials are preliminary and short-term and, except for idiopathic CIDP, uncontrolled. Interpretation of the findings of these uncontrolled studies is complicated by the fact that the natural history of all of these disorders is to show fluctuations. IVIG appears to be a potentially useful and safe agent in the treatment of patients with MG, intractable epilepsy, MS, and CIDP. Its place in the therapeutic approach to these neurological diseases must await the completion of controlled trials. Since other therapeutic modalities have already proven to be useful in several of these disorders, it will be important to determine if IVIG is more efficacious, safer, and more cost-effective. It is also worth considering whether the combination of IVIG and any of these more traditional approaches would provide added therapeutic benefit.


Putative apolipoprotein receptor gene (LRP, A2MR) is not rearranged in either myxoid liposarcoma or lipomas with translocations in 12q13-14. The APR, also known as LRP, gene is highly homologous to the low-density lipoprotein (LDL)-receptor and encodes a cell surface molecule with biochemical properties consistent with function as a lipoprotein receptor. This gene has been mapped to human chromosomal bands 12q13-q14, a region commonly altered in tumors of adipose cells. The proximity of APR to these breakpoints, coupled with its presumed role in lipid metabolism and possible affect on cell proliferation, suggest it as a candidate gene for adipose tissue tumor formation. Pulsed-field gel analysis was used to develop a physical map covering 750 kilobases (kb) surrounding this gene. Examination of myxoid liposarcomas and lipomas bearing the characteristic translocations (12;16)(q13;p11) or (12;variable)(q14;variable), respectively, excluded the breakpoints from within a 750-kb region surrounding the APR gene. These results suggest that APR is not involved directly in the genetic changes that underlie development or progression of these tumors.


Near-haploid clones in a malignant fibrous histiocytoma. Near-haploid solid tumors are very rare. In a storiform-pleomorphic malignant fibrous histiocytoma (MFH) of bone, we found three cell populations: one with a near-haploid, a second with a near-diploid, and a third with a near-tetraploid chromosome number. The near-haploid cells had few structural rearrangements: i(12p) and t(13q21q) in one clone, and these two and an additional t(19;?)(p11;?) in another clone. One structurally normal copy of all chromosomes was also present, except that the only chromosome 13 was involved in the t(13q21q). There were also two near-diploid clones, one without the t(19;?) and one with a single copy of this derivative chromosome. This is the first tumor with i(12p) among bone and soft tissue tumors.


Trisomy 12 in Epstein-Barr virus-transformed lymphoblastoid cell lines of normal individuals and patients with nonhematologic malignancies. Karyotypes of 36 lymphoblastoid cell lines established by Epstein-Barr virus (EBV) transformation of peripheral blood lymphocytes (PBL) of eight normal individuals and 28 patients with various nonhematologic malignancies were analyzed. In seven lines (19.4%), cells with trisomy 12 were noted, with clonality in two of these lines. In two of 11 metaphases with such trisomy, chromosome 12 was involved in structural rearrangements [t(8;12)(q12;p12) and t(12;12)(q11;q24)]. No cells with trisomy 12 were observed in phytohemagglutinin (PHA)-stimulated PBL cultures of these individuals. In 250 individuals (normal and with nonhematologic malignancies) examined in our laboratory in the last 5 years, extra copies of chromosome 12 in PHA-stimulated PBL cultures were observed in only five of 23,216 cells (0.02%). There were no cases of clonality in these samples. The frequency of an extra chromosome 12 was comparable to that of the other chromosomes except 21 and X, whose frequency of occurrence was 0.08% and 0.09%, respectively. These findings should be considered random events in PHA-stimulated PBL. On the contrary, in lymphoblastoid cell lines established by EBV transformation, trisomy of chromosome 12 was the most frequent numerical abnormality. It was observed in 64.7% of all cases with chromosome gains and therefore could not be considered a random occurrence. The specificity of this phenomenon for EBV transformation is supported by the results of cytogenetic analysis of eight lymphoblastoid cell lines established by an alternative procedure in our laboratory [1]. In 400 cells analyzed not a single cell with trisomy 12 was observed. We suggest that EBV transformation might either randomly induce formation of such cells in immortalized B-cell populations or show potentially blastomogenic cells or proneness to their formation in certain individuals who could be predisposed to develop lymphoproliferative diseases, especially chronic lymphocytic leukemia (CLL) in which trisomy of chromosome 12 is the most common alteration.


Supernumerary ring chromosomes in five bone and soft tissue tumors of low or borderline malignancy. Five tumors (two myxoid malignant fibrous histiocytoma, two dermatofibrosarcoma protuberans, and one parosteal osteosarcoma) with ring chromosomes as the sole cytogenetic anomaly or as the only structural rearrangement were observed in a series of 60 karyotypically abnormal, nonlipogenic bone and soft tissue tumors (BST). All five tumors were of borderline or low malignancy. These findings support the suggestion that supernumerary ring chromosomes as the sole structural chromosomal aberration are not associated with any particular histopathologic diagnosis but may characterize a group of BST of borderline or low malignancy.


Enhanced levels of radiation-induced G2 phase delay in ataxia telangiectasia heterozygotes. Ataxia telangiectasia (AT) is a multiform genetic disease characterized by immunodeficiency, cerebellar abnormalities, and cancer predisposition. Heterozygotes also have an increased risk of developing several different cancers. It has been estimated that as many as 18% of all patients with breast cancer, the cancer most clearly associated with AT heterozygotes, may be carriers of the AT gene. We describe an assay for AT heterozygotes that relies on the previous observation that cells from AT homozygotes show a greater and more prolonged radiation-induced accumulation in the G2 phase of the cell cycle than do normal controls. We showed that all 6 A-T heterozygotes show a greater extent of G2 phase delay at different times postirradiation than do controls. The degree of accumulation was less than that observed in AT homozygotes. Only two of 22 controls showed overlap with heterozygotes at 18 hours postirradiation, and that number was reduced to one at the 24-hour point. As a group, AT heterozygotes were intermediate between controls and AT homozygotes at both time points after irradiation. This assay is relatively simple and reliable and can be performed in any laboratory with access to both Epstein-Barr virus (EBV) for transformation of lymphocytes and a fluorescence-activated cell analyzer.


Infectious defective interfering particles of VSV from transcripts of a cDNA clone. The generation of infectious defective interfering (DI) particles of vesicular stomatitis virus (VSV) entirely from cDNA clones is reported. Bacteriophage T7 RNA polymerase was used to direct the transcription of a complete negative-stranded genomic RNA from a cDNA clone of a VSV DI RNA in cells simultaneously expressing the five VSV proteins from separately transfected cDNA clones. The negative-stranded transcript was encapsidated with N protein, replicated by the VSV polymerase, and the replicated RNAs were assembled and budded to yield infectious DI virions. No helper VSV was required. Replication occurred at high levels and was assayed by direct biochemical means. An exact 3' terminus of the initial transcript, which was generated by autolytic cleavage using a ribozyme from hepatitis delta virus, was critical for replication.


SSL2, a suppressor of a stem-loop mutation in the HIS4 leader encodes the yeast homolog of human ERCC-3. Reversion of haploid, His4- yeast containing a stem-loop mutation in the 5' UTR that blocks HIS4 translation initiation identified four unlinked suppressor genes, SSL1-SSL4, which restore His4+ expression. The SSL2 gene encodes an essential 95 kd protein with ATP-dependent helicase motifs. SSL2 protein is 54% identical to the protein encoded by the human gene, ERCC-3, for which a defective form causes xeroderma pigmentosum and Cockayne's syndrome. An SSL2 allele made to resemble the defective ERCC-3 gene confers UV light hypersensitivity to yeast cells. Hence, SSL2 is the functional homolog of ERCC-3. However, the SSL2 suppressor gene does not restore HIS4 expression by removal of the stem-loop from DNA or the mRNA. We propose that SSL2 and ERCC-3 may have two functions, one defined by a UV repair defect, and a second essential function that is related to gene expression.


An MHC interaction site maps to the amino-terminal half of the T cell receptor alpha chain variable domain. We have used cloned T cell receptor (TCR) genes from closely related CD4 T cell lines to probe the interaction of the TCR with several specific major histocompatibility complex (MHC) class II ligands. Complementarity determining region 3 (CDR3) equivalents of both alpha and beta TCR chains are required for antigen-MHC recognition. Our data provide novel information about the rotational orientation of TCR-MHC contacts in that exchange of the amino terminal portion of the TCR alpha chain containing the putative CDR1 and CDR2 regions results in both gain and loss of MHC class II specificity by the resulting receptor. These two TCRs differ primarily in recognition of polymorphisms in the second hypervariable region of the MHC class II alpha chain. These results document the involvement of CDR1 and/or CDR2 of the TCR alpha chain in MHC recognition and suggest a rotational orientation of this TCR to its MHC ligand.


Optimization of electroporation for transfection of human fibroblast cell lines with origin-defective SV40 DNA: development of human transformed fibroblast cell lines with mucopolysaccharidoses (I-VII). To simplify the process of transfection of human fibroblasts and to acquire a suitable number of transformants, we investigated experimental conditions of electric pulse-induced transfection of human fibroblasts using origin-defective simian virus 40 DNA (SV40 (ori-) DNA). Voltage, pulse duration, number of pulses and the concentration of SV40 (ori-) DNA led to the formation of 10 to 30 foci/25 cm2 6 weeks after transfection, using 2 to 3 x 10(6) cells and a square wave pulse generator. Optimal condition was determined to be 2 or 3 pulses at a voltage of 1500 to 2000 V/0.4 cm with 30 microseconds pulse width, using 2 micrograms of linearized SV40 (ori-) DNA. With this approach we developed human transformed fibroblasts cell lines with all types of mucopolysaccharidoses. The transformed fibroblasts grew rapidly and the saturation density exceeded that of the parental cells. All the transformed cell clones expressed T antigen, and deficiency in specific enzymes was conserved. A point mutation which occurred in the human beta-glucuronidase gene in a patient with mucopolysaccharidosis type VII was also conserved.


Monitoring of oxidative free radical damage in vivo: analytical aspects. Free radical damage is an important factor in many pathological and toxicological processes. During the last decade a wide range of methods has been developed to determine free radical damage in various biological fluids and at various stages of development. This review offers an overview of the state of the art of monitoring free radical damage in vivo, with special emphasis on the analytical aspects of non-invasive methods.


Studies on cardiac ingredients of plants. IX. Chemical transformation of proscillaridin by utilizing its 1,4-cycloadducts as key compounds and biological activities of their derivatives. Three aromatic compounds (2-4) possessing a carbomethoxyl group or a dimethoxyphthaloyl group, prepared by the Diels-Alder reaction of the cardiac glycoside, proscillaridin (1), with dimethyl acetylenedicarboxylate and methyl propiolate, were transformed into alcohols, carboxylic acids and amides. The biological activities of the resulting derivatives were evaluated by the use of Na+, K(+)-adenosine triphosphatase (Na+,K(+)-ATPase) from dog kidney and isolated guinea-pig papillary muscle. Although the biological activities of the resulting derivatives were less potent than that of 1, a para-substituted benzylalcohol (5), methylbenzamides (9a and 10a), and ethylbenzamides (9b and 10b) inhibited the activity of Na+,K(+)-ATPase almost as potently as naturally occurring cardiac glycosides such as digoxin and digitoxin.


Synthesis and glutamate-agonistic activity of (S)-2-amino-3-(2,5-dihydro-5-oxo-3-isoxazolyl)-propanoic acid derivatives. (S)-2-Amino-3-(2,5-dihydro-5-oxo-3-isoxazolyl)propanoic acid, (3a) and its analogs (3b--h) were prepared and evaluated for glutamate receptor-agonistic and antifungal activities. Several (S)- and (R)-2-amino-3-isoxazolylpropanoic acid derivatives (3a--d) were synthesized starting with (S)- and (R)-N-tert-butoxycarbonyaspartic acid alpha-methyl esters (4, n = 1) by means of Masamune's chain extension reaction followed by isoxazolone formation with hydroxylamine and subsequent deprotection reactions. Furthermore, (S)- and (R)-N-tert-butoxycarbonylglutamic acid alpha-methyl esters (4, n = 2) were converted to (S)- and (R)-2-amino-4-isoxazolylbutyric acid derivatives (3e-h) via the same sequence of reactions.


Studies on inhibitors of skin tumor promotion. XI. Inhibitory effects of flavonoids from Scutellaria baicalensis on Epstein-Barr virus activation and their anti-tumor-promoting activities. To search for possible anti-tumor-promoters, fourteen flavones obtained from the root of Scutellaria baicalensis were examined for their inhibitory effects on the Epstein-Barr virus early antigen (EBV-EA) activation by a short-term in vitro assay. Among these flavones, 5,7,2'-trihydroxy- and 5,7,2',3'-tetrahydroxyflavone showed remarkable inhibitory effects on the EBV-EA activation, and the effect of the latter on Raji cell cycle was also examined by flow cytometer. These two flavones exhibited remarkable inhibitory effects on mouse skin tumor promotion in an in vivo two-stage carcinogenesis test.


Potentiation of endothelium-dependent relaxations to bradykinin by angiotensin I converting enzyme inhibitors in canine coronary artery involves both endothelium-derived relaxing and hyperpolarizing factors. Studies were designed to investigate the mechanisms underlying the augmentation by angiotensin I converting enzyme (ACE) inhibitors of the endothelium-dependent relaxations evoked by bradykinin. Isometric tension, tissue levels of cGMP, and transmembrane potential were measured in isolated canine coronary arteries as indications of the respective contribution of nitric oxide and endothelium-derived hyperpolarizing factor. In rings of coronary artery with endothelium, relaxations to bradykinin were potentiated by the ACE inhibitors cilazaprilat and perindoprilat. NG-Nitro-L-arginine (NLA), a nitric oxide synthase inhibitor, impaired relaxations to bradykinin. But the presence of ACE inhibitors partially restored this activity. Bradykinin stimulated the production of cGMP, and this was enhanced significantly by ACE inhibitors, indicating an augmented release of nitric oxide. NLA abolished the increase induced by bradykinin irrespective of the presence of ACE inhibitors. Electrophysiological studies revealed that bradykinin elicited an endothelium-dependent hyperpolarization of vascular smooth muscle that was insensitive to NLA and potentiated by ACE inhibitors. The bradykinin-induced hyperpolarization and NLA-resistant relaxations were transient and impaired by potassium depolarization. Thus, production of endothelium-derived hyperpolarizing factor may account for the NLA-resistant relaxations of canine coronary arteries. The relaxations induced by bradykinin were unaffected by the B1 kinin receptor antagonist des-Arg9,[Leu8]-bradykinin either in the absence or in the presence of NLA but were antagonized by the B2 kinin receptor antagonist D-Arg[Hyp3,D-Phe7]-bradykinin. Molecular exclusion chromatography of 125I-labeled [Tyr8]-bradykinin and its degradation products demonstrated that the breakdown of the kinin by isolated coronary arteries was prevented in the presence of perindoprilat.(ABSTRACT TRUNCATED AT 250 WORDS)


Interaction between microvascular alpha 1- and alpha 2-adrenoceptors and endothelium-derived relaxing factor. Intravital microscopy was used to study the effect of endothelium-derived relaxing factor (EDRF) on microvascular adrenoceptor sensitivity in rat cremaster skeletal muscle. NG-Monomethyl L-arginine (L-NMMA, 1-300 microM), an inhibitor of EDRF formation, produced concentration-dependent constriction of arterioles and venules. When an intermediate amount of alpha 1- versus alpha 2-adrenoceptor tone was first produced with bath-added norepinephrine (NE) in the presence of rauwolscine or prazosin, L-NMMA caused constriction with greater potency and efficacy during alpha 2 than during alpha 1 tone. During localized alpha 1 or alpha 2 constriction along an arteriole by perivascular micropipette suffusion of NE in the presence of rauwolscine or prazosin, again, bath-added L-NMMA produced constriction with greater potency during alpha 2 than during alpha 1 constriction. Like L-NMMA, disruption of EDRF release by microembolization caused baseline arteriole constriction and selectively increased alpha 2 sensitivity 75-fold. Although these findings support the hypothesis that endothelial cells possess alpha 2-adrenoceptors that promote EDRF release, a greater susceptibility of alpha 2 than alpha 1 constriction to EDRF inhibition could also account for the results. In support of this latter possibility, alpha 2 constriction was approximately 50-fold more susceptible than alpha 1 constriction to inhibition by the EDRF-like nitrodilator nitroprusside. The similarity in magnitude of this difference in sensitivity with the difference obtained in the embolization experiments does not support the hypothesis that microvascular endothelial cells in skeletal muscle possess EDRF-promoting alpha 2-adrenoceptors. However, these data do suggest that endogenous EDRF release modulates basal arteriole and venule tone and that alpha 2-adrenoceptor constriction is more sensitive than alpha 1 constriction to inhibition by EDRF.


Differences in cardiac calcium release channel (ryanodine receptor) expression in myocardium from patients with end-stage heart failure caused by ischemic versus dilated cardiomyopathy. The molecular basis for the systolic and diastolic dysfunction characteristic of end-stage heart failure in humans remains poorly understood. It has been proposed that both abnormal calcium handling and defects in the contractile apparatus may contribute to the myocardial dysfunction. Two channels, the calcium release channel (CRC) or ryanodine receptor of the sarcoplasmic reticulum (SR), and the slow calcium channel or dihydropyridine receptor (DHPR) of the transverse tubule, play key roles in regulating intracellular calcium concentration and in excitation-contraction (E-C) coupling in the heart. The DHPR serves as the voltage sensor and plasma membrane calcium channel resulting in activation of the CRC during E-C coupling in heart muscle. In this study, we investigated the levels of CRC expression in several forms of end-stage heart failure in humans. A cardiac CRC cDNA was cloned from rabbit and used as a probe for Northern blot analyses to determine mRNA levels in the left ventricles of normal (n = 4) and cardiomyopathic (n = 34) human hearts from patients undergoing cardiac transplantation. Compared with normal patients, patients with ischemic cardiomyopathy (n = 18) showed a 28% decrease in CRC mRNA levels (p less than 0.025) and patients with idiopathic dilated cardiomyopathy (n = 14) a nonsignificant 12% increase. In these same hearts, alpha-actin levels were unchanged in end-stage heart failure, as has been previously reported. This is the first report indicating that the expression of the CRC mRNA is abnormal in end-stage human heart failure.(ABSTRACT TRUNCATED AT 250 WORDS)


Phenotype-related alteration in expression of natriuretic peptide receptors in aortic smooth muscle cells. To elucidate the physiological and pathophysiological roles of the natriuretic peptide family in vascular smooth muscle cells, in which the natriuretic peptide family is implicated in growth inhibition as well as vasorelaxation, we have examined the phenotype-related expression of three kinds of natriuretic peptide receptors in rat aortic smooth muscle cells. The expression of natriuretic peptide receptors at the mRNA level was studied by Northern blot hybridization, and the expression at the protein level was determined by the cGMP production method and receptor binding assay. In intact aortic media, atrial natriuretic peptide (ANP)-A receptor mRNA and ANP-B receptor mRNA were detected, and the potency of cGMP production by ANP was at least two orders of magnitude stronger than that by C-type natriuretic peptide. Clearance receptor mRNA was undetectable, and only a small amount of the clearance receptor was detected by the binding assay in intact aortic media. By contrast, in cultured aortic smooth muscle cells at the first, fifth, and 17th passages, the ANP-B receptor mRNA level markedly increased; meanwhile, the expression of the ANP-A receptor mRNA became undetectable. C-type natriuretic peptide was one order of magnitude more potent than ANP in cGMP production in cultured aortic smooth muscle cells. The clearance receptor density and its mRNA level increased tremendously in these cultured cells. These results demonstrate that the marked phenotype-related alteration occurs in the expression of natriuretic peptide receptors in rat aortic smooth muscle cells.


Mechanisms of vasodilation induced by NKH477, a water-soluble forskolin derivative, in smooth muscle of the porcine coronary artery. To study the mechanism of vasodilation induced by 6-(3-dimethylaminopropionyl) forskolin (NKH477), a water-soluble forskolin derivative, its effects on the acetylcholine (ACh)-induced contraction of muscle strips of porcine coronary artery were examined. [Ca2+]i, isometric force, and cellular concentrations of cAMP and inositol 1,4,5-trisphosphate were measured. NKH477 (0.1-1.0 microM), isoproterenol (0.01-0.1 microM), or forskolin (0.1-1.0 microM) increased cAMP and attenuated the contraction induced by 128 mM K+ or 10 microM ACh in a concentration-dependent manner. These agents, at concentrations up to 0.3 microM, did not change the amount of cGMP. NKH477 (0.1 microM) attenuated the contraction induced by 128 mM K+ without corresponding changes in the evoked [Ca2+]i responses. ACh (10 microM) produced a large phasic increase followed by a small tonic increase in [Ca2+]i and produced a sustained contraction. The ACh-induced phasic increase in [Ca2+]i, but not the tonic increase, disappeared after application of 0.1 microM ionomycin. NKH477 (0.1 microM) attenuated both the increase in [Ca2+]i and the force induced by 10 microM ACh in muscle strips that were not treated with ionomycin and inhibited the ACh-induced contraction without corresponding changes in [Ca2+]i in ionomycin-treated muscle strips. These results suggest that NKH477 inhibits ACh-induced Ca2+ mobilization through its action on ionomycin-sensitive storage sites. In ionomycin-treated and 128 mM K(+)-treated muscle strips, 0.1 microM NKH477 shifted the [Ca2+]i-force relation to the right in the presence or absence of 10 microM ACh. In beta-escin-skinned smooth muscle strips, 0.1 microM NKH477 shifted the pCa-force relation to the right but had no effects on Ca(2+)-independent contraction. We conclude that in smooth muscle of porcine coronary artery, NKH477 inhibits ACh-induced contraction by both attenuating ACh-induced Ca2+ mobilization and reducing the sensitivity of the contractile machinery to Ca2+, possibly by activating cAMP-dependent mechanisms.


Quantification of hydroxyl radical and its lack of relevance to myocardial injury during early reperfusion after graded ischemia in rat hearts. To elucidate the pathophysiological role of the hydroxyl radical (.OH) during the postischemic reperfusion of the heart, we measured the .OH product in the coronary effluent from isolated perfused rat heart during a 30-minute reperfusion period after various ischemic intervals of 5, 10, 15, 20, 30, and 60 minutes. Salicylic acid was used as the probe for .OH, and its derivative, 2,5-dihydroxybenzoic acid (2,5-DHBA), was quantified using high-performance liquid chromatography with ultraviolet detection. 2,5-DHBA was negligible in the effluent from nonischemic hearts, but a significant amount was detected from the hearts rendered ischemic for 10 minutes or longer. The peak of 2,5-DHBA was seen within 90 seconds after the onset of reperfusion in every group. The accumulated amount of 2,5-DHBA was maximal in the group with 15-minute ischemia (6.73 +/- 1.04 nmol/g wet heart wt after 30 minutes of reperfusion); it decreased as the ischemic time was prolonged and was 2.38 +/- 0.84 nmol/g wet wt after 30 minutes of reperfusion in the group with 60-minute ischemia. In the model of 15-minute ischemia/30-minute reperfusion, there was no correlation between the accumulated amount of 2,5-DHBA and functional recovery (+/- dP/dt, heart rate, and coronary flow), lactate dehydrogenase release, and morphological damage. Although treatment with 0.5 mM deferoxamine, an iron chelator, significantly decreased 2,5-DHBA (from 6.73 +/- 1.04 to 2.29 +/- 0.80 nmol/g wet wt after 30 minutes of reperfusion, p less than 0.01), it failed to reduce the postischemic myocardial injury in the group with 15-minute ischemia. The results suggest that .OH production is influenced by the preceding ischemic interval and that .OH does not exert an immediate direct effect on postischemic damage during early reperfusion in the isolated perfused rat heart, although a possibility remains that the small portion of .OH trapped by salicylic acid may not be intimately associated with myocardial injury.


Differential anatomical and cellular patterns of connexin43 expression during postnatal development of rat brain. We have shown previously that connexin43 in the adult rat central nervous system (CNS) is predominantly localized at astrocytic gap junctions. Here we document immunohistochemically the emergence of connexin43-immunoreactive (connexin43-IR) structures and the regional patterns of connexin43 expression during postnatal maturation of the rat brain. On Western blots, connexin43 was detected in brain samples at postnatal day (P) 5, the earliest age studied. Immunohistochemically, most brain regions displayed a characteristic sequence of transient immunoreactive profiles that ultimately gave rise to the uneven distribution of the protein seen in adults. Generally, brains at P1-P5 exhibited long, fibrous connexin43-IR elements which were identified as radial glial cells. This fibrous immunostaining was considerably diminished at P5 and was replaced by short immunoreactive processes which predominated up to P10. These processes had a stellate appearance, emanated from partially stained astrocytic cell bodies and were heterogeneously distributed throughout the developing brain. By P15, there occurred only punctate immunolabelling similar to that seen in adult brain. Some brain regions including the amygdaloid complex, septohypothalamic nucleus, preoptic hypothalamus, zona incerta, ependyma and subfornical organ were exceptional in that they displayed adult immunostaining patterns at early postnatal ages suggesting a precocious maturation of gap junctions in these areas. We conclude that the highly heterogeneous distribution of connexin43-immunoreactivity among defined nuclear structures in adult brain does not reflect an antecedent requirement for connexin43 in early brain morphogenesis, but rather is related to the development of neuronal activity, the establishment of functional circuitry and the contribution of astrocytic gap junctions to glial metabolic coupling and potassium spatial buffering in the mature CNS.


Postnatal blockade of cortical activity by tetrodotoxin does not disrupt the formation of vibrissa-related patterns in the rat's somatosensory cortex. Neuronal activity has been shown to influence pattern formation in the visual system. In the present study, we determined whether or not this was also true in the somatosensory system by silencing the primary somatosensory cortex of rats with tetrodotoxin (TTX) for the first 7-11 days of life. Application of TTX during this period did not prevent the formation of the normal vibrissa-related pattern in S-I as visualized by either staining cortical sections for cytochrome oxidase, demonstration of the pattern with an antibody directed against serotonin, or labelling of thalamocortical axons with the carbocyanine dye, Di-I. These results indicate that neither peripherally evoked nor spontaneous activity are required for qualitatively normal pattern formation in the rat's primary somatosensory cortex.


Fructose 2,6-bisphosphate in developing rat brain. Fructose 2,6-bisphosphate (Fru-2,6-P2) levels and 6-phosphofructo-1-kinase and 6-phosphofructo-2-kinase activities have been studied in rat brain during development from embryonal to adult state. Fru-2,6-P2 increases slightly from day 16 of gestation, reaching a maximum 24 h after birth, remaining quite constant during postnatal development. In contrast with 6-phosphofructo-1-kinase, which increases progressively after the first week of age, 6-phosphofructo-2-kinase remains unaltered throughout the period studied. The role of Fru-2,6-P2 in controlling cerebral glycolysis is discussed.


Activation of cyclic AMP second messenger system stimulates secretion of beta-endorphin from fetal hypothalamic cells. Relatively little is known about physiological regulators of hypothalamic beta-endorphin (END) secretion and mechanisms by which they stimulate secretion. We sought to determine whether activation of the cyclic AMP (cAMP) second messenger pathway was involved in stimulating hypothalamic beta-END secretion from dissociated fetal hypothalamic cells in culture. Forskolin (FSK), a direct activator of adenylate cyclase which stimulates cAMP formation, stimulated immunoreactive (IR)-beta-END secretion. Because FSK can also stimulate independent of increased cAMP formation, we studied dibutyryl cAMP and 8-bromo-cAMP, analogues of cAMP, which also stimulated IR-beta-END secretion. From these studies we conclude: (1) activation of the cAMP second messenger system stimulates IR-beta-END secretion from hypothalamic cells and supports the rationale that endogenous regulators which stimulate this pathway could be involved in the physiological regulation of hypothalamic beta-END secretion; (2) coupling between the cAMP second messenger pathway and stimulation of hypothalamic beta-END secretion which is presumably present at maturity (adulthood) originates at early stages of development (fetal life).


Colposcopy staging and treatment of papillomavirus infection of the cervix. The specificity of colposcopy findings in HPV infections allows the colposcopic differentiation of these lesions. In all of these colposcopically selected cases, additional tests included cytology with immunoperoxidase reaction (IPR), and, recently, virus typing with the use of ViraPap HPV DNA Detection test and ViraType HPV DNA Typing test became also available. When needed, histological examination of the specimen was carried out. In 202 detected and treated cases of HPV infections in females, and in 5 in males, four groups of lesions have been colposcopically distinguished. Group I contains early and fully developed, unsuspected typical papillomas; Group II--atypical papillomas which are usually colposcopically suspected of CIN and/or cancer; Group III--the so-called subclinical forms, i.e. cases with secondary diminution of epithelial transparency, i.e. acetic acid white epithelium, secondary simple punctuation and/or mosaic (flat condylomas) that are visible only in colposcopic magnification and usually suspected of CIN; and Group IV--the so-called latent lesions without any colposcopic findings and only with positive IPR. CIN 3 (including CIS) was determined histologically in five cases of Group II, and CIN 1-2 in ten cases of Group III. HPV virus type 16/18 was found only in cases of CIN 3. The cases of atypical papilloma (Group II) and recently those containing HPV virus type 16/18 were treated by means of surgical conisation or cryosurgery. The remaining cases (Groups I, II, IV) were treated with orally administered Tetracycline and Vitamin A. The effectiveness of this antibiotic/vitamin therapy in the group of typical papilloma and subclinical HPV lesions amounted to 75%.(ABSTRACT TRUNCATED AT 250 WORDS)


Genetic recombination between malignant hyperthermia and calcium release channel in skeletal muscle. Absolute linkage between the gene, on chromosome 19, for the calcium release channel (CRC) of the sarcoplasmic reticulum in skeletal muscle and malignant hyperthermia (MH) has been reported by other workers. In the present study of three Swedish Families informative with respect to this linkage relationship, definite recombinants were found in two families. We conclude that mutations in other genes than the CRC gene can cause the clinical picture of MH. Accordingly, MH appears to be genetically heterogeneous.