Molecular localization of an ion-binding site within the pore of mammalian sodium channels. Sodium channels are the major proteins that underlie excitability in nerve, heart, and skeletal muscle. Chemical reaction rate theory was used to analyze the blockage of single wild-type and mutant sodium channels by cadmium ions. The affinity of cadmium for the native tetrodotoxin (TTX)-resistant cardiac channel was much higher than its affinity for the TTX-sensitive skeletal muscle isoform of the channel (microliters). Mutation of Tyr401 to Cys, the corresponding residue in the cardiac sequence, rendered microliters highly susceptible to cadmium blockage but resistant to TTX. The binding site was localized approximately 20% of the distance down the electrical field, thus defining the position of a critical residue within the sodium channel pore.


Block of Ca2+ wave and Ca2+ oscillation by antibody to the inositol 1,4,5-trisphosphate receptor in fertilized hamster eggs. The concentration of cytoplasmic free calcium (Ca2+) increases in various stimulated cells in a wave (Ca2+ wave) and in periodic transients (Ca2+ oscillations). These phenomena are explained by inositol 1,4,5-trisphosphate (IP3)-induced Ca2+ release (IICR) and Ca(2+)-induced Ca2+ release (CICR) from separate intracellular stores, but decisive evidence is lacking. A monoclonal antibody to the IP3 receptor inhibited both IICR and CICR upon injection of IP3 and Ca2+ into hamster eggs, respectively. The antibody completely blocked sperm-induced Ca2+ waves and Ca2+ oscillations. The results indicate that Ca2+ release in fertilized hamster eggs is mediated solely by the IP3 receptor, and Ca(2+)-sensitized IICR, but not CICR, generates Ca2+ waves and Ca2+ oscillations.


Syntaxin: a synaptic protein implicated in docking of synaptic vesicles at presynaptic active zones. Synaptic vesicles store neurotransmitters that are released during calcium-regulated exocytosis. The specificity of neurotransmitter release requires the localization of both synaptic vesicles and calcium channels to the presynaptic active zone. Two 35-kilodalton proteins (p35 or syntaxins) were identified that interact with the synaptic vesicle protein p65 (synaptotagmin). The p35 proteins are expressed only in the nervous system, are 84 percent identical, include carboxyl-terminal membrane anchors, and are concentrated on the plasma membrane at synaptic sites. An antibody to p35 immunoprecipitated solubilized N-type calcium channels. The p35 proteins may function in docking synaptic vesicles near calcium channels at presynaptic active zones.


Structure and functional expression of an omega-conotoxin-sensitive human N-type calcium channel. N-type calcium channels are omega-conotoxin (omega-CgTx)-sensitive, voltage-dependent ion channels involved in the control of neurotransmitter release from neurons. Multiple subtypes of voltage-dependent calcium channel complexes exist, and it is the alpha 1 subunit of the complex that forms the pore through which calcium enters the cell. The primary structures of human neuronal calcium channel alpha 1B subunits were deduced by the characterization of overlapping complementary DNAs. Two forms (alpha 1B-1 and alpha 1B-2) were identified in human neuroblastoma (IMR32) cells and in the central nervous system, but not in skeletal muscle or aorta tissues. The alpha 1B-1 subunit directs the recombinant expression of N-type calcium channel activity when it is transiently co-expressed with human neuronal beta 2 and alpha 2b subunits in mammalian HEK293 cells. The recombinant channel was irreversibly blocked by omega-CgTx but was insensitive to dihydropyridines. The alpha 1B-1 alpha 2b beta 2-transfected cells displayed a single class of saturable, high-affinity (dissociation constant = 55 pM) omega-CgTx binding sites. Co-expression of the beta 2 subunit was necessary for N-type channel activity, whereas the alpha 2b subunit appeared to modulate the expression of the channel. The heterogeneity of alpha 1B subunits, along with the heterogeneity of alpha 2 and beta subunits, is consistent with multiple, biophysically distinct N-type calcium channels.


Membrane depolarization induces p140trk and NGF responsiveness, but not p75LNGFR, in MAH cells. Nerve growth factor (NGF) is required for the maturation and survival of sympathetic neurons, but the mechanisms controlling expression of the NGF receptor in developing neuroblasts have not been defined. MAH cells, an immortalized sympathoadrenal progenitor cell line, did not respond to NGF and expressed neither low-affinity NGF receptor (p75) nor p140trk messenger RNAs. Depolarizing concentrations of potassium chloride, but none of a variety of growth factors, induced expression of p140trk but not p75 messenger RNA. A functional response to NGF was acquired by MAH cells under these conditions, suggesting that expression of p75 is not essential for this response. Depolarization also permitted a relatively high proportion of MAH cells to develop and survive as neurons in fibroblast growth factor and NGF. These data establish a relation between electrical activity and neurotrophic factor responsiveness in developing neurons, which may operate in the functioning of the mature nervous system as well.


The role of GABAB receptor activation in absence seizures of lethargic (lh/lh) mice. Lethargic (lh/lh) mice, which function as an animal model of absence seizures, have spontaneous seizures that have behavioral and electrographic features and anticonvulsant sensitivity similar to those of human absence seizures. Antagonists of the gamma-aminobutyric acidB (GABAB) receptor suppressed these seizures in lethargic mice, whereas agonists of GABAB receptors exacerbated them. Furthermore, GABAB receptor binding and synaptically evoked GABAB receptor-mediated inhibition of N-methyl-D-aspartate responses were selectively increased in lh/lh mice. Therefore, enhanced GABAB receptor-mediated synaptic responses may underlie absence seizures in lh/lh mice, and GABAB receptor antagonists hold promise as anticonvulsants for absence seizures.


Four year experience with cholangiocarcinoma: a survey of patients, clinical presentation, management and prognosis in two hospitals. Seventeen patients with cholangiocarcinoma diagnosed in Toa Payoh and Tan Tock Seng Hospitals from 1986-90 were studied retrospectively. There was a male preponderance (male:female = 12:5) with a mean age of 58 years (range 28-82 years). All presented with obstructive jaundice. Three had cholangitis. Biliary stones were associated in 3 (18%). Two patients (12%) had choledochal cysts. The level of obstruction was identified at the hilum in 12 (70.5%), lower third in 4 (23.5%) and at a choledochojejunostomy anastomosis in 1 (6%). Ultrasound and percutaneous cholangiography (PTC) were the commonest investigations used. Endoscopic retrograde cholangio-pancreatography (ERCP) was performed in 7 (41%) and computer tomography (CT) of abdomen in 6 (35%). Biochemically, a raised alkaline phosphatase (1.5-9 x normal) was typical. Biliary bypass surgery was performed in 7 (41%); Whipple's procedure in 2 (12%) and drainage only in 6 (35%). Nine operated upon survived an average of 6 months (range 2-11 months) and six by drainage survived an average of 62 days (range 13-155 days). Three (of which two declined treatment) were lost to follow up. Cholangiocarcinoma is an uncommon cancer occurring in the older age group. In younger patients, choledochal cyst seems to be an association. Survival is dismal with palliative treatment.


The value of somatosensory evoked potential testing for upper lumbar radiculopathy. A correlation of electrophysiologic and anatomic data. Patients with lumbar pain syndromes can present with a complex variety of complaints. Most clinicians focus on the lower lumbar nerve roots, but upper lumbar radicular syndromes can provide an especially difficult diagnostic challenge to the spine specialist requiring a multimodal approach to sort out diagnostic complexities. The purpose of this study was to analyze the correlation of somatosensory evoked potential findings with documented spinal pathology demonstrated on morphologic studies, thereby determining whether somatosensory evoked potential testing has a place in spinal diagnosis. The results of this study demonstrated the correlation of somatosensory evoked potential findings with anatomic abnormalities noted on computed tomographic and magnetic resonance imaging scans and discograms. Somatosensory evoked potential testing is recommended not as an isolated test, but as part of an electrophysiologic battery that would also include conventional electromyography.


Prevention of deep vein thrombosis after major knee surgery--a randomized, double-blind trial comparing a low molecular weight heparin fragment (enoxaparin) to placebo. Consecutive patients undergoing knee arthroplasty or tibial osteotomy at four participating hospitals received either enoxaparin, 30 mg subcutaneously every 12 h (n = 66) or an identical-appearing placebo (n = 65). All study medications started the morning after the operation and were continued up to a maximum of 14 days. Patients underwent surveillance with 125I-fibrinogen leg scanning and impedance plethysmography. Bilateral contrast venography was performed routinely at Day 14 or at time of discharge, if sooner. Deep vein thrombosis was detected by venography in 35 of 54 patients (65%) in the placebo group and in 8 of 41 patients in the enoxaparin group (19%), a risk reduction of 71%, P less than 0.0001. For the entire study group, deep vein thrombosis was detected by either venography of non-invasive tests in 37 of 64 patients (58%) in the placebo group and in 11 of 65 patients (17%) in the enoxaparin group, a risk reduction of 71%, P less than 0.0001. Proximal vein thrombosis was found in 19% of the placebo patients and in none of the enoxaparin patients, a risk reduction of 100%, P less than 0.001. Bleeding complications occurred in 5 of 65 patients (8%) in the placebo group and in 4 of 66 patients (6%) in the enoxaparin group, P = 0.71. There were no differences in the amount of blood loss, minimum hemoglobin levels and number of units of packed red cells given between the two treatment groups. We conclude that a fixed dose regimen of enoxaparin, started post-operatively, is an effective and safe regimen for reducing the frequency of deep vein thrombosis after major knee surgery.


ADP receptor antagonists and converting enzyme systems reduce platelet deposition onto collagen. The effect of the ADP receptor antagonists ATP and adenosine 5'-(beta, gamma-methylene)triphosphate (AMP-PCP), and the ADP-utilizing enzyme systems creatine phosphokinase/creatine phosphate (CPK/CP) and pyruvate kinase/phosphoenol pyruvate (PK/PEP) on platelet deposition onto type I collagen was examined. An in vitro perfusion system was used, which allowed continuous visualization of the deposition of fluorescently labelled platelets. This system also provide well-controlled rheology, precise quantification of deposition, and allowed the use of heparinized whole human blood (3 u/ml). Heparinization at this level permits the local generation of thrombin near surface platelet aggregates. The contribution of ADP is thus studied with the combined effects of thrombin, thromboxane A2, and other aggregating agents present. Results from these studies indicate that ATP was capable of inhibiting deposition by 60% at 1 microM and 90% at 5 microM (whole blood conc.). AMP-PCP inhibited deposition in a dose dependent manner with a Ki of approximately 80 microM and a maximum inhibition of 60%. Inhibition by CPK/CP was measured at 20, 40, and 60 u/ml, with approximately 45% inhibition achieved for the latter two concentrations. PK/PEP at 60 u/ml resulted in 70% inhibition. These results support a role for ADP in mediating platelet recruitment in thrombus growth on collagen. Previous work utilizing animal bleeding times supports this conclusion; the present study demonstrates that this role is not dependent upon endothelial or vasoconstrictive effects. Intraplatelet cAMP levels were raised with respect to controls upon exposure to ATP at 8.3 microM (p less than 0.025), and 15 microM (p less than 0.005), as well as AMP-PCP at 42-500 microM (p less than 0.005).(ABSTRACT TRUNCATED AT 250 WORDS)


Transforming growth factor beta (TGF-beta) inhibits expression of fibrinogen and factor VII in a hepatoma cell line. We have investigated the effect(s) of transforming growth factor (TGF)-beta 1 and interleukin (IL)-6 on the expression of fibrinogen and blood coagulation factors VII, IX, X mRNAs in a hepatoma cell line (Hep 3B). The results indicate that TGF-beta induces a decrease of the basal level of fibrinogen and factor VII mRNAs, but does not affect factor X expression. Furthermore, TGF-beta efficiently antagonizes the IL-6 induction of fibrinogen mRNA at late (12-48 h) but not early (6 h) times: this effect is apparently mediated by posttranscriptional mechanism(s). These findings, together with previously reported data on the inhibitory effect of TGF-beta on acute phase genes (e.g., ApoA1 and albumin), suggest a role for TGF-beta in the regulation of liver genes expression. The early stimulatory and late inhibitory effect exerted by IL-6 and TGF-beta respectively on fibrinogen mRNA level may play a role in the regulatory mechanism(s) of clot formation in a variety of conditions.


Non-specific effects of aquaMEPHYTON (vitamin K1) on prothrombin expression in human hepatoblastoma (HepG2) cells. In order to determine the effects of vitamin K1 on prothrombin production, we have treated cultures of human hepatoblastoma cells with an aqueous colloidal suspension of vitamin K1. Dose-response analysis demonstrated increases in secreted prothrombin antigen levels ranging from 3 to 3.7-fold over controls. Time-course analysis demonstrated increases in secreted prothrombin antigen levels over controls up to 6 hours of treatment. Between 6 and 24 hours, secreted prothrombin antigen levels increased at a rate parallel to controls. Vitamin K1 treatment also resulted in a parallel increase in total secreted protein levels. Prothrombin mRNA size (approximately 2.1 kb) and levels (ranging from 390-480 prothrombin mRNA molecules per cell) were determined by Northern and quantitative solution hybridization analysis, respectively, and were unaffected by vitamin K1 treatment. The increases in secreted prothrombin antigen levels most likely result from non-specific effects of vitamin K1 or agents used to emulsify vitamin K1 on protein release from HepG2 cells.


[Seroepidemiology of poliomyelitis in Mexico]. With the massive vaccination campaigns with the inactivated poliomyelitis vaccine starting in 1955 and its oral presentation in 1961, this disease has been controlled in many countries. However, wild polio virus is still transmitted in many developing countries. The study reported in this article had the objectives of estimating the prevalence of antibodies against polio for three types of virus (1, 2 and 3) in the population from 12 to 59 months of age in Mexico and determining the factors associated with the absence of immunity. One section of the National Seroepidemiology Survey (NSS), a study with a representative sample of the Mexican population, included the analysis of 5,260 blood samples for polio seropositivity. These samples were processed using the technique of plaque-reduction-neutralization, with the cut-off for positive titer values at 1:8. The national immunity levels reported for the three types of polio virus were: type 1 (89.8%); type 2 (97.6%); and type 3 (85.4%). The state with the lowest seroprevalence was Campeche, with 59.7 per cent, and the highest observed was Baja California Sur, with 93.0 per cent. The NSS also showed that the immunity level increases with age. There were some differences observed by place of residence; seroprevalences were higher in the urban areas (type one, 93.4%; type two, 98.5% and type three, 88.2%) than in the rural zones (86.6%, 96.8% and 82.9%, respectively). As expected, previous vaccination with three or more doses, referred verbally by the parent or guardian of the child, was associated with higher positivity.(ABSTRACT TRUNCATED AT 250 WORDS)


Critical checking of the radiological diagnosis of "complete remission" and "partial remission" following induction chemotherapy of small-cell lung cancer in the light of postoperative histological examination. A multimodal therapy concept for small-cell lung cancer, which for patients with established pretherapeutic homolateral lymph-node metastases (N2) prescribes induction chemotherapy with subsequent resection as well as supplemental chemo- and radiotherapy, provided the opportunity to evaluate histologically the radiological diagnoses "complete remission" and "partial remission" using resection specimens. In 17 patients a 75% to 100% reduction in tumor size was achieved according to radiological diagnosis. Predictions of "no evidence of disease" or "evidence of disease" were only correct in ten cases. In the remaining seven cases, histology showed the radiological findings to be incorrect. This gives a 77% sensitivity for radiological diagnosis with no specificity. Moreover, differentiation between therapy effect on the primary tumor and on the N2 metastases gives similar results: sensitivity 64% and 67% respectively, specificity 33% and 25% respectively. It is concluded that, particularly after the tumor responds well to therapy, radiological techniques are unsuitable for establishing a diagnosis of "no evidence of disease" or "evidence of disease" in small-cell lung cancer. This is because on the one hand the radiological methods available do not permit clear differentiation between vital tumor tissue and necrosis or fibrosis, while on the other hand groups of vital tumor cells beyond the resolution power of X-ray technology will escape detection.


Breakage and binding of DNA by reaction products of hypochlorous acid with aniline, 1-naphthylamine, or 1-naphthol. Hypochlorous acid (HOCl) is a chemically reactive oxidant and a potent microbicidal agent that is synthesized in phagosomes of inflammatory neutrophils and released into extracellular spaces. Besides reducing pathogenicity by reacting with phagocytized infectious agents, HOCl may damage tissues and yield toxic products upon reaction with various other molecules, including xenobiotics. As model xenobiotics, the substituted aryl compounds aniline, 1-naphthylamine, and 1-naphthol (1-NOH) were investigated herein for their potential to react with HOCl and the transformed into genotoxic products. The compounds were first exposed to HOCl (25-150 microM) in phosphate buffer and afterward used to treat human fibroblasts or purified DNA. DNA single-strand breaks in cells and the binding of HOCl-reacted 1-[14C]NOH to purified DNA were assessed by DNA alkaline elution and scintillation spectrometry, respectively. It was found that neither HOCl nor compounds alone could break cellular DNA. But HOCl-reacted compounds produced up to 400 rad equivalents of DNA breaks. HOCl reaction products of aniline and the model bicyclic aryl compounds differed in their DNA-breaking characteristics. HOCl-reacted 1-[14C]NOH was stable and bound to DNA at up to 124 pmol/mg DNA. Sodium thiosulfate, glutathione, and taurine inhibited the transformation reactions; but only the former two blocked binding of HOCl-reacted 1-NOH to DNA. Ultraviolet spectra showed that HOCl reacted rapidly (less than 1 min) and equally well with 1-NOH at pH 7.2 or at an intraphagosomal pH of 5.0. Reaction concentrations of HOCl in this study were 2- to 11-fold lower than levels generated in vitro by stimulated neutrophils. These results show that certain aryl compounds can react readily with approximated physiological levels of HOCl (-OCl) to form relatively long-lived products that bind DNA and are genotoxic to human cells.


Phthalate ester effects on rat Sertoli cell function in vitro: effects of phthalate side chain and age of animal. Mono(2-ethylhexyl) phthalate (MEHP), the active metabolite of the testicular toxicant di(2-ethylhexyl) phthalate, inhibits FSH-stimulated rat Sertoli cell cAMP accumulation, stimulates basal lactate production, and decreases intracellular ATP levels in vitro. Dibutyl phthalate and dipentyl phthalate but not diethyldimethyl or dipropyl are also age-dependent testicular toxicants in vivo. We therefore examined the effect of animal age and phthalate monoester on the Sertoli cell FSH-stimulated cAMP accumulation, lactate secretion, and ATP levels in order to determine if these effects are part of the mechanism of action of phthalate esters in vivo. MEHP, monobutyl and monopentyl phthalates but not the monoethyl, monomethyl, or monopropyl phthalates inhibited FSH-stimulated cAMP accumulation, a segregation which matches the in vivo toxicity potential of these agents. MEHP and monopentyl, but not monobutyl phthalates, also stimulated Sertoli cell lactate secretion. The effect of the active phthalates on FSH-stimulated cAMP accumulation and lactate secretion is not dependent on age of animal over a range of 13-80 days, suggesting that the age-related toxicity in vivo may be related to differences in metabolism and disposition rather than tissue sensitivity. Since the ED50 of MEHP inhibition of cAMP accumulation and lactate secretion is similar, these two effects may be related to a common initial effect of the active phthalates. Inhibition of intracellular ATP levels is specific for MEHP and is lost with age (greater than 28 days of age) and thus is not likely to be an essential part of the in vivo mechanism of action of phthalate diesters.


Circulating serum levels of interleukin 6 and C-reactive protein after liver transplantation. The study objectives were to investigate serum levels of interleukin-6 and C-reactive protein (CRP) after liver transplantation to correlated measurements with various clinical parameters. Twenty-three patients were studied after orthotopic liver transplantation. Serum IL-6 activity was evaluated by testing its capacity to induce proliferation of the IL-6-dependent hybridoma cell line B9. CRP was assessed by a nephelometric method. Only two of seven patients with acute cellular rejection developed an increase of serum IL-6 and CRP. In contrast to this rejection group, elevated IL-6 levels were observed in 7/9 patients with bacterial infections. Peak values for IL-6 were observed one day and for CRP two days after clinical diagnosis of infection. CMV disease was also associated with markedly increased IL-6 and CRP levels in 5/7 patients. Surprisingly, levels in this condition were approximately in the same range as in bacterial infection. IL-6 and CRP serum levels seen in bacterial infection and CMV disease were significantly higher than those in rejection (P less than 0.001). Serum IL-6 activity was neutralized by an antiserum directed against recombinant human IL-6. Preferential elevations of IL-6 and CRP represent one feature of bacterial and viral infections. Elevation of TNF during rejection as described earlier is only rarely accompanied by increased serum IL-6 levels.


Cytomegalovirus infection and gastric emptying. Gastrointestinal infection due to cytomegalovirus occurs frequently in liver transplant recipients. Upper gastrointestinal cytomegalovirus infection is associated with subjective complaints of nausea, a sense of abdominal fullness, and occasionally emesis and/or dysphagia. In order to determine whether these symptoms reflect a disruption of the normal motility of the stomach, the following study was performed. Eleven individuals who were evaluated for liver transplantation were prospectively recruited and studied as follows: (1) upper gastrointestinal endoscopy with biopsy of the gastric antral mucosa; (2) viral culture of the gastric mucosa; (3) a histologic examination of the gastric mucosa; and (4) a radionuclide gastric emptying study was obtained before and 4-8 weeks following successful liver transplantation. Prior to liver transplantation, none had symptoms of nausea, vomiting, or epigastric fullness. All were culture-negative for cytomegalovirus. All had endoscopic and histologic evidence of portal hypertensive gastropathy but none had antral erosions or ulcers. All demonstrated normal gastric emptying of a liquid meal. Following liver transplantation, 6 remained free of gastric cytomegalovirus while 5 developed a culture-confirmed gastric cytomegalovirus infection. Those that developed a gastric cytomegalovirus infection also had more gastric symptoms, and more gastric histologic abnormalities. Moreover, those with a gastric cytomegalovirus infection demonstrated enhanced gastric retention of a liquid meal (P less than 0.01).


Making muscle in mammals.

Making muscle in mammals. Classical embryology has provided a conceptual basis for our understanding of where muscle comes from. Histological and morphological studies of muscle fibre formation in the foetus and neonate have provided information on how muscle matures. More recent advances in molecular genetics have led to the characterization of muscle structural genes, and to the striking discovery of the MyoD family of myogenic regulatory factors. The question of how myogenesis takes place can now be formulated in terms of gene regulation, and molecular tools can be used to describe this process in the embryo and foetus.


Nerve membrane Na+ channels as targets of insecticides. The mechanisms of action of neuroactive insecticides on the nervous system has been studied for many years. It is now well established that severe neurological symptoms of poisoning with pyrethroids and DDT in mammals and insects are the result of modification of Na+ channel activity. Toshio Narahashi discusses the history, approaches and results of the studies leading to this conclusion. Advanced electrophysiological experiments using voltage clamp and patch clamp, together with ligand-binding and ionic flux experiments, have unveiled unique actions of pyrethroids and DDT of keeping the Na+ channel in the open state for an extremely long period, sometimes as long as several seconds. This modification of Na+ channel properties leads to hyperactivity of the nervous system. These insecticides have also been shown to suppress GABA and glutamate receptor-channel complexes and voltage-activated Ca2+ channels, but the toxicological significance of these actions remains to be seen. The results of these studies provide clues for developing newer insecticides with higher selectivity between mammals and insects and for coping with the problem of insecticide resistance.


The dioxin and peroxisome proliferator-activated receptors: nuclear receptors in search of endogenous ligands. Dioxins and peroxisome proliferators represent two diverse classes of xenobiotic compounds that induce transcription of specific genes encoding cytochrome P-450 drug-metabolizing enzymes. Signal transduction by these chemicals is mediated by two distinct nuclear receptors, one of which has recently been demonstrated to be a member of the steroid hormone receptor superfamily of ligand-activated transcription factors. However, no endogenous ligand has so far been identified for either of these nuclear receptors. Lorenz Poellinger, Martin Göttlicher and Jan-Ake Gustafsson review properties of both these xenobiotic receptor systems and discuss how the molecular details in the receptor activation pathways compare with those of nuclear hormone receptors.


Receptor sites for Ca2+ channel antagonists. Ca2+ channel antagonist drugs inhibit voltage-gated Ca2+ channels in many different cell types. Inhibition of Ca2+ channels in smooth muscle and cardiac muscle cells by these drugs is valuable in the therapy of a wide range of cardiovascular disorders including hypertension, atrial arrhythmia and angina pectoris. Additional uses under evaluation are protection against ischemic damage during myocardial infarction and stroke and in a wide range of other conditions. Further understanding of the sites and mechanisms of action of Ca2+ channel antagonists, as described in this review by Bill Catterall and Jörg Striessnig, will provide new insight into the design of novel therapeutic agents acting on Ca2+ channels and provide further understanding of Ca2+ channel structure and function.


[Disseminated sclerosis and retrovirus]. Multiple sclerosis is a disease characterized by neurologic dysfunction due to focal CNS lesions with demyelination. The cause of the disease is unknown; but it may be due to a virus and/or autoimmune reactions. The latter cause is suspected on account of family- and ethnical studies, the first on account of locally produced antibodies in the cerebrospinal fluid, and also epidemiologic investigations. The newly discovered human retroviruses, especially HTLV-I which is the cause of tropical spastic paraparesis, has been suspected as a possible cause; but this has been disproved by multiple antibody- and PCR-studies. An uncharacterized exogenous or an endogenous retrovirus is still considered to be a possible cause or possibly partial cause of the disease which could be multifactorial.


[Evaluation of the results of ELISA in the quantitative determination of antibodies against infectious bursitis in poultry]. Specific antibodies were investigated in serums of chicks vaccinated with live vaccine and revaccinated with inactivated vaccine against the infectious bursal disease virus, using three methods. An ELISA technique was used to determine antibody titres at a fourfold serum dilution; the reaction was evaluated visually. The results were compared with the titres of neutralizing antibodies and percentage of samples with precipitin activity (Fig. 1). The average values of neutralizing antibody titres and ELISA titres were found to have an analogical pattern; a decrease in maternal antibodies on the first days of chick life and an increase in the antibodies after vaccination, accompanied by an increase in precipitin activity, were typical in these tests. Using the different ELISA technique, 138 samples of fowl serum were examined (Fig. 2). The high correlation, r = 0.85, was found between the ELISA titres determined by visual reading of the reaction within the fourfold serum dilutions and the absorbance values determined at single serum dilution. Applying a spectrophotometer programme, a scale of antibody quantification was made up pursuant to the intensity of immunoenzymatic reaction. For the purposes of method reproducibility, the evaluation was made within the average values of absorbance of positive serum on the one hand and of negative serum on the other. The span of these values was divided into ten equal parts, designated by degrees 0 to 9. Corresponding degrees of positivity were assigned to the examined samples in dependence on the determined value of absorbance (Fig. 3). The correlation r = 0.61 was found between the ELISA values and the titres of neutralizing antibodies.(ABSTRACT TRUNCATED AT 250 WORDS)


Studies on the age resistance of swine to group A rotavirus infection. To determine whether swine become naturally age resistant to group A rotavirus infection, colostrum-deprived, rotavirus-naive newborn pigs that were raised in isolation (n = 34) were studied. Neonatal pigs and pigs 1, 2, 4, 6, 8, 10, and 12 weeks of age were inoculated orally with group A porcine rotavirus or mock inoculum and euthanatized at 24, 31, or 48 hours post-infection. Nine sections of small intestine, cecum, and colon were harvested and immunohistochemically examined for evidence of rotavirus replication within enterocytes. Infectivity was semiquantified by intestinal segment, and a composite score was obtained for each animal. In pigs inoculated at 1 week of age, enterocyte infection was mild and scattered; all other pigs became infected regardless of age or region of intestine, and older animals that became infected had infectivity scores similar to those of younger animals. In a second more limited study, pigs raised in the same isolation environment (n = 11) but previously exposed to virus and demonstrating rotavirus serum antibody had a much lower degree of enterocyte infection at 8, 10, and 12 weeks of age (2, 4, and 6 weeks, respectively, after initial exposure to virus). Age resistance to clinical rotavirus disease in swine is due to factors other than an age-dependent development of resistance of enterocytes to infection, at least through 12 weeks of age.


Immunity to bovine virus diarrhoea virus in calves: the role of different T-cell subpopulations analysed by specific depletion in vivo with monoclonal antibodies. Gnotobiotic calves were injected intravenously with murine monoclonal antibodies (mAb) directed against the BoCD4, BoCD8 or BoWC1 antigens that define the three major T-lymphocyte subpopulations in cattle. This produced a transient, specific depletion of each cell type in the circulation. Calves were then infected intranasally with a non-cytopathogenic biotype of bovine virus diarrhoea virus and the effect of the specific depletion with the mAb on viraemia and shedding of virus from the nasopharynx determined. Depletion of the cells expressing the BoCD4 antigen resulted in an extension of the duration of viraemia and an increase in the titre of virus in blood. No effect on nasopharyngeal shedding was noted. Depletion of either of the other two T-cell subsets that expressed the BoCD8 antigen or the BoWC1 antigen present on the gamma/delta T-cells had no demonstrable effect. These findings are interpreted as showing that the BoCD4+ cells play a pivotal role in controlling a primary infection with this virus but MHC class I restricted BoCD8+ T-cells are not a major effector mechanism. The BoCD4+ cells may be acting directly or be mediators of T-cell help.


Natural killer cells in normal horses and specific-pathogen-free foals infected with equine herpesvirus. Peripheral blood mononuclear cells (PBMC) from an adult horse and from foals demonstrated natural killer (NK)-type cytotoxicity against a range of xenogeneic and allogeneic cell targets. The human tumour cell line, Chang liver was consistently the most susceptible. Chang liver, rabbit kidney (RK-13), equine sarcoid (ES) and embryonic equine kidney (EEK) cells were more susceptible when presented to horse PBMC than monolayer cultures. Embryonic equine lung (EEL) and murine YAC-1 cells conversely, were more susceptible in a trypsinized state. Horse PBMC demonstrated higher levels of NK-type activity against EEK, EEL and RK-13 cells infected with equine herpesvirus 1 (EHV-1) compared with uninfected cells. Similarly, EEK and EEL cells infected with Semliki forest virus (SFV) were more susceptible. Cytotoxicity against EHV-1-infected EEK cells developed faster, between 4 and 8 h of incubation and reaching a maximum at 24 h. By contrast, cytotoxicity against uninfected fibroblasts was not significant until approximately 16 h of incubation with maximum cytotoxicity observed between 32 h and 48 h. Specific pathogen-free (SPF) foals were inoculated with live EHV-1. PBMC isolated from these foals at different days after inoculation did not display appreciably reduced or elevated NK cytotoxicities against Chang liver cells and EHV-1-infected EEK targets, when compared with that of a PBMC reference from a healthy adult horse.


Alteration of neutrophil function in BCG-treated and non-treated swine after exposure to Salmonella typhimurium. Salmonella typhimurium infection in swine causes an enterocolitis followed by a persistent carrier state, but little is known about the mechanisms that allow this organism to colonize and persist in host tissues. Neutrophils provide a first line of defense against invading pathogens such as Salmonella typhimurium. The purpose of this study was to evaluate porcine neutrophil function after in vivo exposure to Salmonella and to determine if the immunomodulator, bacillus Calmette Guerin (BCG), exerts any effect on neutrophil function or on the colonization and persistence of S. typhimurium in the pig. Compared to negative controls, neutrophils from pigs exposed to S. typhimurium exhibited significantly decreased iodination, cytochrome-C reduction, antibody-dependent cell-mediated cytotoxicity, random migration, and chemotaxis (P less than or equal to 0.05). Neutrophil bactericidal activity against S. typhimurium was significantly enhanced. Most of the significant differences were noted in the first two days after exposure to Salmonella. Often the functional alterations were biphasic, peaking again 7-10 days after exposure. BCG alone significantly depressed random migration and cytochrome-C reduction in unstimulated neutrophils. The clinical course, colonization pattern, and persistence of Salmonella were similar between pigs receiving BCG and untreated pigs. These data suggest that S. typhimurium infection causes a depression in oxidative metabolism and motility, yet an increase in overall bactericidal activity against S. typhimurium in circulating porcine neutrophils. It also appears that BCG treatment, as reported here, does not enhance resistance of pigs to S. typhimurium colonization or reduce the number of persistent organisms in the porcine ileum.


Effects of Pasteurella haemolytica A1 leukotoxin on bovine neutrophils: degranulation and generation of oxygen-derived free radicals. To further define the role of Pasteurella haemolytica A1 leukotoxin in the pathogenesis of bovine pneumonic pasteurellosis, its in vitro effects on bovine neutrophils were investigated. Leukotoxin-containing culture supernatant, from P. haemolytica, stimulated a neutrophil respiratory burst as measured by the generation of oxygen-derived free radicals O2- and H2O2. This effect was immediate because preincubation of neutrophils with the culture supernatant for 5 min or longer substantially suppressed this respiratory burst. This suppression was due to cytolysis of the neutrophils. Prolonged incubation of neutrophils with the same culture supernatant caused further cytolysis and degranulation. Heat-inactivated P. haemolytica culture supernatant that had lost its cytotoxic properties failed to stimulate respiratory burst by neutrophils. Furthermore, the respiratory burst, cytolysis and degranulation were abrogated only by leukotoxin-neutralizing monoclonal and polyclonal antibodies, but not by antibodies against the lipopolysaccharide. These studies show that the leukotoxin component in the culture supernatant was responsible for the generation of oxygen-derived free radicals and proteolytic enzymes from neutrophils which may participate in direct lung injury.


Macrophage functions in cats experimentally infected with feline immunodeficiency virus and Toxoplasma gondii. It was suspected that feline immunodeficiency virus (FIV) infection would affect the function of feline macrophages, and that the concomitant infection of cats with FIV and Toxoplasma gondii would cause even greater changes in macrophage function. Sixteen specific-pathogen-free kittens, four per group, were infected either with FIV, T. gondii, both pathogens, or neither pathogen. After the cats had been infected with FIV for 14 weeks (8 weeks after T. gondii infection), peritoneal macrophages were collected. Some macrophages were stimulated with lipopolysaccharide and supernatants were collected for the measurement of interleukin-1 production. Other macrophages were infected with T. gondii in a microbiocidal assay. Peritoneal macrophages from cats infected with FIV had decreased interleukin-1 secretion and increased antimicrobial activity. Co-infection with T. gondii apparently had no effect on these modifications of macrophage activity. Thus, acute FIV infection alone caused significant changes in macrophage functions that were not affected by concomitant T. gondii infection.


[What is reliable in therapy of liver fibrosis?]. Therapy of chronic active liver diseases associated with fibrotic transformation is usually restricted to unspecific antiinflammatory and immunosuppressive agents. However, recent advances in the biochemistry of collagen have allowed to define specific levels of collagen metabolism at which pharmacologic intervention can lead to reduced collagen deposition. The mode of action of some substances which interfere with collagen biosynthesis and degradation is described. However, the efficacy of these agents was tested in vitro exclusively or in animal experiments. Only few agents like colchicine were also studied in clinical trials. Reliable, safe, and specific antifibrotic agents for the clinical management of liver fibrosis do not exist up to now. Advances can be expected, however, from the development of novel inhibitors of prolyl-4-hydroxylase.


Diagnostic strategy for fever of unknown origin in the ultrasonography and computed tomography era. Fever of unknown origin strictly defined by Petersdorf in 1961 may be caused by a whole array of conditions. A rational approach should be based upon the relative frequencies of the different causes and their importance for the health and life of the patient. Risks, discomfort and, to a limited degree, costs must be taken into consideration. The investigation protocol we propose takes into account all these factors and is mainly based upon an extensive literature survey and our personal experience with a series of 199 patients studied in the 1980 s.


HIV-related thrombocytopenia.

HIV-related thrombocytopenia. HIV-related chronic ITP is caused by an accelerated platelet destruction due to adsorption of circulating immune complexes and to specific anti-platelet antibodies, but perhaps also by a defective thrombopoiesis resulting from invasion of the megakaryocytes by the retrovirus. Treatment is needed when platelet numbers drop beneath 20.10(9)/L or when severe bleeding symptoms occur. Steroids, commercially available immunoglobulins for IV use, AZT and anti-Rh immunoglobulins can be administered, although relapses are frequent after withdrawal of the drugs. Recurrences after splenectomy are far less common, but the progression towards AIDS might be accelerated.


Intrathoracic infections with bacteraemia due to Eikenella corrodens as a complication of peritonsillar abscesses: report of a case and review of the literature. A 52-year-old man, without previous disease, presented with dysphagia, dyspnoea, high fever and sore throat after peritonsillar abscesses drainage. Physical and complementary examinations were consistent with pericarditis, mediastinitis, pneumonia and pleuritis. Blood cultures grew Eikenella corrodens resistant to clindamycin and amikacin. We emphasize the pathogenic potential of Eikenella corrodens. To the best of our knowledge, this is the first reported case of this organism as a pathogen in intrathoracic infections after peritonsillar abscesses drainage.


[Hypereosinophilia syndrome. Apropos of 2 cases and literature review]. Idiopathic hypereosinophilic syndrome is characterized by prolonged eosinophilia of undetected cause and multiple organ system involvement (lung, kidney, nervous system, skin,...). Nevertheless, the prognosis has been correlated with heart involvement, which usually results in a restrictive cardiomyopathy with apical obliteration by fibrosis, mural thrombi and mitral and tricuspid regurgitation. This disease has a wide range of severity: some patients suffer from a real myeloproliferative syndrome and may develop blastic transformation while others present only skin involvement or are asymptomatic. Corticosteroids and hydroxyurea are both effective treatments. Interferon alpha seems to be active for the myeloproliferative form of the disease. Cytotoxic activity of activated eosinophil granular proteins may play an important role in tissue damage. The cause of eosinophilic proliferation (primitive malignant proliferation or resulting from a T lymphocyte stimulus) and activation remains uncertain.


Intubating conditions and onset of neuromuscular block of rocuronium (Org 9426); a comparison with suxamethonium. The intubating conditions and neuromuscular blocking profile following 600 micrograms.kg-1 rocuronium (Org 9426) have been investigated in patients under various experimental conditions. They were compared with conditions following 1.5 mg.kg-1 suxamethonium, preceded by a precurarising dose (10 mg) of gallamine, and with those in a control group in the absence of a muscle relaxant. Rocuronium produced good to excellent intubating conditions at 60 as well as at 90 s after administration, even though there was only a partial blockade of the adductor pollicis muscle. Intubating conditions following suxamethonium were comparable with those after rocuronium. Half of the control patients could be intubated. The clinical duration and the recovery time of 600 micrograms.kg-1 of rocuronium were 24(4) and 9(3) min (mean(s.d.)), respectively. Rocuronium may have a major advantage over existing non-depolarising muscle relaxants due to the early presence of excellent intubating conditions. The results indicate that rocuronium may replace suxamethonium in procedures in which rapid sequence induction is required.


A clinical double-blind study of flumazenil, antagonist of benzodiazepines, in loco-regional anesthesia. Flumazenil has been studied for antagonism of the residual sedative effect of midazolam administered as a complement of loco-regional anesthesias. Highly significant differences appeared between the patients receiving placebos and those receiving flumazenil, showing a quick suppression of the lasting sedative effects of midazolam. At the dosage of 1 mg, flumazenil seemed to have a more efficient and complete effect than the 0.5 mg dosage. Tolerance to flumazenil was excellent with the exception of one case of agitation and disorientation, in a patient chronic consumer of benzodiazepines thus this could be considered as a deprivation syndrome. In conclusion, flumazenil should certainly have a place in the pharmacological stores of the anesthesiologist.


Some observations upon the distribution of cytochrome oxidase activity in the globus pallidus of the rat. The histochemical distribution of the enzyme cytochrome oxidase (CO) was explored in the globus pallidus (GP) of the rat, and it was compared with the distribution of acetylcholinesterase (AChE), another well-known enzyme of the basal ganglia in mammals. This neurochemical research illustrates two prominent findings. In the first place, a regional compartmentalization was detected in the pallidal distribution of CO, in which dorsal territories of the GP exhibited a more abundant presence of this enzyme. In addition, the distribution of this mitochondrial enzyme was not uniform all over those dorsal pallidal territories. Thus, the pallidal concentration of CO was gradually decreasing dorsoventrally and lateromedially, and, sometimes, areas highly stained for CO were intermingled with zones in which this enzyme was less conspicuous. In the second place, the pallidal distribution of AChE was the opposite of that found for CO (i.e. more abundant in ventral and medial territories of the GP). The functional significance of these findings is discussed in the light of the heterogeneous hodological neuroanatomy of the GP of rodents.


Oxytocin stimulates translocation of protein kinase C and induces antiviral state in human amnion cells. Association of protein kinase C (PKC) activity to the membrane fraction was observed in oxytocin treated human amnion cells (UAC). In addition, oxytocin was shown to induce an antiviral state and to inhibit multiplication of vesicular stomatitis virus (VSV) in UAC. These observations together with earlier findings indicate that activation of inositol phospholipid breakdown with a consecutive activation of PKC is a common signal transduction pathway in interferon action and hormonal stimulation.


Comparison of haemagglutination inhibition and indirect fluorescent antibody tests to detect certain flavivirus antibodies in equines. Formalinized goose erythrocytes were used in haemagglutination inhibition (HI) and indirect fluorescent-antibody (IFA) tests to detect antibodies to Japanese encephalitis (JE) and West Nile (WN) viruses in equines. Paired serum samples from 31 cases having clinical symptoms of flaviviral infections (JE and WN viruses) and 45 controls were examined. For HI test, formalinized goose erythrocytes were used as such, whereas in IFA test, formalinized goose erythrocytes were first coated with respective viral antigens separately and later used to detect antibodies. By employing HI and IFA tests, paired samples having a titre same or less than two fold rise over the control sera were considered normal for both the viruses. IFA test was found to be a method of choice, due to its sensitivity over HI test.


In vitro interactions of anionic and cationic surfactants with salivary fractions on well-defined solid surfaces. Ellipsometry was used to study the interaction of one anionic (SDS) and one cationic (CTAB) surfactant with films adsorbed from six different salivary fractions obtained after fractionation of whole unstimulated saliva on a Superdex 200 Hiload gel filtration column. Experiments were performed on both hydrophilic silica and hydrophobic methylated silica surfaces. The results of this study indicate that the adhesive and cohesive properties of the films adsorbed from the individual fractions were strongly dependent on the surface characteristics of the substrates and that the outcome of protein/surfactant interactions was dependent on factors such as protein composition, surfactant charge, and substrate characteristics. These interactions probably involve replacement of the adsorbed proteins by surfactants or protein/surfactant complex formation. The anionic surfactant seemed to be more efficient in removing adsorbed salivary proteins than the cationic one.


Type II syndactyly or synpolydactyly.

Type II syndactyly or synpolydactyly. A family with syndactyly type II or synpolydactyly is described. The autosomal dominant inheritance is confirmed by this pedigree. In combination of this anomaly a brachymesophalangia of the fifth finger was inherited by most family members. The duplicated phalanx was resected and the syndactyly separated in the proband with excellent functional and cosmetic results.


Peripheral facial palsy caused by Borrelia burgdorferi and viruses in south-western Finland. In a prospective study from 1983 through 1984, 77 patients (31 men and 46 women with a mean age of 47 +/- 20 years) with peripheral facial palsy of primarily unknown etiology were investigated. Only 2 patients with acute otitis media received antibiotics. Serology of the patients was investigated on days 1 and 14. IgG and IgM antibodies against herpes simplex, varicella-zoster and cytomegalovirus were determined by enzyme immunoassay, and against Epstein-Barr virus by immunofluorescence. In a retrospective analysis, IgM, IgA and IgG antibodies against Borrelia burgdorferi were determined by enzyme immunoassay. Borreliosis was diagnosed in 5 patients and varicella-zoster infection in 7. There was no statistically significant difference in recovery time between the different groups. Follow-up time for the patients with borreliosis was over 5 years. Neither meningeal symptoms nor polyneuropathy was observed in the patients with borreliosis even in the absence of antibiotic therapy.


The use of gamma-linolenic acid in diabetic neuropathy. EF4 is an entirely new approach to the management of diabetic neuropathy. EF4 (providing gamma-linolenic acid or gamolenic acid, GLA) has been shown to reverse existing diabetic neuropathy in trials in seven centres. Diabetic animals and humans have a reduced ability to convert dietary linoleic acid to GLA. GLA and its metabolites are required for normal neuronal structure and function and a normal microcirculation. The lack of GLA and its metabolites may play a major role in the development of the neuropathy. EF4 helps to correct the biochemical defects, restores levels of GLA metabolites towards normal and produces highly significant clinical and neurophysiological improvements in diabetic neuropathy.


Stereospecific and non-stereospecific effects of ibuprofen on human platelet and polymorphonuclear leukocyte functions. This study compares biological activities of racemic ibuprofen (rac-IBU) and its S(+) and R(-) enantiomeres in human platelet and polymorphonuclear cells (PMN). Rac-IBU inhibited cyclooxygenase-related platelet functions (aggregation, thromboxane (TX) B2 formation) in vitro, the S-(+) enantiomer being 40-100-times more active than the R-(-) form. rac-IBU also inhibited PMN functions (O2- generation, beta-glucuronidase release). These effects in PMN were not stereospecific. The data suggest cyclooxygenase-independent actions of ibuprofen in human PMN which may contribute to its antiinflammatory effects in vivo.


Release of eicosanoids and endothelin in an experimental model of adult respiratory distress syndrome. In an experimental model of adult respiratory distress syndrome (ARDS) we have investigated the release of several vasoactive mediators since the rapid and severe increase in pulmonary arterial pressure is known to be a key feature in this condition. Intravenous injection of oleic acid (OA) into rats resulted in the development of a syndrome similar to ARDS characterized by severe hypoxia. As early as 15 min after OA injection elevated levels of 11-deoxy-15-keto-13,14-dihydro-11,16-cyclo-prostaglandin (PG) E2 (DKH2-cyclo-PGE2) as an indicator of endogenous PGE2 formation could be observed. Similarly, elevated levels of cysteinyl-leukotrienes (LT), determined as immunoreactive (ir) LTE4, and ir-endothelin (ET) could be detected. These mediators were found to be elevated in plasma samples for at least 3 h. In addition, rather large amounts of ir-ET could be detected in bronchoalveolar lavage (BAL) fluid samples between 1 h and 3 h. Thus, it seems possible that cysteinyl-LT and peptides of the ET family, both known for their vasoactivity, might be involved in the pathophysiological process of ARDS.


Any physiological role for prostacyclin in regulation of fetal vessel tone? This study investigates the contribution of endogenous prostacyclin and nitric oxide (NO) for maintenance of umbilical artery tone in vitro. Treatment with indomethacin as well as removal of the endothelium was followed by contractions of umbilical arteries and a reduced formation of prostacyclin. In contrast, vessel tone was not affected by inhibition of NO synthesis, using nitro-L-arginine. Thus, prostacyclin rather than nitric oxide appears to be the endogenous endothelium-derived factor in the umbilical circulation that is involved in regulation of vessel tone.


[Giant juvenile angiofibroma: which surgical approach?]. The juvenile angiofibroma is an uncommon, highly vascular, locally invasive, non encapsulated tumor. In spite of its benignity the above mentioned peculiarities make the therapy very hard. The estrogenic hormones and the radiotherapy were used for years but there were many iatrogenic after-effects. The preferred treatment is surgical. Many surgical approaches were used. Everyone has advantages and disadvantages. The choice may be free for tumors of small or medium size. When the tumor is very large, stage III A or more, a single approach isn't enough. A double approach is mandatory; one must be an infratemporal approach (pre o postauricular). We present a case of juvenile angiofibroma stage III B which is a good pattern of the above mentioned statements.


[Malignant mesodermal mixed tumor of the bladder: report of a case]. A 59-year-old male took total cystourethrectomy on July, 1991, since the bladder tumor recurred 2 years and 4 months after transurethral resection. Six months after total cystourethrectomy, an abnormal mass shadow appeared on the right lower lung field. Metastatic lung tumor was strongly suspected from CT scan. Despite chemotherapy, the pulmonary lesion grew rapidly and the patient died. From the autopsy, metastatic lesions were found in the bilateral lung fields, skin (face, head and abdominal wall), pleura, bilateral kidneys, small intestine and lymph nodes (para-aortic and mesenteric). The primary bladder tumor contained histologically transitional cell carcinoma as the epithelial element and sarcomatous changes with osteoid formation as the non-epithelial elements. Thus, the primary lesion was diagnosed as a malignant mesodermal mixed tumor. However, all of the metastatic lesions showed only sarcomatous changes. Only 10 cases of malignant mesodermal mixed tumor of the bladder have been reported in Japan since Fujita's report. In general, total cystectomy is necessary for the treatment of this disease. It has a poor prognosis; 5 of the 10 patients died within one year after operation.


High-resolution CT of bronchioloalveolar carcinoma. Bronchioloalveolar cell carcinoma has a wide spectrum of pathologic and radiologic appearances. Some of the radiologic features are characteristic enough to suggest the underlying pathologic changes. This article illustrates the various manifestations of bronchioloalveolar cell carcinoma on high-resolution CT (1.5-mm collimation reconstructed with a high-spatial-frequency algorithm). Correlations between the CT and pathologic findings are included.


Human papillomaviruses, herpes simplex viruses, and the risk of oral cancer in men. A population-based case-control study was conducted in western Washington state to examine the relations between infection with human papilloma viruses (HPV), herpes simplex viruses (HSV), and risk of oral squamous cell cancer in men. Interviews were completed on 131 oral cancer cases diagnosed between January 1985 and December 1989 and 136 controls frequency matched to cases on age and date of diagnosis who were obtained by random digit dialing. The risk for oral cancer among men with 30 or more sexual partners was 2.4 times that of men with four or fewer partners (95% confidence interval (CI) 1.0-5.9). Men who ever practiced oral sex had lower risk for oral cancer relative to men who never practiced oral sex (relative risk (RR) = 0.4, 95% CI 0.2-0.8). Analyses of exfoliated oral cavity cells for the presence of HPV-6 DNA with polymerase chain reaction revealed that men with an oral HPV-6 infection had 2.9 times the risk for oral cancer of noninfected men (95% CI 1.1-7.3), whereas men with an oral HPV-16 infection had 6.2 times the risk for oral cancer of noninfected men (crude RR = 6.2, 95% CI 0.7-52.2). Relative risks associated with serologically detected HSV-1 and HSV-2 infections were 0.8 (95% CI 0.3-1.7) and 1.8 (95% CI 0.7-4.6), respectively. The authors conclude that HPV-6 is associated with oral cancer. Although men infected with HPV-16 and HSV-2 were at elevated risk, these associations may have been due to chance. The role of specific sexual practices in the transmission of viruses to the oral cavity remains unclear.


Evaluation of the effects of low molecular weight heparin on inflammation and collagen deposition in chronic coxsackievirus B3-induced myocarditis in A/J mice. Coxsackievirus, Group B, type 3 (CVB3) infection of A/J male mice induces chronic myocarditis with increased interstitial fibrosis and collagen deposition. Heparin, a naturally occurring sulfated glycosaminoglycan, has both anti-inflammatory and antifibrotic activities besides its well-known anticoagulant activity. This study determined whether heparin treatment could decrease either cardiac inflammation or fibrosis in chronic CVB3-induced myocarditis. Control mice were either untreated or treated with heparin (4 micrograms/g body weight, subcutaneously 5 times weekly) beginning 2 days before infection of other groups. Additional groups received either virus only (1 x 10(4) plaque-forming units [PFU]), virus followed by heparin beginning 14 days after CVB3 inoculation, or virus and heparin beginning 2 days before CVB3 inoculation. Animals were sacrificed 14, 28, and 58 days after infection. Heparin treatment begun either before or after virus inoculation reduced animal mortality by approximately 20%. Heparin did not alter virus infection or replication in the heart. Histologically, only animals treated with heparin before virus inoculation showed reduced myocardial inflammation, and only at day 58. However, heparin treatment begun either before or after virus infection significantly decreased collagen deposition in the heart (fibrosis).


Immunohistochemical characterization of amyloid proteins in sural nerves and clinical associations in amyloid neuropathy. To test whether immunohistochemical characterization of proteins in amyloid deposits in biopsied sural nerves gives reliable and useful diagnostic information using commercially available reagents, biopsy specimens of sural nerves from 38 patients with amyloid neuropathy were studied. Transthyretin (TTR) was detected in the amyloid deposits of 11 nerves, lambda light chains (LC) in 8 nerves, kappa LC in 7 nerves, and both lambda and kappa LC in 3 nerves. In 9 nerves, the amyloid deposits were too small to allow adequate immunohistochemical characterization of amyloid proteins in serial sections. Evidence that immunohistochemical characterization was correct came from: 1) evaluation of kin, 2) search for monoclonal proteins in the plasma, and 3) sequencing of the gene abnormalities in TTR+ cases. In 9 of 11 TTR+ cases, in which DNA could be obtained, sequencing of the gene showed that each of the 9 cases was heterozygous for a gene mutation; 7 had previously described mutations and 2 undescribed mutations. Therefore, in the nine sporadic cases without plasma monoclonal light chains, the immunohistochemical characterization correctly identified the protein in amyloid as transthyretin. Likewise, there was a high concordance between immunoglobulin light chains in plasma and light chains in amyloid in primary amyloidosis. Evaluation of the type, distribution, and severity of the neurologic symptoms and deficits showed: 1) the sensorimotor and autonomic neuropathy of amyloidosis characteristically affects proximal as well as distal limbs, and 2) the type of amyloidosis probably cannot be determined from the characteristics or severity of the neuropathy alone or from the location or size of amyloid deposits in nerve.


Light and electron microscopic localization of beta-amyloid protein in muscle biopsies of patients with inclusion-body myositis. In 11 of 11 inclusion-body myositis (IBM) patients, including one hereditary case, vacuolated muscle fibers contained large and multiple small inclusions immunoreactive for beta-amyloid protein (beta AP). All IBM muscle biopsies had characteristic cytoplasmic tubulo-filaments (CTFs) by electron microscopy. None of 14 control muscle biopsies contained the beta AP immunoreactive (IR) inclusions characteristic of IBM. On the light microscopy level, beta AP-IR inclusions colocalized with ubiquitin immunoreactivity. By immunogold electronmicroscopy, beta AP immunoreactivity was localized to a) amorphous, poorly defined structures, b) dense floccular material, c) clusters of loosely packed amyloidlike fibrils 6-8 nm in diameter, and d) poorly defined loose fibrillar structures 6-8 nm in diameter. beta AP immunoreactive structures were often in proximity to CTFs, but CTFs themselves never contained beta AP-IR. Our study provides the first demonstration of beta AP accumulations in abnormal human muscle. This finding suggests that in addition to Alzheimer's disease, Down syndrome, and Dutch-type hereditary cerebrovascular amyloidosis, beta AP may play an important role in the pathogenesis of other diseases, including ones outside the central nervous system, for example, IBM.


Analysis of receptor binding displacement curves by a nonhomologous ligand, on the basis of an equivalent competition principle. An exact method for the analysis of receptor-ligand binding data when labeled bound ligand is displaced by a nonhomologous ligand with a different dissociation constant is described. The present method, which is based on an equivalent competition principle for the homologous and the nonhomologous ligand, converts displacement curves into a linear form and is also applicable to situations in which free concentrations of ligand are significantly smaller than the added concentrations as a result of ligand binding. It is shown that the dissociation constant of the nonhomologous ligand is given directly by the concentration of this nonhomologous ligand added and the free concentration of unlabeled homologous ligand required to give the same level of displacement of labeled bound ligand. On the basis of these displacement characteristics, all binding parameters for receptor interaction of the nonhomologous ligand can be obtained and expressed, for example, in a Scatchard plot. The present method, which is referred to as the equivalent competition method, is also evaluated in this study with respect to the effects of nonspecific ligand binding and the presence of multiple receptor classes.


Structural characterization of the mesangial cell type IV collagenase and enhanced expression in a model of immune complex-mediated glomerulonephritis. Secretion of glomerular cell-derived matrix metalloproteinases (MMPs) and their specific inhibitors, TIMP-1,2, may play an important role in the turnover of the glomerular extracellular matrix under basal and pathologic conditions. A 66-68 kd MMP secreted by cultured mesangial cells (MC) with activity against Type IV collagen and gelatin was purified and shown by amino-acid sequence analysis to be identical with a Type IV collagenase/gelatinase secreted by certain transformed tumor cell lines. The expression of the mesangial MMP in vivo was limited within the kidney to a small subset of the intrinsic glomerular mesangial cell population. After induction of acute anti-Thy 1.1 glomerulonephritis, there was a large increment in the number of Type IV collagenase-secreting MC, temporally coincident with the development of mesangial hypercellularity. The expression of the MMP inhibitor protein, TIMP-1, was not changed over this period. Ultrastructural studies localized the mesangial MMP to areas of evolving mesangiolysis and at sites of glomerular basement membrane disruption. Enhanced expression of the mesangial cell-derived Type IV collagenase may contribute to the evolution of glomerular injury in this model of immune complex-mediated glomerulonephritis or may be involved in the extensive matrix remodeling process that accompanies this form of glomerular injury.


Enzyme-based high-performance liquid chromatography supports as probes of enzyme activity and inhibition: the immobilization of trypsin and alpha-chymotrypsin on an immobilized artificial membrane high-performance liquid chromatography support. Immobilized artificial membrane (IAM) HPLC supports have been used to immobilize the enzymes alpha-chymotrypsin and trypsin. The enzymes were trapped in hydrophobic cavities on the support and were not covalently attached to the IAM surface. The resulting IAM-enzyme supports retained the hydrolytic activity of the immobilized enzymes: the IAM-trypsin support catalyzed the hydrolysis of N alpha-benzoyl-DL-arginine-p-nitroanilide (BAPNA), and the IAM-alpha-chymotrypsin support (IAM-ACHT) catalyzed the hydrolysis of a number of substrates, including tryptophan methyl ester. The activities of both supports were decreased by known enzyme inhibitors and the activity of the IAM-ACHT was affected by changes in pH and temperature. When a substrate was chromatographed on an IAM-ACHT HPLC, the hydrolytic activity of the immobilized enzyme could be determined from the resulting substrate/product ratios. These data were obtained either directly from the IAM-ACHT chromatogram or from the chromatogram produced by a coupled column system. The results of this study indicate that IAM-immobilized alpha-chymotrypsin and trypsin can be used as chromatographic probes for the qualitative determination of enzyme/substrate and enzyme/inhibitor interactions.


Specific identification of collagens and their fragments by clostridial and anti-collagenase antibody. A method specific for identification of collagens irrespective of type, species, or tissue origin, and of their derived fragments of molecular weight more than 10,000, is described. The method is based on the low-temperature affinity between clostridial collagenase and almost all types of collagens as well as on the affinity between collagenase and its antibodies. Various collagens or fragments derived from them by treatment with CNBr were separated by SDS-PAGE and immobilized onto a nitrocellulose membrane by a slot-blot technique or electrotransfer. Following binding of clostridial collagenase to a collagen or its fragments at 0 degrees C, the collagen-collagenase complex was fixed with glutaraldehyde. The complex was then allowed to bind anti-collagenase antibody at room temperature. The new complex was subsequently treated with 125I-labeled donkey anti-rabbit IgG and visualized as an autoradiogram. Under the conditions of low temperature used, the collagenase binds to collagens without causing their digestion. This procedure is specific for detection of soluble collagens as well as of insoluble collagens converted to fragments by treatment with CNBr. The method is uniquely suited for detection of fragments of tissue collagens. Also, it may serve as a prototype for methods for detection of other specific polymeric substances.


Pipecuronium-induced neuromuscular blockade during nitrous oxide-fentanyl, enflurane, isoflurane, and halothane anesthesia in surgical patients. This study was designed to determine the capacity of several anesthetics to augment pipecuronium neuromuscular blockade. The potency of pipecuronium was determined with single-bolus administration of 20-50 micrograms/kg in 160 patients. Patients were anesthetized with N2O/O2 (60:40) supplemented with fentanyl (4-5 micrograms/kg), halothane (0.8%), isoflurane (1.2%), or enflurane (1.7%). Neuromuscular blockade was measured by an acceleration-responsive transducer (the Accelograph, Biometer International, Odense, Denmark). Responses were defined in terms of percent depression in first-twitch height and train-of-four response, and the dose-response curves were constructed after probit transformation of the responses. The dose-response curves were found to be parallel for both first twitch height and train-of-four responses. The dose-response lines for the enflurane and isoflurane groups were displaced significantly (P less than 0.01) to the left of the line for the fentanyl-N2O group. The calculated doses producing 50% depression of first twitch height were 21.9, 21.2, 18.9, and 17.8 micrograms/kg for the N2O-fentanyl, halothane, isoflurane, and enflurane groups, respectively. Corresponding calculated doses for 50% depression of train-of-four response were significantly smaller (15.5, 14.4, 13.7, 11.9 micrograms/kg, respectively). The enhancing effects of the volatile anesthetics were reflected by significant prolongation of the clinical duration of neuromuscular blockade by pipecuronium. It is concluded that the potency of pipecuronium is enhanced more by enflurane and isoflurane than halothane or fentanyl-N2O anesthesia.


Anticoagulant-induced shoulder hematoma producing brachial plexus neuropathy--case reports. Hemorrhagic complications from anticoagulants are common and may manifest in any part of the human body. Skin discoloration, pain, tenderness, and soft-tissue swelling may be the main clinical features. The authors present 3 extraordinary cases of brachial plexus neuropathy associated with anticoagulant-induced hemorrhage. The signs, symptoms, important differentials, and clinical treatment are described with regard to the pathologic anatomy.


Opioid and cannabinoid receptor inhibition of adenylyl cyclase in brain. Both opioids and cannabinoids bind to G-protein-coupled receptors to inhibit adenylyl cyclase in neurons. These reactions were assayed in brain membranes, where maximal inhibitory activity occurred in the following regions: mu-opioid inhibition in rat thalamus, delta-opioid inhibition in rat striatum, kappa-opioid inhibition in guinea pig cerebellum, and cannabinoid inhibition in cerebellum. The inhibition of adenylyl cyclase by both cannabinoid and opioid agonists was typical of G-protein-linked receptors: they required GTP, they were not supported by non-hydrolyzable GTP analogs, and they were abolished (in primary neuronal cell culture) by pertussis toxin treatment. The immediate targets of this system were determined by assaying protein phosphorylation in the presence of receptor agonists and App(NH)p, a substrate for adenylyl cyclase. In striatal membranes, opioid agonists inhibited the phosphorylation of at least two bands of MW 85 and 63 kDa, which may be synapsins I and II, respectively. Other experiments determined the long-term effects of this second messenger system. In primary neuronal cultures, opioid-inhibited adenylyl cyclase attenuated forskolin-stimulated pro-enkephalin mRNA levels, thus providing a feedback regulation of opioid synthesis. Finally, in cerebellar granule cells, both cannabinoid and opioid receptors may exist on the same cells. In these cells, agonists which bind to different receptor types may produce similar biological responses.


Migration and chemiluminescence of polymorphonuclear cells and monocytes to Bacteroides sonicates. Recent investigations have demonstrated that various preparations obtained from representatives of the genus Bacteroides are poorly phagocytized by polymorphonuclear cells (PMN) and macrophages. Crude cell sonicates derived from Bacteroides have been examined for their ability to inhibit migration of PMN and monocytes using a modified migration under agarose in vitro assay. B. gingivalis and B. intermedius were found to be inhibitors of such migration while B. asaccharolyticus did not share this property (P less than 0.005). In addition, B. intermedius sonicates were found to inhibit PMN chemiluminescence to known stimulants (P less than 0.001). These data were not found to result from direct sonicate cytotoxicity and therefore lend additional support to the etiologic importance of specific Bacteroides strains in the pathogenesis of acute and chronic dentoalveolar infections.


Antiradical effects in L-propionyl carnitine protection of the heart against ischemia-reperfusion injury: the possible role of iron chelation. L-Propionyl carnitine has been shown to improve the heart's mechanical recovery and other metabolic parameters after ischemia-reperfusion. However, the mechanism of protection is unknown. The two dominating hypotheses are: (i) L-propionyl carnitine can serve as an energy source for heart muscle cells by being enzymatically converted to propionyl-CoA and subsequently utilized in the Krebs cycle (a metabolic hypothesis), and (ii) it can act as an antiradical agent, protecting myocardial cells from oxidative damage (a free radical hypothesis). To test the two possible pathways, we compared the protection afforded to the ischemia-reperfused hearts by L-propionyl carnitine and its optical isomer, D-propionyl carnitine. The latter cannot be enzymatically utilized as an energy source. The Langendorff perfusion technique was used and the hearts were subjected to 40 min of ischemia and 20 min of reperfusion. In analysis of ischemia-reperfused hearts, a strong correlation was found between the recovery of mechanical function and the presence of protein oxidation products (protein carbonyls). Both propionyl carnitines efficiently prevented protein oxidation but L-propionyl carnitine-perfused hearts had two times greater left ventricular developed pressure. The results indicate that both metabolic and antiradical pathway are involved in the protective mechanism of L-propionyl carnitine. To obtain a better insight of the antiradical mechanism of L-propionyl carnitine, we compared the ability of L- and D-propionyl carnitines, L-carnitine, and deferoxamine to interact with: (i) peroxyl radicals, (ii) oxygen radicals, and (iii) iron. We found that none of the carnitine derivatives were able to scavenge peroxyl radicals or superoxide radicals. L- and D-propionyl carnitine and deferoxamine (not L-carnitine) suppressed hydroxyl radical production in the Fenton system, probably by chelating the iron required for the generation of hydroxyl radicals. We suggest that L-propionyl carnitine protects the heart by a dual mechanism: it is an efficient fuel source and an antiradical agent.


Toxicity of 1-methyl-4-phenylpyridinium derivatives in Escherichia coli. Several derivatives of 1-methyl-4-phenylpyridinium (MPP+), i.e., 1-methyl-4-(4'-nitrophenyl)pyridinium (1), 1-methyl-4-(4'-cyanophenyl)pyridinium (2), 1-methyl-4-(3'-nitrophenyl)pyridinium (3), 1-methyl-4-(4'-chlorophenyl)pyridinium (4), 1-methyl-4-(4'-acetamidophenyl)pyridinium (5), and 1-methyl-4-(4'-aminophenyl)pyridinium (6), were synthesized in order to compare their toxicity with that of paraquat (PQ2+) in Escherichia coli. Addition of compounds 1, 2, and 3 to aerobic E. coli cell suspensions caused extracellular ferricytochrome c reduction, which was inhibited by superoxide dismutase in the same manner as that in the case of PQ2+. The rate of the ferricytochrome c (cyt. c) reduction was in the order of PQ2+ greater than 1 greater than 2 greater than 3, which is the same as that of the redox potentials of these compounds. On the other hand, MPP+, 4, 5, and 6, which have more negative potentials, had no effect on the cyt. c reduction. Compound 1 inhibited the growth of E. coli under aerobic conditions, but not under anaerobic conditions. The results show that compound 1 can act as a mediator for production of superoxide (O2-.), which seriously injures E. coli cells. However, though compounds 2 and 3 catalyzed the production of O2-. in E. coli cells, their activity of O2-. production was much lower than that of compound 1 or PQ2+. Thus, compound 3 had no effect on growth or survival of E. coli at 1 mM, while compounds 2 and 4 had both bacteriostatic and bacteriocidal effects which were independent of dioxygen (O2). The results show that the toxic mechanism is different from that of compound 1. MPP+, 5, and 6 had no effect on growth of E. coli. This paper shows that compound 1 is a novel enhancer of intracellular superoxide production, though the mechanism of toxicity of compounds 2 and 4 is not clear yet. The results suggest that the redox potential is a crucial factor for manifestation of the activity.


Additional binding sites for the pyruvate dehydrogenase kinase but not for protein X in the assembled core of the mammalian pyruvate dehydrogenase complex: binding region for the kinase. A standard resolution of the bovine kidney pyruvate dehydrogenase complex yields a subcomplex composed of approximately 60 dihydrolipoyl transacetylase (E2) subunits, approximately 6 protein X subunits, and approximately 2 pyruvate dehydrogenase kinase heterodimers (KcKb). Using a preparation of resolved kinase in which Kc much greater than Kb, E2-X-KcKb subcomplex additionally bound at least 15 catalytic subunits of the kinase (Kc) and a much lower level of Kb. The binding of Kc to E2 greatly enhanced kinase activity even at high levels of bound kinase. Free protein X, functional in binding the E3 component, did not bind to E2-X-KcKb subcomplex. This pattern of binding Kc but not protein X was unchanged either with a preparation of E2 oligomer greatly reduced in protein X or with subcomplex from which the lipoyl domain of protein X was selectively removed. The bound inner domain of protein X associated with the latter subcomplex did not exchange with free protein X. These data support the conclusion that E2 subunits bind the Kc subunit of the kinase and suggest that the binding of the inner domain of protein X to the inner domain of the transacetylase occurs during the assembly of the oligomeric core. Selective release of a fragment of E2 subunits that contain the lipoyl domains (E2L fragment) releases the kinase (M. Rahmatullah et al., 1990, J. Biol. Chem. 265, 14,512-14,517). Sucrose gradient centrifugation yielded an E2L-kinase fraction with an increased ratio of the kinase to E2L fragment. A monoclonal antibody specific for E2L was attached to a gel matrix. Binding of E2L fragment also led to specific binding of the kinase. Extensive washing did not reduce the level of bound kinase. Thus, the kinase is tightly bound by the lipoyl domain region of E2.


The hydroxylation of tryptophan.

The hydroxylation of tryptophan. Products of the chemical hydroxylation of tryptophan by Fenton and Udenfriend reactions are similar to those obtained by ionizing radiation. When tryptophan is exposed to either of these systems, a mixture of four hydroxytryptophans, oxindole-3-alanine, and N-formylkynurenine is formed. This observation indicates that the hydroxyl radical attacks the aromatic nucleus as well as the 2 and 3 positions of the pyrrole ring. During gamma-radiolysis of nitrous oxide-saturated tryptophan solution and in the absence of oxygen or ferric edta, the hydroxyl radical adduct (or hydroxycyclohexadienyl radical) of tryptophan undergoes dimerization and polymerization, which results in a yellow product with maximal absorbance at 425 nm. In the presence of ferric edta, or in a Fenton system, the hydroxyl radical adduct disproportionates, and hydroxylated derivatives are formed. The yields of the hydroxytryptophans are proportional to the concentration of ferric edta to a limiting yield of 54% of the theoretical yield, which is taken to be one hydroxylated product per two hydroxyl radicals. Under these conditions, 4-, 5-, 6-, and 7-hydroxy-derivatives of tryptophan are found in the proportion 4:2:2:3, respectively. The presence of dioxygen during gamma-radiolysis increases the yield of N-formylkynurenine, but does not affect the total yield of hydroxytryptophans. Similarly, tryptophan subjected to the Udenfriend reaction yields 4-, 5-, 6-, and 7-hydroxytryptophan and N-formylkynurenine in approximately equal amounts.


The hydroxylation of phenylalanine and tyrosine: a comparison with salicylate and tryptophan. The hydroxylation of phenylalanine by the Fenton reaction and gamma-radiolysis yields 2-hydroxy-, 3-hydroxy-, and 4-hydroxyphenylalanine (tyrosine), while the hydroxylation of tyrosine results in 2,3- and 3,4-dihydroxyphenylalanine (dopa). Yields are determined as a function of pH and the presence or absence of oxidants. During gamma-radiolysis and the Fenton reaction the same hydroxylated products are formed. The final product distribution depends on the rate of the oxidation of the hydroxyl radical adducts (hydroxycyclohexadiene radicals) relative to the competing dimerization reactions. The pH profiles for the hydroxylations of phenylalanine and tyrosine show a maximum at pH 5.5 and a minimum around pH 8. The lack of hydroxylated products around near pH 8 is due to the rapid oxidation of dopa to melanin. The relative abilities of iron chelates (HLFe(II) and HLFe(III) to promote hydroxyl radical formation from hydrogen peroxide are nitrilotriacetate (nta) greater than ethylenediaminediacetate (edda) much greater than hydroxyethylethylenediaminetriacetate greater than citrate greater than ethylenediaminetetraacetate greater than diethylenetriaminepentaacetate greater than adenosine 5'-triphosphate greater than pyrophosphate greater than adenosine 5'-diphosphate greater than adenosine 5'-monophosphate. The high activity of iron-nta and -edda chelates is explained by postulating the formation of a ternary Fe(III)-L-dopa complex in which dopa reduces Fe(III). The hydroxylations of phenylalanine and tyrosine are similar to that of salicylate (Z. Maskos, J. D. Rush, and W. H. Koppenol, 1990, Free Radical Biol. Med. 8, 153-162) and tryptophan (preceding paper) in that oxidants augment the formation of hydroxylated products by catalyzing the dismutation of hydroxyl radical adducts to the parent compound and a stable hydroxylated product. A comparison of salicylate and the amino acids tryptophan, phenylalanine, and tyrosine clearly shows that salicylate is the best indicator of hydroxyl radical production.


Chlorohydrin formation from unsaturated fatty acids reacted with hypochlorous acid. Stimulated neutrophils produce hypochlorous acid (HOCl) via the myeloperoxidase-catalyzed reaction of hydrogen peroxide with chloride. The reactions of HOCl with oleic, linoleic, and arachidonic acids both as free fatty acids or bound in phosphatidylcholine have been studied. The products were identified by gas chromatography-mass spectrometry of the methylated and trimethylsilylated derivatives. Oleic acid was converted to the two 9,10-chlorohydrin isomers in near stoichiometric yield. Linoleic acid, at low HOCl:fatty acid ratios, yielded predominantly a mixture of the four possible monochlorohydrin isomers. Bischlorohydrins were also formed, in increasing amounts at higher HOCl concentrations. Arachidonic acid gave a complex mixture of mono- and bischlorohydrins, the relative proportions depending on the amount of HOCl added. Linoleic acid appears to be slightly more reactive than oleic acid with HOCl. Reactions of oleic and linoleic acids with myeloperoxidase, hydrogen peroxide, and chloride gave chlorohydrin products identical to those with HOCl. Lipid chlorohydrins have received little attention as products of reactions of neutrophil oxidants. They are more polar than the parent fatty acids, and if formed in cell membranes could cause disruption to membrane structure. Since cellular targets for HOCl appear to be membrane constituents, chlorohydrin formation from unsaturated lipids could be significant in neutrophil-mediated cytotoxicity.


Presence of an essential lysine residue in a GDP-fucose protected site of the alpha 1----3fucosyltransferase from human small cell lung carcinoma NCl-H69 cells. The NCI-H69 cell alpha 1----3fucosyltransferase has been purified from a 0.2% Triton X-100R solubilized enzyme fraction by GDP-hexanolamine-Sepharose affinity chromatography and Superose 12 gel filtration. Photoaffinity labeling experiments with 125I-GDP-hexanolaminyl-4-azidosalicylic acid present in concentrations equivalent to 0.5 and 1 times Ki of the inhibitor for the enzyme indicated that labeling of the 45-kDa protein band could be inhibited by addition of 400 microM GDP-fucose but was not effected by similar concentrations of either GDP-mannose or GDP-glucose. The purified enzyme was applied to studies intended to define catalytically essential amino acid residues of the protein. Incubation of the enzyme in the presence of increasing concentrations of pyridoxal 5'-phosphate was found to result in irreversible inactivation of the enzyme after NaBH4 reduction. The donor substrate, GDP-fucose, was found to protect the enzyme from inactivation. Little or no protection was found for either GDP-mannose or the acceptor substrate nLc4. Pyridoxal 5'-phosphate was shown to behave as a competitive inhibitor with respect to GDP-fucose with a Ki of 105 microM. Labeling with 3H-pyridoxal 5'-phosphate resulted in the incorporation of approximately 8 mol pyridoxal 5'-phosphate per mole subunit. Parallel experiments containing GDP-fucose indicated protection of one site per subunit correlated with GDP-fucose binding. Acid hydrolysis and chromatographic analysis of the 3H-pyridoxylated protein indicated greater than 95% of the 3H label was recovered as pyridoxyl-lysine irrespective of whether GDP-fucose was present or not during labeling. These studies indicate the presence of a catalytically essential lysine residue associated with GDP-fucose binding to this enzyme. This information will be of value in further studies of this and other alpha 1----3fucosyltransferases and may suggest a practical basis for modulation of enzyme activity in the cell.


When are metal ion-dependent hydroxyl and alkoxyl radical adducts of 5,5-dimethyl-1-pyrroline N-oxide artifacts? The formation of the 5,5-dimethyl-1-pyrroline N-oxide (DMPO)/.OH adduct of the spin trap DMPO has been reported to occur through nucleophilic addition of water in the presence of aqueous ferric chloride (K. Makino, T. Hagiwara, A. Hagi, M. Nishi, and A. Murakami, 1990, Biochem. Biophys. Res. Commun. 172, 1073-1080). Due to the serious implications of these findings with respect to many spin trapping studies, the suitability of DMPO as a hydroxyl radical spin trap was studied in typical Fenton systems. Using 17O-enriched water, we show conclusively that nucleophilic addition of water occurs at the nitrone carbon (or C-2 position) of DMPO in the presence of either Fe or Cu ions. Furthermore, our results demonstrate that this nucleophilic reaction is a major pathway to the DMPO/.OH adduct, even during the reaction of Fe(II) or Cu(I) with hydrogen peroxide. Primary alkoxyl adducts of DMPO also form in aqueous solution through nucleophilic addition in the presence of both Fe(III) and Cu(II). Attempts to obtain secondary and tertiary alkoxyl adducts by this mechanism were unsuccessful, possibly due to steric effects. When the reaction is carried out in various buffers, however, or in the presence of metal ion chelators, nucleophilic addition to DMPO from Fe(III) is effectively suppressed. Chelators also suppress the reaction with Cu(II). Hence, under most common experimental conditions in biochemical free radical research, nucleophilic addition to DMPO should not be of major concern.


Evidence against the 1:2:2:1 quartet DMPO spectrum as the radical adduct of the lipid alkoxyl radical. It was reported that the electron paramagnetic resonance (EPR) spectrum of 5,5-dimethyl-1-pyrroline N-oxide (DMPO)/lipid alkoxyl radical exhibited a quartet with 1:2:2:1 relative intensity that is identical to that of DMPO/hydroxyl radical (K. M. Schaich and D. C. Borg, 1990, Free Radicals Res. Commun. 9, 267-278). We repeated these EPR experiments using HPLC separation of radical adducts and isotope substitution. We found that the HPLC/EPR chromatogram of the radical adduct with a 1:2:2:1 quartet obtained by the reduction of methyl linoleate hydroperoxide (MLOOH) with Fe2+ exhibited identical retention time to that of the DMPO/OH radical adduct obtained from the Fenton reaction in two different solvent systems. Upon performing the same reaction in 17O-enriched water, the 17O-hyperfine coupling constants due to DMPO/17OH were identified. Ultimately, approximately 80-90% of the total DMPO/OH is derived from water by an iron-dependent nucleophilic addition reaction. Initially, a water-independent mechanism also significantly contributes to DMPO/OH formation. Regardless of its mechanism of formation, the 1:2:2:1 quartet radical adduct of DMPO formed during the reduction of MLOOH by Fe2+ is in fact DMPO/OH.


Interaction of rabbit muscle enolase and 3-phosphoglycerate mutase studied by ELISA and by batch gel filtration. The interaction of rabbit skeletal muscle enolase and 3-phosphoglycerate mutase was detected by an ELISA test, a batch gel-filtration technique, and fluorescence anisotropy measurements, and the activity of enolase was determined to be a function of mutase concentration. The apparent dissociation constant of this enzyme complex is approximately 1 microM. This value seems to be independent of the presence (in fluorescence anisotropy measurements) or the absence (in activity as well as in ELISA experiments) of fluorescein isothiocyanate used widely as a label for determining the complex formation between enzymes in fluorescence anisotropy measurements.


Purification of interleukin-1 beta converting enzyme, the protease that cleaves the interleukin-1 beta precursor. We have purified the IL-1 beta converting enzyme from the THP-1 cell line using standard chromatographic techniques and obtained the N-terminal amino acid sequence of this novel protein. After stimulation of THP-1 cells with lipopolysaccharide, hydroxyurea, and silica, the protease was solubilized by multiple freeze/thawing. The protein was purified by ion-exchange chromatography, affinity chromatography on blue agarose, gel filtration, and chromatofocusing. The molecular weight of the protein is approximately 22,000 Da and the pI is between 7.1 and 6.8. The overall yield for this procedure was 16% of the activity found in the initial cell lysates. An antiserum raised against a peptide based on the N-terminus was used to precipitate the protease, confirming our identification of the 22,000-Da protein as the IL-1 beta converting enzyme.


Evaluation of the contralateral breast. The role of biopsy at the time of treatment of primary breast cancer. Ninety-five women who underwent blind contralateral breast biopsy during surgical treatment of a known breast cancer primary were studied prospectively. All biopsies were performed between 1981 and 1989. Patients with palpable or mammographic abnormalities prompting the contralateral biopsy were excluded so that the study sample included only truly blind contralateral biopsies. Only two infiltrating carcinomas were found, resulting in a positive biopsy rate of 2.1% for invasive disease. Three additional biopsies showed only lobular carcinoma in situ, a finding that usually does not alter clinical management. One patient with a negative contralateral biopsy developed invasive carcinoma in that breast within 2 years of the biopsy. The authors were unable to identify any subgroup of patients at increased risk of a positive contralateral biopsy. These results suggest that blind biopsy of the contralateral breast performed at the time of the initial treatment of breast carcinoma is not an efficient method of cancer detection. Alternative management strategies are discussed.


GABA receptors in Deiters nucleus modulate posturokinetic responses to cortical stimulation in the cat. The early component of the postural responses which accompany the limb flexion during unilateral stimulation of the motor cortex in the cat is not of reflex origin, but results from a central command. These postural adjustments are characterized by a decreased force under the limb diagonally opposite to the moving one and an increased force under the other two. Since the lateral vestibular nucleus (LVN) exerts an excitatory influence on ipsilateral limb extensor motoneurons, experiments were performed in cats to establish whether the cortical-induced postural changes were mediated through the LVN. This structure is tonically inhibited by GABAergic synapses originating from Purkinje cells of the cerebellar vermis, so that local microinjection into the LVN of GABA agonists or antagonists should either decrease or increase the spontaneous discharge of their neurons. Unilateral microinjection of 0.25 microliters of the GABA-A agonist muscimol or the GABA-B agonist baclofen (at 2-4 micrograms/microliters saline) into the LVN produced a short-lasting episode of ipsilateral postural hypotonia and contralateral hypertonia, during which the cats were unable to stand on the measurement platform. When, shortly after, some recovery of the postural activity appeared, no changes in threshold, latency or amplitude of the cortical-induced flexion movement were observed; however, the early component of the postural responses decreased in the other three limbs. Moreover, the slope of the response curve of the moving limb remained unmodified, while that of the early component of the postural responses, which involved the remaining limbs, decreased following stimulation of the motor cortex at different stimulus intensities. These effects started a few min after the injection and lasted for about 2-3 h. The effects described above were dose-dependent. Moreover, histological controls indicated that the structure responsible for these postural changes corresponded to the middle part of the LVN. The specificity of the results was shown by the fact that unilateral microinjection of 0.25 microliters of the GABA-A antagonist bicuculline or the GABA-B antagonist phaclofen (at 5-8 micrograms/microliter saline) into the LVN produced a postural asymmetry opposite in sign to that elicited in the same experiments by the corresponding agonists. These injections did not modify the amplitude of the cortical-induced limb flexion, but rather enhanced the amplitude of the early component of the postural responses in the other three limbs.(ABSTRACT TRUNCATED AT 400 WORDS)


Hepatocellular carcinoma: MR imaging.

Hepatocellular carcinoma: MR imaging. Thirty patients with hepatocellular carcinoma (HCC) were examined by MRI. Distinctive findings of HCC such as the presence of fatty degeneration, a capsule, mosaic pattern, daughter nodules and tumor thrombi in major veins were noted in 6, 13, 1, 10 and 9 cases or 20%, 43%, 3%, 33% and 30% respectively.


Does the extent of surgery make a difference in high grade malignant astrocytoma? This paper examines the effect of patient age, tumour grade and extent of surgery on the outcome of treatment of 278 patients with high grade malignant gliomas referred to the Queensland Radium Institute between 1980 and 1987. The aim was to determine whether the extent of surgical resection alters survival rates. The extent of surgery had no effect on survival except for those patients with grade 3 tumours in whom a total excision was possible. Those in whom only a biopsy was done did not have a worse prognosis. Grading was found to be of importance, as patients with grade 3 tumours had a better survival than those with grade 4 tumours. In grade 4 tumours, those under 30 years of age had a better survival than those over 30 years, whereas with grade 3 tumours there was a gradation of age effect (under 40 years best, then 40-49 years, and those 50 years and over doing worst.


Computer modeling of two inorganic pyrophosphatases. The yeast Saccharomyces cerevisiae has two inorganic pyrophosphatases that are structurally related. One, PPA1, is a cytoplasmic enzyme. The other, PPA2, is located in the mitochondria and appears to be energy-linked. The sequence similarity of PPA1 and PPA2 is about 66% and the identity is about 50%. All amino acids known to be important for catalysis are conserved, except one glutamate which is substituted by an aspartate in PPA2. The structures of PPA2 and the cytoplasmic PPase from Schizosaccharomyces pombe were modeled based on the three dimensional structure of PPA1. Two cysteines in PPA2 and one in the S. pombe enzyme are located at the catalytic cleft. Four residues form an unique insertion near the entrance of the catalytic cleft in the mitochondrial enzyme.