Multiple heteroreceptors on limbic thalamic axons: M2 acetylcholine, serotonin1B, beta 2-adrenoceptors, mu-opioid, and neurotensin. Ligand binding to many transmitter receptors is much higher in layer Ia of rat posterior cingulate cortex than it is in other layers, and this is where most axons from the anterior thalamus terminate. The present study explores the possibility that a number of receptors may be expressed on axons from limbic thalamic nuclei that terminate in layer Ia. Unilateral thalamic lesions were placed in rats and, 2 weeks later, five ligand binding protocols, coverslip autoradiography, and single grain counting techniques were used to quantify binding in control and ablated hemispheres. Binding to the following receptor subtypes was analyzed: M2 acetylcholine, 3H-oxotremorine-M, or 3H-AF-DX 116 with 50 nM pirenzepine; serotonin1B, 125I-(-)-cyanopindolol with 30 microM isoproterenol; beta 2-adrenoceptors, 125I-(-)-cyanopindolol with 1 microM serotonin and 10 microM atenolol; mu-opioid, 3H-T[r-D-Ala-Gly-MePhe-Gly-ol; neurotensin, 3H-neurotensin. Thalamic lesions reduced binding in two laminar patterns. In one pattern, there was a major reduction in binding in most superficial layers with that in layer Ia ranging from 50 to 70% for binding to M2 muscarinic and serotonin1B receptors. Binding to beta 2-adrenoceptors was also reduced in most superficial layers but to a lesser extent. In the second pattern, reductions were limited to layer I with losses in layer Ia of 20-30% for mu-opioid and neurotensin receptors. In no instance was layer Ia binding completely abolished (i.e., postlesion peaks remained). Since the transmitters for each of the five receptors analyzed in this study are not synthesized by anterior or laterodorsal thalamic neurons, these receptors are heteroreceptors. The greatest postlesion reduction in M2 binding was for AF-DX 116 and so most M2 heteroreceptors are of the "cardiac" subtype. Finally, the diverse population of heteroreceptors on limbic thalamic axons provides for presynaptic modulation by a wide range of transmitter systems and suggests that thalamocortical transmission may not be a simple, unmodulated event.


Activators of protein kinase C increase the phosphorylation of the synapsins at sites phosphorylated by cAMP-dependent and Ca2+/calmodulin-dependent protein kinase in the rat hippocampal slice. Previous studies have shown that activators of protein kinase C (C kinase) produce synaptic potentiation in the hippocampus. For example, the C kinase activator phorbol dibutyrate has been shown to increase transmitter release in the hippocampus. In addition, a role for C kinase in long-term potentiation has been proposed. A common assumption in such studies has been that substrates for C kinase were responsible for producing these forms of synaptic potentiation. However, we have recently shown that phorbol dibutyrate increased the phosphorylated of synapsin II (formerly protein III, Browning et al., 1987) in chromaffin cells (Haycock et al., 1988). Synapsin II is a synaptic vesicle-associated phosphoprotein that is a very poor substrate for C kinase but an excellent substrate for cAMP-dependent and Ca2+/calmodulin-dependent protein kinase. We felt, therefore, that activation of C kinase might lead to activation of a kinase cascade. Thus effects of C kinase activation might be produced via the phosphorylation of proteins that are not substrates for C kinase. In this report we test the hypothesis that activators of C kinase increase the phosphorylation of synapsin II and an homologous protein synapsin I. Our data indicate that PdBu produced dose-dependent increases in the phosphorylation of synapsin I and synapsin II. We also performed phospho-site analysis of synapsin I using limited proteolysis. These studies indicated that PdBu increased the phosphorylation of multiple sites on synapsin I. These sites have previously been shown to be phosphorylated by both cAMP-dependent protein kinase and the multifunctional Ca2+/calmodulin-dependent protein kinase II.(ABSTRACT TRUNCATED AT 250 WORDS)


Evaluation of a protease activation mutant of Sendai virus as a potent live vaccine. A protease activation mutant of Sendai virus, TR-5, was investigated as a candidate for a live vaccine. Vaccination with TR-5 which had been activated by chymotrypsin beforehand (active TR-5) elicited protective immunity against otherwise lethal challenge infection with wild-type Sendai virus in DBA/2, C3H and ICR strains of mice. Less of the active TR-5 was required to confer protection on mice compared with an ordinary ether-inactivated Sendai virus vaccine (split vaccine). The protective immunity elicited by TR-5 lasted longer and the booster effect was more prominent compared to the split vaccine. No seroconversion was observed with contact mice when housed in a cage with mice vaccinated with the active TR-5. The overall results show that the active TR-5 is an effective and safe live vaccine of Sendai virus in mice.


Rapid identification and differentiation of the vaccine strain Rac H from EHV 1 field isolates using a non-radioactive DNA probe. A method for rapid differentiation between the EHV 1 live vaccine strain Rac H and field isolates is described. Total DNA was isolated from virus-infected small scale cell cultures. DNA fragments digested with restriction endonuclease BamHI were separated, transferred and immobilized on filter membranes. A Digoxigenin-labeled probe derived from EHV 1 was used for hybridization. This probe hybridized specifically to sequences of the inverted terminal repeat region which in case of Rac H include a deletion of 0.8 kb. By comparing the different migration patterns after blot hybridization it could be shown that in 65 isolates from cases of abortion the live vaccine strain Rac H was not involved.


Application of the polymerase chain reaction to the detection of bovine viral diarrhea virus infections in cattle. We used the polymerase chain reaction (PCR) technique to detect bovine viral diarrhea virus (BVDV) infections in cattle. Of 120 cattle screened in this study, 29 were scored positive for BVDV with both PCR and conventional virus isolation. Ninety cattle were negative in both assays. One cow was scored positive for BVDV with the PCR but was negative with virus isolation. In dilution experiments PCR analysis was at least 10 times more sensitive than BVDV isolation.


Induction of host cell protein synthesis by human herpesvirus 6. We observed an increase in host cell protein synthesis in human cord blood lymphocytes (CBL) infected with human herpesvirus 6 relative to uninfected cultures. The magnitude of this effect could not be explained by a smaller decrease in cell number in the infected cultures. The induction of host cell protein synthesis by HHV-6 does not appear to be mediated by a stable soluble factor present in the infected cell culture supernatant. When CBL were infected with virus that had been exposed to ultraviolet irradiation (UV) for various intervals, we found that the level of increase in cell number, host protein synthesis, viral DNA and viral antigen was inversely proportional to the length of time of virus exposure to UV. No increase in cell number or host cell protein synthesis was seen in CBL infected in the presence of 50 micrograms/ml phosphonoacetic acid, an inhibitor of HHV-6 DNA replication. These results indicate that components of input virions do not induce the increased protein synthesis and that the induction is dependent on viral DNA replication.


Expression of herpes simplex virus type 1 glycoprotein B in insect cells. Initial analysis of its biochemical and immunological properties. A recombinant baculovirus (vAc-gB1) was constructed which expresses the glycoprotein B (gB) gene of herpes simplex virus type 1 (HSV-1). When Sf9 cells were infected with these recombinant viruses, a protein that was close in size to authentic HSV-1 gB was detected by gB polyclonal antibody. The recombinant gB was found on the membrane of Sf9 cells and was susceptible to tunicamycin, glycosidase F (PNGase F) and partially susceptible to Endo-H. Antibodies raised in mice to this recombinant recognized viral gB and neutralized the infectivity of HSV-1 in vitro. Mice inoculated with the recombinant gB were protected from lethal challenge with HSV-1.


Evolution of the fusion protein gene of human parainfluenza virus 3. The nucleotide sequences of the fusion (F) gene of 15 clinical strains of human parainfluenza virus 3 (HPIV3) isolated between 1959 and 1987 were compared with the F gene sequence of the prototype strain, Wash/47885/57. Nucleotide sequence diversity was greatest in the noncoding regions of the F gene; however, regions believed to function as transcriptional signals were completely conserved. Amino acid sequences were highly conserved and all but a few amino acid substitutions were conservative in nature. Sequence comparisons indicate heterogeneity in HPIV3 F genes; however, a significant proportion of nucleotide changes are maintained after they first appear and seem to be accumulating with time. Phylogenetic analysis suggests that there are 2 lineages of HPIV3 in North America. The two lineages can be distinguished by specific amino acid differences in the F protein, which correlate with differences in antigenic properties and neutralization patterns of HPIV3. The pattern of HPIV3 evolution, based on the analysis of F gene sequences, most closely resembles that of influenza virus B, vesicular stomatitis virus and Newcastle disease virus.


Liver resection for primary neoplasms.

Liver resection for primary neoplasms. Resection is the treatment of choice for almost all hepatic neoplasms. Improvements in operative techniques and perioperative patient management have resulted in low morbidity and mortality. Patients with underlying chronic liver disease in association with hepatic malignancy now can be offered either subsegmental resection or liver transplantation in selected cases. The role of cryosurgery and alcohol injection in patients who are not candidates for resection should become clearer once longer follow-up data are available.


Search for Coxsackievirus B3 RNA in idiopathic dilated cardiomyopathy using gene amplification by polymerase chain reaction. A polymerase chain reaction (PCR) amplification assay was developed to detect Coxsackievirus B3 ribonucleic acid (RNA) in blood and myocardial tissue of explanted hearts from 40 patients who underwent cardiac transplantation and in 1 normal heart. Twenty-one patients were affected by idiopathic dilated cardiomyopathy of different duration and 19 by coronary artery disease. Coxsackievirus B3 in vitro infected Vero cells and cells infected by related human enteroviruses (Coxsackievirus B2, B4, and poliovirus 1) were used as reaction controls. PCR was performed using 4 pairs of primers homologous to Coxsackie-virus B3 sequences. Three sets were located in regions of the genome conserved at nucleotide level between several enterovirus species (replicase gene, 5' noncoding region), while one was located in a Coxsackievirus B3-specific region (VP1 gene). Total RNA was prepared by acid guanidinium isothiocyanate extraction from tissue stored frozen at -80 degrees C. One microgram of total RNA was retrotranscribed with either antisense primer or with random hexanucleotide primers and then subjected to 40 cycles of amplification. PCR products were separated by electrophoresis on a 10% polyacrylamide gel, electrotransferred to a nylon membrane and then hybridized to oligonucleotide probes specific for the coxsackievirus B3 genome radiolabeled with radioactive isotope of phosphorous. All pairs of primers yielded specific amplification products when tested on Coxsackievirus B3-infected Vero cells, with a sensitivity of 1 infected cell out of 10(5) to 10(6) cells starting from 1 microgram total RNA. Primer sets for regions of Coxsackievirus B3 genome highly conserved between related enteroviral species gave positive amplification also when challenged with RNA from cells infected by Coxsackievirus B2, B4 and poliovirus 1.


Ewing's bone sarcoma, paternal occupational exposure, and other factors. To determine risk factors for Ewing's bone sarcoma, the authors interviewed mothers of 43 patients diagnosed between January 1978 and August 1986 and 193 controls in the San Francisco Bay Area, California, regarding medical and occupational history of parents and other factors related to the subjects and their immediate families. Controls were selected by using random digit dial telephone methods. Adjusted relative risk estimates suggest that risks were elevated for children whose fathers were engaged in agricultural occupations during the period from 6 months prior to conception of the subject up to the time of diagnosis for the patients or interview for the controls (relative risk (RR) = 8.8, 95% confidence interval (CI) 1.8-42.7) and for children whose fathers had occupational exposure to herbicides, pesticides, or fertilizers (RR = 6.1, 95% CI 1.7-21.9, p = 0.002). Prior ingestion of poison or an overdose of medication was more common in patients than in controls (RR = 4.4, 95% CI 1.4-13.5). These and other findings should be investigated in larger population-based studies to determine specific factors that may account for the associations.


Prospects for the primary prevention of breast cancer. In this paper, the rationale, stage of development, and known or potential adverse effects of three potential strategies for the prevention of breast cancer are reviewed. Two methods--the use of tamoxifen in postmenopausal women and the use of luteinizing hormone (LH)-releasing hormone agonists in premenopausal women--involve hormonal manipulation. In the premenopausal period, the goal is to reduce the number of ovulatory menstrual cycles a woman experiences in order to reduce her exposure to estrogen and progesterone. Physical activity during adolescence is proposed as a nonhormonal method of accomplishing this. The use of LH-releasing hormone agonists to produce a reversible menopause can also reduce a woman's cumulative exposure to ovarian steroid hormones. Tamoxifen, which is effective in breast cancer therapy, provides endocrine control of estrogen-regulated breast tumor growth. Breast cancer chemoprevention trials using tamoxifen among postmenopausal women have been proposed, and pilot studies are under way.


Cigarette smoking and the risk of anogenital cancer. The association between cigarette smoking and cervical cancer has been demonstrated in numerous prior studies. As part of population-based case-control studies of cancers of the vulva, vagina, cervix, anus, and penis in relation to infection with human papillomavirus, conducted in western Washington State and the province of British Columbia from the mid 1980s until the present time, the authors have collected detailed information on smoking history. The proportion of subjects who were current smokers of cigarettes ranged from slightly over 40% among incident cases of vaginal and cervical cancer to 60% among cases of vulvar and anal cancer. In contrast, only about 25% of controls were current smokers. The adjusted odds ratios (OR) associated with current smoking were substantially elevated (OR = 1.9-14.6) for all cancer sites except cancer of the vagina (OR = 1.3). The risks tended to increase in proportion to the number of cigarettes smoked. For most cancer sites, the odds ratios associated with former smoking were substantially less than those associated with current smoking and diminished with increasing time since cessation of smoking. The authors' data and those of other investigators suggest that cigarette smoking plays a role in the etiology of anogenital cancers and that smoking has a late-stage or promotional effect.


Plasma thrombomodulin as a marker of vascular disorders in thrombotic thrombocytopenic purpura and disseminated intravascular coagulation. Plasma thrombomodulin (TM) levels were significantly elevated at disease onset in patients with thrombotic thrombocytopenic purpura (TTP) and disseminated intravascular coagulation (DIC), but was not in those with essential thrombocythemia and idiopathic thrombocytopenic purpura. However, in patients with TTP and DIC, TM levels decreased significantly after they achieved complete remission. In both TTP and DIC patients, plasma TM levels at onset in those with poor prognosis were higher than that in those with good prognosis. Among DIC patients, the plasma TM level was higher in those with organ failure than in those without, but there were no differences among patients with various underlying diseases associated with DIC. It is speculated that the plasma TM level reflects damage to vascular endothelial cells or organ failure and that it is useful in assessing prognosis for patients with DIC and TTP.


Deficiency of P62, a putative collagen receptor, in platelets from a patient with defective collagen-induced platelet aggregation. Recently, we described a platelet antibody against a putative collagen receptor (P62), which was found in a patient with idiopathic thrombocytopenic purpura (ITP) (Blood 69:1712). We now report a deficiency of the P62 receptor in a young man whose platelets showed defective collagen-induced platelet aggregation. He had a mild bleeding tendency and slight thrombocytopenia. The results of coagulation and fibrinolysis studies were normal. The patient's platelets were partially unresponsive to collagen, although aggregation in response to ADP, thrombin, ristocetin, and calcium ionophore (A23187) was almost normal. Adhesion of his platelets to bovine collagen was markedly reduced. Addition of collagen caused no synthesis of thromboxane (TX)B2 in platelet rich plasma (PRP) from this patient. Furthermore, collagen produced no rise of cytosolic free calcium ([Ca2+]i) in fura2-loaded platelets. In contrast, thrombin caused TXB2 formation and an increase of [Ca2+]i in his platelets. These results suggest defective interaction between the platelets and collagen. The IgG from the ITP-patient induced irreversible aggregation in normal PRP, but caused no aggregation of the young man's platelets. Immunoblot studies showed that normal platelets had antigens with a molecular weight of 62 KDa under reducing conditions and of 57 KDa under nonreducing conditions. In contrast, the young man's platelets had no P62 band, although GPIa/IIa and thrombospondin were normally present. These results indicate that impaired collagen-induced aggregation in the patient's platelets was due to a deficiency of P62 and confirm that P62 may play a crucial role as a collagen receptor in platelet activation.


Expression of high affinity binding sites for erythropoietin on L8057 cells, a mouse megakaryoblastic cell line, associated with cell differentiation. In this study, specific binding sites were examined for erythropoietin (EPO) on the mouse leukemic cell line, L8057. This cell line is megakaryoblastic in origin as evidenced by an enlargement of cell size, multinuclearity, intense activity of acetylcholinesterase, more expression of glycoprotein IIb and IIIa antigen, and higher ploidy distribution after the treatment with 12-o-tetradecanoylphorbor-13-acetate (TPA). The original undifferentiated cells possessed a single class of low affinity binding sites for recombinant human (rh) EPO with a Kd of 3.5 nM. Following the treatment with TPA, high affinity binding sites (Kd; 440 pM) were expressed in addition to the low affinity sites. EPO stimulated the incorporation of 3H-leucine into TPA-treated L8057 cells, and the maximal effect of EPO was observed at the same order as the Kd value of high affinity sites. The present data demonstrates that the expression of high affinity binding sites for EPO is associated with the differentiation of L8057 cells which have megakaryocytic characteristics. Furthermore, protein synthesis stimulated by EPO may be mediated through the high affinity sites.


Acute megakaryoblastic leukemia with translocation t(1;22)(p13;q13) in a 10-week-old infant. A 10-week-old girl without Down syndrome developed an acute megakaryoblastic leukemia (AMKL). Bone marrow aspirates and biopsy showed megakaryoblastic infiltration with myelofibrosis. The diagnosis was made based on the findings that the positive reactions of leukemic cells to platelet peroxidase and to monoclonal antibodies which recognize platelet-specific surface glycoprotein (GP) IIb/IIIa and GP78. The blasts also showed myeloid and monocytoid differentiation antigens. The leukemic cells had a karyotype of 46,XX,t(1;22)(p13;q13). Our case and two other infantile cases reported by other investigators establish the novel association of the t(1;22) with AMKL.


Fibrous minerals in the lungs of mesothelioma patients: comparison between data on SEM, TEM, and personal interview information. To determine and compare the fiber types and size distributions in the lung tissue of mesothelioma patients in Finland, samples from 29 patients with known work history were analyzed with transmission electron microscopy (TEM) and X-ray microanalysis. Compared with the earlier results using scanning electron microscopy (SEM), the fiber concentrations were about three times as high and ranged from 0.1 million to 5,200 million fibers per gram of dry tissue. In 15 patients (52%), crocidolite/amosite were the dominating fiber types, representing more than 70% of all fibers. Anthophyllite asbestos was the most prevalent fiber type in eight patients (28%), and it was found in the samples of 13 patients (45%). One-half of the anthophyllite fibers were longer than 5 microns, whereas other fiber types were somewhat smaller.


Acrylamide monomer and peripheral neuropathy in chemical workers. The relationship between exposure to acrylamide monomer and neurological outcomes was investigated in 82 chemical industry workers. Vibrotactile thresholds were determined quantitatively with a Vibratron II device, using forced choice and method of limits procedures. Symptoms of numbness, limb pain, peeling skin, and sweating hands had significantly higher prevalences in the exposed than in the unexposed group. Signs of peeling skin and sweating hands were significantly more common in the exposed group. Vibration thresholds were not associated with exposure.


Gelsolin gene mutation--at codon 187--in familial amyloidosis, Finnish: DNA-diagnostic assay. Familial amyloidosis, Finnish (FAF), is an autosomal dominant form of systemic amyloidosis with lattice corneal dystrophy and progressive cranial neuropathy as principal clinical manifestations. We have shown that the novel amyloid fibril protein found in these patients is an internal degradation fragment of gelsolin, an actin-binding protein, and that it contains an amino acid substitution, asparagine for aspartic acid at position 15, that is due to a guanine-to-adenine transversion corresponding to codon 187 of human plasma gelsolin cDNA. To test that this mutation cosegregates with the disease high-molecular-weight genomic DNA was isolated from autopsied tissues or lymphocytes of 23 patients, 6 healthy relatives and 20 unrelated healthy control persons. Specific fragments were amplified with the polymerase chain reaction for oligonucleotide hybridization analysis using the slot-blot technique. The guanine-to-adenine transversion was found in all FAF patients tested, but in none of the control subjects. Our results show that the mutation (G to A) cosegregates with the disease phenotype, and that the slot-blot analysis can be used as a diagnostic assay, including prenatal evaluation.


Fatal maternal laryngeal papillomatosis in pregnancy: a case report [corrected]. Although genital condylomata worsen with pregnancy, we are unaware of any reports of fatal maternal laryngeal papillomatosis. A maternal death at 20 weeks' gestation of a patient with chronic laryngeal papillomatosis is described. Pregnant patients with a history of this disorder who have symptoms should be considered candidates for immediate evaluation.


Evaluating the treatment of sexually transmitted diseases at an urban public hospital outpatient clinic. Sexually transmitted diseases occur disproportionately among the poor, are often treated in public hospitals and clinics, and have not been subjected to quality-of-care evaluation. We designed a medical record abstraction system using well-established, specific process-of-care criteria drawn from the medical literature and experts and grouped into three levels of quality: excellent, adequate, and minimal. One hundred seventy-six consecutive patients were identified from the clinic logbook and their medical records abstracted. Deficiencies in history taking, physical examination, laboratory testing, treatment, and public health reporting were identified.


Evaluation of a self-help dietary intervention in a primary care setting. Dietary intervention to reduce fat consumption and increase fiber consumption has been recommended by the National Cancer Institute, but there is little evidence concerning the effectiveness of self-help materials. The purpose of this study was to evaluate such self-help materials, introduced by a nurse in a primary care setting. A randomized controlled trial involving 242 subjects was conducted in two primary care clinics in Chapel Hill, NC, in 1987. Changes in fat and fiber consumption in the intervention and control groups during the 3-month interval between interviews were compared using analysis of covariance. The estimated reduction in fat was 3.8g larger for the intervention group than for the control group, but the confidence interval included zero. For those individuals who had some responsibility for meal preparation there was a larger difference (-6.9g) in favor of the intervention group, although the difference using calorie-adjusted values was -3.8g with a 95% confidence interval (-7.1, -0.4). The differences for fiber change were smaller. We found significant small but consistent differential changes associated with a minimal self-help intervention, but we cannot rule out the possibility of some response bias. Nonetheless, this study demonstrates that the use of self-help materials for dietary change is feasible, and may be effective.


Nutrition: the need to define "optimal" intake as a basis for public policy decisions. Much of the current chronic disease incidence, notably coronary artery disease and certain types of cancer, relates to nutritionally induced metabolic overload. The evidence for this conclusion is based on extensive analytical, descriptive, and metabolic epidemiological investigations as well as critical experimental studies in animals. Dietary assessments within homogenous populations, because of inherent measurement errors and similarities in eating habits, make only limited contributions to this issue. This communication makes recommendations as to an "optimal" diet, especially for fats and fibers, as a goal for effective disease prevention that is within our reach with the appropriate support of an informed public and a cooperative food industry. To facilitate effective public health action, the "optimal" diet is called the 25/25 diet, that is, 25% of calories as fat and 25 g per day of fiber.


Daphnetin: a novel antimalarial agent with in vitro and in vivo activity. Daphnetin is a dihydroxycoumarin that is being used in China for the treatment of coagulation disorders. It is also a chelator and an antioxidant. In vitro, daphnetin causes a 50% inhibition (IC50) of 3H-hypoxanthine incorporation by Plasmodium falciparum at concentrations between 25 and 40 microM. Several related compounds, such as scopoletin, 2, 3-dihydroxybenzoic acid and 3, 4-dihydroxybenzoic acid show no inhibitory activity. The antimalarial activity of daphnetin is inhibited by the addition of iron. Daphnetin does not appear to be an oxidant drug, since it does not spontaneously generate superoxide in vitro. However, it does alkylate bovine serum albumin when incubated in the presence of iron. In vivo, daphnetin significantly prolongs survival of P. yoelli-infected mice.


The epidemiology of hepatitis C virus antibody in Yemen. A cross-sectional survey of 348 subjects without evidence of liver disease was conducted to investigate the prevalence and risk factors for hepatitis C virus antibody (anti-HCV) seropositivity in the Yemen Arab Republic. The mean age of study subjects was 28.7 years (range 3-80), and 61% were males. Using commercial enzyme-linked immunosorbent assays (ELISA), 6.0% (95% confidence interval [CI] 3.8-9.1) of subjects were anti-HCV-positive, 13.5% were hepatitis B surface antigen-positive (HBsAg-positive), and 51.4% were positive for at least one serologic marker of prior hepatitis B infection. Nine (2.6%; 95% CI 1.2-4.9) of the 21 ELISA-positive sera were confirmed to be anti-HCV positive by a recombinant immunoblot assay. Anti-HCV seropositivity was significantly associated with age (odds ratio [OR] 2.0 for each 10-year increase in age) and prior surgery (OR 10.1), but was not associated with a history of prior blood transfusion or markers of hepatitis B infection. These preliminary data suggest that hepatitis C may pose a substantial health threat in Yemen.


Three-dimensional structure of cytidine monophosphatase reactive trans-Golgi elements in spinal ganglion cells of the rat. In order to analyse, at the electron microscope level, the three-dimensional configuration of the trans compartment of the Golgi apparatus rat dorsal root ganglia were treated to demonstrate cytidine monophosphatase (CMPase) activity. The localization of enzymatic activity in the Golgi apparatus varied according to cell types. In type A and C cells, CMPase was exclusively located in the transmost sacculotubular element, whereas in type B cells all the saccules of the stacks forming the Golgi ribbon and the trans-Golgi networks were impregnated. Numerous dense bodies seen at proximity were also CMPase positive. In 3 microns thick sections of type A cells examined at low magnification, the impregnated element was scattered throughout the cytoplasm and never formed a continuous structure. In type B cells, the strongly reactive trans-Golgi networks did not follow the entire length of the impregnated Golgi ribbon but were preferentially located in the concavity of its arched portions. At higher magnification and in all cell types some tubular portions of the trans-Golgi networks took the appearance of spheroidal cage-like structures, the CMPase positive anastomotic tubules forming the bars of the cage. Anastomotic tubules separated from the trans-Golgi networks formed fenestrated spheres, while nearby CMPase-reactive dense bodies exhibited a paler hilus. These observations were taken to indicate that in ganglion cells, some CMPase positive dense bodies, presumably lysosomes, formed by fragmentation of the trans-Golgi networks.


Massive diphenhydramine poisoning resulting in a wide-complex tachycardia: successful treatment with sodium bicarbonate. Diphenhydramine poisoning is characterized most often by anticholinergic effects. Cardiotoxicity and circulatory collapse have rarely been reported after massive ingestions of diphenhydramine and other H1-receptor-blocking agents, although these substances have local anesthetic properties and have been studied as antiarrhythmics. We report the case of a patient who developed a wide-complex tachycardia as a complication of acute diphenhydramine poisoning that responded to IV sodium bicarbonate.


DNA topoisomerase I from calf thymus mitochondria is associated with a DNA binding, inner membrane protein. During purification of the type I DNA topoisomerase from calf thymus mitochondria, two polypeptides, p78 and p63, cofractionate with the enzymatic activity (Lazarus et al., (1987) Biochemistry 26, 6195-6203). The two polypeptides are released from a mitochondrial inner membrane preparation by nonionic detergent lysis and both adsorb strongly to a single-stranded DNA agarose column. We have attempted to characterize the relationship between these two polypeptides and have found the following: (i) the mitochondrial topoisomerase is active in free (monomer) and associated (heterodimer) form; (ii) the catalytic activity resides solely in p78, as adjudged by both the covalent linkage of the enzyme to substrate DNA and the ability of the enzyme to relax supercoils; (iii) at low ionic strength the enzyme is active in monomer form with p78 alone being sufficient for activity; (iv) in high salt, the high molecular weight species is a 140-kDa heterodimer composed of one p78 and one p63; and (v) the two polypeptides are not structurally related as digestion with V8 protease results in distinct proteolytic fragment patterns. These results suggest that p63 may have an important role in the metabolism of the mitochondrial topoisomerase.


The bacterial hemoglobin from Vitreoscilla can support the aerobic growth of Escherichia coli lacking terminal oxidases. Two Escherichia coli mutants that lack both cytochrome o and d terminal oxidases are able to grow with glucose as the carbon source but not with the aerobic substrates succinate or lactate. One of these, GV101, is a deletion mutant of cytochrome o and a point mutation of cytochrome d. The other, GK100, is a total deletion mutant of all the genes for both cytochromes. When these mutants were transformed with a plasmid containing the gene for the bacterial hemoglobin from Vitreoscilla, they were capable of growth in the presence of succinate or lactate and showed aerobic respiration in the presence of these substrates, unlike the parent strains. Cells transformed with a plasmid containing the gene for the hemoglobin but lacking the native promoter did not express the hemoglobin and did not respire. Membrane vesicles prepared from the cells consumed oxygen in the presence of succinate. This succinate-supported respiration decreased with successive washings of the vesicles but was restored by adding E. coli cytosol containing the hemoglobin or by adding the hemoglobin purified from Vitreoscilla. This respiration was inhibited by cyanide.


Anomalous effect of uncouplers on respiratory chain-linked transhydrogenation in Escherichia coli membranes: evidence for a localized proton pathway? Energization of the pyridine nucleotide transhydrogenase in everted membrane vesicles from Escherichia coli JM83 was compared with the process in vesicles of the same strain transformed with the plasmid pDC21 overexpressing this enzyme. Proton translocation was assayed by the quenching of the fluorescence of the probe quinacrine. Agents able to discharge transmembrane proton gradients such as nigericin and the uncouplers 3,3',4',5-tetrachlorosalicylanilide and carbonyl cyanide m-chlorophenylhydrazone inhibited ATP-dependent transhydrogenation of NADP by NADH and discharged transmembrane proton gradients generated by transhydrogenation of AcNAD by NADPH, by oxidation of NADH, and by hydrolysis of ATP. This was observed in everted membrane vesicles of both strains JM83 and JM83pDC21. These strains differed significantly in the response of the NADH oxidation-dependent transhydrogenase. This reaction was inhibited by nigericin and uncouplers in membrane vesicles of JM83 but there was little inhibition or the reaction was stimulated in JM83pDC21, in spite of the discharge of the NADH oxidation-generated proton gradient measured by quinacrine fluorescence in the latter strain. It is proposed that the transhydrogenase is energized by direct or local (nonbulk phase) proton translocation in membranes of this strain. Uncouplers might facilitate these routes but would not discharge them. The generality of these observations was shown using other strains. NADH oxidase activity was severalfold lower in membrane vesicles of JM83pDC21 compared with JM83. The levels of ubiquinone and cytochromes, and the activities of NADH dehydrogenases I and II, and of cytochrome oxidase, were similar in the two strains. It is concluded that the NADH oxidase activity of JM83pDC21 is low because of the reduced rate of collision between electron-transferring complexes of the respiratory chain due to the large amount of transhydrogenase protein in the membranes of this strain. The large amount of transhydrogenase favors direct, nonbulk phase proton transfer. Transhydrogenase activity was stimulated by Ca2+, Mg2+, or Mn2+.


25Mg NMR studies of yeast enolase and rabbit muscle pyruvate kinase. 25Mg NMR spectroscopy was used to study the interactions of the activating cations with their respective binding sites in the enzymes yeast enolase and rabbit muscle pyruvate kinase (PK). Titration of Mg2+ with enolase allows for the calculation of 1/T2 for Mg2+ bound at site I of 1510 s-1 and a quadrupolar coupling constant chi = 0.30 MHz. Titration of Mg2+ with enolase in the presence of 2-phosphoglycerate (PGA) and Zn2+, where Zn2+ binds specifically at site I, gives a 1/T2 for Mg2+ bound at site II of 4000 s-1 (chi = 0.49 MHz). The Mg2+ at site II appears to be more anisotropic than Mg2+ at site I. The titration of site I of the enolase-Mg-PGA-Mg complex with Zn2+ or Mn2+ shows a simple displacement of the Mg2+. No paramagnetic effects by Mn2+ on 25Mg relaxation were observed. Temperature studies of the 25Mg resonance show that fast exchange of the Mg2+ occurs under these conditions. From the lack of a paramagnetic effect, the distance between the cations at sites I and II must be more than 6-9 A. This distance limits the location, hence the function, of the cation at site II for catalytic activity. Titration of Mg2+ with PK gives a 1/T2 for bound Mg2+ of 2200 s-1 (chi = 0.24 MHz). A titration of Mg2+ with PK in the presence of the inhibitor oxalate gives a 1/T2 of 400 s-1. The temperature dependence of 25Mg relaxation in the PK-Mg-oxalate complex is consistent with slow exchange (Ea = 6.1 +/- 1.6 kcal/mol). The enzyme-bound cation is more tightly sequestered by the addition of a ligand that binds directly to the cation. An investigation of the 25Mg relaxation in the PK-Mn-oxalate-Mg-ATP complex, where the Mg2+ is bound to the nucleotide and the Mn2+ was enzyme bound, was not successful due to precipitation of PK under experimental conditions and the short T2 relaxation for 25Mg in this complex. The applications of 25Mg NMR have been useful in partially describing the properties of the bound Mg2+ in these two metal-requiring enzymes.


Increased NADH-dependent production of reactive oxygen intermediates by microsomes after chronic ethanol consumption: comparisons with NADPH. Microsomes from chronic ethanol-fed rats were previously shown to catalyze the NADPH-dependent production of reactive oxygen intermediates at elevated rates compared to controls. Recent studies have shown that NADH can also serve as a reductant and promote the production of oxygen radicals by microsomes. The current study evaluated the influence of chronic ethanol consumption on NADH-dependent microsomal production of reactive oxygen intermediates, and compared the results with NADH to those of NADPH. Microsomal oxidation of chemical scavengers, taken as a reflection of the production of hydroxyl radical (.OH)-like species was increased about 50% with NADH as cofactor and about 100% with NADPH after chronic ethanol consumption. The potent inhibition of the production of .OH-like species by catalase suggests a precursor role for H2O2 in .OH production. Rates of NADH- and NADPH-dependent H2O2 production were increased by about 50 and 70%, respectively, after chronic ethanol consumption. A close correlation between rates of H2O2 production and generation of .OH-like species was observed for both NADH and NADPH, and increased rates of H2O2 production appear to play an important role in the elevated generation of .OH-like species after chronic ethanol treatment. Microsomal lipid peroxidation was elevated about 60% with NADH, and 120% with NADPH, after ethanol feeding. With both types of microsomal preparations, the characteristics of the NADH-dependent reactions were similar to the NADPH-dependent reactions, e.g., sensitivity to antioxidants and free radical scavengers and catalytic effectiveness of ferric complexes. However, rates with NADPH exceeded the NADH-dependent rates by 50 to 100%, and the increased production of reactive oxygen intermediates by microsomes after ethanol treatment was greater with NADPH (about twofold) than with NADH (about 50%). Oxidation of ethanol results in an increase in hepatic NADH levels and interaction of NADH, iron, and microsomes can produce potent oxidants capable of initiating lipid peroxidation and oxidizing .OH scavengers. These acute metabolic interactions produced by ethanol-derived NADH are increased, not attenuated, in microsomes from chronic ethanol-fed rats, and it is possible that such increases in NADH (and NADPH)-dependent production of reactive oxygen species play a role in the development of oxidative stress in the liver as a consequence of ethanol treatment.


Deferoxamine inhibition of Cr(V)-mediated radical generation and deoxyguanine hydroxylation: ESR and HPLC evidence. Electron spin resonance (ESR) and high-performance liquid chromatography (HPLC) techniques were utilized to investigate the effect of deferoxamine on free radical generation in the reaction of Cr(V) with H2O2 and organic hydroperoxides. ESR measurements demonstrated that deferoxamine can efficiently reduce the concentration of the Cr(V) intermediate as formed in the reduction of Cr(VI) by NAD(P)H or a flavoenzyme glutathione reductase/NADH. ESR spin trapping studies showed that deferoxamine also inhibits Cr(V)-mediated .OH radical generation from H2O2, as well as Cr(V)-mediated alkyl and alkoxy radical formation from t-butyl hydroperoxide and cumene hydroperoxide. HPLC measurements showed that .OH radicals generated by the Cr(VI)/flavoenzyme/NAD(P)H enzymatic system react with 2'-deoxyguanine to form 8-hydroxy-2'-deoxyguanine (8-OHdG), a DNA damage marker. Deferoxamine effectly inhibited the formation of 8-OHdG also.


Purification and characterization of bovine interstitial collagenase and tissue inhibitor of metalloproteinases. In this report we describe the purification of bovine interstitial collagenase and provide information on its substrate specificity, kinetic parameters of catalytic activity, and amino terminal protein sequence. In addition, we present a simplified protocol for the purification of bovine tissue inhibitor of metalloproteinases (TIMP). Collagenase was purified by sequential chromatography through heparin-Sepharose, DEAE-Sepharose, and green-agarose, resulting in a product that was greater than 95% pure as judged by polyacrylamide electrophoresis. Typical of other interstitial collagenases, the isolated bovine protein was activated by protease and organomercurial treatment. It also demonstrated a kinetics and substrate specificity similar to those of human collagenase. TIMP was purified by sequential chromatography through heparin-Sepharose and DEAE-Sepharose followed by reverse-phase HPLC. The purified protein had a size, N-terminal sequence, and inhibitor activity similar to those of other mammalian TIMPs. Partial peptide sequences suggested that bovine collagenase and TIMP have strong sequence homology to their human homologues.


Spin trapping study on the kinetics of Fe2+ autoxidation: formation of spin adducts and their destruction by superoxide. The oxidation of Fe2+ was investigated by electron spin resonance spin trapping techniques with N-t-butyl-alpha-phenylnitrone (PBN) and dimethyl sulfoxide. Under pure oxygen, the spin adduct PBN/.OCH3 was rapidly generated by the addition of Fe2+ (0.2-1.2 mM) into phosphate buffer containing ethylenediaminetetraacetate (EDTA), dimethyl sulfoxide, and PBN at pH 7.4, but it decayed. The decay process of PBN/.OCH3 consists of two components. The fast decay was dependent on Fe2+ concentration. Another was due to destruction of the spin adduct by superoxide anion (.O2-), because superoxide dismutase (SOD) markedly prevented the decay. Catalase decreased the yield of PBN/.OCH3. When EDTA was replaced by diethylenetriaminepentaacetic acid (DTPA), both the generation and decay process of PBN/.OCH3 were slow. SOD and catalase effects were similar to those in EDTA. Fe2+ produced PBN/.OCH3 even in the absence of chelators. We could estimate the kinetic parameters by computer simulation, comparing the Fe2+ oxidation in EDTA with that in DTPA. These results demonstrate that Fe2+ reacts with O2 to generate .O2- and then H2O2, which produces .CH3 by reaction with Fe2+ and dimethyl sulfoxide.(.)OCH3 results from the reaction between .CH3 and O2. The adduct PBN/.OCH3 decays by reaction with Fe2+ and .O2-.


Epithelioid component of uterine leiomyosarcoma simulating metastatic carcinoma. A variant of uterine leiomyosarcoma containing two morphologic bell populations is described. A smooth-muscle component with a mitotic rate of 13 per 10 high-power fields, and a second population of cells mimicking metastatic small-cell carcinoma were both present. The second population was composed of small angulated epithelioid cells with hyperchromatic nuclei and barely discernible cytoplasm growing in tight rows, often in single file between fascicles of smooth muscle. DNA flow cytometry showed a large tetraploid aneuploid tumor cell population, and further suggested that the epithelioid cells probably represent tetraploid tumor cells in S-phase. Thus, this morphologic variant of leiomyosarcoma, unlike previously described variants of smooth-muscle tumors, is explained by the variable appearance of the neoplastic cells in different phases of the cell cycle.


Risk factors for the neurologic complications associated with aortic aneurysms. We reviewed the incidence of neurologic complications in 200 consecutive patients with aortic aneurysm or aortic dissection. In this 2-year period, neurologic impairment developed in 18.5% of these patients, and in 10 patients neurologic dysfunction heralded aortic rupture or dissection. Those patients with abnormal neurologic examinations at presentation frequently had aneurysm rupture or dissection and a mortality rate of 54%. Patients with thoracic or thoracoabdominal aneurysms were more likely to have neurologic complications than those with abdominal aneurysms. The most common complications were focal central nervous system ischemia, followed by disorders of consciousness and peripheral nerve complications. In patients who had elective aneurysm resection, female sex, aneurysm location, and intraoperative hypotension were risk factors for focal central nervous system ischemia. We conclude that neurologic complications depend on aneurysmal location, occur at various levels of the nervous system, and frequently develop when the intraoperative mean arterial pressure falls below 55 mm Hg.


Stimulus-response uncoupling in the neutrophil. Adenosine A2-receptor occupancy inhibits the sustained, but not the early, events of stimulus transduction in human neutrophils by a mechanism independent of actin-filament formation. Generation of superoxide anion (O2-) in response to occupancy of neutrophil chemoattractant receptors requires both early events ('triggering') and sustained signals ('activation'). We have previously demonstrated that occupancy of adenosine A2 receptors inhibits O2- generation by neutrophils. In parallel, adenosine-receptor occupancy promotes association of bound N-formylmethionyl-leucyl-phenylalanine (fMLP) receptors with the cytoskeleton, a process associated with termination of neutrophil activation (stimulus-response uncoupling). We undertook this study to determine whether inhibition of neutrophil function by adenosine-receptor occupancy requires intact actin filaments and to examine the effect of adenosine-receptor occupancy on the stimulated generation of intracellular signals involved in neutrophil triggering and activation. Occupancy of adenosine A2 receptors by 5'-N-ethylcarboxamidoadenosine (NECA, 1 microM) significantly increased (130 +/- 1% of control, P less than 0.001, n = 3) association of [3H]fMLP with cytoskeletal preparations. Cytochalasin B (5 micrograms/ml), an agent which disrupts actin filaments, completely blocked association of [3H]fMLP with cytoskeletal preparations, as previously reported. However, NECA markedly increased association of [3H]fMLP with the cytoskeleton even in the presence of cytochalasin B (P less than 0.0002). Moreover, NECA did not significantly affect either the early (30s) or the late (5 min) formation of actin filaments after stimulation by chemoattractant (fMLP, 0.1-100 nM). Cytochalasin B markedly inhibited actin-filament formation by stimulated neutrophils, and NECA did not reverse the effect of cytochalasin B on actin-filament formation. Adenosine-receptor occupancy did not affect the rapid peak in diacylglycerol generation (less than or equal to 15 s) from either [3H]arachidonate- or [14C]glycerol-labelled phospholipid pools. However, as would be predicted if occupancy of the adenosine receptor was a signal for early termination of cell activation, NECA (1 microM) markedly diminished the slow sustained generation of diacylglycerol. These results suggest that adenosine-A2-receptor occupancy does not affect triggering of the neutrophil, but that occupancy of adenosine receptors is an early signal for the termination of neutrophil activation, i.e. the 'premature' finish of signal transduction. Moreover, these data indicate that at least two pathways are available for increasing the association of ligated chemoattractant receptors with the cytoskeleton of neutrophils: F-actin-dependent and -independent.


Domain mapping of the retinal cyclic GMP phosphodiesterase gamma-subunit. Function of the domains encoded by the three exons of the gamma-subunit gene. Retinal rod-outer-segment phosphodiesterase (PDE) is a heterotetramer consisting of two similar, but not identical, catalytic subunits (alpha and beta) and two identical inhibitory subunits (gamma 2). Previously, we have reported that the site of PDE alpha/beta interaction with PDE gamma is located within residues 54-87 [Cunnick, Hurt, Oppert, Sakamoto & Takemoto (1990) Biochem. J. 271, 721-727]. The site for PDE gamma interaction with transducin alpha (T alpha) was found to encompass residues 24-45 of PDE gamma [Morrison, Cunnick, Oppert & Takemoto (1989) J. Biol. Chem. 264, 11671-11681]. In order to identify binding sites and other functional domains of PDE gamma, the three peptides which are encoded by the three exons of the PDE gamma gene were synthesized chemically. These exons encode for residues 1-49, 50-62 and 63-87 of bovine PDE gamma [Piriev, Purishko, Khramtsov & Lipkin (1990) Dokl. Akad. Nauk. SSSR 315, 229-230]. The peptide encompassing residues 63-87 was inhibitory in a PDE assay, whereas peptides 1-49 and 50-62 had no effect. However, both peptides 1-49 and 63-87 bound to PDE alpha/beta in a solid-phase binding assay. Only peptide 1-49 bound to T alpha.GTP[S] (GTP[S] is guanosine 5'-[gamma-thio]triphosphate). These data confirm that the inhibitory region of PDE gamma is encoded by exon 3 (residues 63-87), whereas a separate binding site for PDE alpha/beta and for T alpha.GTP[S] is encoded by exon 1 (residues 1-49). To study further the structure-function relationship of PDE gamma, this entire protein and two mutants were chemically synthesized. One mutant (-CT) lacked residues 78-87, whereas another replaced tyrosine-84 with glycine (TYR-84). Whereas the synthetic PDE gamma inhibited PDE alpha/beta catalytic activity, the -CT and TVR-84 mutants did not. All three synthetic proteins bound to both PDE alpha/beta and and T alpha.GTP[S]. These data confirm the presence of an alternative binding site on PDE gamma and demonstrate the importance of tyrosine-84 in PDE gamma inhibitory activity.


The gene for a novel protein, a member of the protein disulphide isomerase/form I phosphoinositide-specific phospholipase C family, is amplified in hydroxyurea-resistant cells. Cell lines selected in multiple steps for increasing resistance to hydroxyurea have been shown to have corresponding increases in ribonucleotide reductase activity. We have isolated a number of cDNA clones from a cDNA library constructed from a highly hydroxyurea-resistant hamster cell line, 600H, in which the activity of ribonucleotide reductase is elevated more than 80-fold. These clones correspond to genomic DNA sequences amplified in the 600H cell line compared with the V79 parental line. One of these cDNA clones, termed P5, codes for a 50 kDa protein detected by in vitro translation of poly(A)+ RNA isolated by hybridization/selection. The cDNA sequence contains a single open reading frame of 1317 nucleotides which encodes a polypeptide of 439 amino acids. The amino acid sequence deduced from the cDNA insert contains two copies of the 11-amino-acid sequence Val-Glu-Phe-Tyr-Ala-Pro-Trp-Cys-Gly-His-Cys. Duplicate copies of this sequence also occur in the active site of rat and human protein disulphide isomerase (also known as the beta-subunit of human prolyl 4-hydroxylase, tri-iodothyronine-binding protein) and in Form I phosphoinositide-specific phospholipase C, indicating that P5 falls into this newly defined superfamily of proteins. Genomic sequences similar to the cDNA clone are amplified 10-20-fold in hamster cells selected for resistance to increasing concentrations of hydroxyurea, a phenomenon observed earlier with cDNA clones for the M2 subunit of ribonucleotide reductase and ornithine decarboxylase. RNA blots probed with P5 cDNA show two poly(A)+ RNA species which are elevated in hydroxyurea-resistant cells.


Structural gene and complete amino acid sequence of Vibrio alginolyticus collagenase. The DNA encoding the collagenase of Vibrio alginolyticus was cloned, and its complete nucleotide sequence was determined. When the cloned gene was ligated to pUC18, the Escherichia coli expression vector, bacteria carrying the gene exhibited both collagenase antigen and collagenase activity. The open reading frame from the ATG initiation codon was 2442 bp in length for the collagenase structural gene. The amino acid sequence, deduced from the nucleotide sequence, revealed that the mature collagenase consists of 739 amino acids with an Mr of 81875. The amino acid sequences of 20 polypeptide fragments were completely identical with the deduced amino acid sequences of the collagenase gene. The amino acid composition predicted from the DNA sequence was similar to the chemically determined composition of purified collagenase reported previously. The analyses of both the DNA and amino acid sequences of the collagenase gene were rigorously performed, but we could not detect any significant sequence similarity to other collagenases.


The interplay of temperature and protons in the modulation of oxygen binding by squid blood. An extensive set of data relating to the binding of oxygen by haemocyanin from the squid Todarodes sagittatus has been collected under various experimental conditions. The results obtained show that, within the range of physiological pH, the concentration of protons affects mainly the high-affinity state of the molecule without significantly affecting the low-affinity state. As far as the effect of temperature is concerned, the data show a characteristic feature which is very similar to that previously described in the case of haemoglobins from Arctic mammals such as reindeer (Rangifer tarandus) and musk ox. (Ovibos moschatus). The shape of the oxygen equilibrium curve shows strong temperature-dependence, since the overall heat of the binding of oxygen to the low-affinity state of the molecule is strongly exothermic and that to the high-affinity state is very close to zero. The results provide an outline of the intramolecular compromise that, through the interplay of temperature and protons, optimizes the loading and unloading of oxygen under the various environmental conditions experienced by this species of squid.


The role of diacylglycerol in the exocytosis of the sperm acrosome. Studies using diacylglycerol lipase and diacylglycerol kinase inhibitors and exogenous diacylglycerols. When ram spermatozoa were treated with Ca2+ and the ionophore A23187 to induce acrosomal exocytosis, a rise in diacylglycerol (DAG) mass was observed, concomitant with a rapid breakdown of [32P]P1-labelled phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 4-phosphate and a rise in [32P]Pi-labelled phosphatidate. Inclusion of the DAG lipase inhibitor RHC 80267 resulted in further but biphasic increases in DAG; there was an increasing accumulation of DAG with concentrations of RHC 80267 up to 10 microM, whereas higher concentrations produced lessening accumulation. Inclusion of RHC 80267 in the ionophore induction system also resulted in significant accelerations of the onset of exocytosis. In spermatozoa stimulated with Ca2+/A23187 and the DAG kinase inhibitor R59022, a similar increase in DAG levels together with stimulation of acrosomal exocytosis were observed. Preincubation of spermatozoa with sn-1-oleoyl-2-acetylglycerol, rac-1-oleoyl-2-acetylglycerol, sn-1,2-dioctanoylglycerol and sn-1,3-dioctanoylglycerol before treatment with Ca2+/A23187 resulted in a dose-dependent stimulation of exocytosis by all these isomers. Neomycin inhibited Ca2+/A23187-induced generation of DAG together with polyphosphoinositide breakdown, as well as acrosomal exocytosis. Inclusion of exogenous DAG, however, overcame the inhibitory effect of neomycin on exocytosis. Our results suggest that DAG has a key role in acrosomal exocytosis and that it acts as a messenger rather than as a substrate from which other active metabolites are generated. The lack of stereospecificity shown by the exogenous DAGs implies that DAG does not act by stimulating protein kinase C, but the metabolite's actual target in the sperm cell is as yet unclear.


Kinetics of superoxide scavenging by dismutase enzymes and manganese mimics determined by electron spin resonance. This study presents an e.s.r. assay for superoxide dismutase (SOD). Enzymic reactions were studied in which Cu,Zn-SOD, Mn-SOD and Fe-SOD each competed with the spin trap 5,5-dimethyl-1-pyrroline 1-oxide (DMPO) for superoxide anion (O2-) at pH 7.8 O2- from dissolved KO2 (potassium superoxide) in dimethyl sulphoxide was added directly to the enzyme solutions containing DMPO. The results show that, in this competition reaction system, the kinetics of the reactions between the enzymes and O2- follow a function y = f[( SOD]0.5). The rate constant, kSOD = 6.4 x 10(9) M-1. S-1, determined for Cu,Zn-SOD is approximately an order of magnitude larger than those for Mn-SOD and Fe-SOD. A comparative study of reported SOD mimics, including Mn2+, MnO2-desferrioxamine mesylate (Desferal) and MnO2-Desferal-ascorbate, was done. The results show that solutions of these complexes are approximately three orders of magnitude less active than Cu,Zn-SOD and approximately two orders of magnitude less active than Mn-SOD or Fe-SOD. The results also suggest that the reactivity toward O2- in solutions of these complexes originates from the Mn2+ present and not from the MnO2-Desferal complexes.


Inhibition of phosphatidylinositol 4-phosphate kinase by heparin. A possible mechanism for the antiproliferative effects of heparin. Heparin and related glycosaminoglycans are important modulators of vascular smooth muscle cell growth, and may be involved in pathological processes such as atherosclerosis. Since polyphosphoinositide metabolism is a major mechanism for regulating cellular activities, including proliferation, the effects of glycosaminoglycans and polyanionic compounds on the activities of phosphoinositide kinases were characterized. Heparin and heparan sulphate caused dose-dependent inhibitions of rat brain cytosolic phosphatidylinositol 4-phosphate (PIP) kinase activity, with half-maximal inhibitory concentrations of approx. 0.5 and 5 microM respectively. PIP kinase was also inhibited by several dextran sulphates, but was not sensitive to inhibition by keratin sulphate, chondroitin sulphate or hyaluronic acid. Polynucleotides and acidic polypeptides were only weakly inhibitory. Heparin did not alter either the PIP- or the Mg(2+)-dependence of PIP kinase. Addition of heparin to brain membranes suppressed PIP kinase activity without affecting phosphatidylinositol (PI) kinase activity. Heparin interfered with the ability of a GTP analogue to stimulate PIP kinase activity in these membranes, suggesting that it uncouples the kinase from an activating guanine-nucleotide-binding protein. In cultured A-10 vascular smooth muscle cells, heparin caused dose- and time-dependent inhibition of [3H]thymidine incorporation into DNA. Similar treatments with heparin decreased cellular levels of phosphatidylinositol 4,5-bisphosphate (PIP2) without changing PI and PIP levels. Therefore heparin-mediated inhibition of PIP kinase appears to lead to decreases in PIP2 levels which may attenuate cellular proliferation.


Analysis of human hepatic microsomal glucose-6-phosphatase in clinical conditions where the T2 pyrophosphate/phosphate transport protein is absent. The availability of a rare set of human hepatic microsomes in which T2, a pyrophosphate/phosphate transport protein of the glucose-6-phosphatase system, has been shown immunologically to be completely absent, has permitted further characterization of multicomponent glucose-6-phosphatase (EC 3.1.3.9). Pyrophosphatase activity in intact microsomes was found to be totally absent, but was normal in disrupted microsomes. However, Pi did not accumulate within the lumen of the microsomes when glucose 6-phosphate was the substrate. This was not as predicted if there is only one transport protein in the endoplasmic reticulum capable of transporting Pi, produced by glucose-6-phosphatase, out of the lumen. The results suggest that the pyrophosphate/phosphate transport system of human hepatic endoplasmic reticulum must be more complex than previously thought, as it must comprise at least two protein components.


Basic calcium phosphate crystals stimulate cell proliferation and collagenase message accumulation in cultured adult articular chondrocytes. To investigate BCP (basic calcium phosphate) crystal-stimulated mitogenesis and collagenase gene transcription in primary cultures of porcine chondrocytes. The role of protein kinases in BCP crystal-stimulated DNA synthesis was investigated using thymidine incorporation, kinase inhibitors, and protein kinase C (PKC) assays. Northern blot analysis was used to determine the levels of collagenase c-fos and c-jun message. BCP crystals stimulated chondrocyte proliferation in a PKC-dependent manner. Increased levels of collagenase message were preceded by an increased accumulation of c-fos, but not c-jun. BCP crystals could contribute to the abnormal chondrocyte proliferation and collagenase secretion observed in some rheumatic diseases.


Characterization of the subunits and sugar moiety of human placental and leukemic beta-glucuronidase. beta-Glucuronidase purified from human placenta and chronic myelogenous leukemic cells was composed of three components of 18, 64 and 80 kDa, though the relative contents of the components were different between the sources. Analysis of their N-terminal amino-acid sequences showed that the 18-kDa and 64-kDa components were derived from the 80-kDa component by cleavage between Val159 and Gly160. Furthermore, the enzyme was found to be glycosylated at Asn173 and Asn420 with high mannose-type oligosaccharides, based on the electrophoretic mobility of the components as well as the endopeptidic peptides before and after endoglycosidase treatment. The enzyme purified from leukemic cells was poorly phosphorylated by N-acetylglucosamine 1-phosphotransferase as compared to the placental enzyme.


Peripheral noradrenergic function during chronic lithium treatment in the rat. Noradrenergic function was evaluated in the rat vas deferens following chronic treatment with lithium (30 mM in the diet for 2 weeks followed by 50 mM for 1 week). No alteration could be detected in: a) 3H-noradrenaline release evoked by electrical field stimulation, b) alpha 1-adrenoceptor function as assessed by contractile response to noradrenaline, c) active neuronal uptake of 3H-noradrenaline, d) alpha 2-adrenoceptor activity, as assessed by yohimbine-enhancement of evoked release of 3H-noradrenaline. The data argue against a primary action of lithium on the noradrenergic nerve. On the other hand, these findings do not rule out the possibility that the function of CNS noradrenergic neurons could be altered as a result of the known effects of lithium on the phosphatidyl-inositol system, and the greater sensitivity of CNS neurons to depletion of the neuronal membrane inositol pool.


Murine monoclonal antibodies against pneumococcal capsular polysaccharide types 4, 8, 22F and 19A/19F. Monoclonal antibodies (MAbs) were produced against pneumococcal capsular polysaccharides after subcutaneous immunization of BALB/c mice with a 23-valent vaccine (Pneumovax N, Merck, Sharp & Dohme). Selected antibodies were tested in ELISA against individual polysaccharides from 23 different pneumococcal types and in a dot blot assay with heat-killed whole bacteria adhered to nitrocellulose paper. Three MAbs (isotype IgM) were found to be specific for types 4, 8 and 22F, respectively, whereas one (isotype IgA) reacted both with 19A and 19F. Very mild acid hydrolysis of the capsular polysaccharides resulted in loss of reaction with the antibodies.


Vesico-ureteric reflux in the damaged non-scarred kidney. Renal damage without scarring is a rare manifestation of reflux nephropathy, and is infrequently reported in the literature. A retrospective review of a large series of patients with urinary tract disorders at the hospital for Sick Children, Great Ormond Street, identified 12 patients with vesico-ureteric reflux (VUR) with no evidence of scarring but with renal damage as assessed using technetium 99m dimercaptosuccinic acid (99mTc-DMSA) scan. There were 9 simplex kidneys, the differential function was between 33% and 43% (mean 38%). On follow-up to date (mean 6.7 years) all kidneys show stable differential function with neither improvement nor deterioration. Only 8 children had a urinary tract infection. Renal damage without evidence of a focal scar associated with VUR may be a manifestation of growth arrest possibly due to glomerular damage. The sensitivity of 99mTc-DMSA may reveal the permanent nature of the renal damage.


Urinary excretion of brush border antigens and other proteins in children with vesico-ureteric reflux. This study was designed to evaluate the occurrence and the type of proteinuria in 82 children with vesico-ureteric reflux (VUR) with or without renal scars. The urinary excretion of the high molecular weight protein albumin was taken as an index of glomerular alterations and the excretion of retinol-binding protein (RBP), beta 2-microglobulin and brush border antigens (BBA) (measured by monoclonal antibody-based enzyme-linked immunosorbent assay) was taken as an index of tubular alterations. All such markers were increased in children with VUR and were related to the degree of renal function. Patients showing reduced creatinine clearance had very high levels of albuminuria, microproteinuria and BBA, with all these variables reciprocally correlated. In children with normal renal function however, only microproteins (not albumin or BBA) were slightly increased, thus indicating an isolated tubular defect without involvement of the proximal segment of the tubule. However, microprotein excretion did not correlate with the grade of scarring (99mtechnetium-dimercaptosuccinic acid scan), both RBP and beta 2-microglobulin excretion being normal in 75% of children with radioisotopic signs of renal lesions but increased in 17% of children without scars. Therefore, tubular proteinuria identifies different groups of children with VUR but is not related to renal scarring. Prospective studies will define the usefulness of proteinuria as a reliable indicator of renal outcome.


Atrial natriuretic peptide and cyclic 3'5'-guanosine monophosphate as indicators of fluid volume overload in children with chronic renal failure. Plasma atrial natriuretic peptide (ANP) and cyclic 3'5'-guanosine monophosphate (cGMP) were investigated as indicators of fluid volume overload in children and adolescents with chronic renal failure. Plasma ANP and cGMP were measured in both paediatric patients with chronic renal failure (n = 17, mean serum creatinine 371 +/- 242 mumol/l) and those with end-stage renal disease on haemodialysis (n = 18). cGMP was higher in children with chronic renal failure than in 45 healthy controls (1.0 +/- 0.4 vs 2.1 +/- 0.8 nmol/l, P less than 0.01), whereas plasma ANP was similar (26.9 +/- 9.7 vs 34.0 +/- 12.3 pmol/l). Both ANP and cGMP were markedly elevated in children with end-stage renal disease before haemodialysis and fell significantly during dialysis. During dialysis body weight decreased by 1.6 +/- 0.7 kg, corresponding to 4.5 +/- 2.1% of body weight. Plasma ANP correlated positively with plasma cGMP in haemodialysed patients (r = 0.43, P less than 0.05). Reduction in body weight and in mean arterial pressure correlated more closely with plasma ANP than with cGMP. Therefore, elevation of plasma ANP appears to indicate volume overload in children undergoing haemodialysis, but whether it can be used also in children with chronic renal failure requires further investigation.


Detection of HSV-specific DNA in biopsy tissue of patients with erythema multiforme by polymerase chain reaction. Formalin-fixed paraffin-embedded skin biopsies of lesions of erythema multiforme (EM) from 32 patients and 13 controls were examined for the presence of herpes simplex virus (HSV) by polymerase chain reaction (PCR) and for histological findings by direct immunofluorescence and staining with haematoxylin and eosin. HSV-specific DNA was detected in 23 (72%) patients. A history of recurrent skin rash was present in 59% of the PCR-positive cases, while 55% had had suspected HSV infections. Only two PCR-positive specimens were found in patients without a history of recurrent rash and/or previous oral lesions. One biopsy was positive for HSV by conventional cell cultures. There was no significant difference in histology between HSV-related and HSV-negative cases of EM. In the 13 control specimens [bullous pemphigoid (3), dermatitis herpetiformis (2), lichen planus (1), aphthous ulcer (1), fixed-drug eruption (1), varicella-zoster (1), hypereosinophilic syndrome (1), photocontact dermatitis (1), contact dermatitis (1), and cellulitis (1)], no HSV-DNA was detected.


Plasminogen binding sites in normal human skin. Plasminogen is detected in the basal cell layer of the epidermis, keratinocytes can generate plasminogen activators and it is suggested that the generation of plasmin may facilitate keratinocyte division, migration and differentiation. In this study we have investigated the characteristics of plasminogen binding sites in normal human epidermis. It was found that 6-aminohexanoic acid and benzamidine displaced endogenous epidermal plasminogen from the basal layer suggesting that endogenous plasminogen binds initially via the kringle 5 aminohexyl (AH) site. Plasminogen binding sites in epidermis were further investigated by displacing endogenous plasminogen and incubating sections with exogenously added glu-plasminogen, lys-plasminogen and plasmin or the isolated plasminogen fragments kringles 1-3, kringle 4 and kringle 5L. The results suggest that the uptake of plasminogen involves primary interaction with the kringle 5AH site and a secondary interaction with lysine binding sites of kringles 1-3. Cell binding is not dependent upon additional reactions of the plasmin active centre.


Cancer family syndrome associated with multiple malignant melanomas and a malignant fibrous histiocytoma. A 64-year-old female with the cancer family syndrome suffered from multiple malignant melanomas and a malignant fibrous histiocytoma. She has shown unusually long survival which is a feature of these patients.


Effects of staurosporine, PMA and A23187 on human melanocyte cultures with dibutyryl cyclic AMP. Staurosporine, a protein kinase (PK) inhibitor, phorbol-12-myristate-13-acetate (PMA), a PKC activator and A23187 calcium ionophore were added to human melanocyte cultures with or without dibutyryl cyclic AMP (dbcAMP). After 2 days' incubation, changes in various melanogenic factors were examined such as tyrosinase activity and the amount of tyrosinase-related protein (TRP) as well as the morphology of the melanocytes. dbcAMP stimulated all the melanogenic factors. Staurosporine increased tyrosinase activity and amount of TRP and caused morphological changes with the formation of numerous dendrites, regardless of the presence of dbcAMP. In contrast, PMA did not significantly affect tyrosinase activity, TRP content or dendrite formation, with or without dbcAMP. The effects of staurosporine on tyrosinase activity and TRP content were completely inhibited by PMA, but PMA did not significantly affect the staurosporine-induced morphological changes. A23187 inhibited both tyrosinase activity and TRP content, regardless of the presence of dbcAMP, but did not affect the morphology of melanocytes. These findings suggest that tyrosinase activity and TRP content are regulated by adenylate cyclase and Ca2+ and partly by PKC, while the morphological features of melanocytes are affected by intracellular cAMP accumulation and by the inhibition of PKC.


Generalized eruptive histiocytoma.

Generalized eruptive histiocytoma. The case is reported of a 56-year-old female who over 18 months developed a symmetrical eruption of hundreds of bluish-red papules, a small number of which have spontaneously resolved. The clinical and histopathological findings were compatible with the diagnosis of generalized eruptive histiocytoma. The ultrastructural findings in this case suggest that this condition which is in the non-X group of histiocytic disorders is closely related to histiocytosis X.


The molecular biology of retinoic acid receptors: orphan from good family seeks home. A variety of c-DNAs coding for nuclear retinoic acid receptors (RARs) have recently been cloned. These receptors are members of the steroid/thyroid receptor superfamily and are believed to act as ligand-inducible transactivating factors; retinoic acid induces changes in receptor configuration that allows DNA binding and increased gene transcription from specific genes to occur. The retinoic acid receptor family itself may consist of up to 20 separate receptors each with a specific distribution and ligand binding characteristics. The RAR-gamma in the adult is found almost exclusively in the skin but other receptors which are found in a variety of other tissues are also present in skin. Associations of cutaneous disease states with receptor mutants have not yet been reported although some cases of leukaemia may be secondary to retinoic acid receptor gene rearrangements. A variety of approaches to identify the biological function of these receptors based on recombinant DNA technology are already underway.


Analysis of African Burkitt's and high-grade B cell non-Burkitt's lymphoma for Epstein-Barr virus genomes using in situ hybridization. We investigated the association of Epstein-Barr virus (EBV) with African cases of Burkitt's lymphoma (BL) and high grade B cell non-Burkitt's lymphoma (non-BL) occurring in areas where BL is endemic. The presence of EBV genomes was analysed in 24 cases using in situ hybridization with a 35S-labelled EBV probe applied to paraffin sections. EBV DNA was detected in each of 10 cases of BL in which technically satisfactory results were obtained, the virus being homogeneously distributed in all identifiable tumour cells. Two other cases of BL could not be evaluated because of technical problems. In contrast, EBV DNA was not detected in any case of high-grade non-BL (10 centroblastic and two immunoblastic lymphomas). These results confirm previous reports of the strong association of EBV with endemic BL, but suggest that the virus is not important in the pathogenesis of other types of African high-grade B cell lymphoma from regions where BL is endemic.


Central role of tumour necrosis factor, GM-CSF, and interleukin 1 in the pathogenesis of juvenile chronic myelogenous leukaemia. In previous studies on patients with juvenile chronic myelogenous leukaemia (JCML), we found excessive proliferation of malignant monocyte-macrophage elements in the absence of exogenous growth factor, and impaired growth of normal haematopoietic progenitors. In the current study, six newly-diagnosed JCML patients were investigated to characterize the disease further. In co-cultures, JCML cell culture supernatant as well as patient plasma obtained at diagnosis produced a striking reduction in numbers of control marrow BFU-E, CFU-GM, CFU-Meg and CFU-GEMM colonies. Monoclonal anti-tumour necrosis factor alpha neutralizing antibodies (anti-TNF-alpha Ab) abolished these inhibitory properties. In sharp contrast, JCML supernatants exerted a marked growth-promoting effect on autologous JCML cells cultured in clonogenic assays. Anti-TNF-alpha Ab and anti-granulocyte-macrophage colony-stimulating factor neutralizing antibodies (anti-GM-CSF Ab) both reversed the stimulating effect. Recombinant GM-CSF and recombinant TNF alpha produced a profound increase in JCML colonies when tested individually and anti-GM-CSF Ab reversed the TNF-alpha effect. Expression studies of TNF-alpha and TNF-alpha receptor genes of cultured JCML cells demonstrated mRNAs for both. Further, TNF-alpha activity was assayed in a wide variety of cell culture supernatants and in normal and patients' plasma, and only the JCML specimens showed increased TNF-alpha values. Recombinant interleukin-1 alpha (IL-1 alpha) also stimulated JCML colony growth, but polyclonal anti-IL-1 neutralizing antibodies did not suppress JCML colony numbers nor did it reverse the effects of TNF-alpha or GM-CSF. The evidence indicated that the JCML monokine which inhibits normal haematopoiesis is TNF-alpha and that the endogenously-produced TNF-alpha and GM-CSF from JCML cells play an important role in the pathogenesis of the disease by acting as autocrine growth factors. IL-1 alpha also stimulates JCML cell proliferation as an accessory factor and augments the effect of GM-CSF, TNF-alpha or both.


The role of an anti-myeloperoxidase antibody in the diagnosis and classification of acute leukaemia: a comparison with light and electron microscopy cytochemistry. The enzyme myeloperoxidase (MPO) is the hallmark of the myeloid lineage. We have analysed the presence of MPO in blasts from 180 cases of acute leukaemia (103 acute myeloid leukaemia (AML) and 77 acute lymphoid leukaemia (ALL) by means of monoclonal antibodies anti-MPO and immunocytochemistry (alkaline phosphatase anti-alkaline phosphatase method). The aim of the study was to investigate the specificity and sensitivity of this marker compared with MPO cytochemistry by light (LM) and electron microscopy (EM), and with the expression of myeloid antigens. Anti-MPO was positive (greater than 3% blasts) in all but one of the 90 AML positive by LM cytochemistry. Of 13 AML cases negative by MPO cytochemistry, six showed 3-10% blasts reactive with anti-MPO and were also positive with antibodies to CD13 and/or CD33. The presence of MPO was confirmed in four of these by EM. The overall positivity of anti-MPO in AML was 92%. Anti-MPO was negative in all but two ALL (6% and 8% positive blasts). The blasts in these two cases were also CD13, CD33 and MPO positive by EM; both were thus reclassified as biphenotypic. Another two ALL reinterpreted as biphenotypic were negative by MPO cytochemistry and anti-MPO but were MPO positive by EM and with CD13 and/or CD33. We conclude that anti-MPO is a sensitive and specific early marker of myeloid blasts and should be incorporated in the routine immunophenotyping of acute leukaemia.


Okadaic acid--a valuable new tool for the study of signal transduction and cell cycle regulation? Okadaic acid is beginning to find its way into laboratories that study cellular control of gene expression and proliferation. By virtue of its specific inhibitory effects on certain protein phosphatases it seems to be a powerful tool for the analysis of regulatory phosphorylation-dephosphorylation events. Here, some of the important new findings with the use of this drug are summarized, and a few potential drawbacks of its application discussed.


Structural analysis of the operator binding domain of Tn10-encoded Tet repressor: a time-resolved fluorescence and anisotropy study. An engineered Tn10-encoded Tet repressor, bearing a single Trp residue at position 43, in the putative alpha-helix-turn-alpha-helix motif of the operator binding domain, was studied by time-resolved fluorescence and anisotropy. Fluorescence intensity decay data suggested the existence of two classes of Trp-43, defined by different lifetimes. Analysis of anisotropy data were consistent with a model in which each class was defined by a different lifetime, rotational correlation time, and fluorescence emission maximum. The long-lifetime class had a red-shifted spectrum, similar to that of tryptophan zwitterion in water, and a short rotational correlation time. In contrast, the spectrum of the short-lifetime class was blue-shifted 10 nm compared to that of the long-lifetime class. Its correlation time was similar to that of the protein, which showed that Trp in this class was entirely constrained. Trp in this latter class could not be quenched by iodide, whereas most of the long-lifetime class was easily accessible. Presence of disruptive agents, such as 1 M GuCl or 3 M KCl, did not alter markedly the lifetimes but increased the weight of the short-lifetime component. In the same time, the rotational correlation time of this component was dramatically reduced. Taken together, our data suggest that the long-lifetime class could correspond to the tryptophan residues exposed to solvent whereas the short-lifetime class would correspond to the tryptophan residues embedded inside the hydrophobic core holding the helix-turn-helix motif. Destabilization of hydrophobic interactions would lead to an increase in the weight of the latter class for entropic reasons. Analysis of the fluorescence parameters of Trp-43 could provide structural information on the operator binding domain of Tet repressor.


Effects of ouabain on the rotational dynamics of renal Na,K-ATPase studied by saturation-transfer EPR. The interaction of a nitroxide spin-labeled derivative of ouabain with sheep kidney Na,K-ATPase and the motional behavior of the ouabain spin label-Na,K-ATPase complex have been studied by means of electron paramagnetic resonance (EPR) and saturation-transfer EPR (ST-EPR). Spin-labeled ouabain binds with high affinity to the Na,K-ATPase with concurrent inhibition of ATPase activity. Enzyme preparations retain 0.61 +/- 0.1 mol of bound ouabain spin label per mole of ATP-dependent phosphorylation sites, even after repeated centrifugation and resuspension of the purified ATPase-containing membrane fragments. The conventional EPR spectrum of the ouabain spin label bound to the ATPase consists almost entirely (greater than 99%) of a broad resonance at 0 degrees C, characteristic of a tightly bound spin label which is strongly immobilized by the protein backbone. Saturation-transfer EPR measurements of the spin-labeled ATPase preparations yield effective correlation times for the bound labels significantly longer than 100 microseconds at 0 degrees C. Since the conventional EPR measurements of the ouabain spin-labeled Na,K-ATPase indicated the label was strongly immobilized, these rotational correlation times most likely represent the motion of the protein itself rather than the independent motion of mobile spin probes relative to a slower moving protein. Additional ST-EPR measurements of ouabain spin-labeled Na,K-ATPase (a) cross-linked with glutaraldehyde and (b) crystallized in two-dimensional arrays indicated that the observed rotational correlation times predominantly represented the motion of large Na,K-ATPase-containing membrane fragments, as opposed to the motion of individual monomeric or dimeric polypeptides within the membrane fragment. The results suggest that the binding of spin-labeled ouabain to the ATPase induces the protein to form large aggregates, implying that cardiac glycoside induced enzyme aggregation may play a role in the mechanism of action of the cardiac glycosides in inhibiting the Na,K-ATPase.


Structural elements of human parathyroid hormone and their possible relation to biological activities. Human parathyroid hormone (hPTH) and several deletion analogues were examined for the presence of secondary structure using circular dichroism spectroscopy. The spectra of hPTH and the deletion analogues 8-84, 34-53, 53-84, 1-34, 13-34, 1-19, and 20-34, in neutral, aqueous buffer, gave no evidence for extensive secondary structure. An alpha-helical-like spectral contribution was found to arise from a region within peptide 13-34. This spectral contribution was speculated to arise from partial stability of a helix consisting of residues 17-29. Molecular dynamics simulations of peptide 1-34 suggested that this peptide tends to fold with a bend defined by residues 10-14, with the amino-terminal and carboxyl-terminal residues tending to be in more extended forms and the other residues in helical-like conformations. The addition of trifluoroethanol promoted the formation of alpha-helix, mainly in the 1-34 region. The putative helix comprised of residues 17-29 was stabilized by the addition of 10-20% TFE, while a second putative helix proximal to the amino terminus, and comprised of residues 3-11, was stabilized by slightly higher concentrations of TFE. An amphiphilic sequence was identified within the 20-34 fragment. The development of alpha-helix on binding this fragment, and other analogues containing this sequence, to palmitoyloleoylphosphatidylserine vesicles provided experimental evidence for the potential role of this amphiphilic sequence in binding to membranes or to a membrane receptor. The relationships between these alpha-helical regions in 1-34, either potentiated by trifluoroethanol or lipid vesicles, are discussed in terms of different receptor-binding regions within hPTH.


1H NMR assignment and secondary structure of the cell adhesion type III module of fibronectin. The secondary structure of the tenth type III module from human fibronectin has been determined using NMR. This type of module appears many times in a wide variety of proteins. The type III module described here contains an Arg-Gly-Asp sequence known to be involved in cell-cell adhesion. The module was expressed in yeast and characterized by amino acid sequencing and mass spectrometry. 2D and 3D NMR spectroscopy of 15N-labeled protein was used to perform sequence-specific assignment of the spectrum. The secondary structure was defined by patterns of nuclear Overhauser effects, 3JNH-alpha CH spin-spin coupling constants, and amide proton solvent exchange rates. The molecule consists of seven beta-strands in two antiparallel beta-sheets with an immunoglobulin-like fold similar to that predicted for homologous modules in the cytokine receptor super family [Bazan, J. F. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 6934-6938]. The Arg-Gly-Asp sequence is located on a loop between the beta-strands F and G.


14,15N, 13C, 57Fe, and 1,2H Q-band ENDOR study of Fe-S proteins with clusters that have endogenous sulfur ligands. The benefits of performing ENDOR experiments at higher microwave frequency are demonstrated in a Q-band (35 GHz) ENDOR investigation of a number of proteins with [nFe-mS] clusters, n = 2, 3, 4. Each protein displays several resonances in the frequency range of 0-20 MHz. In all instances, features are seen near v approximately 13 and 8 MHz that can be assigned, respectively, to "distant ENDOR" from 13C in natural-abundance (1.1%) and from 14N (the delta m1 = +/- 2 transitions); the nuclei involved in this phenomenon are remote from and have negligible hyperfine couplings to the cluster. In addition, a number of proteins show local 13C ENDOR signals with resolved hyperfine interactions; these are assigned to the beta carbons of cysteines bound to the cluster [A(13C) approximately 1.0 MHz]. Five proteins show resolved, local delta m1 = +/- 2 ENDOR signals from 14N with an isotropic hyperfine coupling, 0.4 less than or equal to A(14N) less than or equal to 1.0, similar to those seen in ESEEM studies; these most likely are associated with N-H...S hydrogen bonds to the cluster. Anabaena ferredoxin further shows a signal corresponding to A(14N) approximately 4 MHz. Quadrupole coupling constants are derived for both local and distant 14N signals. The interpretation of the data is supported by studies on 15N- and 13C-enriched ferredoxin (Fd) from Anabaena 7120, where the 15N signals can be clearly correlated with the corresponding 14N signals and where the 13C signals are strongly enhanced. Thus, the observation of 14N delta m1 = +/- 2 signals at Q-band provides a new technique for examining weak interactions with a cluster.(ABSTRACT TRUNCATED AT 250 WORDS)


Does pyrophosphate bind to the catalytic sites of mitochondrial F1-ATPase? The interactions between the pyrophosphate (PPi) binding sites and the nucleotide binding sites on mitochondrial F1-ATPase have been investigated, using F1 preparations containing different numbers of catalytic and noncatalytic nucleotide-binding sites occupied by ligands. In all cases, the total number of moles of bound nucleotides and PPi per mole of F1 was less than or equal to six. F1 preparations containing either three or two filled noncatalytic sites and no filled catalytic sites (referred as F1[3,0] and F1[2,0]) were found to bind 3 mol of PPi/mol of F1. Tight binding of ADP-fluoroberyllate complexes to two of the catalytic sites of F1 converted the three heterogeneous PPi-binding sites into three homogeneous binding sites, each exhibiting the same affinity for PPi. The addition of PPi at saturating concentrations to F1 containing GDP bound to two catalytic sites (F1[2,2]) resulted in the release of 1 mol of GDP. Furthermore, the addition of PPi to F1 filled with ADP-fluoroberyllate at the catalytic sites resulted in the release of 1 mol of tightly bound ADP/mol of F1. Taken together, these results indicate that PPi binds to specific sites that interact with both the catalytic and the noncatalytic nucleotide-binding sites of F1.


Oxygen-evolving photosystem II preparation from wild type and photosystem II mutants of Synechocystis sp. PCC 6803. We present here a simple and rapid method which allows relatively large quantities of oxygen-evolving photosystem II- (PS-II-) enriched particles to be obtained from wild-type and mutants of the cyanobacterium Synechocystis 6803. This method is based on that of Burnap et al. [Burnap, R., Koike, H., Sotiropoulou, G., Sherman, L. A., & Inoue, Y. (1989) Photosynth. Res. 22, 123-130] but is modified so that the whole preparation, from cells to PS-II particles, is achieved in 10 h and involves only one purification step. The purified preparation exhibits a 5-6-fold increase of O2-evolution activity on a chlorophyll basis over the thylakoids. The ratio of PS-I to PS-II is about 0.14:1 in the preparation. The secondary quinone electron acceptor, QB, is present in this preparation as demonstrated by thermoluminescence studies. These PS-II particles are well-suited to spectroscopic studies as demonstrated by the range of EPR signals arising from components of PS-II that are easily detectable. Among the EPR signals presented are those from a formal S3-state, attributed to an oxidized amino acid interacting magnetically with the Mn complex in Ca(2+)-deficient PS-II particles, and from S2 modified by the replacement of Ca2+ by Sr2+. Neither of these signals has been previously reported in cyanobacteria. Their detection under these conditions indicates a similar lesion caused by Ca2+ depletion in both plants and cyanobacteria. The protocol has also been applied to mutants which have site-specific changes in PS-II. Data are presented on mutants having changes on the electron donor (Y160F) and electron acceptor (G215W) side of the D2 polypeptide.


Synthesis and biochemical characterization of the new sulfhydryl-reactive ATP analogue 8-thiocyano-ATP. Its interaction with Na,K-ATPase and kinases. The synthesis of 8-thiocyano-ATP (CNS8-ATP) is described. At 37 degrees C the ATP analogue inactivates Na,K-ATPase, hexokinase, and pyruvate kinase. In all three cases, inactivation can be prevented by the addition of ATP, thus indicating that CNS8-ATP is recognized within the ATP binding site of the above enzymes. Incubation of the inactivated enzymes with dithiothreitol restores the catalytic activities. Therefore, it is likely that in these enzymes a mixed disulfide (E-S-S8-ATP) is formed between a sulfhydryl in the ATP binding site (E-SH) and the ATP analogue: [formula: see text] From the pseudo-first-order inactivation kinetics, a KD = 2.7 microM with k2 = 0.142 min-1 is calculated for the hexokinase and a KD = 40 microM with k2 = 0.347 min-1 is calculated for the pyruvate kinase interactions with the ATP analogue. At 4 degrees C, Na,K-ATPase recognizes CNS8-ATP with a KD = 8.3 microM. At 37 degrees C, the enzyme becomes inactivated by the ATP analogue in a biphasic manner. Inactivation results in the incorporation of [alpha-32P]8-CNS8-ATP into the catalytic alpha-subunit of the enzyme. Limited tryptic digestion in the presence of 150 mM KCl results in the formation of a radioactive peptide of Mr = 56,000, known to bear the purine binding domain of Na,K-ATPase. The results described in this article verify CNS8-ATP as a sulfhydryl-reactive ATP analogue and characterize this new ATP analogue as a useful tool for structure/function studies on ATP-recognizing enzymes.


Enhanced sensitivity to oxidative stress in Cu,ZnSOD depleted rat erythrocytes. The effects on red blood cells of superoxide dismutase (Cu,ZnSOD) depletion, induced by feeding Wistar rats with a copper deficient diet, were investigated. SOD depleted red blood cells were more sensitive to peroxidation and to hemolysis than normal cells when exposed to tert-butylhydroperoxide (t-BOOH). Membranes isolated from SOD depleted cells showed a lower content of vitamin E and higher (Na+, K+) and Mg2+ ATPase activities. These results support the view that superoxide dismutase plays an important role in cellular oxidative metabolism.


Footprinting of EcoRI endonuclease at high pressure. Hydroxyl radicals generated by irradiation with gamma rays have been used to footprint EcoRI endonuclease with single base pair resolution at pressures up to 144 MPa. At atmospheric pressure (0.1 MPa) a 10 base pair footprint was found. With increasing pressure three types of responses were observed: (1) bases distant from the recognition sequence showed a moderate increase in solvent exposure; (2) the bases at the point of enzymatic activity showed a large increase in cleavage by the hydroxyl radicals; and (3) the two center-most bases exhibited no pressure-induced change in solvent accessibility. The results are interpreted in terms of localized conformational changes of EcoRI.


Expression of insulin-like growth factor-I (IGF-I) and its receptor in the peri-implantation mouse uterus, and cell-specific regulation of IGF-I gene expression by estradiol and progesterone. This study describes the expression of insulin-like growth factor-I (IGF-I) and IGF-I receptor (IGF-IR) genes in the mouse uterus during the peri-implantation period (Days 1-6 of pregnancy), as well as effects of estradiol (E) and progesterone (P) on cell-specific IGF-I gene expression in the uterus of the ovariectomized adult mouse. Northern blot analysis showed that IGF-I mRNA levels were low but readily detectable in the uterus on Day 1 of pregnancy and steadily increased, reaching high levels just before (Day 4) and after initiation of implantation (Days 5 and 6). In general, IGF-IR transcripts were present in low abundance in uterine RNA throughout the peri-implantation period. However, six sizes of uterine IGF-IR transcripts were detected, and the relative abundance of two of these transcripts varied significantly during the peri-implantation period. Cell-specific expression of the IGF-I gene was examined by in situ hybridization to mRNA and immunohistochemical detection of protein. The results indicated that the synthesis of IGF-I on Days 1 and 2 was most predominant in glandular and luminal epithelial cells. However, on Days 3 and 4, stromal cells, and on Days 5 and 6, decidual cells appeared to be the predominant sites of synthesis of this growth factor. Uterine IGF-I gene expression was stimulated by ovarian steroids. Northern blot analysis showed that IGF-I transcripts were rare in the ovariectomized adult mouse uterus, but an injection of P and/or E caused a rapid accumulation of these transcripts. Analysis of the cell-specific expression of uterine IGF-I showed that E induced IGF-I gene expression primarily in epithelial cells, whereas P did so in the stroma. Superimposition of E on the P-primed uterus further stimulated IGF-I expression in the stroma. The results of these studies are consistent with an autocrine/paracrine function of uterine IGF-I, and indicate that ovarian steroids regulate the cell-specific and temporal patterns of expression of this gene in the peri-implantation mouse uterus.