Mu-delta opioid interactions. I: The delta peptide, DPDPE, increases morphine-induced EEG and EEG spectral power. The effects of the selective delta peptide, DPDPE ([2-D-penicillamine,5-D-penicillamine]enkephalin), on morphine-induced EEG and EEG power spectra were assessed. Adult female Sprague-Dawley rats were implanted with cortical EEG electrodes and permanent indwelling ICV and IV cannulae. Rats were pretreated with ICV sterile water or ICV DPDPE at 1.25, 2.5, or 5.0 nmol, 10 min before receiving IV morphine at 3 mg/kg. The treatments produced high voltage EEG bursts and associated behavioral stupor. The EEG data were further analyzed on a Pathfinder II computer and statistical analysis of EEG spectral parameters was performed by using a one-way ANOVA followed by Turkey's HSD. ICV DPDPE at 2.5 nmol and 5.0 nmol significantly increased EEG absolute global spectral power. Furthermore, ICV DPDPE at 2.5 and 5.0 nmol significantly increased the duration of morphine-induced high voltage EEG bursts. These data further indicate an in vivo interaction between mu and delta opioid receptors.


Mu-delta opioid interactions. II: Beta-FNA inhibits DPDPE-induced increases in morphine EEG and EEG spectral power. In the present study, the effects of beta-FNA on DPDPE-induced increases in morphine EEG and EEG power spectra were assessed. Adult female Sprague-Dawley rats were implanted with cortical EEG electrodes and permanent indwelling ICV and IV cannulae. Rats were administered ICV beta-FNA at 20 nmol or ICV sterile water. Then 18-24 h later, rats were administered ICV DPDPE at 2.5 nmol or ICV sterile water followed, 10 min later, by IV morphine at 3 mg/kg. Morphine-induced changes in EEG global (1-50 Hz) spectral parameters, the duration of morphine-induced high voltage EEG bursts, the period of EEG and behavioral excitation, and the latency to onset of slow-wave sleep were statistically analyzed using a one-way analysis of variance. beta-FNA pretreatment significantly decreased morphine-induced total spectral power seen in the DPDPE + morphine group. beta-FNA pretreatment also significantly decreased the duration of morphine-induced EEG bursts, the period of EEG and behavioral excitation, and the latency to onset of slow-wave sleep in the DPDPE + morphine group. These data, therefore, suggest that DPDPE may be increasing the effects of morphine on EEG through delta opioid receptors associated with the mu-delta opioid receptor complex.


The ACTH/MSH(4-9) analogue ORG 2766 stimulates microtubule formation in axons of central neurons of the snail Lymnaea stagnalis. Central nervous systems of the pond snail Lymnaea stagnalis were incubated in vitro in different concentrations of ORG 2766 (10(-9)-2.5 x 10(-4) M) for 10 and 20 h. Quantitative ultrastructural study of cross sections of the cerebral commissure showed that the number of microtubules in large axons had increased after 10 h of incubation by approximately 50% (Experiment 1) and 30% (Experiment 2), respectively. No further increase was observed after 20 h of incubation. (The higher concentrations were studied.) Maximal stimulation was already found at a concentration of 10(-8) M. At a concentration of 10(-9) M control levels were observed. It is concluded that ORG 2766 stimulates microtubule formation already at very low concentrations. It is not clear whether the compound stimulates synthesis of tubulin, induces assembly of microtubules, or causes an increase in stability of microtubules. Nevertheless, ultrastructural data on the morphology of the glial cells indicate that these cells are activated by ORG 2766 treatment, which suggest that ORG 2766 has general trophic effects.


Endothelin-1 (ET)-induced mobilization of intracellular Ca2+ stores from the smooth muscle facilitates sympathetic cotransmission by potentiation of adenosine 5'-triphosphate (ATP) motor activity: studies in the rat vas deferens. Endothelin-1 (ET) enhances nerve-stimulated contractions in epididymal (E) and prostatic (P) halves of the rat vas deferens, in addition to raising the basal tone in E. Whereas the peak increase in basal tone occurs in about 30 s, the maximal enhancement of neurotransmission is observed within 5 min. The latter effect is long lasting and is maintained even after extensive tissue washout. Furthermore, ET potentiates, in a concentration-dependent fashion, the adenosine 5'-triphosphate (ATP) or the adenylylimidodiphosphate (AMP-PNP) but not the noradrenaline (NA)-induced motor activity. The ATP motor response is partially blocked in media without Ca2+ plus 0.1 mM EGTA or following tissue incubation in buffer containing 10-50 nM nifedipine. However, these procedures do not modify significantly the ET-induced potentiation of the ATP contractions. The ET-induced potentiation of the ATP motor response is not modified by tissue preincubation in Ca(2+)-free buffer plus 10-30 microM ryanodine or 5-20 mM caffeine. The ET-induced rise in E basal tension is significantly reduced in the absence of external Ca2+ or by nifedipine; ryanodine does not modify this effect. Surgical denervation of the tissues does not obliterate the ET-induced potentiation of the ATP motor responses nor the ET increase in E basal tension in tissues superfused in Ca(2+)-free media or buffer with 2.5 mM Ca2+. Endothelin-1 does not significantly modify the overflow of 3H-NA, following transmural electrical depolarization of tissue nerve terminals. Hoe 140 did not interfere with the ET activity.


Random cytochrome-C-oxidase deficiency of oxyphil cell nodules in the parathyroid gland. A mitochondrial cytopathy related to cell ageing? Cytochrome-c-oxidase (complex IV) was histochemically studied in oncocytic adenoma (n = 10) and carcinoma of the thyroid gland (n = 3), cystadenolymphomas and oncocytic adenomas of the major salivary glands (n = 9), oncocytic neoplasia of the kidney (n = 1) and in 21 parathyroid glands with primary hyperparathyroidism and adenomatous proliferation (n = 17) and secondary hyperparathyroidism with hyperplasia (n = 4). Only in the parathyroids defects of cytochrome-c-oxidase were found being expressed in all 4 glands with hyperplasia (14 defects) and in 5 of the 17 adenomas (11 defects). All defects were confined to foci with oxyphil cell differentiation, the defect areas varying from 0.09 to 21.10 sq mm in hyperplastic glands and from 0.11 to 13.88 sq mm in adenomas, the size of the oxyphil foci varying from 0.12 sq mm-105.38 sq mm. However, not every oxyphil nodule of a gland was devoid of cytochrome-c-oxidase activity. Of 6 predominantly oxyphil adenomas, 4 showed no defects. No defects were observed either in 2 adenomas without oxyphil cells. Further enzymes of the respiratory chain, succinate dehydrogenase (complex II) and ATP synthetase, (complex V) were devoid of defects. In parathyroids with hyperplasia and oxyphil areas, defects of cytochrome-c-oxidase occurred significantly more often and tended to be larger than in adenomas, statistical analysis revealing a significant correlation between the occurrence of defects and the number of oxyphil foci but not with the total oxyphil area.(ABSTRACT TRUNCATED AT 250 WORDS)


Malignant fibrous histiocytoma and osteosarcoma in association with fibrous dysplasia of bone. Report of three cases. Three cases of bone sarcomas arising in fibrous dysplasia (FD) without prior radiation therapy were reported. One of the tumors was a conventional osteoblastic osteosarcoma (OS) and the other two were malignant fibrous histiocytomas (MFH). The lesion of FD was monostotic in two patients and polyostocic (monomelic) in one. Bone sarcomas and FD were detected simultaneously in all cases. One of the MFH cases died 6 months after diagnosis, and the other MFH case and the OS case are alive without disease 3 years and one and a half years after surgery, respectively. Conservative treatment has been selected in most of the patients of FD. We should closely follow up these patients, because bone sarcomas such as OS and MFH could arise at the site of FD. This is the first report of the cases of MFH of bone arising in FD.


[Granulocyte metabolism in workers at a coke-processing plant]. Acid phosphatase activity examinations and nitrotetrazolium blue reduction test after latex stimulation in peripheral blood neutrophils were performed in the group of 32 coking plant workers. Reference group consisted of 150 men not exposed to any toxic substances. Neutrophils of coking plant workers, comparing with the reference group, revealed decreased activity of acid phosphatase and impaired reduction of nitrotetrazolium blue after latex stimulation. The results suggest the possibility of toxic influence of coking plant environment on the lysosomal enzymes and oxygen-dependent metabolism of neutrophils what subsequently may diminish bactericidal powers.


Morphometry and prognosis in cancer of the pancreatic head. The histological slides of 39 cases of cancer of the pancreatic head were analysed using an interactive image analyser system. Some 14 cases were classified as periampullary, 25 as ductal pancreatic cancer. All cases had undergone radical tumor resection according to Whipple's procedure. Morphometric data, tumor size and metastatic nodal involvement were correlated to prognosis. Univariate statistical analysis showed that the classical differentiation between ductal and periampullary cancer was a weaker prognosticator than morphometric variables. In fact, multivariant statistical analysis showed that the morphometric variable irregularity was the best prognosticator (p = 0.0001). No other variable added significant prognostic information. Irregularity is a newly developed variable describing the nuclear shape corrected for roundness. We conclude that morphometry can be of essential prognostic information for the clinician in cancer of the pancreatic head.


Transmission of infectious diseases in children. A rational approach to the control of pediatric infectious diseases requires an appreciation of their epidemiology and mechanisms of transmission. The setting in which the exposure occurs may have a major influence on the risk of infection. Unfortunately, the epidemiology of many important pediatric pathogens is complex and incompletely understood. Recent advances in diagnostic microbiology and molecular epidemiology may help to solve some of the remaining riddles.


Accumbens GABA-ergic innervation contributes to the stressor-induced locomotor depression in rats. The effect of intra-accumbens injections of drugs changing the function of GABA-A and GABA-B receptor systems on stressor-induced motor depression, was studied in rats. Local injections of picrotoxin and baclofen, but not of midazolam and muscimol, attenuated the inhibitory effect of inescapable footshock on locomotor activity in the open field test, examined 24 h after a single exposure of rats to the stressful event. The results obtained with picrotoxin may be related to the general disinhibitory properties of the convulsant on brain neuronal activity, in a period of time important for consolidation of central processes evoked by inescapable shock. The lack of effects of muscimol and midazolam, further underlines the minor and/or indirect role of accumbens GABA-A receptor-related innervation in the neural processes generated by stressful event. On the other hand, the results obtained with baclofen confirm the reports indicating an inverse relationship between the number of GABA-B receptors in the frontal cortex and the development of helpless behavior in rats. It is also noteworthy that most antidepressant drugs which have been shown to prevent or reverse behavioral deficits after inescapable shock, upregulate GABA-B receptors in the frontal cortex. Hence, it appears that GABA-B receptor-related systems within the nucleus accumbens, may contribute to the footshock-induced behavioral depression, including locomotor inhibition. The reduction of stress effect by baclofen does not seem to reflect changes in fear and anxiety, since the drug was given after the stress session, and the anxiolytic midazolam appeared to be ineffective in this test.


Colonic diverticula. When complications require surgery and when they don't. The value of a high-fiber diet in preventing and treating colonic diverticula is firmly established. Although the diagnosis of diverticulosis is usually made with colonoscopy or barium enema examination, computed tomography has become the test of choice during acute diverticulitis, when the diagnosis cannot be confidently made clinically. Recently developed surgical principles for diverticulitis include radiographically directed drainage with delayed operation for peridiverticular abscess, resection of the site of disease in patients with general peritonitis, and primary anastomosis in most cases requiring urgent intervention. Diverticulosis accompanied by abdominal pain or irregular bowel habits is by itself rarely an indication for surgery. Diverticular bleeding usually resolves spontaneously, but persistent bleeding can usually be successfully treated with segmental colectomy after localization of the bleeding site with colonoscopy or arteriography.


Testing of cyclophosphamide and diethylstilbestrol for their ability to enhance virus survival in normal human cells. A relatively simple assay for determining the capability of chemicals to induce the reactivation of UV-irradiated pseudorabies virus in human embryo cells is presented. Using this assay cyclophosphamide (CP) and diethylstilbestrol (DES) were tested in the presence and in the absence of exogenous metabolizing system. N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) was used for comparison. CP increased the survival of UV-irradiated pseudorabies virus exclusively in the presence of exogenous metabolizing system. In the cells pretreated with CP or MNNG the survival of the virus was dependent on time interval between host cell pretreatment and virus infection. No enhancement of virus survival was obtained in the cells pretreated with DES.


Effects of androgen treatment on adenylate cyclase system in rat hepatic membranes. Effects of androgen treatment of young female rats on glucagon- and catecholamine-sensitive adenylate cyclase activity and adrenergic receptors of hepatic membranes have been studied. Injections of testosterone propionate for 7 days showed a significant decrease in the adenylate cyclase activity responding to isoproterenol and glucagon. The decrease in hormonal stimulation of the enzyme was accompanied with the fall in activation by non-hormonal stimuli, such as forskolin, sodium fluoride, Gpp(NH)p and Mn, without any changes in the number and the affinity of beta-adrenergic receptors of the membrane. These results suggest that androgens exert post-receptor effects by inhibiting the activity of the catalytic unit of adenylate cyclase system in rat hepatic membranes.


Effects of cyclooxygenase and lipoxygenase inhibitors on digoxin-induced arrhythmias and haemodynamics in guinea-pigs. Effects of cyclooxygenase and lipoxygenase inhibitors of digoxin-induced arrhythmias and haemodynamics were studied in guinea-pigs. ECG, mean arterial blood pressure heart rate, pressure rate index and arrhythmias were recorded, starting 15 min before digoxin administration and continuing for 30 min afterwards. The cyclooxygenase inhibitor aspirin (50 mg kg-1) and the dual cyclooxygenase/lipoxygenase inhibitor BW 755C (0.25-10.0 mg kg-1) were found to produce a significant protection against the arrhythmias, whereas aspirin (100 mg kg-1) and CGS 8515 were found to be ineffective. SK&F 104 353, a potent and selective peptidoleukotriene receptor antagonist significantly attenuated the arrhythmias and mortality in a dose-dependent manner. It is concluded that production of both cyclooxygenase and lipoxygenase metabolites could favour the occurrence and/or the maintenance of digoxin-induced cardiac toxicity.


Kava pyrones and resin: studies on GABAA, GABAB and benzodiazepine binding sites in rodent brain. Kava, an intoxicating beverage prepared from the pepper plant Piper methysticum, is widely consumed by the indigenous peoples in the islands of the South Pacific. As the first of a series of studies on the neuropharmacological interactions of kava with CNS receptors we tested purified pyrones and kava resin for activity on GABA and benzodiazepine binding sites in rat and mouse brain membranes. Only weak activity was observed on GABAA binding sites in washed synaptosomal membranes prepared from rat brain and this was abolished by extraction of the membranes with Triton X-100, suggesting that lipid soluble components were involved. No effects were observed on GABAB binding sites in rat brain membranes in vitro. Kava resin and pyrones exerted some weak effects on benzodiazepine binding in vitro but this did not correlate with pharmacological activity. In addition, in ex vivo studies, no effects were observed on [3H]diazepam binding to brain membranes prepared from mice in which selected kava constituents were injected intraperitoneally, whereas similarly administered diazepam (5 mg/kg) inhibited [3H]diazepam binding by greater than 95%. Similar lack of activity was observed in in vivo binding studies; injection of kava resin failed to influence the CNS binding of the benzodiazepine-receptor ligand [3H]Ro15-1788 injected into mice prior to sacrifice. The pharmacological activities of kava resin and pyrones do not appear to be explained by any significant interaction with GABA or benzodiazepine binding sites.


The involvement of sigma and phencyclidine receptors in the action of antipsychotic drugs. An atypical antipsychotic drug clozapine and a selective sigma antagonist BMY 14802 were significantly less effective in the behavioural experiments (against apomorphine, d-amphetamine and MK-801), as well in the radioligand binding studies against 3H-spiperone (dopamine2-receptors) and 3H-haloperidol (sigma receptors) in the rat brain, as compared to a typical antipsychotic compound haloperidol. Contrary to haloperidol and BMY 14802, clozapine was a relatively selective antagonist of MK-801-induced motor excitation in the mouse. A nearly 3-fold lower dose of clozapine was needed to block the effect of MK-801 (6.4 mumol/kg) as compared to the action of amphetamine (17 mumol/kg). Haloperidol and clozapine, but not BMY 14802, antagonized apomorphine-induced aggressiveness in the rat. After long-term treatment (for 15 days) with BMY 14802 (10 mg/kg daily), haloperidol (0.5 mg/kg daily) and clozapine (10 mg/kg daily) the motor depressant effect of apomorphine (0.15 mg/kg) was reversed. Chronic haloperidol treatment, but not administration of BMY 14802 and clozapine, increased the number of dopamine2-receptors in the rat brain. BMY 14802 caused upregulation of sigma receptors in frontal cortex, whereas haloperidol induced the opposite change in cerebellum. Repeated treatment with clozapine significantly augmented the motor stimulating effect of MK-801 in rats. Simultaneously with a behavioural change the density of 3H-TCP binding sites in the rat forebrain was elevated after long-term treatment with clozapine, probably indicating the involvement of PCP binding sites at NMDA channel in the action of clozapine.


Dietary fibre, sugar, starch and amino acid content of kale, ryegrass and seed of rape and field beans as influenced by S- and N-fertilization. In pot experiments, S-deficiency decreased the seed weight of rape from 4.30 to 2.44 g/1000 seeds at highest N-level. N/S ratios varied from 5.8 to 45.0 and from 3.7 to 21.6 in ryegrass and kale. S-deficiency tended to decrease the fat(oil) content. In kale and ryegrass, it was increased by N-applications, but decreased in rape. S-deficiency had very little effect on starch and sugar content, whereas N-deficiency in kale greatly increased starch (eightfold) and sugar content. Soluble (SDF) and insoluble dietary fibre (IDF) content was hardly affected by S-deficiency. In kale and ryegrass N-deficiency decreased SDF-content, but increased IDF-content. Cystine and methionine concentrations (g/16 g N) were affected by S-deficiency, most strongly at high N-levels, where decreases in kale, ryegrass, rape and field beans amounted to respectively 18, 39, 40 and 17% for cystine and 27, 59, 24 and 6% for methionine. Also, lysine, threonine and tryptophan were decreased. S-deficiency increased aspartic acid (asparagine) content of ryegrass (fourfold) and of arginine in rape and kale. In N-balance trials with rats, S-deficiency reduced the biological value (BV) of the protein of kale from 65 to 40 and of field bean seed from 70 to 61. Corresponding decreases in digestible energy (DE) were from 56 to 48 and from 82 to 78, respectively.


[Hyperinsulinism. Causes and mechanisms]. A high plasma insulin concentration in the presence of a normal or high plasma glucose level appears to be a common feature of glucose intolerance, obesity, and hypertension. Hyperinsulinemia has been recognized as a major risk factor for the development of coronary artery disease independent of blood pressure and plasma lipid levels. All these conditions are frequently associated, particularly in aging, a state itself characterized by hyperinsulinemia. This common association has prompted the hypothesis that hyperinsulinemia may be a causative factor rather than the consequence of obesity, diabetes, hypertension, and hyperlipidemia. If that is the case, defining the nature and mechanisms of hyperinsulinemia becomes of primary interest. Insulin resistance is also a striking feature of all of the above mentioned pathologic states. In the presence of a preserved B-cell function, hyperinsulinemia can represent the mechanism designed to overcome the defect in the biological action of the hormone. For instance, there is a clear-cut age-related decline in the body's sensitivity to insulin. In order to compensate for this defect in insulin-mediated glucose metabolism, the B-cell must increase its secretion. On the other hand, a certain degree of insulin resistance can be induced both in animals and man by prolonged euglycemic hyperinsulinemia. Little is known regarding possible primary defects of the B-cell leading to uncontrolled oversecretion of insulin and subsequent insulin resistance. The primary defect, more probably, resides in an alteration of one or more of the steps whereby insulin exerts it own action. In favor of this hypothesis are the observations that insulin resistance segregates in familial clusters and that the first defect found in normoglycemic relatives of insulin-resistant diabetic patients is a reduced transformation of glucose into glycogen. Whatever is the primary defect, it is likely that a correction of insulin resistance might reduce the circulating levels of plasma insulin, possibly playing a beneficial effect on glucose tolerance, body weight, blood pressure and plasma lipid concentration.


The effectiveness of adherence intervention in a colon cancer prevention field trial. Adherence interventions were implemented in a 1-year community-based colon cancer prevention clinical trial (n = 110) using wheat bran fiber and calcium dietary supplements. The adherence promotion strategy was guided by a theoretical model. The adherence intervention contains both a generalized portion given to all participants and an individualized portion given to marginal (50-74% intake) and low (under 50% intake) adherers. A regression model was employed to assess the effectiveness of the interventions both at the first intervention and at subsequent times. The Health Behavior in Cancer Prevention Model-based adherence promotion intervention was associated with retention of participants, both during the run-in period and after randomization (P = 0.05); and maximization of the percentage of the 13.5-g recommended fiber supplement consumed during the trial (92.5%). The positive effects of the adherence intervention were greater with first-time nonadherers and the control group than with the experimental group. The high-fiber group had notably more biological GI effects from the increased fiber intake, more preexisting comorbidities, and lower perceived cognitive and physical health status. Randomized participants had excellent adherence overall. Retention rates in the trial were better than would be expected without the adherence intervention, especially among those participants who may have been at higher risk for dropping out of the study. This suggests that a systematic, theoretically based adherence strategy should be further tested in clinical trial settings in which lower adherence is a problem.


Associations of social status and health-related beliefs with dietary fat and fiber densities. Lower social status groups have higher mortality rates from some diet-related diseases and higher dietary fat and lower dietary fiber intakes. Such dietary patterns have been found to be related to social status, environmental influences, and health-related beliefs and expectations. Associations of social status and diet-related and health-related beliefs and expectations with dietary fat and fiber densities were examined in a population sample of 874 respondents to a postal questionnaire. A food frequency listing of 172 foods was used to assess usual dietary intake. More positive beliefs and expectations were associated with lower dietary fat and higher dietary fiber densities in univariate models; beliefs and expectations differed little between social status groups. In multivariate models, stronger perceptions of external influences on food choices, fewer perceived barriers to eating a healthy diet, and social status were independently associated with low dietary fat density. Diet-related and health-related beliefs and perceptions of external influences on food choices, but not social status, were independently associated with high dietary fiber density. The belief that diet is a major cause of stroke, diabetes, and hypertension was weakly associated with the dietary fiber density of lower social status groups. Social status and perceptions of external influences on dietary choice, as well as personal beliefs, have independent associations with food intake. Although exclusive targeting of lower social status groups is not indicated, interventions to increase dietary fiber intake should address expectations, attitudes, and beliefs about dietary fiber and health and perceptions of external influences on food choices, especially among lower status groups; interventions to lower dietary fat intake should address a broad range of external and social factors, as well as personal beliefs.


Nuclear bodies in the egg cells of a Gyrodactylus species (Platyhelminthes, Monogenea). The presence and ultrastructure of distinctive nuclear bodies in a mature 'egg cell' of a Gyrodactylus sp. are described. These electron-dense granular bodies, which appear in section as open or closed rings or as a solid mass are not membrane-bound and lie in clusters close to the nucleolus. The nuclear bodies are compared with the nuclear-inclusion bodies previously reported in platyhelminths. The possible origin, significance and role of the nuclear bodies in this Gyrodactylus sp. are discussed.


Structural studies of the retroviral proteinase from avian myeloblastosis associated virus. The structure of the retroviral proteinase from avian myeloblastosis associated virus (MAV) has been determined and refined at 2.2 A resolution. This structure is compared with those of homologous proteinases from Rous sarcoma virus (RSV) and human immunodeficiency type 1 virus (HIV). Through comparison with the structure of a proteinase-inhibitor complex from HIV, a model of a complex between MAV proteinase and a peptide substrate has been generated. Examination of this model suggests structural basis for the diverse specifications of viral proteinases.


Structure of inositol monophosphatase, the putative target of lithium therapy. Inositol monophosphatase (EC 3.1.3.25), the putative molecular site of action of lithium therapy for manic-depressive illness, plays a key role in the phosphatidylinositol signaling pathway by catalyzing the hydrolysis of inositol monophosphates. To provide a structural basis from which to design better therapeutic agents for manic-depressive illness, the structure of human inositol monophosphatase has been determined to 2.1-A resolution by using x-ray crystallography. The enzyme exists as a dimer of identical subunits, each folded into a five-layered sandwich of three pairs of alpha-helices and two beta-sheets. Sulfate and an inhibitory lanthanide cation (Gd3+) are bound at identical sites on each subunit and establish the positions of the active sites. Each site is located in a large hydrophilic cavern that is at the base of the two central helices where several segments of secondary structure intersect. Comparison of the phosphatase aligned sequences of several diverse genes with the phosphatase structure suggests that the products of these genes and the phosphatase form a structural family with a conserved metal binding site.


Extended x-ray absorption fine structure studies of a retrovirus: equine infectious anemia virus cysteine arrays are coordinated to zinc. Zinc finger arrays have been established as a critical structural feature of proteins involved in DNA recognition. Retroviral nucleocapsid proteins, which are involved in the binding of viral RNA, contain conserved cysteine-rich arrays that have been suggested to coordinate zinc. We provide metalloprotein structural data from an intact virus preparation that validate this hypothesis. Extended x-ray absorption fine structure (EXAFS) spectroscopy of well-characterized and active preparations of equine infectious anemia virus, compared with a peptide with known coordination and in combination with available biochemical and genetic data, defines a Cys3His1 coordination environment for zinc. The average of the Zn-S distances is 2.30(1) A and that of the Zn-N distance (to histidine) is 2.01(3) A.


Human spumaretrovirus-related sequences in the DNA of leukocytes from patients with Graves disease. Viruses, and more particularly retroviruses, have been postulated to play a role in the pathogenesis of autoimmune diseases. In a search for spumaretrovirus infection markers, we screened a group of 29 patients with Graves disease and a representative healthy population (23 subjects) as a control. Southern blot hybridization under stringent conditions, of patients' DNA extracted from peripheral blood lymphocytes, with a spumaretrovirus-specific genomic probe derived from the human spumaretrovirus (HSRV) prototype, gave a positive signal in 10 cases. Moreover, by PCR, HSRV-related sequences were detected in the DNA of 19 patients (66%). Positive DNA samples in Southern blots were also positive in PCR for all regions tested (gag, bel1, bel2, long terminal repeat). Amplified (gag and bel2) products were cloned and sequenced; they showed high homology with HSRV. On the other hand, all 23 control subjects were negative by both procedures. Sera from both populations were examined for the presence of antibodies reactive with antigens of the spumaretrovirus family. These sera were negative by several immunodetection techniques: ELISA, indirect immunofluorescence, serum neutralization, and Western blotting. These results strongly suggest the existence of an association between Graves disease and the presence of HSRV-related infection markers.


Identification of multiple structural domains regulating viroid pathogenicity. To investigate the role of individual structural domains in viroid pathogenicity and replication, a series of interspecific chimeras was constructed by exchanging the terminal left (TL) and/or pathogenicity (P) domains between tomato apical stunt (TASVd) and citrus exocortis (CEVd) viroids. All six chimeras tested were replicated stably in tomato, and the symptoms exhibited by infected plants were intermediate between those induced by the parental viroids. Quantitative comparisons of symptom development and progeny accumulation revealed that: (i) the TL domain of TASVd contains a determinant required for appearance of severe veinal necrosis in tomato, (ii) the severe epinasty and stunting characteristic of TASVd requires the presence of its TL and P domains, and (iii) the variable (V) and terminal right (TR) domains comprising the right side of the native structure also play an important role in viroid pathogenicity. Chimeras containing the right side of TASVd accumulated to higher levels early in infection, and infected plants developed more severe symptoms than those whose right halves were derived from CEVd. Although the individual contributions of the TL and P domains to symptom induction could not be completely separated from that of viroid titer, the TL domain appears to exert a greater effect upon symptom severity than does the P domain. The TL, P, V, and TR domains of TASVd and CEVd contain three discrete regions of sequence and/or structural variability that may correspond to the pathogenicity determinants uncovered by our genetic analysis.


Methylation in the preinitiation domain suppresses gene transcription by an indirect mechanism. Although the first observations of the inhibitory effect of methylation on gene activity were made almost a decade ago, the mechanism by which methyl groups affect transcription is still obscure. Here we use engineered promoters methylated in vitro in transient transfections to study the mechanism by which methylation mediates promoter repression. The results clearly show that the location of the methyl groups within the promoter region determines the extent of promoter repression. The most effective suppression was observed when methylation was in the preinitiation domain. The results also support a previous suggestion that a mediator protein is involved in the mechanism of promoter inhibition. The suppressor effect of methylation at sequences flanking the TATA box can be partially overcome in the presence of the simian virus 40 enhancer. In addition, results obtained by transient thymidine labeling of Ltk- cells that were transfected with a methylated thymidine kinase gene from herpes simplex virus, at the level of approximately one template per cell, further support the conclusion that methylation affects primarily transcription preinitiation.


Ionizing radiation activates transcription of the EGR1 gene via CArG elements. The present studies have examined the effects of ionizing radiation on control of the early growth response 1 (EGR1) gene. Exposure of human HL-525 cells to x-rays was associated with increases in EGR1 mRNA levels. Nuclear run-on assays showed that this effect is related at least in part to activation of EGR1 gene transcription. Sequences responsive to ionizing radiation-induced signals were determined by deletion analysis of the EGR1 promoter. The results demonstrate that x-ray inducibility of the EGR1 gene is conferred by a region containing six serum response or CC(A+T-rich)6GG (CArG) motifs. Further analysis confirmed that the region encompassing the three distal or upstream CArG elements is functional in the x-ray response. Moreover, this region conferred x-ray inducibility to a minimal thymidine kinase gene promoter. Taken together, these results indicate that ionizing radiation induces EGR1 transcription through CArG elements.


O2- production by B lymphocytes lacking the respiratory burst oxidase subunit p47phox after transfection with an expression vector containing a p47phox cDNA. The respiratory burst oxidase of phagocytes and B lymphocytes is a complicated enzyme that catalyzes the one-electron reduction of oxygen by NADPH. It is responsible for the O2- production that occurs when these cells are exposed to phorbol 12-myristate 13-acetate or other appropriate stimuli. The activity of this enzyme is greatly decreased or absent in patients with chronic granulomatous disease, an inherited disorder characterized by a severe defect in host defense against bacteria and fungi. In every chronic granulomatous disease patient studied to date, an abnormality has been found in a gene encoding one of four components of the respiratory burst oxidase: the membrane protein p22phox or gp91phox, or the cytosolic protein p47phox or p67phox. We report here that O2- production was partly restored to phorbol 12-myristate 13-acetate-stimulated Epstein-Barr virus-transformed B lymphocytes from a patient with p47phox-deficient chronic granulomatous disease by transfection with an expression plasmid containing a p47phox cDNA inserted in the sense direction. No detectable O2- was produced by untransfected p47phox-deficient lymphocytes or by p47phox-deficient lymphocytes transfected with an antisense plasmid. The finding that O2- can be produced by p47phox-deficient B lymphocytes after the transfer of a p47phox cDNA into the deficient cells suggests that this system could be useful for studying the function of mutant p47phox proteins in whole cells.


Dopamine receptor subtypes colocalize in rat striatonigral neurons. Dopaminergic neurons of the substantia nigra provide one of the major neuromodulatory inputs to the neostriatum. Recent in situ hybridization experiments have suggested that postsynaptic dopamine receptors are segregated in striatonigral and striatopallidal neurons. We have tested this hypothesis in acutely isolated, retrogradely labeled striatonigral neurons by examining the neuromodulatory effects of selective dopaminergic agonists on Na currents and by probing single-cell antisense RNA populations with dopamine receptor cDNAs. In most of the neurons examined (20/31), the application of the D1 dopamine receptor agonist SKF 38393 reduced evoked whole-cell Na+ current. The D2 agonists quinpirole and bromocriptine had mixed effects; in most neurons (23/42), whole-cell Na+ currents were reduced, but in others (8/42), currents were increased. In cell-attached patch recordings, bath application of SKF 38393 decreased currents as in whole-cell recordings, whereas quinpirole consistently (6/10) enhanced currents--suggesting that D2-like receptors could act through membrane delimited and non-delimited pathways. Changes in evoked current were produced by modulation of peak conductance and modest shifts in the voltage dependence of steady-state inactivation. Antisense RNA probes of dopamine receptor cDNA Southern blots consistently (5/5) revealed the presence of D1, D2, and D3 receptor mRNA in single striatonigral neurons. These findings argue that, contrary to a strict receptor segregation hypothesis, many striatonigral neurons colocalize functional D1, D2, and D3 receptors.


Guanine nucleotide-binding protein-coupled and -uncoupled states of opioid receptors and their relevance to the determination of subtypes. Opioid receptors are currently classified as mu, delta, and kappa types, but various subtypes have also been proposed. We have investigated whether subtypes exist by using [3H]bremazocine. [3H]Bremazocine binds to twice as many naloxone-sensitive sites as other nonselective opioid agonists, as shown in four membrane types that have very different ratios of mu, delta, and kappa receptor types. [3H]Bremazocine binding is completely inhibited by an excess (in unlabeled form) of other opioid ligands, with Hill coefficients of 0.8-0.95. These paradoxes can be explained if there are high- and low-affinity states of the mu, delta, and kappa receptors and bremazocine binds with similar affinities to both states. We propose that these states are the guanine nucleotide-binding protein (G-protein)-coupled form and the uncoupled form of each receptor. As evidence for this proposal, the [3H]bremazocine binding suffered little or no loss with G-protein-uncoupling treatments, whereas binding of other opioid agonists was fully sensitive. We conclude that [3H]bremazocine offers a tool for the measurement of the total pools of coupled and uncoupled opioid receptors and that much of the previous characterization of opioid receptor subtypes reflects, instead, a significant pool of G-protein-uncoupled opioid receptors.


Short-chain analogs of luteinizing hormone-releasing hormone containing cytotoxic moieties. Five hexapeptide and heptapeptide analogs of luteinizing hormone-releasing hormone (LH-RH) were synthesized for use as carriers for cytotoxic compounds. These short analogs were expected to enhance target selectivity of the antineoplastic agents linked to them. Native LH-RH-(3-9) and LH-RH-(4-9) containing D-lysine and D-ornithine at position 6 were amidated with ethylamine and acylated on the N terminus. The receptor-binding affinity of one hexapeptide carrier AJ-41 (Ac-Ser-Tyr-D-Lys-Leu-Arg-Pro-NH-Et) to human breast cancer cell membranes was similar to that of [D-Trp6]LH-RH. Alkylating nitrogen mustards (melphalan, Ac-melphalan), anthraquinone derivatives including anticancer antibiotic doxorubicin, antimetabolite (methotrexate), and cisplatin-like platinum complex were linked to these peptides through their omega-amino group at position 6. The hybrid molecules showed no LH-RH agonistic activity in vitro and in vivo but had nontypical antagonistic effects on pituitary cells in vitro at the doses tested. These analogs showed a wide range of receptor-binding affinities to rat pituitaries and cell membranes of human breast cancer and rat Dunning prostate cancer. Several of these conjugates exerted some cytotoxic effects on MCF-7 breast cancer cell line.


Acid-induced structural changes in human rhinovirus 14: possible role in uncoating. X-ray diffraction data were collected from human rhinovirus 14 crystals a few minutes after exposure to acid vapor and prior to excessive crystalline disorder. Conformational changes occurred (i) in the GH loop of viral protein (VP) 1, (ii) at the ion binding site on the outer surface of the pentamer center, and (iii) in VP3 and VP4 on the virion's interior in the vicinity of the fivefold axis. Amino acid substitutions in mutants resistant to low pH, or to drugs that inhibit uncoating, were concentrated in the vicinity of the GH loop. It is proposed that the acid-induced changes reflect processes that trigger uncoating.


Growth retardation in glioma cells cocultured with cells overexpressing a gap junction protein. To examine the role of gap-junctional intercellular communication in controlling cell proliferation, we have transfected C6 glioma cells with connexin 43 cDNA. The growth of transfected clones was dramatically reduced compared with nontransfected glioma cells. To further characterize the role of gap junctions in controlling proliferation, we have examined the growth of C6 cells cocultured with transfected cells overexpressing connexin 43. Although C6 cells grew at their normal rate when cocultured with nontransfected C6 cells, when cocultured with connexin 43-overexpressing cells they displayed a dramatic reduction in growth rate. Furthermore, a significant, dose-dependent reduction in cell proliferation was noted when C6 cells were cultured in medium conditioned by transfected cells. This effect correlated with the level of connexin 43 expression. These results suggest that the decreased cell proliferation rate of transfected cells and C6 cells cultured with them is due to the secretion of a growth inhibitory factor(s) and that the secretion of this factor may be linked to the level of gap junctional intercellular communication.


Conservation of structure and function of DNA replication protein A in the trypanosomatid Crithidia fasciculata. Human replication protein A (RP-A) is a three-subunit protein that is required for simian virus 40 (SV40) replication in vitro. The trypanosome homologue of RP-A has been purified from Crithidia fasciculata. It is a 1:1:1 complex of three polypeptides of 51, 28, and 14 kDa, binds single-stranded DNA via the large subunit, and is localized within the nucleus. C. fasciculata RP-A substitutes for human RP-A in the large tumor antigen-dependent unwinding of the SV40 origin of replication and stimulates both DNA synthesis and DNA priming by human DNA polymerase alpha/primase, but it does not support efficient SV40 DNA replication in vitro. This extraordinary conservation of structure and function between human and trypanosome RP-A suggests that the mechanism of DNA replication, at both the initiation and the elongation level, is conserved in organisms that diverged from the main eukaryotic lineage very early in evolution.


Regulation of neurosecretory habituation in PC12 cells: parallel pathways used by cAMP and calcium. Habituation of norepinephrine secretion in PC12 cells serves as a model for understanding the molecular mechanisms of simple memory processes in neurons. Elevation of intracellular cAMP levels by incubation with N-ethylcarboxamidoadenosine (NECA) or forskolin increased norepinephrine secretion in response to depolarization by high potassium or stimulation with acetylcholine. The extent to which cAMP altered norepinephrine secretion was dependent on the timing of its elevation, and it also altered the rate of habituation under certain conditions. However, cAMP increased norepinephrine secretion by a pathway distinct from that governing internal calcium levels, which correlates with habituation in the absence of elevated cAMP. An inverse correlation was found between the ability of calcium to lower NECA-induced cAMP levels and the ability of calcium to cause norepinephrine secretion. A model is proposed in which a single calcium-dependent pathway modulates both norepinephrine secretion and cAMP metabolism.


Translational enhancement of the poliovirus 5' noncoding region mediated by virus-encoded polypeptide 2A. Genetic and biochemical studies have revealed that the 5' noncoding region of poliovirus mediates translation of the viral mRNA by an unusual mechanism involving entry of ribosomes in internal sequences of mRNA molecules. We have found that mRNAs bearing the 5' noncoding region of poliovirus were translated at an enhanced rate in poliovirus-infected mammalian cells at a time when translation of cellular mRNAs was not yet inhibited. This translational enhancement of the polioviral 5' noncoding region was mediated by the expression of virus-encoded polypeptide 2A. This indicates that 2A is a multifunctional protein involved directly or indirectly in the activation of viral mRNA translation, in addition to its known roles in viral polyprotein processing and in inhibition of cellular protein synthesis. Thus, 2A represents an activator of translation of a viral mRNA that is translated by an internal ribosome binding mechanism. A likely consequence of this role of 2A is the efficient translation of viral mRNAs early in the infectious cycle, when host cell mRNAs can still compete with viral mRNAs for the host cell translation apparatus.


Baculovirus-mediated expression of retinoic acid receptor type gamma in cultured insect cells reveals a difference in specific DNA-binding behavior with the 1,25-dihydroxyvitamin D3 receptor. The baculovirus genetic expression system has been used to produce murine retinoic acid receptor (RAR) type gamma in Spodoptera frugiperda insect cells and Manduca sexta insect larvae. A hydroxyapatite binding assay revealed production levels of 300 pmol of unoccupied receptor per mg of protein in insect cells, whereas levels from infected insect larvae were found to average 100 pmol of RAR gamma per mg of protein. The cytosolic preparation from infected insect cells exhibited an equilibrium dissociation constant of 2.1 nM as determined by a retinoic acid saturation analysis plotted by the method of Scatchard. A polyclonal antibody directed against RAR gamma recognized the recombinant receptor protein as a 54,000-Da species. Electrophoretic mobility shift analyses demonstrated that protein extracts from RAR gamma-producing insect cells or larvae are capable of retinoic acid responsive element binding. This contrasts with the specific DNA-binding behavior of the insect cell-produced vitamin D receptor, which requires the presence of a mammalian-derived nuclear accessory protein. This distinction between RAR gamma and the vitamin D receptor suggests a difference in the molecular requirements by these two receptors for specific binding of their respective DNA response elements.


Determination of the orientation of a DNA binding motif in a protein-DNA complex by photocrosslinking. We have developed a straightforward biochemical method to determine the orientation of the DNA binding motif of a sequence-specific DNA binding protein relative to the DNA site in the protein-DNA complex. The method involves incorporation of a photoactivatable crosslinking agent at a single site within the DNA binding motif of the sequence-specific DNA binding protein, formation of the derivatized protein-DNA complex, UV-irradiation of the derivatized protein-DNA complex, and determination of the nucleotide(s) at which crosslinking occurs. We have applied the method to catabolite gene activator protein (CAP). We have constructed and analyzed two derivatives of CAP: one having a phenyl azide photoactivatable crosslinking agent at amino acid 2 of the helix-turn-helix motif of CAP, and one having a phenyl azide photoactivatable crosslinking agent at amino acid 10 of the helix-turn-helix motif of CAP. The results indicate that amino acid 2 of the helix-turn-helix motif is close to the top-strand nucleotides of base pairs 3 and 4 of the DNA half site in the CAP-DNA complex, and that amino acid 10 of the helix-turn-helix motif is close to the bottom-strand nucleotide of base pair 10 of the DNA half site in the CAP-DNA complex. The results define unambiguously the orientation of the helix-turn-helix motif relative to the DNA half site in the CAP-DNA complex. Comparison of the results to the crystallographic structure of the CAP-DNA complex [Schultz, S., Shields, S. & Steitz, T. (1991) Science 253, 1001-1007] indicates that the method provides accurate, high-resolution proximity and orientation information.


Localization of Epstein-Barr virus-encoded RNAs EBER-1 and EBER-2 in interphase and mitotic Burkitt lymphoma cells. The subcellular distribution of the small Epstein-Barr virus-encoded RNAs EBER-1 and EBER-2 has been investigated by using a high-resolution in situ hybridization technique. The distribution patterns in Raji cells of fluorescent oligodeoxynucleotides complementary to each RNA were detected by confocal laser scanning microscopy. Both RNAs were found in the cytoplasm as well as in the nuclei of interphase cells. In contrast, use of the same technique indicated an exclusively nuclear location for cellular U2 RNA. In the cytoplasm distribution of the EBERs was similar to that of the double-stranded RNA-dependent protein kinase, to which these RNAs can bind, and was coincident with the rough endoplasmic reticulum. In cells undergoing mitosis the EBERs became localized around the chromosomes, whereas the protein kinase remained uniformly distributed in the cytoplasm. A cytoplasmic location for EBER-1 and EBER-2 in interphase cells is consistent with the evidence for a role for these small RNAs in translational control.


Basonuclin: a keratinocyte protein with multiple paired zinc fingers. A cDNA clone has been prepared from mRNA of cultured human keratinocytes. The sequence of the cDNA reveals that in the C-terminal two-thirds of the corresponding protein (basonuclin), there are three separated pairs of adjacent zinc fingers. The amino acid sequence of each pair is homologous to that of the single pair of zinc fingers of the Drosophila transcription factor encoded by disco. Near the C-terminal end of basonuclin and on the surface of a putative alpha-helix, there is a stripe of serine residues similar to that of the transcription factor PRDII-BF1. Basonuclin possesses a sequence of six amino acids quite similar to one present in the myogenic family of proteins, including Myf5; this sequence is located in the omega loop of the myogenic proteins but within a zinc finger of the keratinocyte protein. As basonuclin is present mainly in the nuclei of the basal cell layer, its regulatory function is likely to be exerted prior to the process of terminal differentiation.


High density lipoprotein is the major carrier of lipid hydroperoxides in human blood plasma from fasting donors. Analysis of untreated fresh blood plasma from healthy, fasting donors revealed that high density lipoprotein (HDL) particles carry most (approximately 85%) of the detectable oxidized core lipoprotein lipids. Low density lipoprotein (LDL) lipids are relatively peroxide-free. In vitro the mild oxidation of gel-filtered plasma from fasting donors with a low, steady flux of aqueous peroxyl radicals initially caused preferential oxidation of HDL rather than LDL lipids until most ubiquinol-10 present in LDL was consumed. Thereafter, LDL core lipids were oxidized more rapidly. Isolated lipoproteins behaved similarly. Preferential accumulation of lipid hydroperoxides in HDL reflects the lack of antioxidants in most HDL particles compared to LDL, which contained 8-12 alpha-tocopherol and 0.5-1.0 ubiquinol-10 molecules per particle. Cholesteryl ester hydroperoxides (CEOOHs) in HDL and LDL were stable when added to fresh plasma at 37 degrees C for up to 20 hr. Transfer of CEOOHs from HDL to LDL was too slow to have influenced the in vitro plasma oxidation data. Incubation of mildly oxidized LDL and HDL with cultured hepatocytes afforded a linear removal of CEOOHs from LDL (40% loss over 1 hr), whereas a fast-then-slow biphasic removal was observed for HDL. Our data show that HDL is the principal vehicle for circulating plasma lipid hydroperoxides and suggest that HDL lipids may be more rapidly oxidized than those in LDL in vivo. The rapid hepatic clearance of CEOOHs in HDL could imply a possible beneficial role of HDL by attenuating the build-up of oxidized lipids in LDL.


IRS-1 activates phosphatidylinositol 3'-kinase by associating with src homology 2 domains of p85. IRS-1 is an insulin receptor substrate that undergoes tyrosine phosphorylation and associates with the phosphatidylinositol (PtdIns) 3'-kinase immediately after insulin stimulation. Recombinant IRS-1 protein was tyrosine phosphorylated by the insulin receptor in vitro and associated with the PtdIns 3'-kinase from lysates of quiescent 3T3 fibroblasts. Bacterial fusion proteins containing the src homology 2 domains (SH2 domains) of the 85-kDa subunit (p85) of the PtdIns 3'-kinase bound quantitatively to tyrosine phosphorylated, but not unphosphorylated, IRS-1, and this association was blocked by phosphotyrosine-containing synthetic peptides. Moreover, the phosphorylated peptides and the SH2 domains each inhibited binding of PtdIns 3'-kinase to IRS-1. Phosphorylated IRS-1 activated PtdIns 3'-kinase in anti-p85 immunoprecipitates in vitro, and this activation was blocked by SH2 domain fusion proteins. These data suggest that the interaction between PtdIns 3'-kinase and IRS-1 is mediated by tyrosine phosphorylated motifs on IRS-1 and the SH2 domains of p85, and IRS-1 activates PtdIns 3'-kinase by binding to the SH2 domains of p85. Thus, IRS-1 likely serves to transmit the insulin signal by binding and regulating intracellular enzymes containing SH2 domains.


NIP1, a gene required for nuclear transport in yeast. Cytochrome c with a nuclear localization signal added at the N terminus was mistargeted to the nucleus, resulting in a yeast strain deficient in mitochondrial cytochrome c. Reversion of this strain allowed the isolation of temperature-conditional mutants defective in nuclear transport, as demonstrated with one of these mutants, nip1-1, that was shown to be defective in nuclear accumulation of a LacZ protein containing a nuclear localization signal of the yeast ribosomal protein L29. The NIP1+ gene was cloned and shown to encode a 93,143-Da protein. Furthermore, an epitope-labeled NIP1 protein migrated in SDS/polyacrylamide gels with a mass of approximately 100,000 Da and was shown by immunofluorescence to localize mainly in the cytoplasm. NIP1+ was shown to be an essential gene by gene disruption experiments. Intriguingly, NIP1 has a serine-rich acidic N-terminal region that is similar in this regard to the N-terminal region of a previously described nuclear localization signal-binding protein, NSR1.


Zinc rapidly induces a metal response element-binding factor. Metal activation of metallothionein gene transcription is mediated by specific promoter sequences, termed metal regulatory elements (MREs). Nuclear extracts prepared from various human cell lines were assayed for their capacity to bind to a synthetic human MREa (hMREa) oligomer. Electrophoretic mobility-shift assays with extracts from control cells detected a single hMREa-containing complex. Addition to the growth medium of zinc, cadmium, or copper--metals known to induce MT biosynthesis in vivo--resulted in the rapid but reversible appearance of a second distinct hMREa-protein complex in all cell lines studied. This result was not seen when the metals were added directly to the extracts from control cells. DNA-binding protein blotting, UV crosslinking, and electroelution experiments were used to characterize the two hMREa-binding factors, termed BF1 and BF2. MRE-BF1 has an apparent molecular mass of approximately 86 kDa and binds to the hMREa in control cells, whereas MRE-BF2 consists of two molecules of approximately 28 kDa and binds to the hMREa in metal-treated cells. EDTA and o-phenanthroline inhibited binding of both factors to hMREa in a dose-dependent manner, indicating that a metal atom or atoms are essential for interaction of the factors with DNA.


Copper,zinc superoxide dismutase is primarily a cytosolic protein in human cells. The intracellular localization of human copper,zinc superoxide dismutase (Cu,Zn-SOD; superoxide:superoxide oxidoreductase, EC 1.15.1.1) was evaluated by using EM immunocytochemistry and both isolated human cell lines and human tissues. Eight monoclonal antibodies raised against either native or recombinant human Cu,Zn-SOD and two polyclonal antibodies raised against either native or recombinant human Cu,Zn-SOD were used. Fixation with 2% paraformaldehyde/0.2% glutaraldehyde was found necessary to preserve normal distribution of the protein. Monoclonal antibodies were less effective than polyclonal antibodies in recognizing the antigen after adequate fixation of tissue. Cu,Zn-SOD was found widely distributed in the cell cytosol and in the cell nucleus, consistent with it being a soluble cytosolic protein. Mitochondria and secretory compartments did not label for this protein. In human cells, peroxisomes showed a labeling density slightly less than that of cytoplasm.


Model for the role of macromolecular crowding in regulation of cellular volume. A simple model is proposed to account for large increases in transporter-mediated ion flux across cell membranes that are elicited by small fractional changes of cell volume. The model is based upon the concept that, as a result of large excluded volume effects in cytoplasm (macromolecular crowding), the tendency of soluble macromolecules to associate with membrane proteins is much more sensitive to changes in cell water content than expected on the basis of simple considerations of mass action. The model postulates that an ion transporter may exist in either an active dephosphorylated state or an inactive phosphorylated state and that the steady-state activity of the transporter reflects a balance between the rates of phosphatase-catalyzed activation and kinase-catalyzed inactivation. Cell swelling results in the inhibition of kinase relative to phosphatase activity, thereby increasing the steady-state concentration of the active form of the transporter. Calculated volume-dependent stimulation of ion flux is comparable to that observed experimentally.


Human herpesvirus 7 is a T-lymphotropic virus and is related to, but significantly different from, human herpesvirus 6 and human cytomegalovirus. An independent strain (JI) of human herpesvirus 7 (HHV-7) was isolated from a patient with chronic fatigue syndrome (CFS). No significant association could be established by seroepidemiology between HHV-7 and CFS. HHV-7 is a T-lymphotropic virus, infecting CD4+ and CD8+ primary lymphocytes. HHV-7 can also infect SUP-T1, an immature T-cell line, with variable success. Southern blot analysis with DNA probes scanning 58.8% of the human herpesvirus 6 (HHV-6) genome and hybridizing to all HHV-6 strains tested so far revealed homology to HHV-7 with only 37.4% of the total probe length. HHV-7 contains the GGGTTA repetitive sequence, as do HHV-6 and Marek's disease chicken herpesvirus. DNA sequencing of a 186-base-pair fragment of HHV-7(JI) revealed an identity with HHV-6 and human cytomegalovirus of 57.5% and 36%, respectively. Oligonucleotide primers derived from this sequence (HV7/HV8, HV10/HV11) amplified HHV-7 DNA only and did not amplify DNA from other human herpesviruses, including 12 different HHV-6 strains. Southern blot analysis with the p43L3 probe containing the 186-base-pair HHV-7 DNA fragment hybridized to HHV-7 DNA only. The molecular divergence between human cytomegalovirus, on the one hand, and HHV-6 and HHV-7, on the other, is greater than between HHV-6 and HHV-7, which, in turn, is greater than the difference between HHV-6 strains. This study supports the classification of HHV-7 as an additional member of the human beta-herpesviruses.


Evidence for a concerted reaction between lipid hydroperoxides and polypeptides. The events accompanying oxidative modification of low density lipoprotein (LDL) are multiple and complex, and the precise mechanisms remain to be determined. In the present studies, we examined a simple system in which we first prepared large amounts of lipid hydroperoxides (from linoleic acid or from phospholipids containing linoleic acid) by using soybean lipoxygenase (linoleate:oxygen oxidoreductase, EC 1.13.11.12). Linoleoyl hydroperoxide was then incubated with polypeptides in the absence of metal ions. We observed the generation of fluorescent products with a spectrum like that of oxidized LDL. The generation of fluorescent products from incubation of polypeptides with linoleoyl hydroperoxide was manyfold greater than that generated on incubation with preformed 4-hydroxynonenal at the same concentration. Superoxide dismutase (superoxide: superoxide oxidoreductase, EC 1.15.1.1) had no effect on the generation of fluorescent products. Incubation of linoleoyl hydroperoxide with cytochrome c (cyt c) under the same conditions led to progressive reduction of cyt c at a rate determined by the initial linoleoyl hydroperoxide concentration. This reduction was not significantly inhibited by probucol but was inhibited, although never completely, by superoxide dismutase. Even at 100 micrograms/ml, superoxide dismutase inhibited by only 65%. From these results, we are led to suggest a concerted reaction between the peroxy radical and free amino groups of polypeptides or phosphatidylethanolamine to generate fluorescent adducts. During oxidation of LDL or of cell membranes, this mechanism may occur side by side with the conventional Schiff base mechanism.


Dynamics of DNA supercoiling by transcription in Escherichia coli. The relative rotation between RNA polymerase and DNA during transcription elongation can lead to supercoiling of the DNA template. However, the variables that influence the efficiency of supercoiling by RNA polymerase in vivo are poorly understood, despite the importance of supercoiling for DNA metabolism. We describe a model system to measure the rate of supercoiling by transcription and to estimate the rates of topoisomerase turnover in Escherichia coli. Transcription in a strain lacking topoisomerase I can lead to optimal supercoiling, wherein nearly one positive and one negative superturn are produced for each 10.4 base pairs transcribed. This rapid efficient supercoiling is observed during transcription of membrane-associated gene products, encoded by tet (the gene for tetracycline resistance) and phoA (the gene for E. coli alkaline phosphatase), when the genes are oppositely oriented. Replacement of tet by cat, the gene from Tn9 encoding resistance to chloramphenicol, whose gene product is soluble in the cytosol, reduces the efficiency of supercoiling by RNA polymerase. In a wild-type topoisomerase background, both gyrase and topoisomerase I are kinetically competent to relieve superturns produced by transcription. These results suggest that the level of DNA supercoiling in vivo is probably determined by topoisomerase activity, not by transcription.


Function and evolution of a minimal plastid genome from a nonphotosynthetic parasitic plant. Complete nucleotide sequencing shows that the plastid genome of Epifagus virginiana, a nonphotosynthetic parasitic flowering plant, lacks all genes for photosynthesis and chlororespiration found in chloroplast genomes of green plants. The 70,028-base-pair genome contains only 42 genes, at least 38 of which specify components of the gene-expression apparatus of the plastid. Moreover, all chloroplast-encoded RNA polymerase genes and many tRNA and ribosomal protein genes have been lost. Since the genome is functional, nuclear gene products must compensate for some gene losses by means of previously unsuspected import mechanisms that may operate in all plastids. At least one of the four unassigned protein genes in Epifagus plastid DNA must have a nongenetic and nonbioenergetic function and, thereby, serve as the reason for the maintenance of an active genome. Many small insertions in the Epifagus plastid genome create tandem duplications and presumably arose by slippage mispairing during DNA replication. The extensive reduction in genome size in Epifagus reflects an intensification of the same processes of length mutation that govern the amount of noncoding DNA in chloroplast genomes. Remarkably, this massive pruning occurred with a virtual absence of gene order change.


DdPK3, which plays essential roles during Dictyostelium development, encodes the catalytic subunit of cAMP-dependent protein kinase. We have previously reported the analysis of DdPK3, a developmentally regulated putative serine/threonine kinase that shares approximately 50% amino acid sequence identity with metazoan cAMP-dependent protein kinase A (PKA) and protein kinase C, within their catalytic domains. Cells in which the DdPK3 gene has been disrupted do not aggregate but they are able to induce aggregation-stage genes in response to cAMP pulses and the prestalk-specific ras gene DdrasD in response to high continuous levels of cAMP but will not induce prespore gene expression. In this report, we present conclusive evidence that DdPK3 encodes the catalytic subunit of the Dictyostelium PKA. DdPK3 null cells lack kinase activity that phosphorylates a PKA-specific substrate and is specifically inhibitable by recombinant cAMP-dependent protein kinase inhibitor. DdPK3 expressed in Escherichia coli has PKA activity that is inhibitable by protein kinase inhibitor. When Ddpk3 null cells are complemented with DdPK3 expressed from an actin promoter on an extrachromosomal vector (low copy number), PKA activity is restored and the cells proceed to the slug stage but will not culminate, suggesting that properly regulated PKA activity is essential for culmination. Moreover, overexpressing DdPK3 in wild-type cells on integrating vectors (high copy number) from either an actin or prespore-specific promoter results in accelerated development and the ability to form mature spores in monolayer culture in the presence of high cAMP, a developmental potential lacking in wild-type cells.


Genomic sequences with homology to the P element of Drosophila melanogaster occur in the blowfly Lucilia cuprina. We have cloned two DNA elements (Lu-P1 and Lu-P2) from the Australian sheep blowfly Lucilia cuprina that are similar to the transposable P element of Drosophila melanogaster in both structure and sequence but have diverged from it and from each other considerably. Hybridization studies indicate that a third related element probably exists in another, as yet unsequenced, clone. Neither Lu-P1 nor Lu-P2 appears to be active in terms of mobility, and it is not known whether any transposition-competent copies of other related elements occur in the genome of the blowfly. However, the isolation of any P-like sequences from a species outside of the family Drosophilidae allows comparisons to be made of more widely divergent P-related elements than has been possible previously. We are unaware of any report of the presence of multiple P-like family members within a single species. The discovery of Lu-P1 and Lu-P2 in the blowfly fuels the possibility that similar elements may be widespread in insects, and perhaps in other orders of animals.


Ligand-induced phosphorylation of the murine interleukin 3 receptor signals its cleavage. The murine interleukin 3 receptor (mIL-3R) is a heterodimer consisting of a 70-kDa alpha subunit and one of two alternative 120-kDa beta subunits termed beta IL-3 and beta c. beta IL-3 (originally called Aic2A) is capable of binding mIL-3 by itself, whereas beta c (Aic2B) does not bind any ligand on its own but increases the affinity of mIL-3, murine granulocyte/macrophage-colony-stimulating factor, and mIL-5 for their respective alpha subunits. Interestingly, although the mIL-3R does not possess tyrosine kinase activity, its beta IL-3 subunit does become tyrosine phosphorylated upon binding mIL-3. To further investigate the properties of this subunit, we have purified it from the cell line B6SUtA1, which expresses a high level of mIL-3R. Intriguingly, studies comparing the stability of the 140-kDa, tyrosine-phosphorylated form of this subunit with its 120-kDa, non-tyrosine-phosphorylated form reveal that the former is far less stable and is rapidly degraded to a 70-kDa fragment. Mixing experiments demonstrate that the differential stability of the two forms is due to an intrinsic difference in protease susceptibility. Phosphatase studies indicate that the higher protease susceptibility of the tyrosine-phosphorylated beta IL-3 is due to the presence of both phosphotyrosine and phosphoserine residues. Western analyses using an anti-N-terminal mIL-3R beta IL-3 chain antibody reveal that this proteolytic cleavage also occurs rapidly in intact cells following stimulation with mIL-3 and occurs at the cell surface, since it takes place within minutes at 37 degrees C, is observed with purified plasma membranes, and is not inhibited by chloroquine. This degradative step may play an important role in the mechanism of action of mIL-3.


Gene therapy via primary myoblasts: long-term expression of factor IX protein following transplantation in vivo. We have explored the use of primary myoblasts as a somatic tissue for gene therapy of acquired and inherited diseases where systemic delivery of a gene product may have therapeutic effects. Mouse primary myoblasts were infected with replication-defective retroviruses expressing canine factor IX cDNA under the control of a mouse muscle creatine kinase enhancer and human cytomegalovirus promoter. The infected myoblasts were injected into the hindlegs of recipient mice and levels of secreted factor IX protein were monitored in the plasma. We report sustained expression of factor IX protein for over 6 months without any apparent adverse effect on the recipient mice.


Amino acid residues required for fast Na(+)-channel inactivation: charge neutralizations and deletions in the III-IV linker. The cytoplasmic linker connecting domains III and IV of the voltage-gated Na+ channel is thought to be involved in fast inactivation. This linker is highly conserved among the various Na+ channels that have been cloned. In the rat brain IIA Na+ channel, it consists of 53 amino acids of which 15 are charged. To investigate the role of this linker in inactivation, we mutated all 15 of the charged residues in various combinations. All but one of these mutants expressed functional channels, and all of these inactivated with kinetics similar to the wild-type channel. We then constructed a series of deletion mutations that span the III-IV linker to determine if any region of the linker is essential for fast inactivation. Deletion of the first 10 amino acids completely eliminated fast inactivation in the channel, whereas deletion of the last 10 amino acids had no substantial effect on inactivation. These results demonstrate that some residues in the amino end of the III-IV linker are critical for fast Na(+)-channel inactivation, but that the highly conserved positively charged and paired negatively charged residues are not essential.


A cluster of hydrophobic amino acid residues required for fast Na(+)-channel inactivation. The inward Na+ current underlying the action potential in nerve is terminated by inactivation. The preceding report shows that deletions within the intracellular linker between domains III and IV remove inactivation, but mutation of conserved basic and paired acidic amino acids has little effect. Here we show that substitution of glutamine for three clustered hydrophobic amino acids, Ile-1488, Phe-1489, and Met-1490, completely removes fast inactivation. Substitution of Met-1490 alone slows inactivation significantly, substitution of Ile-1488 alone both slows inactivation and makes it incomplete, and substitution of Phe-1489 alone removes inactivation nearly completely. These results demonstrate an essential role of Phe-1489 in Na(+)-channel inactivation. It is proposed that the hydrophobic cluster of Ile-1488, Phe-1489, and Met-1490 serves as a hydrophobic latch that stabilizes the inactivated state in a hinged-lid mechanism of Na(+)-channel inactivation.


Vaccinia virus RNA helicase: an essential enzyme related to the DE-H family of RNA-dependent NTPases. Three distinct nucleic acid-dependent ATPases are packaged within infectious vaccinia virus particles; one of these enzymes (nucleoside triphosphate phosphohydrolase II or NPH-II) is activated by single-stranded RNA. Purified NPH-II is now shown to be an NTP-dependent RNA helicase. RNA unwinding requires a divalent cation and any one of the eight common ribo- or deoxyribonucleoside triphosphates. The enzyme acts catalytically to displace an estimated 10-fold molar excess of duplex RNA under in vitro reaction conditions. NPH-II binds to single-stranded RNA. Turnover of the bound enzyme is stimulated by and coupled to hydrolysis of NTP. Photocrosslinking of radiolabeled RNA to NPH-II results in label transfer to a single 73-kDa polypeptide. The sedimentation properties of the helicase are consistent with NPH-II being a monomer of this protein. Immunoblotting experiments identify NPH-II as the product of the vaccinia virus I8 gene. The I8-encoded protein displays extensive sequence similarity to members of the DE-H family of RNA-dependent NTPases. Mutations in the NPH-II gene [Fathi, Z. & Condit, R.C. (1991) Virology 181, 258-272] define the vaccinia helicase as essential for virus replication in vivo. Encapsidation of NPH-II in the virus particle suggests a role for the enzyme in synthesis of early messenger RNAs by the virion-associated transcription machinery.


Recombination in vitro between herpes simplex virus type 1 a sequences. We have partially purified an activity from extracts of cells infected with herpes simplex virus type 1 that mediates recombination between repeated copies of the 317-base-pair a sequence of herpes simplex virus type 1. Recombination leads to deletion of a lacZ indicator gene situated between two directly repeated copies of the a sequence and is scored by transformation of lacZ- Escherichia coli. The two products of the reaction can be observed directly by restriction enzyme digestion and Southern blot analysis. The recombinase activity is also detectable, but at a lower level, in uninfected cell extracts. The DNA substrate must contain the two a sequences arranged in direct orientation to generate the lacZ deletion. However, when the a sequences are arranged in inverted orientation, an inversion results. A substrate with two homologous sequences of size and G + C content similar to the a sequence undergoes recombination at a much lower frequency. The reaction requires a divalent cation (Mg2+ or Mn2+) but not ATP or any other nucleoside triphosphate. The simple requirements and specificity for the a sequence suggest that the recombination may proceed by a site-specific mechanism.


Characterization of the transcriptional regulatory region of the human dihydrolipoamide dehydrogenase gene. Dihydrolipoamide dehydrogenase (E3; EC 1.8.1.4) is the common component of the three mammalian alpha-ketoacid dehydrogenase complexes and the glycine cleavage system. To study regulation of E3 gene expression, a 12-kilobase clone from a human leukocyte genomic library was isolated, and a 1.8-kilobase fragment containing part of the first intron, the first exon, and 1.5 kilobases of the 5' flanking region of the E3 gene was sequenced. The nucleotide sequence of the E3 promoter region revealed consensus sequences for several DNA binding proteins but no apparent TATA box or Sp1 sites. Although the 1.6-kilobase 5' flanking region has a low percentage of G+C (44%), the nucleotide sequence between +1 and -150 base pairs has a G+C content of 67%. Primer extension analysis showed a major transcriptional start site located 95 nucleotides upstream from the translation initiation codon. A series of 5' deletions from the E3 promoter-regulatory region were ligated to the bacterial chloramphenicol acetyltransferase (CAT) gene, and the resulting constructs were transfected into HepG2 cells. The longest E3 promoter-CAT construct had a relatively high level of CAT enzyme activity, and deletion of a promoter element between -769 and -1223 base pairs resulted in a 3-fold increase in reporter gene expression. These results suggest that the human E3 promoter has characteristics of housekeeping and facultative promoters and that a negative regulatory element is present between 769 and 1223 base pairs upstream from the transcription start site.


The 5' splice site consensus RNA oligonucleotide induces assembly of U2/U4/U5/U6 small nuclear ribonucleoprotein complexes. A short RNA oligonucleotide comprising the 5' splice site consensus sequence (5'SS RNA oligo) efficiently inhibits splicing of mRNA precursors in HeLa cell nuclear extracts. Addition of 5'SS RNA oligo inhibits early, but not late, steps in the splicing reaction, affecting the process of spliceosome assembly. In the presence of 5'SS RNA oligo a majority of U4/U5/U6 triple small nuclear ribonucleoprotein (snRNP) complex present in HeLa nuclear extracts associates with U2 snRNP to form a multi-snRNP complex, which could account for the observed inhibition of splicing by the oligo. This same set of snRNPs has been shown to assemble on pre-mRNAs during in vitro splicing to form splicing complex B. Removal of the 5' end of U1 snRNA, which is complementary to the 5' splice site, does not prevent association of snRNPs into U2/U4/U5/U6 complex in the presence of 5'SS RNA oligo. This suggests that interactions other than U1 snRNA.5'SS RNA oligo base pairing are used in recognition of the oligo sequence. 5'SS RNA oligo-induced assembly of the multi-snRNP complex may thus serve as a model to study the mechanism of 5' splice site recognition during splicing.


Human platelet-derived growth factor A chain is transcriptionally repressed by the Wilms tumor suppressor WT1. Wilms tumor, an embryonic kidney malignancy, accounts for approximately 6% of all pediatric neoplasms. A gene implicated in the genesis of this tumor, the Wilms tumor suppressor gene (WT1), encodes a zinc-finger DNA-binding protein (WT1) that functions as a transcriptional repressor. In certain Wilms tumors, the platelet-derived growth factor A chain (PDGF-A) is overexpressed; it has therefore been suggested that it may play an autocrine role in development of these neoplasms. Since the PDGF-A promoter contains putative binding sites for WT1, we explored the role of WT1 in regulating A-chain expression. The major PDGF-A promoter activity was localized in transient transfection assays to a region spanning from -643 to + 8 relative to the transcription start site. WT1 bound to several sites in this region of the promoter, as demonstrated by gel-shift analysis and DNase I footprinting, and functioned as a powerful repressor of PDGF-A transcription in vivo. Maximal repression (> 50-fold) of the PDGF-A promoter was dependent on the presence of multiple WT1 binding sites in transient transfection assays. Our observations suggest a mechanism for normal downregulation of a growth factor gene and of an autocrine growth process of import in kidney development and other biological systems.


Allelic deletions in the long arm of chromosome 12 identify sites of candidate tumor suppressor genes in male germ cell tumors. Human male germ cell tumors (GCTs) result from malignant transformation of premeiotic or early meiotic germ cells and exhibit embryonal-like differentiation of the three germinal layers. The genetic basis of origin and expression of differentiated phenotypes by GCTs are poorly understood. Our recent cytogenetic analysis of a large series of GCTs has shown that two chromosome 12 abnormalities, an isochromosome for the short arm [i(12p)] and deletions in the long arm [del(12q)], characterize these tumors, which led us to suggest that the deletions represent loss of one or more candidate tumor suppressor genes whose products regulate the normal proliferation of the spermatogonial stem cells. We undertook a molecular mapping of the deletions by comparing germ-line and tumor genotypes of eight polymorphic loci in paired normal/tumor DNA samples from 45 GCT patients. Analysis of loss of constitutional heterozygosity at these loci revealed two regions of frequent loss (> 40%), one at 12q13 and the other at 12q22, identifying the sites of the postulated tumor suppressor genes. One tumor (no. 143A) exhibited a homozygous deletion of a region of 12q22, which included the MGF gene. The KIT and MGF genes have been shown to play key roles in embryonal and postnatal development of germ cells; therefore, we evaluated their expression by Northern blot analysis in a panel of three GCT cell lines and 24 fresh GCT biopsies. Deregulated expression of MGF and KIT, which was discordant between seminomatous and nonseminomatous lesions, was observed.


Hydrolytic elimination of a mutagenic nucleotide, 8-oxodGTP, by human 18-kilodalton protein: sanitization of nucleotide pool. 8-Oxoguanine nucleotide can pair with cytosine and adenine nucleotides at almost equal efficiencies. Once 8-oxodGTP is formed in the cellular nucleotide pool, this mutagenic nucleotide is incorporated into DNA and would cause transversion mutations. The MutT protein of Escherichia coli possesses enzyme activity to hydrolyze 8-oxodGTP to the corresponding nucleoside monophosphate and thus may be responsible for preventing the occurrence of such mutations. Here we show that the human cell has an enzyme specifically hydrolyzing 8-oxodGTP in a fashion similar to that seen with MutT protein. The human 8-oxodGTPase has been found in cell-free extracts from Jurkat cells and purified > 400-fold. Analyses by gel filtration and gel electrophoresis revealed that the molecular mass of the native form of human 8-oxodGTPase is 18 kDa. Mg2+ ion is required for the enzyme action and the optimum pH for the reaction is pH 8.0. The enzyme hydrolyzes 8-oxodGTP to 8-oxodGMP with a Km value of 12.5 microM. dGTP and dATP are also degraded to dGMP and dAMP, respectively, with Km values 70 times greater than that for 8-oxodGTP. dTTP and dCTP are not hydrolyzed. These properties of the human 8-oxodGTPase are similar to those observed with the E. coli MutT protein, suggesting that the function of protecting the genetic information from the threat of endogenous oxygen radicals is widely distributed in organisms.


Molecular cloning of DNA encoding a calmodulin-dependent phosphodiesterase enriched in striatum. A murine cDNA for the 63-kDa calmodulin-dependent phosphodiesterase (CaM-PDE), PDE1B-1, was isolated by using polymerase chain reaction with degenerate primers followed by the cloning of a full-length cDNA from a whole-brain phage library. The nucleotide sequence of 2986 base pairs contains an open reading frame encoding a protein of 535 amino acids (M(r) = 61,231) with a predicted isoelectric point of 5.54. The deduced protein sequence shows approximately 60% identity with that of the 61-kDa isoform (PDE1A2), consistent with the proposal that these proteins arise from two separate genes [Novack, J. P., Charbonneau, H., Bentley, J. K., Walsh, K. A. & Beavo, J. A. (1991) Biochemistry 30, 7940-7947]. Southern blot analysis suggests high nucleotide-sequence conservation of the PDE1B1 gene among mammalian and avian species. A single approximately 3600-nucleotide mRNA transcript was seen in all brain regions, with striatum containing 4- to 30-fold higher levels than other areas. In nonneural tissues, low amounts of PDE1B1 mRNA were detected in lung, spleen, thymus, and testis; hybridization to several larger mRNA species was also seen in thymus and testis. By using nucleic acid probes for PDE1B1, the mechanisms that control its highly selective gene expression can now be studied at the molecular level.


Cytoskeletal integrity in interphase cells requires protein phosphatase activity. Phosphorylation by protein kinases has been established as a key factor in the regulation of cytoskeletal structure. However, little is known about the role of protein phosphatases in cytoskeletal regulation. To assess the possible functions of protein phosphatases in this respect, we studied the effects of the phosphatase inhibitors calyculin A, okadaic acid, and dinophysistoxin 1 (35-methylokadaic acid) on BHK-21 fibroblasts. Within minutes of incubation with these inhibitors, changes are seen in the structural organization of intermediate filaments, followed by a loss of microtubules, as assayed by immunofluorescence. These changes in cytoskeletal structure are accompanied by a rapid and selective increase in vimentin phosphorylation on interphase-specific sites, and they are fully reversible after removal of calyculin A. The results indicate that there is a rapid phosphate turnover on cytoskeletal intermediate filaments and further suggest that protein phosphatases are essential for the maintenance and structural integrity of two major cytoskeletal components.


T-cell activation by the CD28 ligand B7 is required for cardiac allograft rejection in vivo. Organ graft rejection is a T-cell-dependent process. The activation of alloreactive T cells requires stimulation of the T-cell receptor/CD3 complex by foreign major histocompatibility complex (MHC)-encoded gene products. However, accumulating evidence suggests that, in addition to T-cell receptor occupancy, other costimulatory signals are required to induce T-cell activation. Previously, the CD28 receptor expressed on T cells has been shown to serve as a surface component of a signal transduction pathway that can provide costimulation. In vitro, interaction of CD28 with its natural ligand B7 expressed on the surface of activated B cells or macrophages can act as a costimulus to induce proliferation and lymphokine production in antigen receptor-activated T cells. We now report evidence that stimulation of T cells by the CD28 ligand B7 is a required costimulatory event for the rejection of a MHC-incompatible cardiac allograft in vivo. These results demonstrate that the B7/CD28 activation pathway plays an important role in regulating in vivo T-cell responses.


Study of the effect of lithium on lymphokine-activated killer cell activity and its antitumor growth. The in vitro effect of lithium on lymphokine-activated killer cell (LAK) activity and its in vivo antitumor growth were observed. LAK activity was enhanced when LiCl was added during LAK cell induction, and this enhancement was observed both in human peripheral blood mononuclear cell and in mouse splenocytes used as LAK precursors. Cholera toxin, which can increase intracellular levels of cAMP, decreased LAK cell activity. However, lithium partially reversed this inhibitory effect, indicating that lithium increased LAK cell activity by decreasing cAMP levels. D-Sphingosine, an inhibitor of protein kinase C, and EGTA, a calcium chelator, both inhibited the LAK cell activity. However, their inhibitory effects could not be reversed by lithium because lithium was added in the culture in combination with one of these inhibitors during LAK cell induction. By using slot blot analysis, the effect of lithium on the expression of tumor necrosis factor-alpha mRNA of LAK cells was analyzed. Lithium increased the level of tumor necrosis factor-alpha mRNA when both lithium and interleukin 2 were added to induce LAK cells. The in vivo antitumor effect of lithium has also been studied. Using a mouse melanoma experimental model, the effect of lithium on tumor growth was also observed. Both lithium alone and interleukin 2/LAK had an antitumor effect, whereas the treatment of interleukin 2/LAK in combination with lithium had the strongest inhibitory effect on tumor growth, since this treatment resulted in reduction of tumor size and prolongation of survival in tumor-bearing mice. Therefore, it is hopeful that lithium can be used as a new immunomodulator for cancer immunotherapy and immune diseases.


Epidermal growth factor up-regulates intestinal Na+/H+ exchange activity. The present studies were designed to examine the regulation of Na+/H+ exchange activity by epidermal growth factor (EGF) in an in vitro system. Na+/H+ exchange activity was determined in brush-border membranes isolated from rat jejunal enterocytes incubated with epidermal growth factor and a number of second messengers. EGF at physiological concentrations stimulated Na+/H+ exchange activity without affecting vesicle size. The stimulation of Na+/H+ activity was the result of increasing Vmax of Na+/H+ (6.0 +/- 0.4 compared with 3.3 +/- 0.27 nmol/mg protein/5 sec, P < 0.01). Km values of the Na+/H+ exchanger in brush-border membrane from cells stimulated with EGF and controls were similar (16.0 +/- 3.0 vs 13.0 +/- 3.0, respectively). Na+/H+ activity was inhibited by phorbol esters, calmodulin, and cyclic AMP. The effects of EGF, calmodulin, cyclic AMP, and phorbol esters were dependent on ATP, because depleting the cells from ATP masked the effects on Na+/H+ exchange activity. The results suggest that EGF stimulates Na+/H+ exchange activity in the enterocytes. This stimulation is most likely not via activation of the phosphatidylinositol pathway.


Hypothesis for the receptors of human blood platelet aggregation and its inhibition by structure-activity relationship. We tried to clarify the size and the common charge distribution of the inhibition or stimulation of human platelet aggregation by structure-activity relationship. Numerous inhibiting and stimulating agents were able to enter the receptors. Inhibitory receptor had recess of 14 x 12.5 A in diameter. Stimulatory receptor had recess of 11 x 12 A in diameter. In the recess, there were three charges, two negative and one positive in the inhibitory receptor, and one negative and two positive in the stimulatory receptor, respectively. Charge distributions and conformation of inhibiting or stimulating agents were similar for the inhibitory agents, prostaglandin I2 (PGI2), PGD2, PGE1 adenosine and isoproterenol and conformation of the stimulating agents, thromboxane A2 (TXA2), platelet activating factor (PAF), adenosine diphosphate (ADP) and adrenaline. Each molecule had 3-10 inhibiting and stimulating conformations. The ratio of the number of conformations for inhibition and stimulating of platelet aggregation was highest for PGI2 which showed the strongest inhibitory activity. TXA2 was opposite in both respects.


Naloxone-induced hypoalgesia: lack of involvement of the GABA-benzodiazepine receptor complex. Previous evidence has demonstrated that repeated daily administration of the opiate receptor antagonist naloxone prior to assessment of pain sensitivity provokes the development of a nonopioid form of hypoalgesia. The present experiments assessed whether the GABA-benzodiazepine receptor complex may be involved in the mediation of this effect. Male Wistar rats were administered 10 mg/kg naloxone prior to hot-plate tests (48.5 degrees C) for pain sensitivity for 8 consecutive days. Control animals were administered saline prior to, and naloxone 2-4 h after, assessment of pain reactivity. Beginning on the fourth or fifth day of this regimen, animals tested under the influence of naloxone displayed longer paw-lick latencies than controls. Preadministration of the GABAA agonist muscimol (1.0-5.0 mg/kg) and GABAA antagonist bicuculline (0.25-1.0 mg/kg) failed to affect paw-lick latencies in naloxone-tested and control rats. The GABAB receptor agonist baclofen (1.0-5.0 mg/kg) and the benzodiazepine receptor agonist diazepam (1.0-5.0 mg/kg) both elevated paw-lick latencies to the same degree in both groups of animals. These results suggest that the GABA-benzodiazepine receptor complex is not involved in the mediation of naloxone-induced hypoalgesia.


Cardiovascular responses to naloxone challenge in opiate-dependent individuals. Vagally mediated tachycardia appears to be a common response to abused drugs and, therefore, has implications for abuse liability. To test the specificity of this common factor, we determined whether the tachycardia to naloxone in opiate-dependent individuals has a significant vagal component. Naloxone challenge (0.4 mg, IM) in 19 opiate-dependent men and women was associated with highly reliable tachycardia, but no significant change in vagal tone index, a noninvasive measure of parasympathetic inhibitory control of the heart. We conclude that tachycardia during naloxone-precipitated withdrawal is not vagally mediated. Thus, there is some degree of specificity to the common factor of vagally mediated tachycardia to abused drugs because it was ruled out in at least one drug (naloxone) with aversive subjective effects.


Quantitation of rodent catalepsy by a computer-imaging technique. Catalepsy is usually defined as a behavioral state in which an animal maintains an unnatural posture for an extended period of time. While numerous laboratory models have been developed for assessing catalepsy, a common problem encountered with most procedures is the difficulty in quantitating immobility. Measurement of catalepsy is still frequently subjective in nature. To eliminate this subjectivity, a computer-based technique was developed for quantitating catalepsy in mice and rats as measured on the elevated ring. The system consisted of a video camera that was focused on either three mice or two rats. Their behavior was recorded during a 5-min session on videotape that was subsequently transmitted to a Macintosh II microcomputer via a Scion Image-Capture 2 board. A modification of the NIH Image 1.17 public domain program allowed the image of the rat to be transformed to a purely black or white image by assigning pixel values of either 0 or 256. The subsequent captured image was preprocessed in an identical manner and each pixel was subtracted from its corresponding pixel in the previous frame. Thus, changes in animal posture between the two frames can be quantitated. One subtraction cycle (acquisition, bilevel processing, and subtraction) was repeated at an average rate of approximately one per second. To quantitate immobility by image analysis, each frame was subtracted from the previous frame during a 5-min session. The resulting data were sorted according to the magnitude of movement (number of changed pixels) and plotted vs. time.(ABSTRACT TRUNCATED AT 250 WORDS)


Sex differences in sensitivity to pentylenetetrazol but not in GABAA receptor binding. Female rats have a higher threshold than males for seizures induced by the convulsant pentylenetetrazol, a GABAA receptor-chloride channel complex blocker. No sex difference was observed for the anticonvulsant activities of ethanol or diazepam to protect against pentylenetetrazol seizures. Ovariectomy reduces the pentylenetetrazol seizure threshold of females to that of males. In contrast, females have a lower threshold than males to electroshock seizures. Pentylenetetrazol receptors were compared in males and females and gonadectomized animals by binding of several radioligands to the GABAA receptor complex. No differences were found for these four groups of animals in the binding of [3H]flunitrazepam to the benzodiazepine sites and [35S]t-butyl bicyclophosphorothionate ([35S]TBPS) to the chloride channel/convulsant sites in membrane homogenates, nor for allosteric modulation of binding by GABA, the steroid anesthetic alphaxalone, or the benzodiazepine Ro 5-4864. In tissue section autoradiography, no difference was observed for these same assays nor for the binding of [3H]muscimol in the presence and absence of alphaxalone in several major regions. We conclude that circulating female sex hormones, possibly neurosteroid metabolites of progesterone, known to interact directly with the GABAA receptor complex, are involved in the sex differences in pentylenetetrazol seizure susceptibility.