Decreased urokinase receptor expression by overexpression of the plasminogen activator in a colon cancer cell line. There is now ample evidence that the proteolytic action of urokinase (UK) is potentiated by a specific cell surface receptor. The present study was undertaken to determine the role of UK as a modulator of its binding site. GEO colonic cells, which secrete low levels of UK (approximately 2.5 ng/ml per 72 h per 10(6) cells) and display approx. 10(4) receptors per cell, the majority of which are vacant, were transfected with an exogenous UK gene driven by the RSV long terminal repeat (LTR) promoter (pRSVUK). Several UK-overexpressing pRSVUK clones were identified by an e.l.i.s.a., Northern blotting and Southern blotting, and analysed for receptor numbers after an acid pretreatment which dissociates receptor-bound UK. pRSVUK GEO clones, expressing high levels of UK, consistently bound 50-75% less radioactive di-isopropylfluorophosphate (DFP)-UK than clones harbouring the selectable marker gene neo only or control GEO cells. Cross-linking experiments with a radioactive N-terminal fragment of UK which binds to the receptor showed a decreased amount of a binding protein of approx. 51 kDa in representative pRSVUK-transfected cells. Saturation and Scatchard analysis indicated that this reduction in radioligand binding reflected a 40-70% decrease in the number of UK receptors, rather than a change in the dissociation constant. The reduction in receptor display could be accounted for by a decrease in the amount of steady-state mRNA encoding the receptor. Radioactive DFP-UK binding to pRSVUK GEO clones, which display two-thirds less receptors than their neo counterparts, could be restored to control levels (untreated cells harbouring neo) by cultivating them in the presence of an antibody which inhibits the interaction of UK with its receptor. These data suggest that for one colonic cell line at least, UK reduces the expression of its own binding site via an autocrine stimulation of its cell surface receptor.


Structure and evolution of insulins: implications for receptor binding. Insulin is a member of a family of hormones, growth factors and neuropeptides which are found in both vertebrates and invertebrates. A common 'insulin fold' is probably adopted by all family members. Although the specificities of receptor binding are different, there is a possibility of co-evolution of polypeptides and their receptors.


[Physiopathology of atopic dermatitis].

[Physiopathology of atopic dermatitis]. This pathological sequence, although partly hypothetical, allows consideration of many points involved in therapy. These treatments arouse interest as to how they behave on the causal mechanisms. Thus, should symptomatic treatments that act on pruritus, xerosis, skin inflammation, of which the effect may be only transitory be offered, or active therapy on the initial factors which may have a much more radical effect on the illness?


The significant conservative and variable regions of the homologous protein sequences. A method of identification of significant conservative and variable regions in homologous protein sequences is presented. A set of aligned homologous sequences is divided into two groups consisting of m and n most related sequences. Each pair of sequences from different group is compared using unitary similarity matrix. The superposition of pairwise comparisons scanned by a window of 10 amino acid residues gives intergroup local variability profile (VP). Area S of the figure between the VP and its mean value line is compared with averaged area S(r) of 1000 VPs of artificial homologous protein families. The difference (S-S(r)) given in standard deviation units sigma r is believed to be the amino acid substitution overall irregularity along the homologous protein sequences OI = (S-S(r))/sigma r. If OI greater than 2, the real VP extrema containing the surplus of area S-(S(r) + 2 sigma r) are cut off. The cut off stretches are likely to be significant conservative and variable regions. The significant conservative and variable regions of six homologous sequence families (phospholipases A2, cytochromes b, alpha-subunits of Na, K-ATPase, L- and M-subunits of photosynthetic bacteria photoreaction centre and human rhodopsins) were identified. It was shown that for artificial homologous protein sequences derived by k-fold lengthening of natural proteins the OI value rises as square root of k. To compare the degree of substitution irregularity in homologous protein sequence families of different length L the value of standard substitution overall irregularity for L = 250 is proposed.


Tetraplex formation of d(GGGGGTTTTT): 1H NMR study in solution. The oligonucleotide d(G5T5) can in principle form a fully matched duplex with G.T pairing and/or a tetraplex. Non-denaturing gel electrophoresis, circular dichroism and NMR experiments show that the tetraplex is exclusively formed by this oligomer in solution. In the presence of its complementary strand d(A5C5) at low temperature, d(G5T5) forms the tetraplex over the normally expected Watson-Crick duplex. However, when d(G5T5) and d(A5C5) are mixed together in equimolar amounts and heated for several minutes at 85 degrees C, and then allowed to cool, the product was essentially the Watson-Crick duplex. The lack of resolution in the 500 MHz 1H NMR spectra and the presence of extensive spin diffusion do not allow us to derive a quantitative structure for the tetraplex from the NMR data. However, we find good qualitative agreement between the NOESY and MINSY data and a theoretically derived stereochemically sound structure in which the G's and T's are part of a parallel tetraplex.


Molecular dynamics studies on nucleoside 2',3'-cyclic phosphates. 2',3'-cyclic nucleotides are intermediates and substrates of Ribonuclease (RNase)-catalysed reactions. The characterization of the equilibrium conformation as well as the flexibility inherent in these molecules helps in understanding the enzymatic action of RNases. The present study explores parameters like phase angle, glycosydic torsion angle and hydrogen bond to find possible interrelationship between them through Molecular Dynamics (MD) simulations on 3'-GMP,3'-UMP, A greater than p, G greater than p, U greater than p, C greater than p, GpA greater than p and UpA greater than p. Interesting results of the effect of cyclisation and other constraints such as hydrogen bond between certain groups on the equilibrium ribose conformation have emerged from this study.


The effect of long-term alcohol intake on brain NGF-target cells of aged rats. It was reported that chronic exposure to ethanol causes a loss of hippocampal pyramidal cells and of brain cholinergic neurons in both laboratory animals and humans. In the present study, it was hypothesized that nerve growth factor (NGF), a trophic agent for the survival and maintenance of basal forebrain cholinergic neurons (FCN), might be affected by the neurodegenerative events which occur during ethanol consumption. To test this hypothesis, we used aged rats (14 months) exposed for 16 weeks to 40 g/kg per day of undiluted wine. Our experiments showed that chronic alcohol consumption causes a reduction of NGF in the hippocampus (HI) and of choline acetyltransferase (ChAT) activity in both the septum and the HI and a reduction in the distribution of NGF-receptors (NGF-R) in the septum and nucleus of Meynert. Intracerebral injection of NGF in alcohol-exposed rats results in a return to normal levels of ChAT enzymatic activity and NGF-R expression. These experiments indicate that the damaging effect of alcohol on the FCN is also associated with impairment of central NGF-target structures.


Expression cloning of TGF-beta receptors. Using a powerful expression cloning method in COS cells, we have cloned the TGF-beta types II and III receptors. The type III TGF-beta receptor is a membrane-bound proteoglycan with a core protein of about 110 kDa. Stable expression of the type III receptor in L6 myoblasts leads to an apparent increase in the ability of the type II receptor to bind iodinated TGF-beta 1. The cloned type II receptor has a predicted protein core of about 60 kDa with a cysteine-rich extracellular domain, a single transmembrane domain, and a functional serine/threonine kinase domain that is homologous to the activin receptor and to the C. elegans protein daf-1. These results implicate serine/threonine phosphorylation as an important mechanism of TGF-beta action.


TGF-beta receptors.

TGF-beta receptors. The nature and role of cell surface proteins that bind members of the TGF-beta family has been investigated. TGF-beta, activins, and BMPs each bind to receptors of 55 kDa (type I) and 70 kDa (type II). In the TGF-beta system, these receptors are implicated in the mediation of multiple responses. A member of the type II receptor family has been cloned that encodes four alternatively spliced versions of a transmembrane serine/threonin kinase receptor related to the recently cloned mouse activin receptor and C-elegans daf-1 gene. Inhibitors of serine/threonine kinase activity block transcriptional and growth inhibitory responses to TGF-beta. In addition to the signaling receptors, many cell types express the TGF-beta binding proteoglycan betaglycan. Betaglycan has been purified, molecularly cloned, and shown to bind TGF-beta via its core protein and basic fibroblast growth factor via its heparan sulfate chains. In addition to receptors I and II and betaglycan, some cells express a newly identified set of membrane proteins that specifically bind either TGF-beta 1 or TGF-beta 2. Three of the four isoform-restricted binding proteins are bound to the membrane via phospholipid anchors. Like betaglycan, these proteins might function to regulate the interaction between TGF-beta and their target cells.


Abnormal response of osteoblasts from Hyp mice to 1,25-dihydroxyvitamin D3. To further explore the hypothesis of an osteoblast inappropriate response to 1,25-(OH)2D3 in hypophosphatemic vitamin D-resistant rickets (HYP), osteoblasts were isolated from Hyp mice, the animal model for human HYP, and their response to a physiologic dose of 1,25-(OH)2D3 (10(-10) M) was investigated with respect to alkaline phosphatase (ALP) activity and cell proliferation, and compared to that of normal osteoblasts. Cells in secondary culture were incubated for 72 h while in log phase, with or without 1,25-(OH)2D3, at various medium phosphate (P) concentrations ranging from 0.5 to 4.5 mM. Stimulation of ALP activity and inhibition of cell proliferation was induced by 10(-10)M 1,25-(OH)2D3 in normal cells exposed to medium P concentration corresponding to serum levels observed in normal mice (2.1-2.7 mM P). By contrast, Hyp cells failed to respond to 1,25-(OH)2D3 in that range of P concentrations. Stimulation of ALP activity and inhibition of proliferation of mutant cells were evident at higher medium P concentrations (over 3 mM). 1,25-(OH)2D3 at the supraphysiologic level of 10(-9)M had no consistent effect on ALP activity in normal and Hyp mouse osteoblasts, but inhibited cell proliferation in cultures of both genotypes at all P concentrations tested. These results indicate that extracellular P modulates the action of 1,25-(OH)2D3 on osteoblasts, and that this modulation was altered in osteoblasts from Hyp mice. The failure of Hyp cells to respond to a physiologic dose of 1,25-(OH)2D3 upon normal P concentration may reflect the abnormal response of bone to 1,25-(OH)2D3 observed in Hyp mice and HYP patients.


Herpes simplex virus infection in cancer patients: prevention and treatment. Herpes simplex virus (HSV) infection is common in patients receiving cytotoxic therapy for cancer. Almost all infections result from reactivation of latent virus during treatment-induced immunosuppression. Typical, self-limited orolabial or genital ulceration does not always require laboratory diagnosis or treatment, but HSV may present in an atypical fashion in cancer patients and cause more severe and prolonged mucocutaneous infection or visceral disease. The presence of antibodies to HSV identifies patients at risk for recurrent HSV infection. The treatment of choice is acyclovir, which may also be used to prevent infection in high-risk patients. Acyclovir resistance has been reported in patients with profound and prolonged immune deficiency, but remains rare in patients treated for cancer.


Overexpression of multiple oncogenes related to histological grade of astrocytic glioma. The expression of the c-erbB-1, c-myc, Ha/N-ras and c-fos oncogenes was investigated in 62 astrocytomas of low, intermediate and high grades by immunogold silver histochemistry. Elevated expression of c-erbB-1 was observed in 95%, 48% and 86% of low, intermediate and high grade tumours respectively, c-myc in 5%, 33% and 76% respectively, Ha/N-ras in 0, 43% and 71% respectively and c-fos in 55%, 48% and 52% respectively. Controls included normal brain and tumour sections immunoreacted with pre-immune serum or with antisera absorbed with synthetic peptides. Analysis of co-overexpression revealed that low grade tumours co-overexpressed a maximum of two of these genes, intermediate grade tumours a maximum of three of these genes, while co-overexpression of all four genes was observed in some high grade tumours. Co-overexpression of c-erbB-1 and c-fos was frequently observed in low grade astrocytomas and may be predictive of non-progression. On the other hand, there was a statistically significant increase in the number of tumours overexpressing Ha/N-ras or c-myc with increasing grade of tumour, suggesting that overexpression of these two oncogenes may be indicative of progression.


Biological and clinical results of a neuroimmunotherapy with interleukin-2 and the pineal hormone melatonin as a first line treatment in advanced non-small cell lung cancer. The metastatic non-small cell lung cancer (NSCLC) still remains an untreatable disease, and the role played by chemotherapy has yet to be defined. The new immunotherapeutic strategies, such as interferon and IL-2, seem to be also less effective, since they generally determine only a stabilisation of disease. On the basis of previous experimental results suggesting a synergistic action between IL-2 and the pineal neurohormone melatonin (MLT), a study was started to evaluate the clinical efficacy and toxicity of a neuroimmunotherapeutic combination consisting of IL-2 plus MLT as a first line therapy in metastatic NSCLC. The study included 20 patients (adenocarcinoma: 10; epidermoid cell carcinoma: 7; large cell carcinoma: 3). MLT was given orally at a dose of 10 mg day-1 at 8.00 pm every day, starting 7 days before the onset of IL-2 administration. IL-2 was given subcutaneously at a dose of 3 x 10(6) IU m-2 every 12 h for 5 days/week for 4 weeks, corresponding to one cycle of immunotherapy. In responder patients or in those with stable disease, a second cycle was given after a rest-period of 21 days. A partial response was achieved in 4/20 (20%) patients. Ten other patients had a stable disease (50%), whereas the last six patients progressed. Toxicity was low in all cases. This study shows that the neuroimmunotherapeutic therapy with IL-2 and the pineal hormone MLT may represent a new effective and well tolerated treatment in metastatic NSCLC, with results comparable to those obtained with chemotherapy, but with an apparent lower biological toxicity.


Plasminogen receptors on rat colon carcinoma cells. Cells from rat carcinoma cell lines PROb (giving progressive tumours) and REGb (giving regressive tumours) have cell surface receptors which bind specifically rat plasminogen and plasmin. Affinity for Pg was found to be higher in PROb (Kd = 10(-7) M) than in REGb cells (Kd = 5.10(-7) M) but with a concomitant decrease in the number of binding sites, 0.9 x 10(6)/cell (range from 0.6 to 1.2 x 10(6)) in PROb vs 3.6 x 10(6)/cell (range 1.2 to 6 x 10(6)) in REGb cells. The number and the affinity of binding sites varied in an opposite way in PROb and REGb cells. The difference in affinity parameters was unrelated to the degree of invasiveness of tumour cells in syngenetic rats. Bound plasmin retained its enzymatic activity, which indicates that its binding does not involve the catalytic active site. In cell solubilisates plasminogen receptor appeared as one major band situated in the area of 50-60 kDa.


Loss of lung function associated with exposure to silica dust and with smoking and its relation to disability and mortality in South African gold miners. The data from a lung function study on 2209 white 45-54 year old South African gold miners in 1968-71 and at a five year follow up examination, were analysed to establish the actual loss of lung function associated with exposure to silica dust and with smoking. Ex-smokers were excluded from the analysis. Of the remaining 1625 subjects, 1249 had the five year follow up test of lung function. The estimated excess loss of lung function for a 50 year old gold miner, associated with 24 years of underground dust exposure of an average respirable dust concentration of 0.30 mg m-3 (14.4 ghm-3) was 236 ml of FEV1 (95% confidence interval (95% CI 134-337) and 217 ml of FVC (95% CI 110-324). By comparison, the effect of smoking one packet of cigarettes a day over 30 years was associated with an estimated loss of 552 ml of FEV1 (95% CI 461-644) and 335 ml of FVC (95% CI 170-500). The cumulative dust exposure was not associated with the longitudinal loss of FEV1 or FVC when the initial FEV1 and FVC were adjusted in the models. According to the predicted values, however, gold miners appear to have a greater loss of lung function from 50 to 55 years of age than that predicted for a general population.


Extreme airborne asbestos concentrations in a public building. Fibre concentrations of asbestos were measured in the air of a communal dining room in which the damaged ceiling had a sprayed on coating of insulation containing asbestos. The average concentration of crocidolite asbestos fibres was 4 f/cm3, 20 times the highest air concentration that appears to have been reported previously for a public building. It is concluded that although air concentrations of asbestos fibres in public buildings containing asbestos insulation materials are usually low, high concentrations can occur. This may have implications for the risk of exposed persons developing diseases associated with asbestos.


Evidence for a tonic inhibitory role of nifedipine-sensitive calcium channels in aldosterone biosynthesis. This study investigated the effects of the calcium channel blockers nifedipine (a dihydropyridine) and verapamil (a papaverine derivative), on aldosterone production utilizing isolation of the early and late phases of aldosterone biosynthesis. Pregnenolone production (the early phase of aldosterone biosynthesis) was assessed in trilostane-treated bovine glomerulosa cells, used to inhibit the conversion of pregnenolone onwards to aldosterone. Conversion of exogenous corticosterone to aldosterone, an index of late phase activity, was assessed using aminoglutethimide to inhibit endogenous aldosterone production. Low concentrations of nifedipine, 10(-11)-10(-9) M, stimulated basal total aldosterone biosynthesis by enhancing the late phase although the early phase was inhibited. In the presence of 12 mM potassium (K+), which is less effective in stimulating aldosterone production than lower K+ concentrations, aldosterone production was enhanced by nifedipine, 10(-8) M, by an effect on the late phase. At K+ 6 and 8 mM, nifedipine, 10(-4) M, inhibited the early phase. Nifedipine 10(-5) inhibited angiotensin II (AII)-stimulated total aldosterone biosynthesis by independent effects on the early and late phases. Verapamil, 10(-4) M, inhibited total and early phase aldosterone production at K+, 4 mM and inhibited both phases at K+, 8 mM, stimulation was not observed using verapamil. Verapamil, 10(-4) M, also inhibited AII-stimulated aldosterone production. Basal and AII-stimulated pregnenolone production were inhibited by verapamil, 10(-4) M (basal) and 10(-6) M (AII-stimulated). These studies using nifedipine have revealed subtle calcium-dependent mechanisms involved in the tonic inhibition of activity in the late phase of aldosterone biosynthesis and the reversal of the inhibitory effect of high K+ concentrations also on the late phase. In addition, the data reported indicate that both AII and K+ independently enhance activity in the early and late phases of aldosterone production by calcium-dependent mechanisms.


Low sodium intake enhances sensitivity of 11-deoxycortisol and deoxycorticosterone to ACTH in ACTH-suppressed normal subjects. Continued administration of ACTH to patients with hypopituitarism produced normal increases in steroids dependent on microsomal cytochrome P450(21) and P450(17 alpha) but reduced responses of steroids dependent on mitochondrial cytochrome P450(11 beta-18). To explore possible mechanisms and to determine whether this dissociation occurs with short-term ACTH suppression, we have examined the steroid responses to ACTH after 1 h in 12 normal subjects after equilibration on sodium intakes of 124 mmol/d [normal sodium diet (NSD)], 22 mmol/d [low sodium diet (LSD)], and 240 mmol/d [high sodium diet (HSD)] before and during continued ACTH suppression with dexamethasone (DEX). Two distinct patterns of steroid responses were observed. Deoxycorticosterone (DOC) responses were initially reduced during LSD-DEX but eventually returned to the NSD-control (NSD-CONT) values; in contrast 18-hydroxydeoxycorticosterone and corticosterone remained suppressed. 11-Deoxycortisol and 21-deoxycortisol showed patterns similar to DOC, with a return to normal ACTH responses on LSD-DEX. Basal cortisol levels were reduced and the ACTH response was unchanged by LSD. HSD-DEX reduced basal levels of all steroids as well as their ACTH responses. LSD and/or increased activity of the renin-angiotensin system have a significant impact on 17 alpha- and 21-hydroxylation functions in the zona fasciculata to maintain a normal ACTH response of microsomally dependent steroids under these conditions. In contrast, on HSD-DEX with the renin-angiotensin system suppressed, there is generalized impairment of steroid responses to ACTH.


Effects of hydrochlorothiazide, amiloride, and lisinopril on the metabolic response to adrenaline infusions in normal subjects. Twelve healthy male volunteers were given adrenaline infusions, 0.05 microgram/kg body weight/min over 120 minutes in order to achieve serum adrenaline concentrations comparable with those seen in acute myocardial infarction. The infusions were given on four occasions, at intervals of at least 4 weeks. Before the infusions the subjects were given, in random order, 14 days of pretreatment with placebo, hydrochlorothiazide 50 mg once daily, amiloride 10 mg once daily, or lisinopril 20 mg once daily. The adrenaline infusion induced a drop in serum potassium of the same magnitude in all four groups, with the lowest absolute value after hydrochlorothiazide because of the lowest pre-adrenaline level. The infusion-induced decreases in serum calcium and magnesium were of the same magnitude in all groups, with the absolute calcium being least low in the hydrochlorothiazide group because of the highest preinfusion value. Preinfusion serum urate was highest after hydrochlorothiazide and fell during the adrenaline infusion in all groups, although not significantly. Blood glucose increased during the adrenaline infusion in all groups, but significantly more after hydrochlorothiazide and amiloride than after lisinopril. Heart rate increased during the adrenaline infusion in all groups but least after lisinopril. QTc preinfusion was longer after hydrochlorothiazide than after amiloride and placebo, but the infusion-induced prolongation of QTc was of the same magnitude in all pretreatment groups. Since our results were obtained in short-term experiments in normal subjects, their clinical relevance is questionable, but they support the view that ACE inhibitors may have certain metabolic advantages over diuretics.


Anti-free-radical and neutrophil-modulating properties of the nitrovasodilator, nicorandil. The nitrovasodilator, nicorandil, is a clinically effective antianginal agent. We tested whether nicorandil may also possess anti-free-radical characteristics, since the nicotinamide moiety of its molecular structure is a known hydroxyl radical scavenger. In vitro production of hydroxyl radicals by hypoxanthine plus xanthine oxidase in the presence of iron produced a marked degradation of deoxyribose. Nicorandil and the structural analogs, nicotinic acid and nicotinamide, produced significant inhibition of deoxyribose breakdown at concentrations equipotent to the classical hydroxyl radical scavenger, mannitol. Nicorandil also produced a concentration-dependent inhibition of superoxide anion production by canine neutrophils that were activated with either phorbol myristate acetate (PMA) or opsonized zymosan. This inhibition could not be mimicked by the analog, nicotinamide. While equimolar concentrations of nitroglycerin produced less inhibition of superoxide anion generation in opsonized zymosan-activated neutrophils than that observed with nicorandil, nitroglycerin did not alter free-radical production in PMA-stimulated neutrophils. Glyburide, the ATP-sensitive potassium-channel blocker, did not reverse the action of nicorandil on neutrophils. Thus, nicorandil is a uniquely different nitrovasodilator with anti-free-radical and neutrophil-modulating properties.


Damage to mitochondrial respiration chain is related to phospholipase A2 activation caused by tumor necrosis factor. Tumor necrosis factor (TNF) has been shown to be cytotoxic to tumor cell lines in vitro, but the mechanism by which TNF exerts its cell growth-regulatory effects is not known. In this report, we used various inhibitors to investigate the sequence of events that lead to cytotoxic effects of TNF on L.P3 cells, a highly sensitive, murine fibroblast cell line. Our results indicate that mitochondrial respiration chains are damaged by a hydroxyl radical at an early stage of the cell lysis after TNF treatment. This event is followed by the activation of phospholipase A2, and finally leads to cell lysis.


Controlled drinking by chronic drunkeness offenders--a British experience. Twenty-seven of the 235 drunkenness offenders treated at the Manchester Detoxification Centre and followed-up for 2 years attempted controlled drinking for varying lengths of time and for different stated reasons, even though the treatment goal at the Centre was total abstinence. Only nine of the twenty-seven were able to sustain controlled drinking for periods of up to twelve weeks. The overall sub-group controlled drinking performance was 10.2% which compared favourably with the 10% overall abstinence performance of all patients followed-up. It was observed that there was little argument in favour of offering the option of controlled drinking as an alternative to the traditional treatment goal of total abstinence. It was advocated however that while total abstinence must remain the treatment goal of therapist, controlled drinking as an alternative goal may be suitable for carefully selected individual alcohol dependent persons and an important future research area should be the criteria for selecting such individuals.


Interleukin-2 and interferon-alpha in the treatment of patients with advanced non-small-cell lung cancer. The role of combination chemotherapy in the treatment of advanced non-small-cell lung cancer is controversial. At best, a small survival benefit can be achieved. Therefore, other treatment modalities are needed. On the basis of the promising treatment results with interleukin-2 (IL-2) -containing immunotherapy in renal cell cancer and melanoma, we performed a phase I-II study with IL-2 and interferon alpha (IFN-alpha). Eligible patients were treated with IL-2 18 x 10(6) IU/m2/day by continuous intravenous infusion (c.i.v.) for 3 days. On the same days, 5 x 10(6) U/m2/day IFN-alpha was given intramuscularly. After a rest period of 4 days, patients at the first dose level received IL-2 2.4 x 10(6) IU/m2/day c.i.v. for a period of 28 days, followed by 14 days' rest, 14 days' treatment, 7 days' rest, and a final treatment for 14 days. Patients at the second dose level were treated according to the same schedule, in which the dose of IL-2 was increased to 3.6 x 10(6) IU/m2/day. During low-dose IL-2 treatment, patients received IFN-alpha 5 x 10(6) U/m2/day on days 1 and 4 of each week. Eleven patients were admitted to the study, six at the first and five at the second dose level. Median age was 54 years; all patients had a performance status of 0 or 1. The most important adverse effects included anorexia, fatigue, nausea, and headache, which were not dose limiting. In the 11 patients treated, no responses were seen. Nine patients developed progressive disease during the first 5 weeks of treatment. We concluded that this regimen of IL-2 and IFN-alpha is ineffective.


The persistence of chlamydial inclusions in clinically quiescent trachoma. Most of the trachoma patients seen at the Guinness Eye Clinic in Kaduna, Northern Nigeria today are in the healed or quiescent stages of evolution and disease intensity is largely trivial. A significant proportion however, seem to have recrudescence of the active stages, a phenomenon that is usually attributed to reinfection. Giemsa cytology of conjunctival scrapings obtained from a cross section of our patients however indicates that a significant proportion still harbour inclusions in spite of clinical inactivity and that some of these inclusion positive cases demonstrate no obvious host inflammatory response, suggesting persistence of infection and host immunocytological tolerance. This brings up the possibility of a spontaneous self perpetrating reactivation cycle which may account for the multicyclicity of trachoma without a reinfecting source in susceptible individuals.


Evidence from directed mutagenesis that aspartate 170 of the D1 polypeptide influences the assembly and/or stability of the manganese cluster in the photosynthetic water-splitting complex. To identify amino acid residues that influence the assembly or stability of the manganese cluster in photosystem II, we have generated site-directed mutations in the D1 polypeptide of the cyanobacterium, Synechocystis sp. PCC 6803. Indirect evidence has suggested that the D1 polypeptide provides some of the ligands that are required for metal binding. Mutations at position 170 of D1 were selected for characterization, since an aspartate to asparagine mutation (DN170D1) at this position completely abolishes photoautotrophic growth, while retention of a carboxylic acid at this position (aspartate to glutamate, DE170D1) supports photoautotrophic growth. Photosystem II particles were purified from control, DE170D1, and DN170D1 cells by a procedure that retains high rates of oxygen evolution activity in control particles [Noren, G.H., Boerner, R.J., & Barry, B.A. (1991) Biochemistry 30, 3943-3950]. Spectroscopic analysis shows that the tyrosine radical, Z+, which normally oxidizes the manganese cluster, is rapidly reduced in the DE170D1 mutant, but not in the DN170D1 mutant. A possible explanation of this block or dramatic decrease in the rate of electron transfer between the manganese cluster and tyrosine Z is an alteration in the properties of the metal center. Quantitation of manganese in these particles is consistent with aspartate 170 influencing the stability or assembly of the manganese cluster, since the aspartate to asparagine mutation results in a decrease in the manganese content per reaction center. Photosystem II particles from DN170D1 show a 60% decrease in the amount of specifically bound manganese per reaction center, when compared to control particles. Also, we observe a 70% decrease in the amount of specifically bound manganese per reaction center in partially purified DN170D1 particles and at least an 80% decrease in the amount of hydroxylamine-reducible manganese in DN170D1 thylakoid membranes. Single-turnover fluorescence assays and steady-state EPR measurements demonstrate that the remaining, endogenous manganese does not rapidly reduce tyrosine Z+ in the DN170D1 mutant. Additional evidence that aspartate 170 influences the assembly or stability of the metal site comes from analysis of the DE170D1 mutant. Although this mutant assembles a functional manganese cluster, as assessed by oxygen evolution and spectroscopic assays, the properties of the manganese site are perturbed.


p-Chloroamphetamine induces serotonin release through serotonin transporters. p-Chloroamphetamine (PCA) interacts with serotonin transporters in two membrane vesicle model systems by competing with serotonin for transport and stimulating efflux of accumulated serotonin. In plasma membrane vesicles isolated from human platelets, PCA competes with [3H]imipramine for binding to the serotonin transporter with a KD of 310 nM and competitively inhibits serotonin transport with a KI of 4.8 nM. [3H]Serotonin efflux from plasma membrane vesicles is stimulated by PCA in a Na(+)-dependent and imipramine-sensitive manner characteristic of transporter-mediated exchange. In membrane vesicles isolated from bovine adrenal chromaffin granules, PCA competitively inhibits ATP-dependent [3H]serotonin accumulation with a KI of 1.7 microM and, at higher concentrations, stimulates efflux of accumulated [3H]serotonin. Stimulation of vesicular [3H]serotonin efflux is due in part to dissipation of the transmembrane pH difference (delta pH) generated by ATP hydrolysis. Part of PCA's ability to stimulate efflux may be due to its transport by the vesicular amine transporter. Flow dialysis experiments demonstrated uptake of [3H]PCA into chromaffin granule membrane vesicles in response to the delta pH generated in the presence of Mg2+ and ATP. In plasma membrane vesicles, no accumulation was observed using an NaCl gradient as the driving force. We conclude that rapid nonmediated efflux of transported PCA prevents accumulation unless PCA is trapped inside by a low internal pH.


Differential interaction of lecithin-retinol acyltransferase with cellular retinol binding proteins. Esterification of retinol (vitamin A alcohol) with long-chain fatty acids by lecithin-retinol acyltransferase (LRAT) is an important step in both the absorption and storage of vitamin A. Retinol in cells is bound by either cellular retinol binding protein (CRBP), present in most tissues including liver, or cellular retinol binding protein type II [CRBP(II)], present in the absorptive cell of the small intestine. Here we investigated whether retinol must dissociate from these carrier proteins in order to serve as a substrate for LRAT by comparing Michaelis constants for esterification of retinol presented either free or bound. Esterification of free retinol by both liver and intestinal LRAT resulted in Km values (0.63 and 0.44 microM, respectively) similar to those obtained for esterification of retinol-CRBP (0.20 and 0.78 microM, respectively) and esterification of retinol-CRBP(II) (0.24 and 0.32 microM, respectively). Because Kd values for retinol-CRBP and retinol-CRBP(II) are 10(-8)-10-(-10) M, these similar Km values indicated prior dissociation is not required and that direct binding protein-enzyme interaction must occur. Evidence for such interaction was obtained when apo-CRBP proved to be a potent competitive inhibitor of LRAT, with a KI (0.21 microM) lower than the Km for CRBP-retinol (0.78 microM). Apo-CRBP(II), in contrast, was a poor competitor for esterification of retinol bound to CRBP(II). Apo-CRBP reacted with 4 mM p-(chloromercuri)benzenesulfonic acid lost retinol binding ability but retained the ability to inhibit LRAT, confirming that the inhibition could not be explained by a reduction in the concentration of free retinol.(ABSTRACT TRUNCATED AT 250 WORDS)


Phosphorescence and optically detected magnetic resonance measurements of the 2'AMP and 2'GMP complexes of a mutant ribonuclease T1 (Y45W) in solution: correlation with X-ray crystal structures. Phosphorescence and ODMR measurements have been made on ribonuclease T1 (RNase T1), the mutated enzyme RNase T1 (Y45W), and their complexes with 2'GMP and 2'AMP. It is not possible to observe the phosphorescence of Trp45 in RNase T1 (Y45W). Only that of the naturally occurring Trp59 is seen. The binding of 2'GMP to wild-type RNase T1 produces only a minor red shift in the phosphorescence and no change in the ODMR spectrum of Trp59. However, a new tryptophan 0,0-band is found 8.2 nm to the red of the Trp59 0,0-band in the 2'GMP complex of the mutated RNase T1 (Y45W). Wavelength-selected ODMR measurements reveal that the red-shifted emission induced by 2'GMP binding, assigned to Trp45, occurs from a residue with significantly different zero-field splittings than those of Trp59, a buried residue subject to local polar interactions. The phosphorescence red shift and the zero-field splitting parameters demonstrate that Trp45 is located in a polarizable environment in the 2'GMP complex. In contrast with 2'GMP, binding of 2'AMP to RNase T1 (Y45W) induces no observable phosphorescence emission from Trp45, but leads only to a minor red shift in the phosphorescence origin of Trp59 in both the mutated and wild-type enzyme. The lack of resolved phosphorescence emission from Trp45 in RNase T1 (Y45W) implies that the emission of this residue is quenched in the uncomplexed enzyme. We conclude that local conformational changes that occur upon binding 2'GMP remove quenching residues from the vicinity of Trp45, restoring its luminescence.(ABSTRACT TRUNCATED AT 250 WORDS)


Early steps in cytochrome c folding probed by time-resolved circular dichroism and fluorescence spectroscopy. The kinetics of protein folding for horse ferricytochrome c was investigated by stopped-flow methods, using far-UV circular dichroism (CD), near-UV CD, and tryptophan fluorescence to probe the formation of secondary structure and tertiary interactions. In the far-UV region of the CD spectrum (222 nm), 44% of the total change associated with refolding occurs within the dead time of the stopped-flow experiment, indicating that a significant amount of helical secondary structure is formed in less than 4 ms. The remaining changes in the ellipticity at 222 nm occur in two kinetic phases with time constants of about 40 ms and 0.7 s, respectively. In contrast, there is no evidence for rapid changes in the ellipticity at 289 nm: an aromatic CD band, which is indicative of the formation of a tightly packed core, only begins to appear in a 400-ms step and is completed in a final 10-s phase. The fluorescence of a single tryptophan at position 59, which becomes quenched upon folding via nonradiative energy transfer to the heme group, provides complementary information on the condensation of the polypeptide chain during refolding. The fluorescence-detected stopped-flow folding kinetics of ferricytochrome c exhibits a 35% decrease in fluorescence during the dead time, suggesting that a substantial decrease in the average tryptophan-heme distance occurs on a submillisecond time scale. The subsequent fluorescence changes exhibit two prominent phases with time constants of about 20 and 300 ms, followed by a minor 5-s phase.(ABSTRACT TRUNCATED AT 250 WORDS)


Modified, large-scale purification of the cytochrome o complex (bo-type oxidase) of Escherichia coli yields a two heme/one copper terminal oxidase with high specific activity. The cytochrome o complex is a bo-type ubiquinol oxidase in the aerobic respiratory chain of Escherichia coli. This complex has a close structural and functional relationship with the eukaryotic and prokaryotic aa3-type cytochrome c oxidases. The specific activity, subunit composition, and metal content of the purified cytochrome o complex are not consistent for different preparative protocols reported in the literature. This paper presents a relatively simple preparation of the enzyme starting with a strain of Escherichia coli which overproduces the oxidase. The pure enzyme contains four subunits by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Partial amino acid sequence data confirm the identities of subunit I, II, and III from the SDS-PAGE analysis as the cyoB, cyoA, and cyoC gene products, respectively. A slight modification of the purification protocol yields an oxidase preparation that contains a possible fifth subunit which may be the cyoE gene product. The pure four-subunit enzyme contains 2 equivs of iron but only 1 equiv of copper. There is no electron paramagnetic resonance detectable copper in the purified enzyme. Hence, the equivalent of CuA of the aa3-type cytochrome c oxidases is absent in this quinol oxidase. There is also no zinc in the purified quinol oxidase. Finally, monoclonal antibodies are reported that interact with subunit II. One of these monoclonals inhibits the quinol oxidase activity of the detergent-solubilized, purified oxidase. Hence, although subunit II does not contain CuA and does not interact with cytochrome c, it still must have an important function in the bo-type ubiquinol oxidase.


Cooperative gating of chloride channel subunits in endothelial cells. New methods are described to detect subconductance levels and to analyse ion channel gating. These methods are applied to simulated and experimental data. Single chloride channel records from inside-out membrane patches excised from human umbilical venous endothelial cells (HUVEC) exhibit, in addition to the full closed and full open configurations, intermediate subconductance levels which are multiple of an elementary conductance of 112.5 pS. Analysis of transitions from one state to another and the comparison of real data with simulated data leads to the proposal of a cooperative model of gating for the observed subunits of a chloride channel.


Cis-allosteric effects of cytoplasmic Na+/K+ discrimination at varying pH. Low-affinity multisite inhibition of cytoplasmic K+ in reconstituted Na+/K(+)-ATPase engaged in uncoupled Na(+)-efflux. In liposomes with reconstituted shark Na+/K(+)-ATPase the effect of cytoplasmic K+ was investigated in the absence of extracellular alkali ions. During such conditions the Na+/K(+)-ATPase is engaged in the so called uncoupled Na+ efflux mode in which cytoplasmic Na+ activates and binds to the enzyme and becomes translocated without countertransport of K+ as in the physiological Na+/K+ exchange mode. In this uncoupled flux mode only low-affinity inhibition by K+cyt is found to be present. The inhibition pattern is consistent with a model in which cytoplasmic K+ exhibit mixed inhibition of Na+ activation, probably by binding at the three cytoplasmic loading sites on E1ATP (E1A). With determined intrinsic binding constants for cytoplasmic Na+ to this form of KS1, KS2, KS3 = 40 mM, 2 mM, 2 mM the inhibition pattern can be simulated assuming three K+cyt sites with equal affinity for Ki = 40 mM, similar to KS1 for the first Na+cyt site. The discrimination between cytoplasmic Na+ and K+ is therefore enhanced by allosteric interaction initiated from the cis-side due to binding of the first Na+, as opposed to K+, which induces the positive cooperatively in the successive Na+ bindings. pH is found to influence the pattern of K+cyt inhibition: A lowering of the pH potentiates the K+cyt inhibition, whereas at increased pH the inhibition is decreased and transformed into a pure competitive competition.


Occlusion of Rb+ after extensive tryptic digestion of shark rectal gland Na,K-ATPase. Na,K-ATPase from rectal glands of Squalus acanthias has been subjected to proteolysis with trypsin. The E1- and E2-forms of the enzyme can be distinguished from the inactivation patterns at low trypsin concentrations, as previously seen with kidney enzyme. Extensive degradation by trypsin in the presence of 5 mM Rb+ yields membrane fragments with a 19 kDa peptide as the major proteolytic fragment of the alpha-subunit. The sequence of the N-terminal 40 residues of this peptide is almost identical to that of a similar proteolytic fragment isolated by Capasso et al. (Capasso, J.M., Hoving, S., Tal, D.M., Goldshleger, R. and Karlish, S.J.D. (1992) J. Biol. Chem. 267, 1150-1158) using kidney Na,K-ATPase. Rb+ occlusion can be fully retained under these circumstances, supporting the findings with kidney enzyme that only minor parts of the alpha-subunit are required to form a functional occlusion-site.


Direct modification of low density lipoprotein by the spin trap 3,5-dibromo-4-nitrosobenzenesulfonic acid. We have previously reported that the spin trap alpha-phenyl-tert-butyl nitrone (PBN) inhibited the oxidative modification of low density lipoprotein (LDL) (Kalyanaraman, B., Antholine, W.E. and Parthasarathy, S. (1990) Biochim. Biophys. Acta 1035, 286-292). In the present study, we report that 3,5-dibromo-4-nitrosobenzenesulfonic acid (DBNBS), a water-soluble spin trap, also inhibited the oxidation of LDL as measured by the formation of thiobarbituric acid reactive substances (TBARS). However, when compared with LDL incubated without DBNBS, the DBNBS-incubated LDL showed increased negative charge on agarose gel electrophoresis and was avidly degraded by mouse peritoneal macrophages. Despite the suggestion of biological modification, there was no decrease in lysine-amino groups in DBNBS-incubated LDL. Furthermore, reductively methylated LDL in which more than 85% of the amino group of lysines was blocked, was also modified by DBNBS. A sulfonic acid analog of PBN failed to modify LDL in a similar manner, suggesting that the presence of sulfonic acid alone does not ensure modification. When LDL was incubated with DBNBS, radical adducts associated with both lipid and protein were detected by electron paramagnetic resonance (EPR) technique. It is suggested that DBNBS may bind to the apoprotein B100 and lipids of LDL by a lysine-independent mechanism resulting in increased recognition and degradation by macrophages. The present work offers a novel approach for rapid modification of LDL.


The effects of the phospholipase A2 inhibitors aristolochic acid and PGBx on A23187-stimulated mobilization of arachidonate in human neutrophils are overcome by diacylglycerol or phorbol ester. Aristolochic acid and PGBx, two structurally unrelated, protein-targeted inhibitors of isolated phospholipases A2, are effective antagonists of calcium ionophore A23187-stimulated mobilization of [3H]arachidonate from human neutrophils. We now report that preincubation of neutrophils with oleoylacetylglycerol (OAG, 15 microM) substantially reverses the inhibitory effect of 200 microM aristolochic acid (from 70 to 24% inhibition). Similarly, OAG increases the IC50 for PGBx from 2.5 to greater than 20 microM. The effects of OAG on inhibition by either aristolochic acid or PGBx are dose-dependent, with an ED50 of 2.5 microM. Protection against inhibition by either aristolochic acid or PGBx is also observed with phorbol myristate acetate (PMA, ED50 3 nM), but not 4-alpha-phorbol didecanoate. Aristolochic acid and PGBx do not inhibit PMA-stimulated superoxide generation, and are thus not protein kinase C inhibitors. Furthermore, neither aristolochic acid nor PGBx inhibit diglyceride generation through the phospholipase D/phosphatidate phosphohydrolase pathway. A23187-stimulated [3H]arachidonate mobilization is increased by 20-50% when neutrophils are preincubated with OAG or PMA. The present results indicate that OAG and PMA also modulate the A23187-stimulated [3H]arachidonate mobilization so as to render it less sensitive to inhibitors of phospholipase A2.


Puromycin reaction of the A-site bound peptidyl-tRNA. AcPhe2-tRNA(Phe) which appears in ribosomes after consecutive binding of AcPhe-tRNA(Phe) at the P sites and EF-Tu-directed binding of Phe-tRNA(Phe) at the A sites is able to react quantitatively with puromycin in the absence of EF-G. One could readily explain this fact to be the consequence of spontaneous translocation. However, a detailed study of kinetics of puromycin reaction carried out with the use of viomycin (inhibitor of translocation) and the P-site test revealed that, apart from spontaneous translocation, this peptidyl-tRNA could react with puromycin being located at the A site. This leads to the conclusion that the transpeptidation reaction triggers conformational changes in the A-site ribosomal complex bringing the 3'-end of a newly synthesized peptidyl-tRNA nearer to the peptidyl site of peptidyltransferase center. This is detected functionally as a highly pronounced ability of such a peptidyl-tRNA to react with puromycin.


Effect of the terminal phosphate derivatization of beta- and alpha-oligodeoxynucleotides on their antisense activity in protein biosynthesis, stability and uptake by eucaryotic cells. Inhibition of polypeptide chain elongation with the mRNA-complementary (antisense) oligonucleotide has been realized through a RNase H independent mechanism. Nuclease resistant complementary non-natural alpha-17-mer oligonucleotide did not inhibit cell-free protein biosynthesis of beta-globin in the wheat germ system because it did not elicit RNase H activity. Linkage of alkylating group [4-(N-2-chloroethyl-N-methyl)-aminobenzyl]-methylamine to the 5'-terminus of the alpha-oligomer led to the formation of its covalent adduct with mRNA which could not be translated in vitro. Linkage of hydrophobic residues to the terminal phosphates of natural oligonucleotides increased their stability against nucleases and uptake by human cancer cells. A porphyrin, substituted in the meso-position by aromatic groups, gave a rise to an approximately six-fold increase of uptake and cholesterol a 30-100-fold increase. Eighty percent of bound derivatives were found in cytoplasmic cellular fractions.


Hematopoietic stem cell deficiency resulting from cytomegalovirus infection of bone marrow stroma. Cytomegalovirus (CMV) recurrence from latency is a major risk factor in bone marrow transplantation (BMT). Owing to the immunodepletive treatment, ablation of the immune control of latent CMV is responsible for recurrence and cytopathogenic spread of the virus in vital tissues. There is increasing evidence for reconstituting bone marrow being itself a target tissue of CMV. By inhibiting post-transplantation hematopoiesis, CMV is causal for maintenance of the immunocompromised state, which leads to a prolonged phase of persistent virus replication. Based on results in a murine model of BMT and concurrent CMV infection, we discuss possible mechanisms of CMV-mediated bone marrow graft failure. It is concluded that an irremediable damage of bone marrow stroma by CMV is responsible for a reduced rate of regeneration of the marrow-repopulating, pluripotent stem cell.


Cytomegalovirus and marrow function.

Cytomegalovirus and marrow function. Infection with cytomegalovirus (CMV) continues to be one of the most common complications following allogeneic bone marrow transplantation. A proportion of patients with CMV infection also experience neutropenia. To investigate the possible role of CMV in the suppression of hematopoiesis, we have examined the effect of CMV on the growth of isolated myeloid progenitors and on the production of myeloid cells in the long-term bone marrow culture (LTMC) system. In these studies, various isolates of CMV were added either directly to cultures of progenitors or to LTMC established from normal CMV-seronegative donors. In the first system, myelosuppression is manifested by a reduction in the number of colonies that grow. In the second system, myelosuppression is manifested by a reduction in the number of myeloid cells produced and released into the culture supernatant. Analysis of the data observed indicated that myelosuppression could in some cases be attributed to direct infection of myeloid progenitors. In other cases stromal cells were infected. In the latter cases, myelosuppression was then caused by an alteration in cytokines produced by the stromal cells. These observations made in vitro raise the possibility that comparable mechanisms may be responsible for the myelosuppression observed with CMV infection in vivo. To pursue this possibility we proposed to detect the CMV genome in defined subpopulations of marrow cells isolated from infected patients. Given the technical restrictions imposed by the small sample size available from patient marrow aspirations, our initial attempts to develop on appropriate technique involved isolation of cells from CMV-seropositive normal bone marrow donors. Using the polymerase chain reaction we were able to amplify CMV DNA contained within marrow cells of some healthy CMV-seropositive marrow donors.


Myelopoiesis in vitro is suppressed by hepatitis A virus. Perturbations of hematopoietic regulation ranging from transient granulocytopenia to rare cases of bone marrow failure are associated with infections due to hepatitis A virus (HAV). In an attempt to elucidate the pathogenetic mechanisms we had previously established that HAV has a direct suppressive effect on human bone marrow progenitors (CFU-GM, -GEMM, BFU-E). These studies were extended to long-term bone marrow cultures (LTBMC): Inoculation of bone marrow mononuclear cells with HAV did not interfere with the establishment of an adherent stromal layer, nor did the inoculation of already established layers cause any morphologically recognizable changes to the stroma. In contrast, a significant and progressive decline of the CFU-GM content in the culture supernatants was demonstrated. HAV antigen was detected by APAAP stain in a subpopulation of stromal cells, and sequential estimations of virus titers in the supernatants provided evidence for viral replication in primary bone marrow cultures. Interferon-gamma and tumor necrosis factor-alpha levels of infected cultures did not differ from those of uninfected controls. These findings argue for a direct suppression of (pre-) CFU-GM by HAV in a model system (LTBMC) lacking an immune defense which would limit viral replication.


Intensification of GVHD prophylaxis interferes with the effects of pretransplant herpes virus serology on the occurrence of grades II-IV acute graft-versus-host disease. The effects of pretransplant herpes virus serology on the occurrence of grades II-IV acute graft-versus-host disease (GVHD) were studied in 262 recipients and their HLA-identical family donors. In 131 recipients on standard GVHD prophylaxis (either methotrexate or cyclosporin A) significant effects were observed for donor HSV serology (seropositivity associated with increased risk for GVHD) and donor EBV serology (seronegativity associated with increased risk). However, these effects were nonsignificant in the other 131 recipients on intensified GVHD prophylaxis (i.e., methotrexate combined with cyclosporin A, in vivo anti-T-cell monoclonal antibodies, or various procedures to reduce the T-cell numbers in the transplants.


Influence of human cytomegalovirus on immune reconstitution after bone marrow transplantation. HCMV infection diagnosed by the highly sensitive polymerase chain reaction (PCR) technology in blood, urine and skin biopsies of patients after bone marrow transplantation (BMT) correlated with the reconstitution of peripheral blood lymphocytes and dermal immunohistological alterations to evaluate the interaction of viral infection with the recovery of the immune system, as well as with the induction or aggravation of graft-versus-host disease (GVHD). In a prospective study 73% of 63 patients showed viremia at a median time of 25 days after BMT. Only 44% of these cases that also presented with a higher frequency of acute GVHD symptoms developed HCMB disease later on. In the skin, similar immunohistological alternations, as well as frequent primary local HCMV infection before the development of cutaneous signs of GVHD, was found, suggesting the direct involvement of anti-HCMV immune responses in the induction of GVHD-associated organ lesions.


Correlation of pretransplant viral serology and complications of bone marrow transplantation. Latent herpes viruses such as herpes simplex virus, cytomegalovirus (CMV), and varicella zoster virus are often reactivated after bone marrow transplantation, giving rise to infections. In contrast, Epstein-Barr virus infections rarely occur. Significant mortality is induced especially by pneumonitis, most often caused by CMV. Immunosuppression and pancytopenia caused by CMV increase the risk of bacterial infections and invasive fungal infections. Herpes viruses may increase the risk of acute and chronic graft-versus-host disease (GVHD). Thus, immunity to several herpes viruses was associated with an increased risk of acute GVHD. Seropositivity for CMV in recipient and donor increased the risk of chronic GVHD. Herpes viruses were also associated with a decreased risk of leukemic relapse. CMV infection, asymptomatic CMV infection, and seropositivity for several herpes viruses were associated with a reduced incidence of relapse in different reports. In spite of this possible antileukemic effect, leukemia-free survival was unaffected by herpes virus immunity in recipients or donors.


Risk factors for viral reactivation following bone marrow transplantation. The identification of risk factors for viral reactivation following transplantation is essential in order that antiviral prophylactic regimes may be allocated rationally. In the pretransplant period qualitative serological assessment of recipient and donor is informative with regard to the risk of post-transplant reactivation and disease. In addition, the quantitative level of herpes simplex virus (HSV) IgG in the pretransplant recipient is predictive of post-transplant HSV excretion, although this relationship does not exist for other viruses of the herpes group. We discuss the value of surveillance cultures for cytomegalovirus in the post-transplant period in predicting the development of CMV disease, and how this may allow effective intervention to prevent what is a major cause of morbidity and mortality in the transplant population.


Prevention of viral infections after bone marrow transplantation. After bone marrow transplantation, a number of viral infections contribute to the morbidity and mortality of the procedure. Established preventive measures to avoid primary infection and reactivation of herpes-and cytomegaloviruses are outlined. Possible future strategies against these viruses (e. g., monoclonal antibodies, transfer of T-lymphocytes) and the possible role of improved diagnostic tools are briefly discussed.


Prevention and treatment of CMV infection after allogeneic bone marrow transplant. CMV infection is the major infectious complication following bone marrow transplantation. It is most often related to reactivation of latent infection in patients who were CMV seropositive before BMT. The incidence and severity have recently been modified by the use of preventive and curative treatments. Prevention of CMV infection with the transfusion of seronegative blood products is useful only when donor and recipient are seronegative. High-dose acyclovir has been shown effective in one randomized study. A multicenter study is currently being performed in Europe to confirm this result. Intravenous gammaglobulins seemed to lower the number of patients who incur interstitial pneumonitis but not the incidence of viremia. They also decreased the incidence of gram-negative sepsis and severe GVH and improved survival. The treatment is based on the use of gancyclovir. Several studies show that gancyclovir is more effective in asymptomatic patients with viral isolation from blood or bronchoalveolar lavage. The addition to gancyclovir of high-dose gammaglobulin improves survival in symptomatic patients with interstitial pneumonitis. This progress in the prevention and treatment of CMV infection has improved the overall results of allogeneic bone marrow transplantation.


Epstein-Barr virus infection in allogeneic marrow grafting: lessons for transplant physicians and virologists. The relationship between Epstein-Barr virus (EBV) and the host is profoundly disturbed by allogeneic bone marrow transplantation (BMT) because EBV resides in the recipient's hematopoietic system, which has to be destroyed in the majority of cases, and in the donor's hematopoietic system, i.e., the marrow graft. We have shown that EBV may be eradicated from some BMT recipients and that the virus may be transferred with the marrow graft. During the immediate post-transplant period oropharyngeal EBV excretion may occur which, by infecting passing B lymphocytes, may act as co-factor for acute graft-versus-host disease and help the virus to survive, despite the temporary depletion of its reservoir. The coexistence of totally different EBV strains in BMT recipients but not in healthy, untransfused controls, suggests that superinfection may by possible in case of immunodeficiency; alternatively, transfer of the virus by the reservoir itself (the B lymphocytes) might be the only effective route for superinfection. The generation of 'variant' strains during viral replication may form the basis of the vast polymorphism between wild-type EBV isolates in the population.


An exploratory analysis of survival with AIDS using a nonparametric tree-structured approach. We illustrate an analysis with classification and regression trees applied to survival data. Through this application, we provide a description of the opportunistic diseases and sociodemographic factors that contribute to survival among people with human immunodeficiency virus disease. The analyses are based on 43,795 cases reported to the Centers for Disease Control between January 1, 1984, and December 31, 1987. We used vital status as of December 31, 1989, to estimate mortality rates. We identified Kaposi's sarcoma and opportunistic diseases causing central nervous system damage (cryptococcosis, primary lymphoma of the brain, cytomegalovirus disease, and progressive multifocal leukoencephalopathy) as important predictors of death. In addition, advanced age at diagnosis (50+), race (white/other), and history of illicit drug use were found to be important determinants. Estimates of the cumulative probability of survival for subgroups of individuals defined by the tree structure illustrate the effect of these determinants on mortality. For the purpose of comparison, two proportional hazards models were also fit to the data using factors identified in the tree structure as the determinants of interest. This application illustrates the utility and limitations of both this new technique and proportional hazards models for epidemiologic research.


[Lipolysis in model membranes in the presence of positively charged soluble proteins]. Phospholipase A2 hydrolysis of neutral and negatively charged lipid membranes modified by positively charged proteins has been studied using liposomes composed of either dioleoylphosphatidylcholine (DOPC) or dioleoylphosphatidylglycerol (DOPG) alone or their equimolar mixture in the presence of cytochrome c, histone H1, cytochrome b5, and polylysine. Twenty minutes after the reaction had been initiated, DOPC hydrolysis was 58%, while that in the equimolar mixture with DOPG was 35%. DOPG hydrolysis was more complete in binary mixtures of liposomes. The same was observed for liposomes in the presence of cytochrome c. Hydrolysis of phospholipids in binary liposomes in the presence of histone H1 was 3 times faster than that in protein-free liposomes. In the presence of polylysine the rate of DOPG hydrolysis was decreased. The results obtained are suggestive of electrostatic interactions between hydrophilic proteins and negatively charged phospholipids, the phospholipase A2 catalytic activity being affected by these interactions.


[Dalargin-binding proteins of synaptic membranes from the rat brain: isolation and comparison of their properties with opiate receptor properties]. The proteins isolated from rat brain synaptic membranes were studied by affinity chromatography on dalargin-omega-aminohexyl-Sepharose 4B and specific elution with DAGO (Tyr-D-Ala-Gly-N-Me-Gly-ol). These proteins were shown to bind specifically 3H-naloxone (Kd = 6.6 nM; Bmax = 690 pmol/mg of protein). SDS electrophoresis of the dalargin-binding proteins termed as DBPDAGO revealed one major protein band with M(r) of 42 kDa and two minor bands with M(r) of 29 and 67 kDa. The glycoprotein component was found in DBPDAGO; their isoelectric properties were established (pI 5.4). The close similarity of DBP properties with those of isolated brain opiate receptors suggest them to be opiate receptor components.


[Formation of superoxide radicals in membranes of subcellular organelles in regenerating liver]. A correlation between the changes in the rates of superoxide radical generation, upsilon, in microsomes, mitochondria, and nuclei and the Cu, Zn- and Mn-SOD activities in rat liver during the first 5 days after partial hepatectomy, has been studied. Level of upsilon in microsomal and mitochondrial membranes in the regeneration process was reduced. The Cu, Zn- and Mn-SOD activities changed in an extreme and antibate manner: the former was characterized by a minimum, whereas the latter-by a maximum with an extreme on the 3rd day after surgery. Analysis of the correlation between the values of upsilon in the nuclear membranes and cell cycle stages (on a literary basis) revealed that the upsilon was decreased 2 times on the stage of DNA synthesis. When mitosis was at maximum, upsilon showed a 4-5-fold increase in comparison with the control, the Cu, Zn-SOD activity being essentially unchanged. A role of SOD and O2-. in cell division is postulated. O2-. is assumed to play a role in gene expression, disassembly, and regeneration of the nuclear membrane; that of SOD is thought to consist in regulation of the proliferative activity.


[Oxidative modification and inactivation of superoxide dismutase by hypochlorite]. The inactivating effect of hypochlorite on Cu, Zn-superoxide dismutase (SOD) from bovine erythrocytes has been studied. According to SDS gel electrophoresis and isoelectric focusing data, oxidation is associated with the degradation of the polypeptide chain, formation of aggregates, and appearance of new isoforms. These protein fractions differ from native SOD by the electric charge and molecular mass but possess a catalytic activity. Modified SOD isoforms occur as a result of intramolecular crosslinking of amino groups and aldehydes which is confirmed by the appearance of fluorescence maxima in the longwave region characteristic of such links. It is assumed that the mechanism of SOD inactivation is coupled to the oxidation of amino acids located outside the active center of the enzyme.


[Features of the interaction of Glu- and Lys-forms of plasminogen with native and partially hydrolyzed fibrin]. Glu- and Lys-plasminogen interaction with native and desAABB-fibrin obtained from fibrinogen partially hydrolyzed by plasmin was studied. It was found that native fibrin adsorbs 6 times more Lys-plasminogen as compared to the native form of the proenzyme. The range of the Lys-plasminogen binding does not change, if part of the fibrinogen molecules hydrolyze down to X-fragments. At the same time, the appearance in the system of 1% Xi-fragments leads to a 6-fold increase in the Glu-plasminogen binding. The amount of adsorbed Glu-plasminogen reaches the level of Lys-plasminogen adsorption both in the native and partially hydrolyzed fibrin. It was found that kringle K 1-3 or 6-aminohexanoic acid at saturating for high-affinity lysine-binding sites concentrations do not influence the Glu-plasminogen binding to native fibrin but inhibit it when the partially purified form is used. It is assumed that the manyfold increase of the Glu-plasminogen binding to partially hydrolyzed fibrin is due to the alteration of the proenzyme conformation at the initial steps of fibrin hydrolysis during the formation of Xi fragments.


Murine pregnancies predisposed to spontaneous resorption show alterations in the concentrations of leukotriene B4 and prostaglandin E2. Female CBA/J mice mated with DBA/2 males exhibit an increased spontaneous resorption rate (30-35%) in their first pregnancy. Second pregnancies show a decreased resorption rate (15-20%). In contrast, resorption in CBA/J females mated with BALB/c males (identical to DBA/2 at the H-2 major histocompatibility locus) occurs with a frequency of 5-10%. Resorption is preceded by fetoplacental infiltration of natural killer (NK)-like cells and a deficiency in a lipophilic NK-suppressive activity. The eicosanoids leukotriene B4 (LTB4) and prostaglandin E2 (PGE2) are known to modulate NK activity in vitro. We measured the concentrations of LTB4 and PGE2 in extracts of individual fetoplacental units at Day 8 of gestation from (1) primigravid CBA/J x DBA/2 resorption-prone matings (RES); (2) second CBA/J x DBA/2 matings (SEC); and (3) primigravid CBA/J x BALB/c control matings (CON). We detected a significant decrease in the mean concentration of LTB4 in RES fetoplacental units (176.4 +/- 11.8 pg/ml; n = 42) compared with CON and SEC fetoplacental units (570.2 +/- 45.5 pg/ml; n = 21 and 420.2 +/- 59.5 pg/ml; n = 39, respectively). To confirm that the LTB4 deficiency is associated with decreased NK suppression in RES matings, we supplemented RES extracts, in vitro, with exogenous LTB4 (0-500 pg/ml). The effect of the addition of LTB4 to RES extracts was biphasic. Addition of LTB4 in the range of 30-125 pg/ml increased the extract's NK suppressive capacity, whereas LTB4 alone either stimulated NK activity or was without effect. These results suggest a critical role for LTB4 in averting NK-mediated early spontaneous fetal resorption.


[The melanotic neuroectodermal tumor of childhood. Tumor identification with immunohistochemical methods. A case report]. The authors report a case of a melanotic neuroectodermal tumor of infancy occurring in the maxilla of a 6-month-old female infant. The tumor displayed a biphasic cellular pattern with small neuroblast-like cells and large melanocytic cells showing a highly characteristic immunophenotype. Both tumor cell types immunostained for neuron specific enolase (NSE) and vimentin. The melanocytoid cells reacted additionally with the anti-keratin antibody Kl 1 and with HMB 45, an antibody specific for melanocytic antigens. The melanotic neuroectodermal tumor of infancy usually runs a benign course, however, local recurrence and metastasis may occur. The course of the disease cannot be predicted from morphological findings.


A sequence-dependent 1H-NMR study on the formation of beta-turns in tetrapeptides containing charged residues. The importance of side-chain charge interactions in the formation of beta-turns was studied. Sixteen protected NAc-tetrapeptide amides were studied, namely the variants of DEKS: NEKS, EEKS, DDKS, DQKS, NQKS, DERS, NERS, EERS, DDRS, NDRS, DQRS, and DKES. Three tetrapeptides--NPDM, NSDM, and NDDS--were also studied as they have a high probability of forming beta-turns, based on statistical predictions. The results indicate that a small proportion of type I beta-turn exists in solutions of DEKS and DERS in methanol/water (60/40), while NEKS has an even smaller population of this turn. The other tetrapeptides are present in solution only in the extended conformation. These results clearly show the importance of the salt bridge between the side chains of K2 and E3 or R2 and E3, as well as the importance of the charge on the side chain of the first residue in stabilizing the beta-turn. The relevance of statistical predictions for beta-turns in short peptides is discussed.


Studies in feline long-term marrow culture: hematopoiesis on normal and feline leukemia virus infected stromal cells. To study the effects of feline leukemia virus (FeLV) on the hematopoietic microenvironment, a two-step feline long-term marrow culture (LTMC) system was developed and characterized. The adherent, stromal layer of these cultures is composed of fibroblastoid cells (50% to 80%), macrophages (10% to 30%), fat cells (10% to 20%), and large, polygonal cells that express muscle actin (1% to 2%). When fresh, enriched marrow mononuclear cells (MMNC) were added to 3-week-old irradiated stromal cultures, nonadherent erythroid progenitors (BFU-E) and granulocyte/macrophage progenitors (CFU-GM) could be detected for up to 5 and 12 weeks, respectively. LTMC stromal layers established from marrow cells from cats viremic with either a nonpathogenic strain of FeLV (FeLV-A/61E) or the anemogenic strain FeLV-C/Sarma were morphologically equivalent to uninfected LTMC stromal layers, although more than 80% of the stromal cells expressed FeLV gag protein. When FeLV-infected stromal cultures were recharged with uninfected MMNC, altered patterns of hematopoiesis were observed, compared with recharged, uninfected stromal cultures. In cultures with infected stroma, fewer nonadherent cells (NAC), nonadherent BFU-E, and nonadherent CFU-GM were detected during the first 4 to 5 weeks after recharge. In contrast, greater numbers of NAC and nonadherent CFU-GM were found from weeks 5 to 12 after recharge. When FeLV-infected stromal cultures were recharged with MMNC from a cat heterozygous for the X-chromosome-linked enzyme glucose-6-phosphate dehydrogenase (G-6-PD), the percentage of nonadherent CFU-GM expressing the domestic type G-6-PD isoenzyme remained stable over time (mean % domestic [%d], 53% +/- 3%), and was equivalent to that of nonadherent CFU-GM maintained in uninfected cultures (mean %d, 56% +/- 3%), indicating that clonal drift or clonal selection was not responsible for the enhanced maintenance of CFU-GM. Furthermore, as only 10% to 20% of recharged hematopoietic cells became infected with FeLV in vitro, it is unlikely that the altered pattern was due to progenitor infection. We hypothesize that the increase in NAC and nonadherent CFU-GM in FeLV-infected cultures resulted from enhanced growth factor production by stromal cells. The two-step LTMC system may facilitate the characterization of stromal-derived factors that affect progenitor cell engraftment and proliferation.


Rapid ultrastructural changes in the dense tubular system following platelet activation. The dense tubular system (DTS) functions to regulate platelet activation by sequestering or releasing calcium, similar to the sarcotubules of skeletal muscle. In resting platelets, the DTS exists as thin elongated membranes. Within 10 seconds of the addition of thrombin, platelets show a major ultrastructural change in their DTS: from the thin elongated form to a rounded vesicular form. These morphologic changes were demonstrated with two different stains and two different fixation methods. Platelets exposed to the calcium ionophore A23187 showed the same ultrastructural changes in the DTS. In contrast, the DTS remains in a thin elongated form when platelets are stimulated by the protein kinase C activators phorbol 12-myristate 13-acetate (PMA) and oleoylacetylglycerol (OAG). These morphologic changes may be related to the discharge of calcium from the DTS because this is stimulated by thrombin and A23187, but not by PMA. Preincubation of the platelets with the intracellular calcium chelator 5,5'-dimethyl-bis-(0-aminophenoxy)-ethane-N,N,N',N tetra acetic acid (BAPTA) largely prevented both the thrombin-induced rise in intracellular calcium and the changes in DTS morphology, suggesting that the changes in DTS morphology are secondary to the increase in cytosolic calcium. The results provide a morphologic correlate to existing biochemical evidence showing that the DTS is involved early during paltelet activation.


Interferon-gamma gene expression in human B-cell lines: induction by interleukin-2, protein kinase C activators, and possible effect of hypomethylation on gene regulation. Human interferon-gamma (IFN-gamma) is an important immunomodulatory protein produced predominantly by T cells and large granular lymphocytes (LGLs). Whereas large amounts of data have been accumulated regarding IFN gamma gene expression in these two cell types, little information about IFN gamma expression in other cell types exists. In this study, we have analyzed the production of IFN gamma by the Epstein-Barr virus (EBV)-positive B-cell line, JLP(c), derived from a patient with Burkitt's lymphoma, and another human B-cell line, PA682BM-1, which was derived from an acquired immunodeficiency syndrome patient. Southern blot analysis indicates the presence of an Ig heavy chain gene rearrangement, but no rearrangement of the T-cell receptor beta chain gene or IFN gamma gene in these B-cell lines. Both cell lines were found to express surface IgD and other B-cell surface markers, thus confirming their B-cell lineage. Analysis for surface Ig, cytoplasmic Ig, and secreted Ig indicates that the two cell lines are in relatively early stages of the B-cell differentiation pathway. We now report that PA682BM-1 can be triggered by the protein kinase C (PKC) activators, phorbol 12-myristate 13-acetate (PMA) and (-)Indolactam-v, to secrete IFN gamma, whereas JLP(c) cells spontaneously produce low levels of IFN gamma that can be enhanced by PKC activators and interleukin-2 (IL-2). After activation of the cell lines with IL-2, (-)Indolactam-v, and PMA, increases in cytoplasmic messenger RNAs (mRNAs) of IFN gamma and the IL-2 receptor chains were also observed. The induction of IFN gamma mRNA and protein by IL-2 was completely blocked by a monoclonal antibody to IL-2 receptor p75 (beta chain), but not by the monoclonal antibody to p55 (alpha chain). Analysis of IFN gamma genomic DNA indicates that the gene is not amplified, but that hypomethylation in the 5' noncoding region of the IFN gamma gene has occurred in the B-cell line from the Burkitt's lymphoma patient that spontaneously produces IFN gamma. This finding suggests that the methylation state of the promoter region may play an important role in the control of IFN gamma gene expression in B cells.


Simultaneous mobilization of Mac-1 (CD11b/CD18) and formyl peptide chemoattractant receptors in human neutrophils. Mobilization of a distinct subset of specific granules provides a physiologically important mechanism to recruit Mac-1 (CD11b/CD18) from an intracellular pool to the external surface of the neutrophil plasma membrane, where the functionally active heterodimer mediates several adherence-dependent processes that are crucial for adequate host defense and cellular inflammatory responses. We observed similar characteristics for translocation of Mac-1 and neutrophil formyl peptide receptors (FPR) and hypothesize that the readily accessible pools of both Mac-1 and FPR are colocalized within this specific granule subset. Plasma membrane levels of both FPR (assessed with 3H-FMLP) and Mac-1 (assessed by fluorescence-activated cell sorter analysis of fluorescein isothiocyanate [FITC]-Mo-1-labeled cells) were markedly downregulated in cells prepared at low temperature from blood cooled to 4 degrees C immediately after removal from the circulation. Levels of both FPR and Mac-1 remained low on cells held at 4 degrees C. Upon warming, spontaneous upregulation of Mac-1 and FPR occurred with similar kinetics and temperature dependency. Translocation of both Mac-1 and FPR was markedly potentiated by exposure of cells to either fluoride ion (which has been shown by others to specifically elicit exocytosis of gelatinase-rich and vitamin B-12 binding protein-poor granules) or granulocyte-macrophage colony-stimulating factor (GM-CSF), a cytokine that markedly potentiates the neutrophils' host defense capabilities. Levels of both FPR and Mac-1 on F-- or GM-CSF-treated neutrophils exceeded those present on cells incubated at 37 degrees C for extended time intervals, indicating that stimulated translocation may involve mobilization of an additional granule subset. Scatchard analysis showed that only low-affinity FPR were translocated during spontaneous and stimulus-dependent upregulation. To directly compare FPR levels on the surface of cells displaying varying levels of Mac-1 within a single cell suspension, cells were labeled with FITC-Mo-1 and sorted into subpopulations based on fluorescence intensity. After sorting, the individual populations were held at 4 degrees C to prevent further spontaneous upregulation, concentrated by centrifugation, and assayed for FPR levels. Under a variety of conditions, FPR levels correlated with Mac-1 (CD11b) expression on cell populations selected on the basis of CD11b fluorescence intensity. Analysis of subcellular fractions obtained from disrupted neutrophils before and after upregulation provided additional support for the hypothesis that Mac-1 and FPR are colocalized within a readily accessible subset of neutrophil granules.(ABSTRACT TRUNCATED AT 400 WORDS)


Scaffold-associated regions: cis-acting determinants of chromatin structural loops and functional domains. It has been proposed that scaffold-associated regions are DNA elements that form the bases of chromatin loops in eukaryotic cells. Recent evidence supports a role for these elements as cis-acting 'handlers' of both structural and functional chromatin domains.


Diversity of axonal growth-promoting receptors and regulation of their function. Growth-promoting receptors for substrate-bound molecules are usually found to belong to the integrin, immunoglobulin, or cadherin families of glycoproteins. New members of each of these families have been identified in the past year, and advances have been made in our understanding of their functional regulation.


Agrin and the organization of the neuromuscular junction. Agrin is a component of the synaptic extracellular matrix and may regulate the organization of acetylcholine receptors and other synaptic molecules in both synapse regeneration and development. Analyses of cDNAs encoding agrin define a number of structural domains, including regions of homology to laminin, Kazal protease inhibitors, and epidermal growth factor repeats.


Determination of enalapril and its active metabolite enalaprilat in plasma and urine by gas chromatography/mass spectrometry. The method for the simultaneous determination of angiotensin-converting enzyme (ACE) inhibitor enalapril and its active metabolite enalaprilat in plasma and urine was developed by gas chromatography/mass spectrometry. Enalapril and enalaprilat in plasma and urine were extracted and cleaned up by using Sep-Pak C18 and silica cartridges. Derivatization was carried out using diazomethane and trifluoroacetic anhydride. Detection by selected ion monitoring was selected to m/z 288 (enalaprilat) and 302 (enalapril). The detection limit of enalapril and enalaprilat was 200 pg/mL in plasma and 2 ng/mL in urine. This method was applied to the pharmacokinetic analysis of enalapril and enalaprilat in body fluids.


Pallidal substrate of morphine-induced locomotion. Bilateral microinjections of morphine hydrochloride (5.0; 7.5; 10.0 micrograms/0.5 microliters/side) or saline were infused into 3 different regions (dorsal, medial, ventral) of the rat globus pallidus, to examine their effects on locomotor activity. Locomotor activity of each rat was measured 45 min before and 90 min after saline or morphine pallidal microinjections. Morphine induced a dose-dependent increase in locomotion. This increase in locomotion was also significantly different between the 3 pallidal regions. Pretreatment with naloxone (1 mg/kg, sc) inhibited the morphine (7.5 micrograms) hyperlocomotion elicited from all three pallidal areas. The results suggest that the entire pallidum serves as substrate of morphine hyperlocomotion mediated by opiate receptors.


Zn-ligation in inorganic deposits in the wall of the human aorta determined by X-ray absorption spectroscopy. X-ray absorption spectra were recorded at the K edge of Zn from inorganic deposits present in the human aortic wall. The interatomic distances and coordination numbers obtained indicate that Zn atoms probably occupy selected Ca positions in the lattice of the octacalcium phosphate crystals localized at the surface of the deposit.


Gastric adenocarcinoma presenting as an eyelid and conjunctival mass. A 50-year-old white man complained of "inflammation" of his right eyelid since January 1989. In June 1990, he had undergone a gastrectomy for an adenocarcinoma of the stomach. In August 1990, his right eye showed a painless, firm infiltration of the upper and lower lid, ulceration and loss of eyelashes of the upper lid and a reddish, subepithelial thickening of the medial bulbar conjunctiva. Biopsies of the right upper lid and nasal bulbar conjunctiva disclosed a metastatic, poorly differentiated adenocarcinoma of the stomach (signet ring cell carcinoma) both in the lid and conjunctival biopsies. Carcinoembryonic antigen (CEA) was detected in the tumor cells. Electron microscopic examination revealed tumor cells with signs of secretory activity. Although metastases to the eyelids and conjunctiva are rare, they may precede the clinical manifestation of the primary tumor for months or even years. In patients with chronic, recalcitrant lesions of eyelids or conjunctiva, especially if accompanied by loss of eyelashes, a primary or secondary malignancy should be excluded by early biopsies and histopathological examination.


Paralytic poliomyelitis in England and Wales, 1985-91. To ascertain all cases of paralytic poliomyelitis in England and Wales during 1985-91 and to determine the source of infection in each case. Descriptive study of cases reported between 1985 and 1991. All health districts in England and Wales. Patients normally resident in England and Wales whose clinical features were consistent with paralytic poliomyelitis or with laboratory evidence of recent poliovirus infection and compatible symptoms. Clinical, epidemiological, and laboratory features in identified cases. Of 54 suspected cases of poliomyelitis, 33 were excluded, leaving 21 cases, of which 13 were vaccine associated (nine recipient and four contact) cases, five were imported cases, and three were cases whose source of infection was unknown. No cases due to indigenous wild polioviruses were identified; two were imported cases due to wild viruses. One patient died during the acute phase of the illness, and two children with previously unrecognised severe congenital immune deficiency died between one and two months after the onset of paralysis after the first or second dose of oral polio vaccine. The estimated risk of vaccine associated paralysis is 1.46 per million for the first dose, 0.49 for the second, zero for the third and fourth doses, and 0.33 for the fifth. Indigenous wild poliovirus seems to have been eradicated, although wild virus may be imported; improved surveillance of suspected cases including immediate notification and characterisation of the virus to ensure that eradication is maintained is essential.


Prenatal stress alters brain catecholaminergic activity and potentiates stress-induced behavior in adult rats. Previous studies demonstrated that throughout the preweaning period prenatally stressed rats have an overactive hypothalamic-pituitary-adrenal (HPA) system. This increased HPA activity was accompanied by an increase in defensive behavior. This study examined whether these alterations in HPA activity and defensive behavior continued into adulthood. Brain catecholamines in the cerebral cortex and locus coeruleus were also measured in prenatally stressed and control rats. Shock-induced levels of defensive freezing were significantly higher in prenatally stressed rats than in controls. However, plasma ACTH and corticosterone concentrations did not differ between groups either in the basal state or after exposure to foot shock. Concentrations of norepinephrine (NE) in the cerebral cortex and locus coeruleus region were significantly reduced in prenatally stressed rats. In addition, concentrations of NE metabolites were significantly elevated in prenatally stressed rats, suggesting an increased turnover of brain NE. Prenatally stressed rats also had, in the locus coeruleus region, significantly reduced dopamine (DA) levels but elevated concentration of DA metabolites. Results indicate that prenatal stress produces an increased behavioral responsiveness to stress that is evident in early life and continues into adulthood. The early hyperactivity of the HPA system in prenatally stressed rats, however, appears to normalize in adulthood. The increased turnover in brain catecholamines measure in the cerebral cortex and locus coeruleus region of prenatally stressed rats may be associated with the heightened expression of stress-induced behavior.


Phorbol ester enhances excitatory amino acid-induced dopamine release from mesencephalic cell cultures. The hypothesis that protein kinase C activation can modulate excitatory amino acid-induced dopamine release was tested by investigating effects of phorbol esters, direct activators of protein kinase C, on dopamine release stimulated by N-methyl-D-aspartate (NMDA) and non-NMDA sub-types of excitatory amino acid agonists in fetal rat mesencephalic cell cultures. The phorbol ester, 12-O-tetradecanoyl phorbol-13-acetate (TPA), enhanced dopamine release evoked by NMDA, kainate, quisqualate and by K+ depolarization. Release in the presence of NMDA and TPA was completely abolished by the NMDA antagonist, MK-801. TPA enhancement of NMDA-stimulated dopamine release was likely due to protein kinase C activation by the phorbol ester since (1) the NMDA response was enhanced by nanomolar concentrations of TPA, (2) two phorbol esters capable of activating protein kinase C enhanced the NMDA response while an inactive phorbol ester did not, (3) staurosporine, a potent protein kinase C inhibitor, blocked TPA enhancement of the NMDA response. TPA enhancement of NMDA-stimulated dopamine release was not blocked by H8, an inhibitor with high affinity for cyclic nucleotide dependent kinases, while forskolin, a direct activator of adenylate cyclase, had no effect on NMDA-stimulated release, indicating a lack of involvement of cAMP-dependent kinase in the TPA effect. TPA enhanced NMDA-stimulated release both in the presence and absence of Mg2+, indicating that TPA enhancement was not due to reversal of a Mg2+ blockade of the NMDA receptor.(ABSTRACT TRUNCATED AT 250 WORDS)


Regional variations in particulate cyclic AMP dependent-protein kinase binding activity in the gerbil hippocampus following transient forebrain ischemia by [3H]cyclic AMP binding. Changes in the binding of [3H]cyclic AMP as an indicator of particulate cyclic AMP-dependent protein kinase (AMP-DPK) binding activity following transient forebrain ischemia were studied in the gerbil using in vitro autoradiography. [3H]Cyclic AMP binding in the strata pyramidale and lacunosum-moleculare of the hippocampal CA1, the stratum pyramidale of the CA3, and the dentate gyrus decreased transiently in the early postischemic phase but then recovered. However, [3H]cyclic AMP binding in the strata pyramidale and radiatum of the CA1, the granular layer of the dentate gyrus, and the upper layer of the cortex decreased again 7 days after ischemia. In the CA4 subfield and the lower layer of the cortex, the binding showed no significant alterations after ischemia. Administration of pentobarbital prior to the induction of ischemia prevented the decrease in [3H]cyclic AMP binding in the CA1 subfield 6 h and 7 days after ischemia, and showed protective effects against neuronal death of the CA1 pyramidal cells 7 days after ischemia. These results indicate that marked alteration of intracellular signal transduction precedes neuronal damage in the hippocampal CA1 subfield. Furthermore, postischemic reduction of [3H]cyclic AMP binding in the histologically intact cerebral cortex, CA3, and dentate gyrus may be the reflection of cellular dysfunction after energy failure.


Possible role of surface potential in the gating mechanism of Ca2+ channels in cat adrenal chromaffin cells: studies with fura-2 microfluorometry. The cytosolic free Ca2+ concentration ([Ca]in) in isolated cat chromaffin cells was measured by fura-2 microfluorometry. During 30 mM KCl depolarization or sucrose substitution for NaCl, a reduction in external Ca2+ concentration under optimal conditions paradoxically caused a rise in [Ca]in and, in separate experiments, in catecholamine secretion. The results support a previously suggested role of surface potentials in the gating mechanism of Ca2+ channels.


Transneuronal labeling of spinal interneurons and sympathetic preganglionic neurons after pseudorabies virus injections in the rat medial gastrocnemius muscle. The distribution of retrogradely and transneuronally labeled neurons was studied in CNS of rats 4 days after injections of the Bartha strain of pseudorabies virus (PRV) into the medial gastrocnemius (MG) muscle. Tissue sections were processed for immunohistochemical detection of PRV. Retrogradely labeled cells were identified in the ipsilateral MG motor column in the caudal L4 and the L5 spinal segments. In order to evaluate the efficacy of PRV retrograde cell body labeling, the number of PRV retrogradely labeled neurons in the MG motor column was compared to the number labeled with two conventional retrograde cell body markers--Fluoro-Gold and cholera toxin-HRP. A ratio of 1:3 representing medium-sized (less than 30 microns) versus large neurons (greater than 30 microns) was found in the Fluoro-Gold dye experiments; a 1:2 ratio was seen in the PRV experiments. In contrast, when cholera toxin-HRP was used as a retrograde marker, mainly large neurons were labeled; the medium-to-large cell body ratio was 1:10 suggesting cholera toxin-HRP may have a greater affinity for the terminals of alpha-motoneurons as opposed to gamma-motoneurons. Transneuronally labeled cells were identified in the L1-L6 spinal gray matter, intermediolateral cell column (T11-L2), lateral spinal nucleus and medial part of lamina VII in C4 and C5 spinal segments, brainstem (caudal raphe nuclei, rostral ventrolateral medulla, A5 cell group, paralemniscal nucleus, locus coeruleus, subcoeruleus nucleus, red nucleus) and paraventricular hypothalamic nucleus. In the L5 spinal cord, transneuronally labeled neurons were seen in the ipsilateral spinal laminae I and II and bilaterally in spinal laminae IV-VIII, and X. Similar results were obtained in rats that had chronic unilateral L3-L6 dorsal rhizotomies indicating most of the labeling was due to retrograde transneuronal cell body labeling. In order to determine whether PRV was transported into the spinal cord by the dorsal root axons, the ipsilateral dorsal root ganglia (DRGs) were examined for PRV immunoreactivity; none was found. However, using the polymerase chain reaction, viral DNA was shown to be present in the ipsilateral DRGs indicating that some of spinal cord cell body labeling may have resulted from anterograde transneuronal labeling, as well.


Quantitative autoradiographic localization of [125I]neuropeptide Y receptor binding sites in rat spinal cord and the effects of neonatal capsaicin, dorsal rhizotomy and peripheral axotomy. Using in vitro quantitative receptor autoradiography the present study reports on the distribution and possible changes of [125I]neuropeptide Y (NPY) binding sites in the rat spinal cord following neonatal capsaicin treatment, dorsal rhizotomy and sciatic nerve section. In control spinal cord the highest density of [125I]NPY binding sites was noticed in the superficial layers of the dorsal horn whereas low-to-moderate densities of [125I]NPY binding sites were detected in the deeper dorsal horn and in the ventral horn. In comparison with control rats, neonatally treated capsaicin rats showed a significant (P less than 0.001) bilateral decrease in [125I]NPY binding sites in the superficial layers of the dorsal horn. Unilateral dorsal rhizotomy and unilateral sciatic nerve section also exhibited a significant (P less than 0.05) depletion in [125I]NPY labeling in the superficial layers of the dorsal horn ipsilateral to the surgery. These results suggest that a certain proportion of [125I]NPY receptor sites is located on the primary afferent fibers of the superficial layers of the dorsal horn. This peptide thus could play an important role in the modulation of nociceptive transmission by acting directly on primary afferent terminals.


Quantitative autoradiography of hemicholinium-3 binding sites in human amygdala. We report the binding properties and subnuclear localization of [3H]hemicholinium-3 binding sites in human amygdala using quantitative autoradiography. Specific binding was saturable and high affinity (apparent Kd 2-11 nM). Binding was highest in the basolateral nucleus which receives dense cholinergic innervation from the basal forebrain. Binding closely approximated acetylcholinesterase reactivity. These data support [3H]hemicholinium-3 as a quantitative marker for cholinergic terminals in human brain.


Alpha 2-adrenoceptor density in forebrain areas of Zucker rats: no effect of genetic obesity or antiglucocorticoid treatment. The density of alpha 2-adrenoceptors was determined in different forebrain regions of lean and obese Zucker rats by para-[125I]iodoclonidine ([125I]PIC) receptor autoradiography. No significant differences were observed between the Zucker phenotypes in the density of alpha 2-adrenoceptors in any brain areas examined. Four days treatment with the antiglucocorticoid mifepristone (RU 38486) did not affect [125I]PIC-binding. It is concluded that the hyperphagia and abnormal corticosterone secretion of genetically obese Zucker rats do not appear to be associated with changes in alpha 2-adrenoceptor density in brain.


Dopamine inhibits voltage-activated calcium channel currents in rat pars intermedia pituitary cells. Several lines of evidence suggest that dopamine acts as a neurotransmitter that inhibits both hormone secretion and electrical activity in pituitary intermediate cells (melanotrophs). In this study we examined the effects of exogenously applied dopamine on voltage activated calcium currents recorded with the whole-cell mode of the patch-clamp technique from short-term primary cultures of melanotrophs. Two types of calcium currents were distinguished by their voltage dependence and kinetics of inactivation similar to the low voltage-activated currents (LVA; or T-type) and high voltage-activated currents (HVA; N&L-types) of calcium currents. Exogenously applied dopamine (2-20 microM) reversibly reduced both LVA and HVA types of calcium currents. Evidence for these results came from experiments in which LVA and HVA calcium currents were separated by stepping to different membrane potentials from a fixed holding potential (Vh) or by changing Vh. These results suggest that dopamine can regulate the entry of calcium into melanotrophs by acting on at least two different populations of calcium channels thereby affecting hormone secretion and electrical activity.