Monoclonal antibodies to toxin II from the scorpion Androctonus australis Hector: further characterization of epitope specificities and neutralizing capacities. The epitope specificities of two previously prepared monoclonal antibodies (mAb) to the toxin II from Androctonus australis Hector were characterized. Neither mAb 4C1 nor mAb 3C5 was able to recognize any of the 58 overlapping synthetic heptapeptides which cover the whole sequence of toxin II. Thus, both mAbs probably recognize conformation-dependent epitopes at the surface of the toxin. Experiments were designed to check whether or not the two mAbs, or their Fab fragments, were able to bind simultaneously to the toxin. The results indicated that the epitopes recognized by the two antibodies are probably close together at the surface of the toxin, thus preventing the simultaneous binding of both mAbs to a single toxin molecule. Given the proximity of the two epitopes and the fact that mAb 4C1 is known to be a neutralizing antibody, the capacity of mAb 3C5 to inhibit the toxic effects of the toxin was re-evaluated in C57BL/6 mice. A clear, but weak, neutralizing effect was found, consistent with the low affinity binding of the mAb in the proximity of a neutralizing site of the toxin.


Glutamate receptor channels: novel properties and new clones. Glutamate is the principal excitatory neurotransmitter in the brain. Glutamate activates cation-selective receptor channels carried by nearly every neuron and by glial cells. Bernd Sommer and Peter Seeburg describe how our concepts concerning the molecular and functional design of ionotropic glutamate receptors are rapidly progressing, with the recent discovery of novel receptor properties and new subunits following the landmark cloning of the first receptor subunit by M. Hollmann and his colleagues. New properties currently revealed by the cloned receptor channels may guide physiologists in characterizing the elementary steps in synaptic transmission, help neurologists to define the role of glutamate receptors in acute and chronic neuropathologies, and enlighten all neuroscientists whose models for learning and memory involve the idiosyncracies of particular channel subtypes.


[Beta-HCG-producing ovarian dysgerminoma]. A patient with stage 2C pure dysgerminoma which produced free beta-HCG subunit is described. The importance of excluding elements of other germ cell tumors is stressed. HCG-positive dysgerminomas should be treated like other pure dysgerminomas, and beta-HCG will in this case be a useful monitor of the effect of treatment.


[Palliative treatment of rectal and sigmoid cancer with Nd-YAG laser]. Nd-YAG laser treatment was employed as palliative treatment in 47 patients with colorectal cancer. Thirty-eight patients (81%) experienced good symptomatic effect from the treatment. Four complications occurred: three cases of perforation and one case of moderate bleeding. There was no mortality. It is concluded that laser treatment is a good palliative method in patients with colorectal cancer, especially in elderly patients with complicating diseases and in patients with local recurrence or symptoms from nonresected tumours.


The effect of acute distension on vasoactive intestinal polypeptide (VIP), neuropeptide Y (NPY) and substance P (SP) immunoreactive nerves in the female rat urinary bladder. The effect of acute distension on vasoactive polypeptide (VIP)-, neuropeptide Y (NPY)- and substance P (SP)-immunoreactive nerves in the wall of the urinary bladder was investigated. At the age of 3 months, 25 female albino rats underwent forced diuresis combined with balloon obstruction to achieve maximal distension for 3 h. A modified, indirect immunofluorescence detection method was applied 2 days, 7 days and 21 days after distension. A marked, extensive depletion of VIP, NPY- and SP-immunoreactive nerves was observed after distension. This disturbance was reversible, and increased fluorescence of VIP-, NPY- and SP-immunoreactive nerve fibres compared with control specimens was seen in bladder specimens taken even as soon as 21 days after distension. This transient depletion of peptidergic innervation may partly explain the prolonged voiding problems that often occur after acute urinary retention. The depletion of sensory nerves containing SP shortly after distension may explain the transient benefit obtained from distension therapy in patients with painful bladder disease. It is suggested that the increased SP activity during the recovery phase may be related to neurogenic inflammation.


Androgen concentrations in expressed prostatic secretions: no correlation with tissue levels. In an attempt to determine whether the androgen profiles of the prostate fluid (EPS) mirror the concentrations in the prostate tissue, we have measured testosterone and dihydrotestosterone (DHT) in EPS and correlated their levels to the concentrations found in hyperplastic prostate tissue (BPH) obtained from the same 19 patients. Although androgen concentrations in EPS were very high (testosterone = 106.7 +/- 81.9 ng/g dry weight; DHT = 54.2 +/- 11.2 ng/g dry weight), they did not reflect the concentrations measured in BPH tissue (testosterone = 7.56 +/- 1.54 ng/g dry weight; DHT = 10.54 +/- 0.63 ng/g dry weight). Additionally the accumulation of tissular DHT usually associated with BPH was not mirrored in the EPS specimens which demonstrated a relatively higher concentration of testosterone when compared to DHT. In EPS, there was also a very strong correlation between testosterone and DHT concentrations (r = 0.89; P less than 0.01). We have also measured zinc concentrations in EPS and BPH tissue but were unable to detect any relationship between the two parameters. Significantly, however, EPS zinc concentrations showed a close correlation with EPS testosterone (r = 0.73; P less than 0.01) and DHT (r = 0.69; P less than 0.01) concentrations.


Modulation of in vitro cell growth of human and murine urothelial tumor cell lines under the influence of interleukin-3, granulocyte-, macrophage- and granulocyte-colony-stimulating factor. To elucidate possible growth-modulating effects of interleukin 3 (IL-3), granulocyte-macrophage-colony-stimulating factor (GM-CSF) and granulocyte-colony-stimulating factor (G-CSF), human transitional cell carcinoma (TCC) cell lines T24, RT112, EJ and 647 V were solitarily and continuously exposed to these hematopoietic growth factors at concentrations of 1-100 ng/ml. The murine line MBT-2 was used as a negative and the colon carcinoma cell line HTB38 as a positive control, because of species specificity and known proliferation in response to growth factors, respectively. In the T24 TCC-line solitary and continuous exposure to IL-3, GM-CSF and G-CSF at the highest concentration of 100 ng/ml led to a significant proliferation of cell growth in vitro. Significant proliferation in the RT112 line was only achieved with continuous exposure to IL-3 and GM-CSF (100 ng/ml); G-CSF failed to induce growth modulation in the RT112 line. No significant proliferative effect of any of cytokines administered was observed in the 647V line. Exposure of the EJ line to cytokines at the highest activity levels had a proliferative effect only in suboptimal growth conditions.


Cytological detection of copper for the diagnosis of inherited copper toxicosis in Bedlington terriers. The reliability of a cytological examination of impression smears of the liver was investigated in 89 Bedlington terriers by using the rubeanic acid staining method for copper and comparing the results with the results of a histological examination. Histological examination revealed copper accumulation in the liver of 24 dogs. The cytological method had a sensitivity of 0.96 for detecting copper toxicosis and a specificity of 1.0. When a grading system for the amount of copper was applied, the coefficient of correlation (r) between the cytological and histological techniques was 0.917 (P less than 0.0001). It was concluded that a cytological examination of impression smears stained with rubeanic acid for copper offers a rapid and reliable method for the detection of copper toxicosis in Bedlington terriers.


[Side effects of administration of gamma globulin preparations in patients with primary agammaglobulinemia]. Eight patients with adverse reactions were observed in a group of 20 primary agammaglobulinaemic patients treated with gammaglobulin derivatives. In four cases only sporadic reactions were observed, but in 2 cases repeated reactions made gammaglobulin therapy impossible. In the remaining two patients a change of gammaglobulin derivatives made adequate therapy possible. Possible causes of adverse reactions during gammaglobulin treatment and their prevention are discussed.


Lung cancer mortality in Wisconsin: year 2001. Lung cancer is the major cause of cancer mortality in Wisconsin, with an estimated 2,300 deaths in 1991. The low 5-year survival rate of about 10% in lung cancer is primarily attributed to advanced inoperable stage at diagnosis, limited response to therapy, and a high incidence of metastatic spread. Since smoking is the major etiologic factor associated with lung cancer, this is a largely preventable disease. Many current smokers may already have severe bronchopulmonary dysplasia or neoplastic changes that may manifest many years later. We report on a model developed to evaluate the effect of improved therapy for lung cancer patients in the next decade. Based on this model, Wisconsin's projected lung cancer mortality is estimated at 2,117 in the year 2001, without improved treatment, resulting in nearly 25,000 "years of potential life lost." According to this model, a 10% reduction in lung cancer mortality plus a 12-month increase in palliation in the year 2001 will "regain" more years of potential life lost than the current mortality for stomach, cervical, or uterine cancer. Thus, improved cure and palliation for lung cancer patients represents an important goal for Wisconsin's medical community.


[Serum cholinesterases as activity parameters in chronic inflammatory bowel diseases]. Significantly decreased levels of serumcholinesterase (CHE) were found in acute Crohn's disease (= CD) (3.2 +/- 1.0 KU/L) and acute ulcerative colitis (= UC) (3.54 +/- 1.6 KU/L) as compared to patients with mild or quiescient disease (CD: 5.5 +/- 1.1 KU/L; UC: 5.59 +/- 0.94 KU/L) and healthy controls (5.69 +/- 1.3 KU/L). Suppression of CHE was most evident in Crohn's colitis (2.98 +/- 1.0 KU/L) and extensive UC (2.96 +/- 1.28 KU/L). Intraindividual comparison showed an increase of CHE-levels during treatment with steroids and salicylates. There was no significant correlation to the reduced bodyweight-levels in severe IBD. Best correlations were seen between CHE/albumin (CD: r = +0.61; UC: r = +0.73) and CHE/hematocrit (CD: r = +0.50; UC: r = +0.61) in severe inflammatory bowel disease. The results of a discriminant analysis showed that CHE-levels can predict the degree of activity correctly in the majority of patients with CD and UC. It is suggested that the decrease of serumcholinesterase reflects an inhibition of liver synthesis as an acute phase response-induced by endotoxins and cytokines.


Occurrence of keratinolytic fungi and related dermatophytes in soils in Cairo, Egypt. 120 soil samples collected from various sites of Cairo were processed for the isolation of keratinophilic fungi by "ToKaVa" hair baiting technique. 22 species belonging to 6 genera were isolated viz.: Chrysosporium tropicum, C. indicum, C. keratinophilum, C. queenslandicum, C. merdarium, C. anamorph of Arthroderma curreyi, C. pannicola, C. lobatum, C. anamorph of Renispora flavissima, C. pseudomerdarium, Microascus mangini, Malbranchea gypsea, Ml. State of Uncicarpus reesii, Ml. State of Coccidioides immitis, Microsporum gypseum, Mr. distortum Mr. audouinii, Mr. fulvum, Trichophyton mentagrophytes, T. terrestre, T. verrucosum and Epidermophyton floccosum. The frequency of occurrence of the isolated fungi was determined. Microsporum gypseum, Chrysosporium tropicum and Chrysosporium indicum were the most frequent species recovered from soil. Most species of keratinophilic fungi were isolated from university, public garden and zoo garden. The distribution of the isolates are discussed.


[Immunobiological properties and therapeutic effectiveness of preparations from Klebsiella bacteriophages]. The purified preparations of Klebsiella bacteriophages, viz. the monovalent preparation of K. pneumoniae bacteriophage and the polyvalent bacteriophage preparation for the treatment of infections caused by K. ozaenae, K. rhinoscleromatis scleromatis and K. pneumoniae sensu lato, have been obtained. The bacteriophage preparations have proved to be nontoxic and safe for laboratory animals after the intraperitoneal injection of these preparations followed by the pathomorphological study of the internal organs of the animals. The clinical study of the newly developed bacteriophage preparations in the course of the treatment of purulent inflammatory diseases in 109 patients has revealed that the preparations are not reactogenic and exhibit sufficient effectiveness in the therapy of ozena, rhinoscleroma and Klebsiella infections with different localization of the infectious process.


[Value of prostate-specific antigen as prognostic factor of metastatic cancer of the prostate]. This paper examines the behaviour of PSA in 70 patients with metastatic prostate cancer. PSA serum concentration, prior to therapy, was directly related to the tumoral mass and inversely related to the histological degree, which does not constitute a prognostic factor with regard to the disease evolution. Within 3 months of therapy, PSA concentration decreased more noticeably in clinically responding patients, down to less than 10 ng/ml in 93% of them. Also, this level represented a prognostic factor with regard to the free-of-progression interval of the disease.


[Medical treatment of benign prostatic hyperplasia in patients with high surgical risk]. Eight patients with prostate adenoma and high surgical risk were given leuprolide in depot. Prostate volume decreased by 44.7% after 3 months of treatment and by 58.9% at six months. Two of the 7 patients were resident vesical catheter carriers, 28% re-started spontaneous miction.


Taurine and niacin offer a novel therapeutic modality in prevention of chemically-induced pulmonary fibrosis in hamsters. The bleomycin (BL)-hamster model of interstitial pulmonary fibrosis (IPF) is generally associated with increased lung lipid peroxidation, measured as malondialdehyde equivalent (MDAE), calcium and collagen content; and superoxide dismutase (SOD), prolyl hydroxylase (PH) and poly(ADP-ribose) polymerase activities. We found that combined treatment with taurine in drinking water (1%) and niacin IP (250 mg/kg) daily, significantly decreased the BL-induced increases in lung MDAE and calcium content, and SOD, PH and poly(ADP-ribose) polymerase activities. This treatment almost completely ameliorated the BL-induced increases in the lung collagen accumulation as well. Findings of a similar nature were also demonstrated when taurine (2.5%) and niacin (2.5%) were supplemented in the diet of hamsters used in the same BL model of IPF. The diet supplemented with taurine (2.5%), niacin (2.5%), or taurine (2.5%) + niacin (2.5%) also reduced AD-induced increases in lung collagen accumulation, phospholipids, MDAE and SOD activity. It was concluded that diet supplemented with taurine and/or niacin would completely or partially ameliorate chemically-induced pulmonary fibrosis.


The short term effect of peripherally administered brain-gut peptides on gastric acid secretion in rats. Certain brain gut-peptides are known to either stimulate or inhibit gastric acid secretion in several species after direct injection into the central nervous system. However there is inconsistency of published results on the gastric acid secretory response to some of these peptides after peripheral administration in different experimental systems. Seven peptides, namely neurotensin (NT), substance P, cholecystokinin (CCK), thyrotropin releasing hormone (TRH), human calcitonin (hCT), rat calcitonin-gene-related peptide (rCGRP) and bombesin, all known to modulate gastric acid secretion after central administration, were initially screened for activity after peripheral (subcutaneous) injection of 10 micrograms/kg body weight in a single rat model. Peptides showing an effect were retested at lower doses. Despite the inherent variability of the gastric acid secretory response in the non-anaesthetized pylorus ligated rat, a standardized experimental design confirmed that reproducible and statistically valid results could be obtained. The technical feasibility of using a one hour collection period as might be appropriate for short acting peptides was demonstrated by the significant dose dependent inhibitory activity of salmon calcitonin. In this model, NT and substance P had no significant effect on either volume or concentration of acid secreted, CCK showed a slight stimulation of acid output, and TRH, hCT, rCGRP and bombesin all inhibited acid output; CGRP and bombesin were active at 10 and 100-fold lower doses. The potent and inhibitory activity of bombesin in this system is in disagreement with other publications reporting no effect or variable stimulatory effect in rats. Time and dose dependent responses in our rat system indicate that this apparent discrepancy may be explained by the short duration of action of bombesin.


Formation of histamine-releasing activity from albumin by medium conditioned by endotoxin-stimulated rat peritoneal macrophages. Incubation of bovine serum albumin (BSA), rat serum albumin or rat plasma with medium conditioned by endotoxin stimulated rat peritoneal macrophages produced an activity that released histamine from isolated rat serosal mast cells. The amount of histamine-releasing activity (HRA) produced increased with the length of the incubation period, with the concentration of albumin, with the number of macrophages stimulated, and with the duration of exposure of the macrophages to endotoxin. Moreover, the formation of the HRA showed a dependency on the pH of the incubation medium with an optimum at pH 4.5. Boiling the medium conditioned by stimulated macrophages before its incubation with albumin or including the acid protease inhibitor, pepstatin with the conditioned medium prevented the formation of HRA. The generation of HRA was not inhibited by pretreatment of the macrophages with the inhibitor of protein synthesis, cycloheximide. Media from macrophages not stimulated with endotoxin failed to generate HRA. Histamine release from mast cells in response to the HRA was inhibited by pretreatment of the cells with antimycin A and deoxyglucose or by preincubation in Ca-free Locke's solution containing a calcium chelating agent. When injected intradermally into anesthetized Evan's Blue treated rats, the generated HRA produced a change in vascular permeability that was prevented by the H1 antagonist, diphenhydramine. Treatment of the HRA with carboxypeptidase A reduced its ability to stimulate histamine release from mast cells. Histamine-Releasing Peptide (HRP), a neurotensin-related octapeptide, shown previously by us to be formed by the action of cathepsin D or pepsin on albumin, was identified by radioimmunoassay in acid:acetone extracts of the histamine-releasing activity. It is concluded that the formation of HRA is due to the actions of enzymes released from macrophages acting on albumin. It is suggested that such histamine-releasing activity could be formed during the later stages of the inflammatory response and that HRP is one of the peptides present.


The efficacy of cyclosporin A, FK-506 and prednisolone to modify the adoptive transfer of experimental allergic encephalomyelitis (EAE). The in vitro potency of the immunosuppressants Cyclosporin A (CsA), FK-506 and Prednisolone was assessed using the adoptive transfer model of EAE in the Lewis rat. Co-culture of encephalitogen-sensitised splenic leukocytes with Prednisolone did not inhibit the transfer of disease to naive histocompatible recipients despite significant suppression of neuroantigen-stimulated leukocyte proliferation by the drug. The addition of CsA (100 nM) to cultures inhibited the induction of adoptive EAE but a lower dose of the agent (10 nM) did not prevent the development of clinico-histopathological signs of disease. FK-506 (1 nM) was 100 times more effective than CsA at suppressing adoptive EAE thus emphasising the usefulness of the model in determining the relative efficacy of compounds to modify cell-dependent autoimmune disease.


Distribution of CA 125 in adenocarcinomas. An immunohistochemical study of 481 cases. To determine the distribution of CA 125 in adenocarcinomas, formalin-fixed, paraffin-embedded tissue from 481 cases of adenocarcinoma from a variety of primary sites were studied using a monoclonal antibody to CA 125 (B27.1) and an avidin-biotin immunohistochemical technique. Positive reactivity was most common in adenocarcinomas of the endocervix, ovary, and endometrium (61% to 94%). However, relatively frequent positive reactions also were seen in adenocarcinomas of the pancreas (48%) and bile ducts (56%). Adenocarcinomas of the breast, lung, thyroid, distal esophagus/stomach, and liver (hepatocellular carcinoma) showed positive reactions in 7% to 20% of cases. Staining of rare tumor cells was seen in 2 of 45 colonic adenocarcinomas and in 1 of 61 prostatic adenocarcinomas. No reactivity was seen in the 54 renal cell carcinomas studied. Although CA 125 is most commonly present in gynecologic adenocarcinomas, it is also produced by some adenocarcinomas from many other sites. Immunostaining for CA 125 may be helpful in ruling out renal cell carcinoma in certain clinical settings.


A rare lymphoepithelial cyst of the pancreas. Reported is a case of a rare lymphoepithelial cyst of the tail of the pancreas that developed in a young man with no symptoms. To the authors' knowledge, it is the first case described to be entirely situated within the pancreas, thus confirming its pancreatic origin. Biologic behavior appears to be entirely benign, and the primary importance of its recognition is in the distinction from cystic malignant neoplasms.


The heart in Tangier disease. Severe coronary atherosclerosis with near absence of high-density lipoprotein cholesterol. Cardiac necropsy findings are described in a 72-year-old man with Tangier disease whose plasma total cholesterol levels averaged 70 mg/dL, low-density lipoprotein cholesterol level was 45 mg/dL, and high-density lipoprotein cholesterol level was 1.4 mg/dL, and who had coronary artery bypass grafting for severe atherosclerotic coronary artery disease. At necropsy, 24 of the 72 (33%) 5-mm segments of the 4 major (right, left main, left anterior descending, and left circumflex) native coronary arteries and 4 of the 27 (15%) 5-mm segments of the saphenous vein aortocoronary bypass conduits were narrowed by more than 75% in cross-sectional area by atherosclerotic plaques. The plaques were composed primarily (91% to 97%) of fibrous tissue. Oil red O staining, polarized light microscopy, and electron microscopy revealed cholesterol deposits in the plaques and in the walls of coronary arteries, saphenous vein grafts, and aorta. Such deposits also were found in foam cells of histiocytic origin, fibroblasts in all four cardiac valves, and in Schwann cells of cardiac nerves.


[Substance P: immuno-allergic implications]. Substance P is a decapeptide which forms part of the group known as neuropeptides, that is, peptides released by some neurones such as the slow-conducting C fibres and the rapid-conducting A-delta fibres. These neurones belong to the category of non-adrenergic non-cholinergic nerves (NANC), which perform their action through a mechanism known as "axonic reflex". This mechanism is an antidromic stimulation which produces a secretion of neuropeptides, especially substance P. It is known that substance P, once released, is able to exert a number of actions including among others inflammatory and bronchospastic activities and a stimulation of the immunologic system. Other effects attributed to this substance are an increase in capillary permeability and oedema and the perpetuation of certain conditions such as asthma, rhinitis and chronic urticaria or hives. The production, metabolism and actions of this neuropeptide are described.


Systemic polyclonal B-immunoblastic proliferation with marked peripheral blood and bone marrow plasmacytosis. The clinical and pathologic features of a case of acute systemic polyclonal B-immunoblastic proliferation characterized by pronounced peripheral blood and bone marrow plasmacytosis and infiltration of the hepatic portal areas by immunoblasts, plasma cells, and lymphocytes are reported. Clinical and laboratory findings during the acute phase and long-term follow-up support the diagnosis of a benign process, possibly related to Pseudomonas aeruginosa septicemia. The patient experienced a dramatic clinical recovery on administration of high-dose intravenous corticosteroids. Pathologists should be aware of this entity so as not to confuse it with non-Hodgkin's lymphoma or a form of plasma cell dyscrasia.


The study cellular subpopulations in peripheral blood from a normal reference group population (blood donors). The spectacular development of monoclonal antibodies against cellular antigens an technology such as flow cytometry allow the investigation of cellular subpopulations that until now have been unknown. At the same time, the functional study of these subpopulations becomes of maximal interest, as this information could have future applications for pathological processes. Due ti this basic need for information, we have studied diverse lymphocytic subpopulations in a normal population that serves as a reference group, using the following antigens: CD3, CD4, CD8, CD20, CD45RA, CD25, LAM1, CD29, CD11b and CD23. Some of these subpopulations had not been previously studied in a normal reference group.


Filtration dynamics and natriuretic response to volume expansion in humans. We used differential solute clearances and a theoretical analysis of glomerular ultrafiltration and dextran sieving to characterize the hemodynamic response of nine healthy humans to infusion of isoncotic, 5% albumin in saline or saline vehicle alone. During albumin infusion (10.2 +/- 0.2 ml.kg-1.30 min-1) plasma volume increased by 18%, but oncotic pressure rose by only 0.8 mmHg. Despite the hypervolemia, renal blood flow (RBF) declined by 140 ml/min and glomerular filtration rate (GFR) declined by 16 ml/min during the infusion. RBF increased progressively postinfusion, exceeding baseline by 135 ml/min after 4 h; GFR was restored to baseline. Although oncotic pressure declined by 2 mmHg, a similar transient decline in GFR (-13 ml/min) was associated also with infusion of saline vehicle alone (9.4 +/- 0.3 ml.kg-1.30 min-1), which increased plasma volume by 9%. Sieving coefficients of dextrans (radius 32-42 A) were lowered during and after either infusion, a phenomenon that we compute to reflect a reduction in glomerular pore size. Assuming that the transcapillary hydraulic pressure difference was not lowered, we calculate that there was a simultaneous depression of the ultrafiltration coefficient (Kf) during volume expansion with saline and possibly also to a lesser extent with albumin. The hypofiltration during either infusion delayed the onset of a natriuretic response until the filtered sodium load was restored to baseline in the postinfusion period. We propose that the net effect of changes in intracapillary pressures and Kf during volume expanding infusions is to transiently lower GFR, thereby preventing the human kidney from mounting an immediate natriuretic response to acute hypervolemia.


Dual action of phosphonoformic acid on Na(+)-phosphate cotransport in opossum kidney cells. Phosphonoformic acid (PFA, foscarnet) was found to exert both an inhibitory and a stimulatory effect on Na(+)-dependent Pi transport in opossum kidney (OK) cells. When added in the uptake media, PFA produced a dose-dependent inhibition of Na(+)-Pi cotransport. PFA had no effect on the Na(+)-dependent transports of methyl-alpha-D-glucopyranoside (AMG) or L-alanine or on amiloride-sensitive Na(+)-H+ antiport. The inhibition of Na(+)-Pi cotransport was competitive [inhibitory constant (Ki) = 6.0 mM], reversible by dilution, and solute specific. When OK cells were incubated with PFA for longer time periods (1-15 h), the Na(+)-Pi uptake measured after removal of PFA was significantly increased, i.e., "upregulated." The extent of Na(+)-Pi cotransport upregulation was dependent on time (greater than or equal to 30 min) and dose of PFA (2-10 mM). The increase in Na(+)-Pi cotransport by upregulation with PFA was due to higher apparent Vmax with no change in apparent Michaelis constant (Km) for Pi and was solute specific: uptakes of AMG or L-proline were not changed. Removal of PFA from culture medium resulted in a fast reversal of upregulation. Upregulation was not inhibited by cycloheximide, actinomycin D, or cordycepin. Solute-specific increase of Na(+)-Pi cotransport was also found when measured in apical membrane vesicles isolated from OK cells. Thus PFA exerts a dual action on the Na(+)-Pi cotransporter of OK cells: 1) acute, competitive inhibition and 2) after prolonged exposure it increases Na(+)-Pi uptake, probably by insertion of Na(+)-Pi cotransporters into apical membrane.


Characteristics of calcium currents in rabbit portal vein myocytes. The properties of voltage-dependent Ca2+ channels were studied in isolated portal vein myocytes using the whole cell voltage-clamp method. Ca2+ currents (ICa) were identified based on their activation and inactivation potential, their dependence on external Ca2+ ([Ca2+]o), their suppression by organic or inorganic Ca2+ channel blockers, their augmentation by BAY K 8644, and their insensitivity to tetrodotoxin or alterations in external Na+ ([Na+]o). Changing the holding potential from -90 to -40 mV decreased ICa from 4.6 +/- 0.6 to 2.0 +/- 0.3 pA/pF at 0 mV but did not shift its voltage dependence significantly. The voltage dependence of steady-state inactivation and activation was represented by Boltzmann distributions with the following parameters: inactivation, half-maximal voltage (V0.5) = -32 +/- 7 mV and slope factor (k) = 6.1 +/- 0.2 mV; activation, V0.5 = -15 +/- 4 mV and k = 5.6 +/- 0.6 mV. Doubling the [Ca2+]o increased ICa and shifted the voltage dependence of its activation and inactivation by approximately 10 mV toward more positive potentials without altering the window currents. Substituting Na+, Ba2+, or Sr2+ for Ca2+ as the charge carrier through the Ca2+ channel slowed the rate of its inactivation and shifted its voltage dependence toward more negative potentials. Divalent selectivity of the Ca2+ channel showed an apparent concentration dependence: at 2 mMISr less than IBa = ICa, while at 10 mM ICa less than ISr = IBa. Because 50-100 microM ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid abolished the apparent concentration dependence of the divalent ion selectivity, this phenomenon was attributed to a high Ca2+ selectivity of the channel. Our data support the presence of only one type of Ca2+ channel in rabbit portal vein myocytes with characteristics similar to the L-type Ca2+ channel described in other cells, but with somewhat different divalent selectivity, holding potential, and [Na+]o dependence.


Familial thrombocytopenia with micromegakaryocytes. Chronic thrombocytopenia was noted in two siblings and a first cousin. The initial impression was of immune thrombocytopenic purpura (ITP) with decreased megakaryocytes. One patient had splenectomy for presumed chronic ITP but showed no improvement. Bone marrow buffy coat slides were examined in the three children with thrombocytopenia, four normal controls, and five children with "classic" acute ITP. Megakaryocyte size, maturation, and ploidy were determined with Wright-Giemsa and Feulgen-stained material. Mean megakaryocyte diameters were 23.1 microns in the three related patients, 30.8 microns in normal controls, and 63.1 microns in children with "classic" acute ITP. Many "micromegakaryocytes" were noted in the three related children with chronic thrombocytopenia. An exhaustive family history was obtained, which showed multiple points of consanguinity. These patients represent an apparently new autosomal recessive disorder of megakaryocytopoiesis, characterized by disturbed megakaryocyte ploidization and maturation. More sensitive recognition of micromegakaryocytes should be attempted in children with atypical chronic thrombocytopenia, familial history of thrombocytopenia, or patients who have ITP and who have not responded to initial therapy.


Impairment of cerebral biogenic amine synthesis in a patient receiving high-dose methotrexate. A transient acute neurologic syndrome occurred in a 15-year-old boy receiving high-dose methotrexate (MTX) for acute lymphoblastic leukemia. Cerebrospinal fluid analysis revealed a decrease of homovanillic acid and 5-hydroxyindoleacetic acid concentrations with a rise of biopterin levels. These findings suggest that MTX can induce a transient alteration of the metabolism of tetrahydrobiopterin leading to the defect of biogenic amine neurotransmitter synthesis.


A study of keratin expression in benign familial chronic pemphigus. We studied keratin expression in the involved and uninvolved skin of six benign familial chronic pemphigus (BFCP) patients, using monoclonal antibodies specific for various keratin polypeptides and immunohistopathologic techniques. Normal and psoriatic (i.e., hyperproliferative) skin specimens served as controls. The uninvolved BFCP epidermis showed keratin profiles identical to that of normal epidermis. In the acantholytic epidermal segments of the involved BFCP skin, some of the lower suprabasal acantholytic cells failed to express keratin polypeptides 10 and 11 (Moll's catalog). This delay in expression of suprabasal keratins was not accompanied by an expression of hyperproliferative keratin polypeptide 16. Also, some of the lower suprabasal acantholytic cells of the involved BFCP epidermis retained staining by the antikeratin KS-1A3 antibody, which in the normal, psoriatic, and uninvolved epidermis was limited to the basal cell layer. Staining for keratin polypeptide 18 was negative in the epidermis of all four types of specimens. We believe that the delay in suprabasal keratin expression in the involved BFCP epidermis was more likely secondary to the acantholysis (i.e., "arrest of differentiation" due to acantholysis) rather than due to a primary defect in keratin expression.


Apocrine type of cutaneous mixed tumor with follicular and sebaceous differentiation. Eight cases of apocrine (tubular branching lumina) type cutaneous mixed tumors with follicular and sebaceous differentiation are presented. All eight tumors arose on facial skin; six patients were male and two were female. The lesions showed a cystic or nodular clinical appearance and were surgically excised. Histopathological examination confirmed the diagnosis of apocrine type of cutaneous mixed tumor in each case. Follicular differentiation consisted of (a) keratinous cysts with infundibular keratinization (infundibular differentiation); (b) hair bulbs with papillary mesenchyma, matricial differentiation with basophilic, transitional, and shadow cells, trichohyaline granules, vellous hair shafts, and clear cells of the outer root sheath (anagen differentiation); and (c) epithelial columns composed of inner cells with plump oval nuclei and scant cytoplasm, and similar cells at the periphery that were arranged in a palisade, resembling the inferior segment of a normal hair follicle in telogen. Sebaceous differentiation was represented by mature sebaceous cells, either as single cells or as small islands, within epithelial tracts of the tumor. The proportion of the areas showing these different types of differentiation varied among lesions, but some follicular differentiation was always present, whereas three cases lacked sebaceous differentiation. Immunohistochemical analysis in three cases with respect to their eccrine or apocrine differentiation showed contradictory results as in a previously reported series of cutaneous mixed tumors. The presence of follicular and sebaceous differentiation in the apocrine (tubular branching lumina) type of cutaneous mixed tumor is a confirmation of the apocrine nature of this neoplasm as well as an expression of the common embryologic derivation of all elements of the folliculosebaceous-apocrine unit.


Expression of Ulex europaeus agglutinin I lectin-binding sites in squamous cell carcinomas and their absence in basal cell carcinomas. Indicator of tumor type and differentiation. Lectins bind tightly to carbohydrate moieties on cell surfaces. Alterations in lectin binding have been reported to accompany epidermal cell differentiation, marking alterations in membrane sugars during this process. The presence of UEA I (Ulex europaeus agglutinin I) L-fucose-specific lectin-binding sites has been used as a marker for terminally differentiated (committed) keratinocytes. In this article, we report the presence of UEA-I-binding sites on squamous keratinocytes of well-differentiated squamous cell carcinomas, with patchy loss of UEA I positivity on poorly differentiated cells of squamous cell carcinomas, suggesting a possible use for this technique in the rapid assessment of less differentiated areas within the squamous cell tumor. The absence of UEA-I-binding sites on basal cell carcinomas may be related to an inability of cells comprising this tumor to convert the L-D-pyranosyl moiety on basal cells to the L-fucose moiety, resulting in an inability of basal cell carcinoma cell to undergo terminal differentiation into a committed keratinocyte.


Food allergy: identification of the major IgE-binding component of peach (Prunus persica). Since peaches are a relatively common cause of food allergy, we set out to identify the allergens involved. With the use of a panel of 48 sera from patients allergic to peach, we demonstrate that most of the allergenicity of that fruit is confined to the skin, rather than to the flesh of peaches, and corresponds to a protein doublet with an estimated molecular weight of 8-10 kD.


Circulating elevated levels of soluble CD23, interleukin-4, and CD20+CD23+ lymphocytes in atopic subjects with elevated serum IgE concentrations. Circulating IgE protein levels, leukocyte counts, lymphocyte subsets, IL-4, and soluble CD23 levels were quantitated in 43 atopic and 19 nonatopic subjects. Mean values of IgE protein levels, total eosinophil counts, CD20+CD23+ cells (B cells with low-affinity IgE receptor), IL-4 and sCD23 levels were elevated in atopic patients compared with nonatopic controls. The results suggest that sCD23, IL-4, and CD20+CD23+ lymphocytes may play a role in the increased production of IgE in atopic subjects in a manner similar to that observed by other investigators in prior in vitro studies.


Soy hypersensitivity in children with food allergy. To evaluate humoral (IgE antibodies) and clinical (positive challenge test) soy hypersensitivity prevalence, we studied 317 children (271 boys and 100 girls) with a median age of 5 months (range 1-120) who visited the Division of Allergy and Clinical Immunology of the Pediatric Department of the University of Roma "La Sapienza" because of histories and symptoms suggestive of food allergy. Atopic dermatitis (AD) was present in 247/317 children (78%), diarrhea in 19 (6%), urticaria in 22 (7%), and rhinitis and/or asthma in 29 (9%). All children underwent diagnostic procedures including family and personal history, physical examination, PRIST, and RAST to cows milk (CM), egg, wheat, soy, and Dermatophagoides pteronyssinus (Dpt). Open challenge tests to soy were performed in the hospital under observation and with emergency equipment at hand. The prevalence of humoral sensitization to CM was 54%, to egg 46%, to Dpt 35%, to wheat 24%, and to soy 22%. Only five children had IgE only to soy; six to soy and egg; and 58 to soy, CM, and egg. Only ten children (3%) had positive challenge to soy and only five of them had IgE to soy. RAST had a sensitivity of 0.69, a specificity of 0.83, a negative predictive value of 0.77, and a positive predictive value of only 0.06.


A pore transport model for pulmonary alveolar epithelium. Hydrodynamic heteropore flow models for transport of solutes across alveolar epithelial tissue have been developed. A two-size cylindrical pore model and a similar parallel-plate model were formulated, tested and used to predict effective pore sizes from literature data on transport in bullfrog, canine and rat lungs. The best fit equivalent pore-size estimates were obtained using a modified, nonlinear least squares procedure, with alveolar surface area to volume ratio (S/V) and small-pore area fraction of total pore area as parameters. Small-pore and large-pore width estimates of 4 nm (84% of total flow area) and 10 nm, respectively, with an average deviation of 20% from experimentally derived permeabilities were obtained from the bullfrog alveolar epithelium parallel-plate pore model (13 solutes, diameters 0.3 to 2.8 nm). The equivalent cylindrical pore model diameter estimates were 5 nm and 10 nm, with small-pore area fraction and percentage deviations similar to the parallel-plate model estimates. Eighty-eight percent of the bulk water driven by a sucrose osmotic gradient was predicted to be transported through the small pores. The rat alveolus parallel-plate pore model (6 solutes) yielded small-pore and large-pore widths of 0.4 nm and 50 nm, respectively. Clearance rate-constant data for dextran macromolecules (3,000 to 250,000 Daltons), using a single parallel-plate pore model, resulted in a pore width estimate of 98 nm for canine alveoli with an average deviation of the predicted rate constants of 18% from literature experimental values. In all cases tested, the parallel-plate pore model predicted lower small-pore size estimates than did the cylindrical pore model, and both models had appreciably smaller percentage deviations from experimental data than previous models.


Comparison of the effects of intragastric infusions of equal volumes of water, dioctyl sodium sulfosuccinate, and magnesium sulfate on fecal composition and output in clinically normal horses. A Latin square design was used to compare the effects of laxatives and a corresponding volume of water on gastrointestinal tract function in 4 healthy horses. Horses were intragastrically infused with each of the following: dioctyl sodium sulfosuccinate (DSS; 50 mg/kg of body weight); magnesium sulfate (0.5 g/kg--low dosage); magnesium sulfate (1.0 g/kg--high dosage); and an equal volume of water (6 L) given as a control infusion. From 5 to 33 hours after the high dosage of magnesium sulfate, feces were slightly softer than usual in all horses. In 1 horse, DSS caused mild colic, hyperpnea, and diarrhea from 0.3 to 3 hours after administration. After all laxative treatments and the control infusion, fecal output, fecal water, number of defecations, and fecal water percentage were greater during the first 6 and 12 hours, compared with each subsequent 6-hour period (P less than 0.05). The high dosage of magnesium sulfate had greater effect on fecal output and fecal water than did the low dosage and control infusion (P less than 0.05). However, this effect preceded arrival of the liquid transit marker, polyethylene glycol, and magnesium at their highest concentrations in feces by 12 to 18 hours. Compared with the control infusion, none of the laxative treatments affected excretion of polyethylene glycol and plastic particulate markers, nor did they increase water consumption. It was concluded that the response to intragastric infusions may involve reflex mechanisms in the gastrointestinal tract and that these responses could be used for treatment of colon impactions.(ABSTRACT TRUNCATED AT 250 WORDS)


Hemostatic defects associated with two infusion rates of dextran 70 in dogs. We investigated changes in hemostatic function after infusion of 6% dextran 70 (high molecular weight dextran) at 2 rates. Six healthy dogs underwent 3 regimens: 20 ml of dextran/kg of body weight administered in 1 hour (trial A), 20 ml of dextran/kg administered in 30 minutes (trial B), and 0.9% sodium chloride solution as a control administered over 1 hour to achieve hemodilution equivalent to that for 20 ml of dextran/kg (trial C). Before and at 2, 4, 8, and 24 hours after the start of trials A and B, we measured PCV, total solids (TS) concentration, amount of von Willebrand factor antigen (vWf:Ag), factor VIII coagulant activity (VIII:C), prothrombin time, activated partial thromboplastin time (APTT), platelet retention in a glass bead column, and buccal mucosa bleeding time (BMBT). Values were not obtained at 8 and 24 hours for trial C. Saline-induced changes in hemostasis were significant (P less than 0.05) from baseline throughout the sample collection period. Significant differences (P less than 0.05) between trial A and control were observed for vWf:Ag, VIII:C, BMBT, APTT, TS, and PCV values at 2 hours, and for VIII:C at 4 hours. Significant differences (P less than 0.05) between trial B and control were observed for APTT, TS, and PCV values at 2 hours, and for vWf:Ag, VIII:C, BMBT, APTT, TS, and PCV values at 4 hours. During trials A and B, mean values of analytes infrequently deviated from reference intervals, and clinical signs of bleeding were not observed in any dog.(ABSTRACT TRUNCATED AT 250 WORDS)


L-696,229 specifically inhibits human immunodeficiency virus type 1 reverse transcriptase and possesses antiviral activity in vitro. L-696,229 (3-[2-(benzoxazol-2-yl)ethyl]-5-ethyl-6-methyl-pyridin-2 (1H)-one) is a specific inhibitor of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) activity that possesses antiviral activity in cell culture (W.S. Saari, J.M. Hoffman, J.S. Wai, T.E. Fisher, C.S. Rooney, A.M. Smith, C.M. Thomas, M. E. Goldman, J. A. O'Brien, J. H. Nunberg, J. C. Quintero, W. A. Schleif, E. A. Emini, and P. S. Anderson, J. Med. Chem. 34:2922-2925, 1991). In the present study, the RT-inhibitory activity and antiviral properties were characterized in detail. The inhibition of RT activity was template-primer dependent with 50% inhibitory concentrations of 0.018 to 0.50 microM and was noncompetitive with respect to deoxynucleoside triphosphates. L-696,229 inhibited RT activity in a mutually exclusive manner with respect to either phosphonoformate or azidothymidine triphosphate and was a weak partial inhibitor of the RNase H activity associated with HIV-1 RT. The compound did not significantly inhibit other retroviral or cellular polymerases at 300 microM.L-696,229 inhibited the spread of HIV-1 infection in cell cultures with all cell types and viral isolates tested, including human peripheral blood mononuclear cells and a virus isolate resistant to azidothymidine.


Identification of the human immunodeficiency virus reverse transcriptase residues that contribute to the activity of diverse nonnucleoside inhibitors. The reverse transcriptase (RT) of human immunodeficiency virus type 1 (HIV-1) is potently inhibited by a structurally diverse group of nonnucleoside compounds. These include pyridinone derivatives, tetrahydroimadazo[4,5,1-j,k][1,4]-benzodiazepin-2(1H)-one and -thione, and BI-RG-587 (nevirapine). The compounds act noncompetitively, by an unknown mechanism, with respect to template-primer and nucleotide substrates. Despite a high degree of similarity between the HIV-1 and HIV-2 RTs, the HIV-2 enzyme is totally insensitive to these inhibitors. Using a novel method for joining DNA sequences, we have exploited this difference between the two enzymes to identify the regions of the RT that contribute to the compounds' inhibitory activities. The relative in vitro sensitivities of HIV-1/HIV-2 chimeric and site-specific mutant enzymes were determined. Sensitivity to inhibition was largely, though not exclusively, dependent upon the RT region defined by amino acid residues 176 to 190, with specific contributions by residues 181 and 188. The region defined by residues 101 to 106 was found to functionally interact with the domain from 155 to 217. In addition, the functional equivalence of the three inhibitor groups was shown.


Effector-assisted refolding of recombinant tissue-plasminogen activator produced in Escherichia coli. Recombinant tissue-plasminogen activator (r-tPA), expressed in Escherichia coli cells in an aggregated form, was solubilized with a strong chaotrope in the absence of any reducing agent. The solubilized molecule was reactivated by a procedure that was developed to mimic the physiological conditions optimal for the functional folding and activity of the native protein. The use of partially purified fibrinogen, as a source of fibrin (the effector), is shown to facilitate the reactivation process and increase its yield by at least a factor of two. The yield of the process is also shown to be particularly dependent on the recombinant protein concentration. At a concentration level of 3-3.7 mg r-tPA/L in the reactivation mixture, up to a 90% yield of activity was obtained. Purification of the activated form of r-tPA was achieved with a two-step column-chromatography scheme. This included a gel filtration step on a Sephadex G-50 column followed by an affinity chromatography step on a lysine-sepharose column. The product was composed of roughly equal amounts of one-chain and two-chain t-PA. The feasibility of using a two water-soluble polymeric phase system, with a centrifugal partition chromatography (CPC), in scaling up the reactivation process or the purification step was also evaluated.


[Enterovesical fistula secondary to leiomyosarcoma]. We report an unusual case of an enterovesical fistula caused by leiomyosarcoma infiltrating the intestinal wall and urinary bladder. The patient had previously presented recurrent urinary tract infection for one year. Although he has residual tumor, so far this elderly patient has survived for three years post-operatively without further antineoplastic treatment.


Use of endotracheal silicone stents for relief of tracheobronchial obstruction. In this article we describe our initial experience with bifurcated and longitudinal silicone stents that can be inserted entirely endoscopically. A total of 10 patients were stented; half had upper airways obstruction resulting from malignant disease and half had anastomotic obstruction after single-lung (3 patients), double-lung (1 patient), or heart-lung transplantation (1 patient). All patients derived immediate relief of life-threatening stridor. Stents were in place for between 5 days and 2 1/2 years (mean, 232.9 days). In the patients with malignant disease, the stents have provided effective relief from stridor for the remainder of their lives. In the transplant recipients, the medium-term results are encouraging, with the stents providing effective relief from stridor, although the longitudinal stents have been associated with distal migration, requiring that the stents be replaced on up to five occasions. The stents have not been associated with infection in the nonimmunosuppressed patients, and during the relatively short follow-up period there has been no tissue reaction to the material.


The hypothalamo-cerebellar projection in the rat: origin and transmitter. Hypothalamic neurons projecting to cerebellum were identified by retrograde tracing with wheat germ agglutinin-horseradish peroxidase (WGA-HRP) in the rat. Selective D-[3H]aspartate labelling was used to investigate whether any of these connections may use excitatory amino acids as transmitters. The WGA-HRP experiments revealed that the hypothalamo-cerebellar fibers have their main origins in the lateral, dorsal and posterior hypothalamic areas, and the tubero-mammillary nucleus, while smaller numbers of cells were observed in tuber cinereum, the anterior hypothalamic area, and the periventricular and paraventricular nuclei. After injections of D-[3H]aspartate into the cerebellar cortex, intense labelling of the olivocerebellar climbing fiber system was observed, but hypothalamic cells were not retrogradely labelled with this selective tracer. The absence of D-[3H]aspartate labelling indicates that hypothalamo-cerebellar neurons lack specific uptake mechanisms for excitatory amino acids, but it does not entirely preclude the possibility that some of these hypothalamic neurons may use such transmitters. Many cerebellar projecting cells were located in the tubero-mammillary nucleus, which is known to contain histaminergic and GABAergic neurons, and it was concluded that part of the hypothalamo-cerebellar pathways may use histamine and/or GABA as transmitters. The transmitter remains unknown for other parts of the hypothalamo-cerebellar pathways.


Cloning and expression of an amylase gene from Streptococcus bovis in Escherichia coli. An amylase gene was identified in a Streptococcus bovis 033 lambda gtWES lambda B genomic library. Using a starch overlay and a Congo red-iodine staining procedure, amylase positive clones could be identified by zones of clearing. Ten amylase positive clones were identified using this procedure. The clone chosen for further study, lambda SBA105, contained an insert of approximately 7.5 kb. The insert was mapped, and subcloning localized the amylase gene to a region of approximately 3.1 kb. Cloning of the 3.1 kb amylase fragment into pUC18 in both orientations revealed that the amylase gene was transcribed from its own promoter. Amylase activity was expressed by the Escherichia coli subclones and was found to be largely associated with the cytoplasmic fraction. Southern hybridization of genomic DNA from the amylolytic strains, S. bovis 033, S. bovis 077, Butyrivibrio fibrisolvens 194 and 195 revealed a single hybridizing band in S. bovis 033 DNA only. This indicates that the amylase gene from S. bovis may differ from the amylases of these other amylolytic bacteria.


Detection of glycoproteins in Borrelia burgdorferi. The presence of carbohydrates on proteins of Borrelia burgdorferi, the causative agent of Lyme disease, was investigated by using a digoxigenin labeling method together with Schiff staining and N-glycosidase F assay. The two major outer surface exposed proteins of 31 kDa and 34 kDa showed to be glycosylated and gel filtration high pressure liquid chromatography (HPLC) of proteins of B. burgdorferi metabolically labeled with 14C-N-acetylglucosamine revealed the incorporation of the carbohydrate into the glycosyl residue of these proteins.


Identification of the hepatocyte mitogen in bovine spleen as heparin-binding growth factors. Growth promoting activity for rat hepatocytes in bovine spleen was identified as three heparin-binding growth factors. All the features tested, such as heparin affinity, molecular mass, cross reactivity with antibody, and partial amino acid sequence, indicated that one of the three factors was identical to FGF-1 (fibroblast growth factor-1, acidic FGF), another one was related to FGF-2 (fibroblast growth factor-2, basic FGF), whereas it was more potent for hepatocytes than the FGF-2 purified from bovine brain. The third one was eluted from heparin-Sepharose column at 0.75M NaCl, of which activity was not abolished by anti-FGF-1 or FGF-2 antibodies. In addition, the mitogenic effect of this factor was synergistic with that of HGF (hepatocyte growth factor), a known potent hepatocyte mitogen, suggesting that it is a novel growth factor for hepatocytes.


Expression of TAPA-1 in preimplantation mouse embryos. TAPA-1 is a member of a new family of evolutionarily conserved transmembrane proteins which may be involved in regulation of cell growth and/or cell signalling. We have examined the temporal pattern of TAPA-1 RNA expression during mouse development. Using a sensitive reverse transcription/polymerase chain reaction assay, we show that TAPA-1 RNA is present in oocytes, fertilized eggs and cleavage stage embryos.


Molecular cloning of rabbit CAP-50, a calcyclin-associated annexin protein. CAP-50 is a member of annexin family proteins which binds specifically to calcyclin in a Ca2+ dependent manner (Tokumitsu. H., Mizutani. A., Minami. H., Kobayashi. R., and Hidaka. H. (1992) J. Biol. Chem. 267,8919-8924). The cDNA representing the rabbit form of this protein has been cloned from rabbit lung cDNA library. Sequence analysis of two overlapping clones revealed a 81-nucleotides 5'-nontranslated region, 1512-nucleotides of open reading frame, a 672-nucleotides 3'-nontranslated region, and a poly(A) tail. Authenticity of the clones was confirmed by comparison of portions of the deduced amino acid sequence with eight sequences of proteolytic peptides obtained from rabbit lung protein. CAP-50 cDNA encodes a 503 residue protein with a calculated M(r) of 54,043 and shows that the protein is composed of four imperfect repeats and hydrophobic N-terminal region. C-terminal region including four imperfect repeats shows 58.1% identity with human synexin (annexin VII), 48.0% identity with annexin I, 47.4% identity with annexin II, 60.1% identity with annexin IV, 54.5% identity with annexin V. Hydrophobic N-terminal region composed of 202 amino acid residues is not homologous with other annexin proteins suggesting that CAP-50 is a novel member of annexin family proteins.


Template. Phosphorothioate oligonucleotides duplexes as inhibitors of HIV-1 reverse transcriptase. We have investigated the interaction between a number of 14 mers phosphorothioate oligonucleotides and HIV-1 reverse transcriptase. Two methods were used to measure the affinity of the analogs for the enzyme. In the first, the oligonucleotide or its duplex with Poly(rl) were used as inhibitors of the enzyme using Poly(rA).(dT)14 as template primer. In the second, the oligonucleotides or their duplexes were used to displace a fluorescent template primer complex of known affinity from its binding site on reverse transcriptase. The two methods gave the same relative order of affinity. Phosphorothioate oligodeoxyribonucleotides had a much higher affinity than oligo(dC)14 and it was increased on hybridization. Quantitatively similar results were obtained for S(dC)14 or its analog with bases in the alpha-configuration. Of the analogs tested, only S(dC)14 showed priming activity.


Adhesion promotes transcellular leukotriene biosynthesis during neutrophil-glomerular endothelial cell interactions: inhibition by antibodies against CD18 and L-selectin. Eicosanoid formation by transcellular routes can amplify the levels and types of lipid mediators within a local milieu. To evaluate the role of adhesion in this process, we assessed the influence of mAb against adhesion molecules on LTC4 generation by PMN-endothelial cell interaction. Transcellular LTC4 generation was initiated by addition of fMLP to coincubations of GM-CSF-primed PMN and TNF-activated endothelial cells cultured from kidney glomeruli. Both PMN-endothelial cell adhesion and transcellular LTC4 generation were inhibited by mAb against leukocyte L-selectin and CD18. These results indicate that cytokine-treated PMN and endothelial cells generate LTC4 via transcellular routes by receptor-triggered mechanisms. They suggest that adhesion promotes transcellular eicosanoid biosynthesis and that adhesion molecules may also be targets for blockade of transcellular biosynthesis of lipid mediators.


Rapamycin inhibits the phosphorylation of p70 S6 kinase in IL-2 and mitogen-activated human T cells. Phosphorylation of 40S ribosomal protein S6 is regulated in part by the mitogen-activated p70 S6 kinase (p70s6k). Following the addition of IL-2 to the IL-2 dependent human cell line Kit225, or mitogenic activation of resting human T cells, a rapid phosphorylation of p70s6k was observed by immunoblotting. Rapamycin (RAP), a potent suppressor of T-cell proliferative responses, markedly inhibited the phosphorylation of p70s6k induced by IL-2 in Kit225 cells or by the mitogens added to resting T cells. Other immunosuppressants such as cyclosporin A or an FK506 analogue were without effect. Moreover, the effect of RAP was restricted to p70s6k; it did not inhibit the phosphorylation of p90rsk, another kinase which utilizes the S6 protein as a substrate. These data indicate for the first time that RAP may target the pathway leading to p70s6k phosphorylation during human T-cell proliferation.


Mapping the orientation of an antigenic peptide bound in the antigen binding groove of H-2Kb using a monoclonal antibody. The major histocompatibility complex class I molecules are receptors for intracellular peptides, both of self and non-self origin. When non-self peptides (eg., pathogen derived) are bound to the class I molecules, they form ligands for T cell receptors resulting in antigen specific lysis of the infected cells by cytotoxic T lymphocytes. Therefore, an understanding of the process of antigen recognition requires the precise definition of the structural features of the bimolecular complex formed by a single well defined antigenic peptide bound to the class I molecule. A strategy using antibodies was developed to probe the structural features of the H-2Kb containing a defined peptide in the antigen cleft. We report that the binding surface area of a Kb specific monoclonal antibody (28-13-3s) includes residues in the alpha 1 (Gly56 and Glu58) and alpha 2 (Trp167) helices of Kb thus, binding across the antigen binding groove. When cells treated with the antigenic peptide of vesicular stomatitis virus, N52-59, and its alanine substituted analogs were tested for 28-13-3s binding, it was found that position 1 of the peptide also forms a part of the antibody binding site. This finding strongly supports the positioning of the N-terminus of N52-59 proximal to pocket A, thus, assuming an orientation parallel to the alpha 1 helix.


Alteration of RNA I metabolism in a temperature-sensitive Escherichia coli rnpA mutant strain. E. coli strain A49 carries the themosensitive mutation in the rnpA gene encoding the protein component of RNase P, a tRNA-processing enzyme. Two small RNAs were highly accumulated in the A49 carrying derivatives of ColE1-type plasmids, at nonpermissive temperature. Characterization of these RNAs showed that they were the processed or degraded products derived from RNA I, which is the negative controller of ColE1-type plasmid replication. These derivatives of RNA I only differ in size at the 5' ends. The data of their degradation and synthesis kinetics suggest that they are intermediates of RNA I metabolism.


A model system for tumor angiogenesis: involvement of transforming growth factor-alpha in tube formation of human microvascular endothelial cells induced by esophageal cancer cells. Tumor growth is dependent on angiogenesis, which is thought to be mediated through growth factors, such as transforming growth factor-alpha (TGF-alpha) and -beta (TGF-beta), epidermal growth factor (EGF), and basic fibroblast growth factor (bFGF), produced by tumor cells. We have developed a model system for tumor angiogenesis in vitro: tube formation of human omentum microvascular endothelial (HOME) cells in type I collagen gels when these cells are co-cultured with tumor cells. Exogenously added TGF-alpha induced tube formation of HOME cells in collagen gel. In contrast, TGF-beta inhibited the TGF-alpha-induced tube formation of endothelial cells. We investigated whether tube formation could be induced in HOME cells in collagen gel when the HOME cells were co-cultured with three esophageal cancer cell lines, TE1, TE2, and TE5. TE1 and TE2 cells expressed both TGF-alpha and TGF-beta mRNA, but the level of TGF-alpha mRNA in TE2 was found to be much lower than in TE1 cells. TE5 did not express either TGF-alpha or TGF-beta. The tube formation of HOME cell was induced when they were co-cultured with TE1 cells, while both TE2 and TE5 cell lines induced tube formation at much lower rates than TE1. TE1-induced tube formation of HOME cells was specifically blocked by co-administration of anti-TGF-alpha-antibody, but not by anti-bFGF-antibody. The present study suggests that, in our model system, esophageal tumor angiogenesis is partly controlled by TGF-alpha, possibly through a paracrine pathway.


Platelet-derived growth factor activation of gastric mucosal calcium channels. Gastric mucosal calcium channel complex was isolated from the solubilized epithelial cell membranes by affinity chromatography on wheat germ agglutinin. The complex following labeling with [3H]PN200-100 was reconstituted into phospholipid vesicles which exhibited active 45Ca2+ uptake. The channels responded in a dose dependent manner to dihydropyridine calcium antagonist, PN200-110, which at 0.5 microM exerted maximal inhibitory affect of 66% on 45Ca2+ uptake, while a 52% enhancement in 45Ca2+ uptake occurred with a specific calcium channel activator, BAY K8644. On platelet-derived growth factor (PDGF) binding in the presence of ATP, channels showed an increase in protein tyrosine phosphorylation of 55 and 170kDa subunits of calcium channel. Such phosphorylated channels following reconstitution into vesicles displayed a 78% greater 45Ca2+ uptake. The results point towards the importance of PDGF in the regulation of gastric mucosal calcium homeostasis.


Enhanced photorelaxation in aorta, pulmonary artery and corpus cavernosum produced by BAY K 8644 or N-nitro-L-arginine. Segments of endothelium-denuded aorta, pulmonary arterial rings and strips of corpus cavernosum from rabbits were superfused with Krebs medium. Photorelaxation elicited by ultraviolet light (366 nm) was significantly enhanced by either BAY K 8644 (20 nM) or N-nitro-L-arginine (100 and 500 microM) and was associated with increased cyclic GMP. This action of both drugs was greater in pulmonary artery than aorta and corpus cavernosum and persisted in vascular rings for 90 min after drug removal. The effect was significantly attenuated by hemoglobin (10 microM) but was unaltered by superoxide dismutase (30 u/ml). The mechanism of such photosensitization is presently unclear.


Solubilization of ribosomes in reverse micelles. Pig liver ribosomes have been solubilized in reverse micelles constituted by bis (2-ethyl hexyl) sodium sulfosuccinate (AOT) in isooctane and 3.6% water, v:v. The micellar ribosomal solutions are transparent, show no significant scattering and permit direct spectroscopic observation of the ribosomes to be made. Ultraviolet absorption and circular dichroic spectra have been recorded and indicate that the ribosomes maintain in the micellar environment their structural integrity. Some possible applications of these micellar systems are discussed.


Serotonin stimulates a Ca2+ permeant nonspecific cation channel in hepatic endothelial cells. The contraction of hepatic endothelial cell fenestrae after exposure to serotonin is associated with an increase in intracellular Ca2+ which is derived from extracellular Ca2+, is inhibited by pertussis toxin and is not associated with activation of phosphoinositol turnover or cAMP. Using cell-attached and excised patches in primary cultures of rat hepatic endothelial cells, we identified a serotonin-activated channel with conductance of 26.4+/-2.3 pS. The channel was also permeant to Na+, K+ and Ca++ but not to anions. In cell-attached patch recordings, addition of serotonin to the bath significantly increased channel activity with Ca2+ or Na+ as the charge-carrying ions. This channel provides a mechanism whereby serotonin can raise the cytosolic Ca2+ concentration in hepatic endothelial cells.


Modulation of K+ channels by hydrogen peroxide. External application of hydrogen peroxide (H2O2) was found to inhibit the time-dependent fast inactivation process of three cloned voltage-gated K+ channels expressed in Xenopus oocytes: KShIIIC, KShIIID and HukII. As expected from kinetic models where some channels are still opening while a significant fraction of channels is already inactivated there was a large increase in current magnitude concomitant to inactivation block. The channels otherwise functioned normally. The effects of H2O2 were specific (other cloned voltage-gated K+ channels were not affected), and reversible, the currents returned to normal upon removal of the H2O2. H2O2 is produced during normal metabolism; it could act as a modulator of excitability through effects on K+ channels if effective local concentrations are reached in neuronal regions close to the channel. KShIIIC and KShIIID currents are very similar to an O2-sensitive K+ current present in type I cells of the carotid body which is believed to underlie the modulation of excitability of these cells by changes in arterial O2 pressure. H2O2 has been proposed as an intermediary between O2 and cellular response in the carotid body; our results provide support for this model.


The suppression of hepatic cytochrome P4504A mRNA mediated by the interferon inducer polyinosinic acid.polycytidylic acid. Interferon and interferon inducers are well known to depress the cytochrome P450-dependent hepatic mixed-function oxidase system and cause a decrease in the capacity of the liver to metabolize drugs and xenobiotics. In this study we have shown that the interferon-mediated changes in an induced form of hepatic cytochrome P450 (CYP4A) are mediated via a depression in the levels of mRNA as assessed by Northern blot and slot blot analyses using a 20-base synthetic oligodeoxyribonucleotide hybridization probe. Rats were pretreated with clofibrate to maximize CYP4A mRNA levels prior to the administration of polyinosinic acid.polycytidylic acid (poly IC), an alpha/beta interferon inducer. Hepatic CYP4A mRNA levels were decreased by 49 and 30% at 6 and 24 hr, respectively, following poly IC administration. In hepatic microsomes cytochrome P450 and functional CYP4A as measured by lauric acid hydroxylation, were not affected at 6 hr, but were depressed by 39 and 27%, respectively, 24 hr following poly IC administration. These results suggest that interferon depresses induced levels of hepatic drug metabolism by lowering the level of cytochrome P450 mRNAs and subsequent synthesis of cytochrome P450 apoproteins.


Neuropharmacological mechanisms of capsaicin and related substances. Capsaicin activates poorly myelinated primary afferent neurons, many of which are polymodal nociceptors. Activation is accompanied by membrane depolarization and the opening of a unique, cation-selective, ion channel which can be blocked by the polyvalent dye ruthenium red. The capsaicin-induced activation is mimicked by resiniferatoxin, a potent analogue, and by low pH. Activation is mediated by a specific membrane receptor which can be selectively and competitively antagonized by capsazepine. Repetitive administration of capsaicin produces a desensitization and an inactivation of sensory neurons. Several mechanisms are involved including receptor inactivation, block of voltage activated calcium channels, intracellular accumulation of ions leading to osmotic changes, and activation of proteolytic enzyme processes. Systemic and topical capsaicin produces a reversible antinociceptive and anti-inflammatory action after an initial undesirable algesic effect. Capsaicin analogues, such as olvanil, have similar properties with minimal initial algesic activity. Antinociception produced by capsaicin does not involve neurotoxicity, sensory neuropeptide depletion or activity at peripheral receptors; rather, systemic capsaicin produces antinociception by activating capsaicin receptors on afferent nerve terminals in the spinal cord. Spinal neurotransmission is blocked by a prolonged inactivation of sensory neurotransmitter release. However, local or topical applications of capsaicin block C-fibre conduction and inactive neuropeptide release from peripheral nerve endings. These mechanisms account for localized antinociception and the reduction of neurogenic inflammation, respectively.


Expression of the cytochrome P4502E and 2B gene families in the lungs and livers of nonpregnant, pregnant, and fetal hamsters. Members of the cytochrome P4502E and 2B gene families have been implicated in the activation of nitrosamines to reactive species capable of binding to cellular macromolecules and initiating tumor formation in various rodent species. This study was initiated to determine the relative prevalence of these isozymes and their response to ethanol during pregnancy and late gestation. Nonpregnant and pregnant hamsters were given a 10% ethanol solution in their drinking water for 10 days (gestation days 5-15) prior to being killed. RNA blot analysis of liver and lung tissue from nonpregnant, pregnant, and fetal hamsters demonstrated tissue-specific expression of CYP2E and 2B in adult and fetal animals. The levels of RNA expression of both P450s in fetal hamsters were less than 30% of nonpregnant adult values. In pregnant hamsters, the hepatic levels of CYP2E and 2B RNAs were decreased compared to nonpregnant animals. In contrast, the pulmonary levels of CYP2B RNA were increased in pregnant versus non-pregnant hamsters, while no effect of pregnancy on the levels of CYP2E RNA was seen. Although rats contain a single CYP2E1 gene transcript. Northern analysis demonstrated the presence of 1.8 and 2.8 kb bands in both liver and lung tissue of the hamster. Pretreatment with ethanol had little effect on the levels of either P450 RNA species in the lungs or livers of nonpregnant, pregnant, and fetal hamsters. These results demonstrate differences in the levels of expression of members of the CYP2E and 2B gene families during pregnancy and late gestation compared to nonpregnant adult hamsters. Fetal animals, like the adults, apparently respond to ethanol treatment by altering the levels of these P450 isozymes at the post-transcriptional level.


Transmembrane signalling in T cells.

Transmembrane signalling in T cells. Transmembrane signalling to activate T cells to proliferate and differentiate is a complex multistep process. It is the focus of much current interest, mostly because a selective and well-controlled inhibition of the process will allow regulation, or at least modulation, of the immune response. Here, Sándor Damjanovich and colleagues review the contributions of Hungarian scientists to the understanding of signalling in lymphocytes in particular, and cell activation in general.


Synthetic peptides in the search for T- and B-cell epitopes. In this article, Eva Rajnavölgyi describes two aspects of the rigorous application of organic chemistry to the task of synthesizing peptides that induce immune responses. First, the development of viral peptides that activate T and B cells without the need for carrier molecules and secondly, the production of a new generation of immunologically inert carrier molecules.


Allorecognition by NK cells: nonself or no self? The issue of antigen recognition by NK cells is complex, fascinating and, as yet, unresolved. This article reviews recent research on the repertoire of human NK cell clones for the recognition of different allogeneic cells, and summarizes the studies, most of which have been performed in mice, that implicate the MHC in NK cell recognition. It goes on to provide a common conceptual framework within which these different systems may be understood.


Image analysis of Feulgen-stained NIH/3T3 cells transformed with DNA of 4-nitroquinoline 1-oxide treated human breast epithelial cells. The nuclear phenotypes of Feulgen-stained NIH/3T3 cells transformed with 4-nitroquinoline 1-oxide (4NQO) treated, human breast epithelial cell (HBEC) DNA were studied by scanning microspectrophotometry and image analysis and compared with data obtained for nontransformed cells and for NIH/3T3 cells under ras oncogene transfecting situations. The Feulgen-DNA content of the individual nuclei (NQ1, NQ2, and NQ3 phenotypes) of the transformed cells was found not to be deeply affected, although presence of chromatin structures resembling double minutes could be verified in part of the metaphases of the transformed cells. On the other hand, the chromatin supraorganization of these cells showed some changes involving increased (NQ2, NQ3) or decreased (NQ1) levels of condensation. The changes in chromatin packing states, however, were of small magnitude compared with those reported for NIH/3T3 cells transfected with a c-H-ras oncogene or an N-ras-containing MCF-7 cell DNA. It was assumed that the transformation of the NIH/3T3 cells is not always necessarily accompanied by high levels of chromatin condensation. The transformation of the NIH/3T3 cells induced by the 4NQO-treated HBEC DNA and particularly the changes in chromatin condensation in these transformed cells could not be attributed merely to a ras activation elicited by the carcinogen. It is suggested that a more complex transforming mechanism is involved, probably owing to the fact that a whole genomic DNA of the 4NQO-treated HBEC has been used for transfection.


Effects of cholinergic drugs on cell interactions during fertilization and early development of the sea urchin Paracentrotus lividus. In the present work we tested the effects of cholinergic drugs on the early development of P. lividus. Fertilization was performed in the presence of cholinergic drugs, and the embryos were fixed after 2 hours, when the controls (untreated) completed the second segmentation cleavage. We identified four classes of stages in the development of the embryos affected by the drugs, i.e.: unhatched zygotes, first cleaved stage, second cleaved stage (= unaffected), and anomalous embryos. Statistical analysis indicated that that drugs with analogous action mechanism caused similar alterations in the distribution of the treated embryos in these four classes. This finding supports the hypothesis of the presence of a complete "pre-nervous" cholinergic system, with a true cholinergic role. It is tempting to speculate that this system is involved in the first cell to cell interactions during early segmentation and possibly in the block to polyspermy.


Spatial distribution of active genes implicated in the regulation of insulin-like growth factor stimulatory loops in human decidual and placental tissue of first-trimester pregnancy. We have previously shown that the insulin-like growth factor-2 (IGF-2) gene is partially coexpressed with the IGF-1 and -2 receptor genes in proliferative cytotrophoblasts of the human extraembryonic tissue. Here we show that high levels of IGF-2 gene expression are not restricted to the embryonic tissue but can also be found in the decidua compacta. The IGF-2 gene is thus expressed at high levels in the mesenchymal stroma of the decidua to establish potentially short-range communication with primarily IGF-1 receptor-positive mesenchymal stroma cells. Conversely, the glandular and surface epithelia coexpress the IGF-1 receptor and IGF-1 genes, while the IGF-2 gene is not detected above background levels. The potential control mechanisms of these cell-cell signalling pathways were investigated by the analysis of the spatial distribution of active IGF binding proteins (IGFBP) genes. The IGFBP-3 gene is coexpressed with the IGF-2 gene in proliferative cytotrophoblasts of the embryonic placenta. While active IGFBP-1 and -2 genes in our hands cannot be detected in the embryonic placenta, all three IGFBP genes are expressed in complex and overlapping patterns in the decidua compacta. The results are discussed in terms of how the various IGFBP genes may operate in different cell types to restrict IGF local stimulatory pathways.


Expression of the tumour suppressor gene p53 in oral cancer. In this preliminary series, the product of the tumour suppressor gene p53 was detected in 12/15 cases of oral squamous cell carcinoma (SCC) and in two cases of leukoplakia. The tumours either expressed the mutant form of p53 throughout the specimen or contained focal areas of positive cells. p53 expression was commonly observed in tumours obtained from patients who were heavy smokers and drinkers suggesting that alterations in the p53 gene may be one of the sites of genetic damage in this group of patients.


Molecular and channel-forming characteristics of gramicidin K's: a family of naturally occurring acylated gramicidins. The gramicidin K family is a set of naturally occurring acylated linear peptides in which a fatty acid is esterified to the ethanolamine hydroxyl of either gramicidin A or C, and possibly also to gramicidin B (Koeppe, R. E., II, Paczkowski, J. A., & Whaley, W. L. (1985) Biochemistry 24, 2822-2826). These acylated gramicidins form membrane-spanning channels in planar lipid bilayers and therefore constitute a model system with which to study the structural and functional consequences of acylation on membrane proteins. This paper serves to characterize further the channels formed by acylated gramicidins A and C and to demonstrate that these channels are structurally equivalent to the channels formed by the standard gramicidins. We also present additional evidence for the ester linkage in the natural acylated gramicidins A and C and identify the fatty acyl chains.


Influence of acylation on the channel characteristics of gramicidin A. The influence of acylation on the conductance, average duration, and channel-forming potency of channels formed by gramicidin A analogues was investigated using single-channel and multichannel techniques. Lauroyl-, myristoyl-, palmitoyl-, stearoyl-, and oleoylgramicidin A were prepared by covalent coupling of that fatty acid to the C-terminal ethanolamine group. Acylation of gramicidin A does not affect the single-channel conductance or the minichannel frequency in diphytanoylphosphatidylcholine/n-decane black lipid membranes. However, the average duration of all acylgramicidin channels was increased approximately 5-fold as compared to unmodified gramicidin A, which has a duration of 0.9 s at 200-mV applied potential. Somewhat surprisingly the rate of channel formation of the acylgramicidins is decreased relative to gramicidin A: lauroyl- and stearoylgramicidin are approximately 200 times less effective in channel formation as compared to gramicidin A. We conclude that channels formed by the acylgramicidins and by gramicidin A are structurally and conformationally equivalent.


Molecular cloning and tissue distribution of alternatively spliced mRNAs encoding possible mammalian homologues of the yeast secretory pathway calcium pump. Rat stomach and testis cDNAs corresponding to two alternatively spliced mRNAs encoding variants of a P-type ion-transport ATPase that closely resembles the yeast secretory pathway Ca2+ pump have been isolated and characterized. A partial kidney cDNA was identified previously using an oligonucleotide probe corresponding to part of the sarcoplasmic reticulum Ca(2+)-ATPase [Gunteski-Hamblin, A., Greeb, J., & Shull, G.E. (1988) J. Biol. Chem. 263, 15032-15040]. In the present study, we first isolated and characterized a stomach cDNA that contains the entire coding sequence. The 919 amino acid enzyme has the same apparent transmembrane organization and contains all of the conserved domains present in other P-type ATPases. Northern blot analyses demonstrate that 3.9- and 5-kilobase mRNAs corresponding to the cDNA were present in all tissues examined, suggesting that the protein it encodes performs a housekeeping function. Rat testis also contained a 3.7-kilobase mRNA that hybridized with a probe from the 5' end of the stomach cDNA but did not hybridize with a probe from the 3' end. Cloning and characterization of cDNAs corresponding to the smaller testis mRNA revealed that it is derived from the same gene but encodes a variant of the enzyme in which the C-terminal residue, Val-919, is replaced by the sequence Phe-919-Tyr-Pro-Lys-Ile-923. Similarity comparisons show that the two enzymes are more closely related to the known Ca2+ pumps than to other P-type ATPases.(ABSTRACT TRUNCATED AT 250 WORDS)


Active site studies of human immunodeficiency virus reverse transcriptase. The active site of human immunodeficiency virus reverse transcriptase (HIV1-RT) was probed using three group-specific reagents: phenylglyoxal (PG), N-ethylmaleimide (NEM), and pyridoxal 5'-phosphate (PLP). The inactivation of HIV1-RT by arginine-specific PG was found to be completely protected against by adding primer-template. The potential active site arginine was localized to position 277 in the primary structure, suggesting that the polymerase domain of the enzyme should be considered as extending at least this far from the N terminus. The sulfhydryl-modifying reagent NEM completely inhibits NY5-HIV1-RT, which contains a cysteine at position 162, and such inhibition is protected against by primer-template. However, it does not strongly inhibit LAV-HIV1-RT, in which C162 is replaced by S162, indicating that while C162 may be at or near the active site or interact allosterically with primer-template, it is not essential for activity. The lysine-specific reagent PLP was found to be a noncompetitive inhibitor with respect to both primer-template [poly(rA).oligo(dT)] and dTTP. The latter result differentiates HIV1-RT from other RTs, for which PLP has been shown to be a competitive inhibitor with respect to dTTP.


Mapping function to structure in a channel-blocking peptide: electrostatic mutants of charybdotoxin. Electrostatic interactions between charybdotoxin (CTX), a specific peptide pore blocker of K+ channels, and a Ca(2+)-activated K+ channel were investigated with a genetically manipulable recombinant CTX. Point mutations at certain charged residues showed only small effects on the binding affinity of the toxin molecule: Lys11, Glu12, Arg19, His21, Lys31, and Lys32. Replacement by Gln at Arg25, Lys27, or Lys34 strongly decreased the affinity of the toxin. These affinity changes were mainly due to large increases of toxin dissociation rates without much effect on association rates, as if close-range interactions between the toxin and its receptor site of the channel were disrupted. We also found that the neutralization of Lys27 to Gln removed the toxin's characteristic voltage dependence in dissociation rate. Mutation and functional mapping of charged residues revealed a molecular surface of CTX which makes direct contact with the extracellular mouth of the K+ channel.


Analysis of side-chain organization on a refined model of charybdotoxin: structural and functional implications. The spatial organization of side chains on a refined model of charybdotoxin is presented. First, the structural role of two groups of well-defined, low-accessible side chains (Thr3, Val5, Val16, Leu20, Cys33 and Leu20, His21, Thr23, Cys17, Cys35) is discussed. These side chains are conserved in three out of the five known scorpion toxins acting on K+ channels. Interestingly, they are not conserved in scyllatoxin which presents a slightly different secondary structure organization. Second, the spatial organization of all positively charged residues is analyzed. Comparison with the results presented by Park and Miller [(1992) Biochemistry (preceding paper in this issue)] shows that all functionally important positive residues are located on the beta-sheet side of the toxin. These results are different from those obtained by Auguste et al. [(1992) Biochemistry 31, 648-654] on scyllatoxin, which blocks a different type of K+ channel. This study shows, in fact, that functionally important positive residues are located on the helix side of the toxin. Thus, charybdotoxin and scyllatoxin, which present the same global fold, interact with two different classes of K+ channels by two different parts of the motif.


Structure-function analysis of DNA polymerase-beta using monoclonal antibodies: identification of a putative nucleotide binding domain. DNA polymerase-beta was purified from Novikoff hepatoma and used as an antigen in an in vitro immunization system to produce monoclonal antibodies. These reagents surprisingly showed cross-reactivity to a number of proteins, including several DNA polymerases. Nearly all of these proteins possess nucleotide binding sites, which suggested the potential value of using the monoclonals to elucidate structure-function relationships within polymerase-beta. Furthermore, these antibodies were able to partially neutralize (40-50%) polymerase-beta activity, and this effect could be blocked by dNTP1 but not by dNMP or rNTP. The limited neutralization phenomenon is at least partially explained by the weak binding affinity of these antibodies. Scatchard analysis of immunoprecipitation data predicted a Kd of 1.8 x 10(-8) M. Epitope mapping studies showed that the region of polymerase-beta recognized by one of the monoclonal antibodies is within residues 235-335, and sequence homology studies indicated that the epitope is probably located in the region of amino acids 283-320. At least a portion of this area, namely residues 301-308 and 311-315, appears to be part of a nucleotide binding domain which has sequence homology with a portion of the highly conserved ATP binding site in adenylate kinase.


Importance of asparagine-61 and asparagine-109 to the angiogenic activity of human angiogenin. Two distinct regions of angiogenin are critical for angiogenic activity: a catalytic site capable of cleaving RNA and a noncatalytic site, encompassing residues 60-68, which may bind to an endothelial cell-surface receptor [Hallahan, T. W., Shapiro, R., & Vallee, B. L. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 2222-2226]. We have now shown that Asn-61 is an essential residue within the cell-binding site and that in addition a segment containing Asn-109 is part of this site. Both asparagines undergo nonenzymatic deamidation during long-term storage or treatment at alkaline pH. While the isolated desamido-61 and desamido-109 derivatives retain nearly full enzymatic activity, their angiogenic activity on the chicken embryo chorioallantoic membrane is markedly attenuated and they do not inhibit angiogenin-induced neovascularization. Tryptic peptide mapping and Edman degradation demonstrate that the isolated deamidated derivatives primarily contain isoaspartic rather than aspartic acid at the positions in question (83% for desamido-61, greater than 99% for desamido-109). Aspartic acid replacement of Asn-61 and Asn-109 by site-directed mutagenesis results in the same ribonucleolytic and angiogenic activities as those of the spontaneous deamidation products. However, the aspartyl derivatives differ strikingly from their isoaspartyl counterparts in that they do inhibit angiogenin-induced angiogenesis. These results indicate that the combination of ribonucleolytic activity and receptor-binding capacity is not sufficient for angiogenic activity and that Asn-61 and Asn-109 within the noncatalytic site are required for some additional function, as yet undefined.(ABSTRACT TRUNCATED AT 250 WORDS)


Characterization of gramicidin A in an inverted micellar environment. A combined high-performance liquid chromatographic and spectroscopic study. We have investigated the conformational adaptability of gramicidin A incorporated into reverse micelles of sodium bis(2-ethylhexyl)sulfosuccinate (AOT)/isooctane/water, a so far unexplored "host" membrane-mimetic model system for this peptide. A high-performance liquid chromatographic strategy previously developed for the study of gramicidin in phospholipid vesicles and normal micelles [Bañó et al. (1989) FEBS Lett. 250, 67; Bañó et al. (1991) Biochemistry 30, 886] has been successfully extended to this system. The method has permitted the separation of peptide conformational species, namely, double-stranded dimers and monomers, and an accurate quantitation of their proportion in the inverted micellar environment. It has been demonstrated that, once inserted in the micelle, the double-stranded dimers undergo a dissociation process toward a thermodynamically stable monomeric configuration, whose monomerization rate constant (k1) is dependent in a bell-shaped manner on the water:surfactant mole ratio, w0. A tight correlation between k1 and the double-stranded dimer backbone conformation has been found from the comparison of chromatographic and circular dichroism data. In addition, fluorescence experiments indicate that the peptide tryptophans are in a rather nonpolar environment, with a restricted accessibility to water-soluble quenchers such as acrylamide.


Human inter-alpha-trypsin inhibitor: full-length cDNA sequence of the heavy chain H1. Inter-alpha-trypsin inhibitor (ITI), called inter-alpha-inhibitor, is a 220 kDa serine proteinase inhibitor found in human serum. It is composed of at least three distinct polypeptide chains. These chains, named H1, H2 and L, are an independently synthesized and proteolytically processed precursor protein. Only the complete structure of H2 and L has been established so far. We used a PCR-based cloning approach and a cDNA screening library to isolate the full-length cDNA H1. The amino acid sequences of the two heavy chains deduced from the cDNA are highly similar (40% identity). Nevertheless, the structure of the signal peptide and propeptide in the N-terminal region is different in these two chains. A complex posttranslational cleavage at both ends of H1 and H2 may be proposed prior to assembly of the ITI chains.


Different patterns of gene expression of topoisomerase II isoforms in differentiated tissues during murine development. The expression of DNA topoisomerase II alpha and beta genes was studied in murine normal tissues. Northern blot analysis using probes specific for the two genes showed that the patterns of expression were different among 22 tissues of adult mice. Expression levels of topoisomerase II alpha gene were high in proliferating tissues, such as bone marrow and spleen, and undetectable or low in 17 other tissues. In contrast, high or intermediate expression of topoisomerase II beta gene was found in a variety of tissues (15) of adult mice, including those with no proliferating cells. Topoisomerase II gene expression was also studied during murine development. In whole embryos both genes were expressed at higher levels in early than late stages of embryogenesis. Heart, brain and liver of embryos two days before delivery, and these same tissues plus lung and thymus of newborn (1-day-old) mice expressed appreciable levels of the two genes. Interestingly, a post-natal induction of the beta gene expression was observed in the brain but not in the liver; conversely, the expression of the alpha gene was increased 1 day after birth in the liver but not in the brain. However, gene expression of a proliferation-associated enzyme, thymidylate synthase, was similar in these tissues between embryos and newborns. Thus, the two genes were differentially regulated in the post-natal period, and a tissue-specific role may be suggested for the two isoenzymes in the development of differentiated tissues such as the brain and liver. Based on the differential patterns of expression of the two isoforms, this analysis indicates that topoisomerase II alpha may be a specific marker of cell proliferation, whereas topoisomerase II beta may be implicated in functions of DNA metabolism other than replication.


Molecular cloning of rat glucose-dependent insulinotropic peptide (GIP). A cDNA clone encoding glucose-dependent insulinotropic peptide (GIP) was identified that consisted of 34 bp of 5' untranslated sequence, an open reading frame of 432 bp and 115 bp in the 3' untranslated region. The deduced amino acid sequence revealed a 144 amino acid preprohormone consisting of a 43 amino acid N-terminal extension including a signal peptide, a 42 amino acid hormone, and a 59 amino acid C-terminal extension. Rat GIP differs from the human hormone by two amino acid substitutions: arginine for histidine at position 18 and leucine for isoleucine at position 40. A single mRNA from small intestine of approximately 800 bases was identified on Northern blot analysis in equivalent amounts in proximal and distal small intestine.


A serine, threonine and proline-rich region near the carboxyl-terminus of a rat intestinal mucin peptide. Further sequencing of a cDNA encoding the C-terminal region of a rat intestinal mucin peptide reveals a region corresponding to 258 amino acids enriched in serine, threonine and proline, but no typical mucin-like tandem repeat structures. Between this region and a previously described stretch of 4.5 degenerate S,T,P-rich tandem repeats, there is a 42 amino acid cysteine-rich segment. The discontinuity of cysteine-rich and S,T,P-rich areas near the C-terminus has not been observed in other mammalian mucin structures reported to date.


The primary structure of chicken ribosomal protein L37a. The amino acid sequence of chicken ribosomal protein L37a was deduced from the nucleotide sequence of a recombinant cDNA and its genomic DNA. Chicken ribosomal protein L37a has 92 amino acids and a molecular mass of 10,247 Da including the initiator methionine. The protein contains a typical Cys2Cys2 zinc finger motif, which may be involved in protein-RNA interaction.


Supportive care of neurologic complications. Approximately one cancer patient in five will suffer from injuries to either the central or peripheral nervous system. These complications occur preferentially in the advanced stages of neoplastic disease, and their management is an important aspect of the supportive care of these patients. The most common neurologic complications seen in patients with cancer are related to metastases or to local tumor spread. Antineoplastic treatments, mainly chemotherapy or radiotherapy, account for more than 10% of these complications. A much smaller percentage (probably less than 1%) are paraneoplastic manifestations. In this review, we discuss the most recent developments in the management of neurologic complications specifically related to cancer as well as some less specific problems such as thromboembolic complications and the use of steroids.


Kaposi's sarcoma.

Kaposi's sarcoma. In the era before the acquired immunodeficiency syndrome, Kaposi's sarcoma was a rare cutaneous event. Most reported cases usually originated from one of two geographic regions, an induced immunosuppressed state, or sporadically with various neoplasms that have been known to cause immunosuppression. Since the first cases described with acquired immunodeficiency syndrome in 1981 from New York and San Francisco, this uncommon disorder has had an increased incidence that crosses geographic boundaries. This article describes the current understanding of the pathogenesis of Kaposi's sarcoma and treatments used to ameliorate this disorder.