The efferent control of the organs of balance and equilibrium in the toadfish, Opsanus tau.

All vertebrates are endowed with a vestibular efferent system (EVS) consisting of somata within the central nervous system with long axons exiting the brain to innervate the labyrinth. Behaviorally relevant stimuli related to feeding and/or aggressive behaviors and conditions leading to enhanced attentional states or alerting activate the EVS. Increased EVS activity modifies the resting rate and response dynamics to motion of vestibular afferents. This modification is nonuniform across the fiber spectrum of the semicircular canals, for example, affecting the more-sensitive, low-spontaneous-activity cells more profoundly than their less-sensitive counterparts. The cellular bases for EVS effects are excitatory axoaxonic synapses upon primary afferents and axosomatic inhibitory synapses upon hair cells.


Cholinergic innervation of the cerebellum of the rat by secondary vestibular afferents.

The cholinergic innervation of the cerebellar cortex of the rat was studied by immunohistochemical localization of choline acetyltransferase, radiochemical measurement of ChAT activity, and double labeling of ChAT-positive neurons with HRP injected into the cerebellum. ChAT immunohistochemistry revealed large mossy fiber rosettes as well as finely beaded terminals with different morphological characterization, laminar distribution within the cerebellar cortex, and regional differences within the cerebellum. Large "grapelike" ChAT-positive mossy fiber rosettes that were distributed primarily in the granule cell layer were concentrated, but not exclusively located, in three separate regions of the cerebellum: (1) the uvula-nodulus (lobules 9 and 10); (2) the flocculus, and (3) the anterior lobe vermis (lobules 1 and 2). Regional differences in ChAT-positive afferent terminations in the cerebellar cortex demonstrated by immunohistochemistry were confirmed by regional biochemical measurements of ChAT activity. Using ChAT immunohistochemistry in combination with HRP injections into the uvula-nodulus, we have studied the origin of the cholinergic projection. The caudal medial vestibular nucleus and to a lesser extent the nucleus prepositus hypglossus contain ChAT-positive neurons that were double labeled following HRP injections into the uvula-nodulus. We conclude that (1) there is a prominent cholinergic mossy fiber pathway to the vestibulocerebellum, (2) this pathway originates primarily in the caudal third of the medial vestibular nucleus, and (3) this cholinergic pathway likely mediates secondary vestibular information related to postural adjustment.


Tacrolimus (FK 506)--a new therapeutic agent for severe recalcitrant psoriasis.

Psoriasis, a disease of unknown etiology, is in some patients severe, extremely debilitating, and unresponsive to conventional therapies, including UV-B, oral psoralen with long-wave UV radiation in the A range (PUVA), oral retinoids, and methotrexate. We report the results from our study of seven patients with refractory psoriasis who were treated with the new immunosuppressive drug, tacrolimus (FK 506). All seven patients showed a dramatic resolution of psoriasis that remained in remission as long as they received full-dose therapy. Serial skin biopsy specimens demonstrated a rapid disappearance of the inflammatory infiltrate and a slower resolution of the epidermal changes. Tacrolimus was well tolerated during the 5.5 to 14 months of observation. Side effects, including nephrotoxicity and hypertension, were controlled by appropriate modification of drug dosage. Tacrolimus, a new immunosuppressive agent, is effective in treating patients with severe recalcitrant psoriasis. The mechanism of its action in psoriasis is unknown, but it may be related to its ability to modulate immune function. Further studies will establish criteria for patient selection and drug dosage, to maximize efficacy of this agent in psoriasis, while minimizing its toxicity.


Frequency of bullous pemphigoid-like antibodies as detected by western immunoblot analysis in pruritic dermatoses.

Ninety-seven patients suffering from a pruritic dermatosis were screened for the detection of bullous pemphigoid (BP) antibodies (ab) using the Western immunoblot (WB) analysis technique. Twenty-four patients (25%) reacted at least twice with the BP antigen on WB analysis at a 1/10 dilution: seven had typical BP, four had papular BP, 10 had pruritus and prurigo of unknown origin, two had eczema, and one had lichen planus. This corresponds to 13% of BP-type ab in patients who did not fulfill criteria for BP. Therefore, we did the following: (1) tested a control group of 24 subjects; (2) assessed the reproducibility of the WB method by retesting the same serum samples on different epidermal extracts; and (3) estimated the BP ab titer. None of the 24 control subjects had detectable BP ab, and reproducible results were obtained in all groups when serum samples were retested at the 1/10 dilution. Although 86% (6/7) of patients with BP had BP ab titers of 1/100 or greater, only 60% (6/10) of the group with pruritus and prurigo and 33% (1/3) of the group with eczema reached such titers. These results indicate that due to its sensitivity, the WB method can detect low titers of BP ab in patients with pruritic dermatoses who did not fulfill criteria for BP, and therefore we question the specificity of this method.


A 2-year prospective follow-up study of children and adolescents with disruptive behavior disorders. Prediction by cerebrospinal fluid 5-hydroxyindoleacetic acid, homovanillic acid, and autonomic measures?

A 2-year prospective follow-up study of 100% (N = 29) of a sample of children and adolescents with disruptive behavior disorders found that the baseline lumbar cerebrospinal fluid monoamine metabolite concentration and autonomic nervous system activity predicted some subsequent outcomes. The 5-hydroxyindoleacetic acid concentration significantly predicted severity of physical aggression during follow-up. The skin conductance level significantly predicted institutionalization. Correlations were in predicted directions with lower cerebrospinal fluid 5-hydroxyindoleacetic acid concentrations and autonomic activity correlated with poor outcome. Moreover, in multivariate analyses, which included nonlaboratory measures as predictors, cerebrospinal fluid and autonomic measures still contributed significantly to the prediction. However, hypothesized predictions of cerebrospinal fluid 5-hydroxyindoleacetic acid concentrations for suicide attempts and of low autonomic nervous system activity for arrests were not supported thus far. Patients are still at risk; consequently, these results must be considered preliminary. Nonetheless, the results suggest that further investigation of relationships between biological factors and outcome of children with disruptive behavior disorders is warranted.


Cerebrospinal fluid monoamine and adrenal correlates of aggression in free-ranging rhesus monkeys.

Clinical and preclinical studies involving several different mammalian species and research paradigms suggest a negative correlation between aggression and central serotonin activity. To test the generalizability of laboratory findings in rhesus monkeys that show a negative correlation between cerebrospinal fluid 5-hydroxyindoleacetic acid concentrations and aggression, we obtained cisternal cerebrospinal fluid and blood plasma samples from monkeys living in naturalistic conditions. During a semiannual trapping, 28 juvenile and adolescent male rhesus monkeys were chosen from a population of 4200 provisioned, free-ranging rhesus monkeys living on Morgan Island, a sea island located off the coast of South Carolina. Based on direct observations of participation or avoidance of aggressive behavior and examinations of apparent fight wounds, 18 monkeys were selected for cerebrospinal fluid taps and blood samples. The remaining 10 monkeys were selected at random. Descriptions of aggressive behavior and the number of old scars and recent wounds were carefully transcribed, and a photograph showing wounds and scars was obtained for each animal. Using the transcriptions and photographs, researchers experienced in rhesus monkey behavior, but blind to the subjects' monoamine and hormone concentrations, were asked to rank the monkeys from the most to the least aggressive. The results showed a significant negative correlation between high rankings for aggression and cerebrospinal fluid 5-hydroxyindoleacetic acid concentrations. There was evidence that aggression was associated with stress, in that cerebrospinal fluid, norepinephrine, and plasma corticotropin and cortisol concentrations were positively correlated with high rankings of aggression.


Relationship between central and peripheral serotonin indexes in depressed and suicidal psychiatric inpatients.

Serious suicidal behavior, affective disorders, and a variety of other psychopathologic behaviors and syndromes have been found to correlate with measures of the serotonin system. Clinical studies have employed a range of serotonin indexes, including the cerebrospinal fluid level of 5-hydroxyindoleacetic acid, the prolactin response to serotonin agonists, such as fenfluramine hydrochloride, and platelet serotonin-related proteins or serotonin content. Many of these indexes are correlated with suicidal behavior, but the interrelationship of these biologic measures has been uncertain. We studied the relationship of a series of serotonin indexes in patients in whom these measures were correlated with suicidal behavior. A positive correlation was found between cerebrospinal fluid 5-hydroxyindoleacetic acid and the maximal prolactin response to fenfluramine but not with platelet serotonin2 receptor indexes. The fenfluramine-stimulated maximal prolactin response correlated with platelet serotonin2 receptor number, particularly in older patients. We conclude that cerebrospinal fluid 5-hydroxyindoleacetic acid measurements cannot be replaced but can be complemented by less invasive procedures, such as a fenfluramine challenge test or platelet serotonin2 measures, in the study of the relationship of the serotonin system to psychiatric disorders.


Marked reduction in indexes of dopamine metabolism among patients with depression who attempt suicide.

Cerebrospinal fluid studies have reported that low concentrations of the dopamine metabolite homovanillic acid are associated with suicidal behavior in depression. Although only a small proportion of homovanillic acid in the urine derives from the brain, we decided to examine 24-hour urinary outputs of homovanillic acid in relation to suicidal behavior in depression. Patients with depression who had attempted suicide had significantly smaller urinary outputs of homovanillic acid, dihydroxyphenylacetic acid, and total body output of dopamine (sum dopamine) than did patients with depression who had not attempted suicide. Patients with depression who reattempted suicide during 5-year follow-up had significantly smaller urinary outputs of homovanillic acid and sum dopamine than did patients who did not reattempt suicide, patients who never attempted suicide, and normal control subjects, and had significantly smaller outputs of dihydroxyphenylacetic acid than patients who never attempted suicide or control subjects. These data suggest that urinary outputs of homovanillic acid may be peripheral correlates of suicidality in depression. These data add to data on the low levels of homovanillic acid in cerebrospinal fluid in suggesting that diminished dopaminergic neurotransmission may play a part in suicidal behavior in depression.


Structural and computational investigations of the conformation of antigenic peptide fragments of human polymorphic epithelial mucin.

Human polymorphic epithelial mucins (PEM) are complex glycoproteins that are associated with breast and ovarian carcinomas. The PEM core protein consists of variable numbers of a tandem repeat sequence which contains a short antigenic hydrophilic region (Pro1-Asp-Thr-Arg-Pro-Ala-Pro7). High-field n.m.r. studies undertaken on antigenic 20- and 11-amino acid fragments of the PEM core protein in dimethyl sulphoxide have identified a type-I beta-turn to be present in the region Pro1-Asp-Thr-Arg4. This region includes and overlaps the identified type-I (Asp2-Thr-Arg4) and type-II (Arg4-Pro-Ala6) epitopes of anti-PEM monoclonal antibodies. The studies indicate that the beta-turn is stabilized by the presence of a salt-bridge interaction between Asp-2 and Arg-4. In order to probe the conformations accessible to the PEM peptides a computational study was undertaken independently on the peptide Pro-Asp-Thr-Arg-Pro using a modified Metropolis Monte Carlo algorithm. This study identified the n.m.r.-observed salt-bridge type-I beta-turn as the major low-energy conformer. These results suggest that this structural motif may be involved in the immune recognition of PEM.


Isolation and characterization of a membrane protein from rat erythrocytes which inhibits lysis by the membrane attack complex of rat complement.

The membrane attack complex (MAC) of complement in humans is regulated by several membrane-bound proteins; however, no such proteins have so far been described in other species. Here we report the isolation and characterization of a rat erythrocyte membrane glycoprotein of molecular mass 21 kDa which inserts into cell membranes and is a potent inhibitor of the rat MAC. This protein, here called rat inhibitory protein (RIP), was first partially purified by column chromatography from a butanol extract of rat erythrocyte membranes. Monoclonal antibodies (Mabs) were raised against RIP and used for its affinity purification. Affinity-purified RIP was shown to inhibit in a dose-dependent manner the cobra venom factor (CVF)-mediated 'reactive' lysis of guinea pig erythrocytes by rat complement. Conversely, the anti-RIP MAbs 6D1 and TH9 were shown to markedly enhance the CVF-mediated lysis of rat erythrocytes by rat complement. RIP acted late in the assembly of the MAC (at or after the C5b-8 stage) and was releasable from the membranes of rat erythrocytes by phosphatidylinositol-specific phospholipase C. These features, together with its size, deglycosylation pattern and N-terminal amino acid sequence, lead us to conclude that RIP is the rat homologue of the human MAC-inhibitory protein CD59 antigen.


Human platelet glycoprotein IIIb binds to thrombospondin fragments bearing the C-terminal region, and/or the type I repeats (CSVTCG motif), but not to the N-terminal heparin-binding region.

Major blood membrane platelet glycoprotein IIIb (GPIIIb), also termed GPIV or CD365, has been identified as a receptor for thrombospondin (TSP), collagen and Plasmodium falciparum-infected erythrocytes. The aim of the present study was to identify region(s) of TSP involved in binding of GPIIIb. Proteolytic fragments of TSP (M(r) 140 kDa, 120-18 kDa and 27 kDa on SDS/PAGE under reducing conditions) were purified by f.p.l.c. and identified by N-terminal gas-phase sequencing, e.l.i.s.a. and Western blots using monoclonal antibodies directed against defined domains of TSP. The 140 kDa and 120-18 kDa fragments (C-terminal region), but not the 27 kDa fragment (N-terminal region), were shown to bind to GPIIIb by using e.l.i.s.a. and affinity-chromatography systems. TSP binding to a GPIIIb-affinity column was Ca(2+)-dependent and reduced by 45% in the presence of EDTA. Moreover, TSP was only partially eluted with EDTA from a Ca(2+)-equilibrated GPIIIb column. A fragment of 68 kDa, obtained by further digestion of the 140 kDa fragment, bound to the GPIIIb-Sepharose affinity column. This fragment, or stalk-like region, bears the TSP type I repeats that show sequence similarity to regions on properdin, Plasmodium falciparum proteins and antistasin. Peptides (CSVTCG or SVTCGGGV) representing these repeats bound isolated GPIIIb in a Ca(2+)-independent way, but did not completely inhibit the GPIIIb and TSP interaction. These studies indicate that GPIIIb binds to the TSP via the C-terminal region and/or the CSVTCG motif, but not to the N-terminal region. Interaction between GPIIIb and the TSP C-terminal region or the CSVTCG motif is respectively Ca(2+)-dependent and -independent.


Molecular biology of the 2-haloacid halidohydrolase IVa from Pseudomonas cepacia MBA4.

The structural gene (hdl IVa) for the Pseudomonas cepacia MBA4 2-haloacid halidohydrolase IVa (Hdl IVa) was isolated on a 1.6 kb fragment of Ps. cepacia MBA4 chromosomal DNA. The recombinant halidohydrolase was expressed in Escherichia coli and Pseudomonas putida and the structural gene was subcloned on to the tac expression vector pBTac1. High-level expression from the tac promoter was seen to be temperature-dependent, a consequence of the nucleotide sequence adjacent to the fragment encoding the halidohydrolase. The nucleotide sequence of the fragment encoding the Hdl IVa was determined and analysed. Three ATG codons were identified in one of the open reading frames and the one corresponding to the start of the hdl IVa structural gene was determined by comparison of the predicted amino acid sequences with the experimentally determined N-terminal sequences of halidohydrolase IVa. The hdl IVa gene encoded a 231-amino acid-residue protein of M(r) 25,900. The sequence and predicted structural data are discussed and comparison is made with sequence data for other halidohydrolases.


The antigenicity of the carbohydrate moiety of an insect glycoprotein, honey-bee (Apis mellifera) venom phospholipase A2. The role of alpha 1,3-fucosylation of the asparagine-bound N-acetylglucosamine.

A rabbit polyclonal antiserum raised against honey-bee (Apis mellifera) venom phospholipase A2 (PLA2) contains antibodies that react exclusively with its glycosylated variants and cross-react with plant glycoproteins. The interaction of anti-(horseradish peroxidase) antiserum with PLA2 suggests the existence of a carbohydrate determinant common to both glycoproteins. E.l.i.s.a. binding and inhibition experiments, employing glycoproteins and glycopeptides of plant and animal origin with known N-glycan structures, in combination with chemical and enzymic deglycosylation, identified alpha 1,3-fucosylation of the asparagine-bound N-acetylglucosamine as the antigenic determinant. This fucose residue is present in the N-glycan of PLA2 and is frequently found in plant glycoproteins, whereas mammalian glycoproteins lack this modification.


Ca2+ transport by digitonin-permeabilized Leishmania donovani. Effects of Ca2+, pentamidine and WR-6026 on mitochondrial membrane potential in situ.

The use of low concentrations of digitonin allowed the quantitative determination of the mitochondrial membrane potential of Leishmania donovani promastigotes in situ using safranine O. L. donovani mitochondria were able to build up and retain a membrane potential of a value comparable with that of mammalian mitochondria. The response of promastigotes mitochondrial membrane potential to phosphate, carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP), valinomycin and Ca2+ indicates that these mitochondria behave similarly to vertebrate mitochondria with regard to the properties of their electrochemical proton gradient. When L. donovani promastigotes were permeabilized with digitonin in a reaction medium containing MgATP, succinate and 3.5 microM free Ca2+, they lowered the medium Ca2+ concentration to the submicromolar level (0.05-0.1 microM). The presence of 1 microM-FCCP decreased by about 75% the initial rate of Ca2+ sequestration by these permeabilized cells. This FCCP-insensitive Ca2+ uptake, probably by the endoplasmic reticulum, was completely inhibited by 500 microM-vanadate. On the other hand, when vanadate instead of FCCP was present, the initial rate of Ca2+ accumulation was decreased by about 25% and the Ca2+ set point was increased to 0.7 microM. The succinate-dependence and FCCP-and Ruthenium Red-sensitivity of the Ca2+ uptake detected in the presence of vanadate indicate that this uptake is probably by the mitochondria. This interpretation was further supported by the Ruthenium Red-sensitive decrease in the mitochondrial membrane potential caused by Ca2+ addition. The anti-leishmanial cationic drugs pentamidine and WR-6026 also induced a rapid collapse of the mitochondrial inner membrane potential of L. donovani promastigotes.


Thrombospondin receptor expression in human neutrophils coincides with the release of a subpopulation of specific granules.

The extracellular matrix (ECM) protein thrombospondin (TSP) binds specifically to polymorphonuclear leucocyte (PMN) surface receptors and promotes cell adhesion and motility. TSP receptor expression increases 30-fold after activation with the synthetic chemotactic peptide, N-formylmethionyl-leucylphenylalanine (FMLP) or the Ca2+ ionophore A23187, in combination with cytochalasin B. The expression of TSP receptors was correlated with the exocytosis of both specific and azurophil granules. Newly expressed TSP receptors are not derived from easily mobilized specific granules since agents that trigger some specific granule release [phorbol myristate acetate (PMA), FMLP or ionophore A23187 alone] do not increase TSP receptor expression. In this study we used the anion-channel blocker, 4,4'-di-isothiocyanatostilbene-2,2'-disulphonic acid (DIDS) to investigate the source of these newly expressed receptors. When PMNs were exposed to cytochalasin B and FMLP or to cytochalasin B and ionophore A23187 in the presence of 30-100 microM-DIDS, TSP receptor expression increased coincidently with vitamin B12-binding protein release from specific granules. Under these same conditions, the release of the azurophil granule component, myeloperoxidase, was significantly inhibited. Using agonists that cause release of specific granules, or both specific granules and azurophil granules, we determined that DIDS blocked the release of PMA-mobilized specific granules and cytochalasin B plus FMLP- or cytochalasin B plus ionophore A23187-mobilized myeloperoxidase-containing azurophil granules but not specific granules mobilized by cytochalasin B plus FMLP or cytochalasin B plus ionophore A23187. These results suggested that PMNs contain at least two subpopulations of specific granules: one that is easily mobilized, lacks TSP receptors and is inhibitable by DIDS, and one that is difficult to mobilize, contains a large pool of TSP receptors and the release of which is enhanced in the presence of DIDS.


Cell cycle progression is associated with distinct patterns of phosphorylation of Op18.

Op18 is a highly conserved major cytosolic phosphoprotein which has been implicated in signal transduction in a wide variety of cell types. Freshly isolated peripheral blood lymphocytes (PBL) constitutively express low levels of mostly unphosphorylated Op18. Following mitogenic stimulation of PBL, Op18 synthesis is induced at a time when cells are entering S-phase. In this study we have characterized Op18 phosphorylation during progression of freshly isolated PBL through the cell cycle. Transition from G0 to G1 following activation with OKT3 was associated with an increase in a phosphorylated form designated Op18c. Progression of cells through G1 into S resulted in an increase in phosphorylated Op18 forms, designated Op18a and Op18b, which paralleled new Op18 synthesis. Transition of cells into G2 + M resulted in the appearance of the more acidic phosphorylated forms Op18d and Op18e. Calphostin C, a specific inhibitor of protein kinase C, dramatically decreased all forms of phosphorylated Op18 in OKT3 treated Jurkat cells. Our results suggest that Op18 phosphorylation is mediated in part by PKC activation as well as by other kinases yielding different phosphorylated forms at specific stages of the cell cycle.


Formation of salivary-mucosal pellicle: the role of transglutaminase.

The present investigation was carried out to identify salivary components of mucosal pellicles in vivo and explore further the mechanism of interaction between salivary molecules and buccal epithelial cells. By using specific antisera and immunoprotein blotting, high-(MG1) and low-(MG2) molecular-mass salivary mucins, amylase, salivary cystatins and proline-rich proteins were detected within mucosal pellicle in vivo. In addition, the data indicated that the mucins and proline-rich proteins could be cleaved into lower-molecular-mass products, whereas the proline-rich proteins could also be cross-linked into higher-molecular-mass complexes. The role of buccal epithelial cell transglutaminase in these interactions was further studied by utilizing purified iodinated amylase, neutral cystatin SN and acidic proline-rich proteins 1 and 3 (APRP1 and 3). After incubation with buccal epithelial cells in vitro 125I-labelled APRPs appeared to undergo a greater degree of cross-linking than 125I-labelled cystatin SN, as determined by SDS/PAGE/autoradiography. Amylase did not appear to be cross-linked at all. Recovery of 125I-labelled APRPs and 125I-labelled cystatin SN with epithelial cell envelopes after repeated extraction suggested that both molecules were cross-linked to envelope proteins, but that 125I-labelled APRPs were cross-linked to a greater degree than 125I-labelled cystatin SN. Cross-linking in buccal epithelial cell preparations was inhibited by an excess of methylamine hydrochloride, a transglutaminase substrate. In a further assessment of amylase, cystatin and APRPs as transglutaminase substrates, only APRP3 and a partially purified preparation of APRPs acted as an amine acceptor for the cross-linking of [14C]methylamine by purified transglutaminase, as determined by SDS/PAGE/fluorography. This reaction was completely inhibited by excess EDTA. The combined data from this study suggest that during mucosal pellicle formation multiple components of saliva adsorb to buccal epithelial cell surfaces, and that, within this group, selected components are enzymically cross-linked by an epithelial transglutaminase and/or proteolytically cleaved into smaller fragments.


Isolation of a human cDNA encoding a 25 kDa FK-506 and rapamycin binding protein.

Recently, the nearly complete peptide sequence of a 25 kDa rapamycin and FK-506 binding protein that had been isolated from calf thymus, brain, and spleen was reported (1). Based upon the amino acid sequence of this bovine protein, bFKBP25, we have isolated from a JURKAT cDNA library the cDNA encoding the human homolog, hFKBP25. Translation of the open reading frame contained within this cDNA clone yields a sequence that, in its C-terminal half, is 41% identical to the major human FK-506 binding protein, hFKBP12, and 43% identical to hFKBP13. The N-terminal half of hFKBP25 is unrelated to any known protein.


HIV-1 and HIV-2 reverse transcriptases: a comparative study of sensitivity to inhibition by selected natural products.

One hundred and fifty six pure natural products, which had previously been tested against HIV-1 reverse transcriptase, were evaluated for HIV-2 reverse transcriptase inhibitory activity. Compounds that lacked effect in the HIV-1 reverse transcriptase system were found also to be inactive against HIV-2 reverse transcriptase. However, compounds belonging to the benzophenanthridine and protoberberine classes of alkaloids, certain flavonoids, the iridoid, fulvoplumierin, and the ansamycin antibiotic, daunomycin, exhibited similar potencies in both enzyme systems. In contrast, HIV-2 reverse transcriptase was observed to be four-fold more sensitive toward the inhibitory effects of the ipecac alkaloids, O-methylpsychotrine sulfate heptahydrate and psychotrine dihydrogen oxalate. Such differences in susceptibilities to inhibitors may indicate subtle dissimilarities in enzyme structure and function.


High cell density-dependent resistance and P-glycoprotein-mediated multidrug resistance in mitoxantrone-selected Chinese hamster cells.

Mitoxantrone (MIT) resistance has been studied in a colony selected from the CHO AA8 parental line in one step under a low degree of selective pressure (9 nM). The cells of the clonal isolate AA8/MIT C1(0) were sensitive to 9 nM MIT at low cell density but able to grow at high density. Parental AA8 cells were not able to grow under the latter condition. Decreased MIT accumulation (-20%) was observed at this step (step 0) in the absence of overexpression of mdr RNA coding for the drug efflux pump P-glycoprotein. Furthermore, AA8/MIT C1(0) did not exhibit cross resistance to vincristine, Adriamycin and etoposide at low cell density. During subsequent controlled growth for 2 months at high cell density in the presence of 9 nM drug, an additional selection occurred leading to a 4-fold MIT-resistant subline AA8/MIT C1(+). This subline was characterized at this step (step I) and after an additional 4 months of culture in the presence of 9 nM MIT (step II). Analysis of mdr gene expression and gene copy number showed an increase in mdr RNA and a pattern of mdr gene amplification which changed between step I and II. AA8/MIT C1(+)II exhibited a classical multidrug resistance phenotype with decreased accumulation of [14C]MIT and cross-resistance to vincristine, Adriamycin and etoposide. The ability to form the cleavable complex in the presence of etoposide in DNA topoisomerase II-containing nuclear extracts was identical in AA8/MIT C1(+)II and AA8 cell lines. These results demonstrate a new sequence of events in MIT resistance: low level of drug resistance at high cell density followed by mdr gene amplification.


Further characterization of reversal of signs of induced cotton effects of dicumarol derivatives-alpha 1-acid glycoprotein systems by protriptyline.

The interaction of dicumarol derivatives and protriptyline with respect to the binding to alpha 1-acid glycoprotein (AGP) has been investigated by circular dichroism (CD), equilibrium dialysis and ultrafiltration. Investigation of the induced CD spectra of dicumarol derivatives bound to AGP indicated that the conformations of these compounds were different when bound to AGP. Though all the dicumarol derivatives, protriptyline and AGP formed a ternary complex, interaction modes were different, depending upon the substituent groups at position 3 of the dicumarol molecule. On the basis of the protriptyline effect on the CD spectra of all dicumarol derivatives bound to AGP, the compounds were classified in the following way: (1) Dicumarol, ethylidenebis 4-hydroxycoumarin and propylidenebis 4-hydroxycoumarin caused reversal of the sign of ellipticity. This interaction was explained by cooperative binding. (2) Butylidenebis 4-hydroxycoumarin and pentylidenebis 4-hydroxycoumarin generated new band and disappeared ellipticity of the original Cotton effect. This interaction was also explained by the cooperative binding mode. (3) Ethylbiscoumacetate which generated the CD band similar to that of dicumarol in the absence of protriptyline, reversed the sign of the CD spectrum only at 325 nm. The interaction was anticooperative in nature. (4) Benzylidenebis 4-hydroxycoumarin represented type four which had no change in the CD spectrum by the addition of protriptyline. This interaction was explained by the two-state model accompanying the conformational change of AGP. These results suggested that all compounds, except for benzylidenebis 4-hydroxycoumarin, induced negative Cotton effects at 325 nm by taking the same asymmetrical perturbation by the addition of protriptyline and the interaction was carried out according to model 2. An attempt to study the interaction mechanism of two or more drugs with regard to the binding to protein using these models is thought to help in understanding drug-protein interactions.


CD7- T cells in rheumatoid arthritis.

Rheumatoid arthritis (RA) is characterized by decreased expression of CD7 in the peripheral blood and in the synovium. The present study was designed to identify the basis for and functional consequences of this decreased expression. Peripheral blood lymphocytes from normal controls and from patients with RA or systemic lupus erythematosus (SLE), and T cell lines derived from rheumatoid synovium, were evaluated using 3-color fluorescence-activated cell sorter analysis. Normal subjects and most SLE patients expressed homogeneous, bright CD7 on CD4+, CD45RA+ cells, whereas RA patients demonstrated a significantly increased proportion of CD7- cells. T cell lines derived from rheumatoid synovium demonstrated a striking deficiency of CD7 on CD4+, CD45RA- cells. CD4+, CD45RA+ cells from RA patients changed phenotype after in vitro activation to CD45RA negativity, with up-regulation of CD7. CD7-, CD4+, CD45RA- cells were assessed for their ability to induce pokeweed mitogen-driven IgM and IgM-rheumatoid factor synthesis, and they were found to be potent helper/inducer cells. An increased population of CD7-, CD4+ cells in peripheral blood was found to predict a low response to recall antigens. The low expression of CD7 in RA may explain some of the immune abnormalities which may contribute to the pathogenesis of this disease.


Phenotypic and functional similarities between 5-azacytidine-treated T cells and a T cell subset in patients with active systemic lupus erythematosus.

Antigen-specific CD4+ T cells treated with DNA methylation inhibitors become autoreactive, suggesting a novel mechanism for autoimmunity. To test whether this mechanism might be involved in systemic lupus erythematosus (SLE), phenotypic markers for the autoreactive cells were sought. Cloned normal T cells were treated with the DNA methylation inhibitor 5-azacytidine (5-azaC) and studied for altered gene expression. T cells from patients with active SLE were then studied for a similar change in gene expression, and cells expressing the marker were tested for autoreactivity. 5-azaC-treated normal T cells had increased CD11a (leukocyte function-associated antigen 1 alpha) expression relative to other membrane molecules. A T cell subset with similar CD11a expression was found in patients with active SLE. This subset contained cells that spontaneously lysed autologous macrophages, with a specificity similar to that of 5-azaC-treated cells.


Restricted epitope recognition by precipitin-negative anti-La/SS-B-positive sera.

To determine whether anti-La/SS-B-positive sera that are precipitin negative show a distinct B cell epitope pattern. Serum reactivity was tested with recombinant La/SS-B fusion proteins. Among the 18 precipitin-negative anti-La/SS-B-positive sera, reactivity was confined to the full-length recombinant protein (La33.3) in 8 (44%); 5 of 18 (28%) reacted only with La33.3 and with the first 107 N-terminal amino acids (LaA), and 4 (22%) reacted with La33.3, LaA, and the middle region of the La molecule (LaC; amino acids 111-242). One serum reacted with La33.3 and LaC. None of the 18 precipitin-negative sera was positive on a carboxy-terminal fragment (LaL2/3; amino acids 346-408). In contrast, all 26 precipitin-positive anti-La/SS-B-positive sera reacted with La33.3, LaA, and LaC, and 92% reacted with LaL2/3. Rheumatoid factor and serum IgG levels were significantly lower in the precipitin-negative group, providing further evidence of a distinct serologic subset. The restricted epitope recognition by these sera may explain the lack of precipitin formation and may represent an early autoantibody response to La/SS-B.


Lymphokine-activated killer cell and natural killer cell activities in patients with systemic sclerosis.

To determine the ability of T lymphocytes and natural killer (NK) cells from patients with systemic sclerosis (SSc) to respond to cytokines and to generate immune effector cells. The numbers and percentages of peripheral blood T and NK cells were examined by 2-color flow cytometry, and NK and lymphokine-activated killer (LAK) cell function were measured in 4-hour 51Cr-release assays, in 34 patients with SSc. The patients were categorized into 3 subgroups: 10 had diffuse cutaneous disease of less than or equal to 3 years disease duration, 11 had diffuse cutaneous SSc of greater than 3 years duration, and 13 had limited cutaneous disease. Baseline and activated NK and T cell numbers and NK activity were normal in SSc patients. However, mean LAK activity was significantly depressed in all SSc subgroups. Decreased LAK cell function, despite normal numbers of circulating T and NK cells, indicates that SSc patients have poor ability to produce effector cells in response to interleukin-2.


Effect of saffron on cell colony formation and cellular nucleic acid and protein synthesis.

A concentrated extract of saffron was prepared from the flowers of Crocus sativis. The effect of this extract on the ability of HeLa cells to form colonies, and on cellular DNA, RNA and protein synthesis was examined. Incubation of cells with extract for 3 h resulted in significant inhibition of colony formation and cellular nucleic acid synthesis with 50% inhibition at concentrations of approximately 100-150 micrograms/ml. In contrast there was no inhibition of cellular protein synthesis at concentrations of extract as high as 400 micrograms/ml.


Reduced-size liver transplantation for infants and children.

Pediatric liver transplantation, although successful, was limited by the lack of available size-matched organs. Up to 25% to 50% of children died on the waiting list. RLT was initiated and developed to help alleviate donor shortage. Over time, RLT has proven to be an acceptable option for children requiring hepatic transplantation. In experienced centers survival rates are comparable to OLT, and long-term organ function is equally good. Future application of this technique may also provide more innovative approaches to pediatric liver transplantation.


Rapid differentiation of Pseudomonas pseudomallei from Pseudomonas cepacia.

The Minitek disc system was utilized for the differentiation of Pseudomonas pseudomallei, the causative agent of melioidosis, from Ps. cepacia. The system was simple to use, inexpensive, and furnished rapid, clear-cut test results after 4 h. This procedure is suitable for differentiating soil bacteria presumptively identified as Ps. pseudomallei, Ps. cepacia or flavobacteria, and for the rapid confirmation of the presumptive identification of either Ps. pseudomallei or Ps. cepacia obtained by commercial identification-kit systems in the clinical laboratory.


Modification of cell proliferation with inhibitors.

Recent developments on mechanisms that control cell multiplication, using molecular biology, are renewing interest in inhibitors and activators. A great deal of information has been gained in the past through the use of chemicals that modify passage through the cell cycle. The kinds of inhibitors, their sites of action that disrupt functions essential for proliferation, their usefulness in synchronizing cultures and, importantly, their therapeutic value, have been the subject of many investigations.


Selectins and other mammalian sialic acid-binding lectins.

Several recently discovered mammalian cell adhesion proteins recognize and bind to sialic acid-containing ligands. Reports concerning the molecular specificities of these interactions have been intriguing but somewhat confusing, partly because of pitfalls in methodology or interpretation. Nevertheless, these protein-carbohydrate recognition phenomena are important in the normal biology of blood cells and in the pathophysiology of many diseases.


Immunochemical characterization of Citrobacter strain PCM 1487 O-specific polysaccharide- and core oligosaccharide-protein conjugates.

O-specific-polysaccharide and core oligosaccharide were isolated from Citrobacter strain PCM 1487 lipopolysaccharide and purified. The polysaccharide was selectively devoid of 4-deoxy-D-arabinohexose residues. Covalent conjugates of the modified O-specific polysaccharide and of the core oligosaccharide with tetanus toxoid were prepared. Immunochemical characterization of these conjugates proved that they are strong immunogens. Using monospecific rabbit antisera raised against the conjugates, the antigenic relationships between lipopolysaccharides of various strains of Citrobacter, Shigella sonnei, and Shigella flexneri were studied by quantitative microprecipitin and quantitative microprecipitin inhibition and immunoblotting tests.


[Measurement of the distance among CK-4, Hox 3.1 and Hox 3.3 genes on mouse chromosome 15 by pulse field gel electrophoresis].

Pulse field gel electrophoresis (PFGE) and Southern hybridization analysis were used to measure the maximum distance between the mouse type II cytokeratin-4 gene and the homeobox-containing gene Hox 3.3 to be 130kb, and that between Hox 3.3 and Hox 3.1 genes to be 50kb. Ck-4, Hox 3.1 and Hox 3.3 genes are mapped on mouse chromosome 15, and the order of loci is CK-4-Hox 3.3-Hox 3.1. A restriction map of Mlul, Notl, SacIl, Sal1 and Sfil restriction enzymes on a DNA fragment of 1,100 kb in length on which those three genes located was constructed.


Effectiveness of two palliative support teams.

The palliative care of 227 consecutive patients by two support teams was measured according to 17 key indicators in the Support Team Assessment Schedule (STAS), an instrument previously developed and validated for use in these settings. Mean time in care was 71 days (range 1-547); 56 per cent of patients died at home, 26 per cent in hospital, 18 per cent in a hospice. Totalled ratings (sum of 15 items, excluding two items owing to missed ratings) improved in 83 per cent of cases, remained unchanged in 3 per cent and deteriorated in 13 per cent. The main problems which the STAS identified at referral were family anxiety, symptom control, patient anxiety and communication between patient and family. Fifteen of the 17 items showed significant improvements (Wilcoxon Z ranged from -3.18 to -8.20, p less than 0.00005) between referral ratings and ratings for the last week of the patient's life; family anxiety and spiritual needs did not. Patient anxiety and symptom control, although improved, also remained relatively severe at death. These results demonstrate the value of measuring key indicators and indicate areas where improvement in palliative care is needed.


Monoclonal antibodies to the C4 region of human immunodeficiency virus type 1 gp120: use in topological analysis of a CD4 binding site.

We have raised antisera and monoclonal antibodies (MAbs) to the C4 region of HIV-1 gp120, using an antigen chimaera of poliovirus as immunogen. These MAbs and sera, together with MAbs to the same region raised by other methods, fall into three groups defined by their abilities to bind to recombinant gp120 and/or the immunogenic peptide. In some cases, the amino acids recognized by the MAbs have been identified by pep-scan and by solution phase peptide inhibition of binding to recombinant gp120. Our results indicate that the amino acids WQEVGKAMYA are exposed on the surface of recombinant gp120. Antibodies to these amino acids on recombinant gp120 compete for soluble CD4 binding in vitro, but only weakly neutralize HIV.


Synergistic neutralization of HIV-1 by human monoclonal antibodies against the V3 loop and the CD4-binding site of gp120.

Two distinct regions or epitope clusters of human immunodeficiency virus type 1 (HIV-1) gp120 have been shown to elicit neutralizing antibodies: the V3 loop and the CD4-binding site. We have isolated neutralizing human monoclonal antibodies (HuMAbs) against conserved epitopes in both of these regions. In this study, we demonstrate that an equimolar mixture of two of these HuMAbs, one directed against the V3 loop and the other against the CD4-binding site, neutralizes HIV-1 at much lower concentrations than does either of the individual HuMAbs. Mathematical analysis of this effect suggests cooperative neutralization of HIV-1 by the two HuMAbs and demonstrates a high level of synergy, with combination indices (CIs) of 0.07 and 0.16 for 90% neutralization of the MN and SF-2 strains, respectively. The dose reduction indices (DRIs) for each of the two HuMAbs at 99% neutralization range approximately from 10 to 150. A possible mechanism for this synergism is suggested by binding studies with recombinant gp160 of the MN strain; these show enhanced binding of the anti-CD4 binding site HuMAb in the presence of the anti-V3 loop HuMAb. These results demonstrate the advantage of including both V3 loop and CD4-binding site epitopes in a vaccine against HIV-1 and indicate that combinations of HuMAbs against these two sites may be particularly effective in passive immunotherapy against the virus.


HIV-1 env, nef, and gag-specific T-cell immunity in mice: conserved epitopes in nef p27 and gag p25 proteins.

Cellular immunogenicity of env gp160, nef p27, and gag p55 proteins of human immunodeficiency virus type 1 (HIV-1) was studied in mice immunized with vaccinia virus recombinants. Proliferative responses of spleen cells were comparable against env gp160, nef p27, and gag p25 recombinant proteins. No specific activity was observed against gag p18 protein. Env, nef, and gag-specific T-cell lines were generated by repeated stimulation of immune spleen cells with recombinant HIV-1 proteins. They were CD4 positive, proliferative, and also cytotoxic against HIV-transfected target cells. Specificity of the T-cell response against nef and gag protein was analyzed with synthetic peptides. Peptides nef 15, nef 16, and gag AM-30 were, respectively, reactive in nef- and gag-specific proliferative and cytolytic assays. The three peptides described have a relatively conserved amino acid sequence among HIV isolates and appear broadly immunoreactive among species.


Increased risk for lung cancer and for cancer of the gastrointestinal tract among Geneva professional drivers.

A historical prospective cohort study of 6630 drivers from the Canton of Geneva was carried out to evaluate mortality and incidence of cancer in this occupation. The study population was all men (of all vocations) who held in 1949 a special licence for driving lorries, taxis, buses, or coaches and all new licence holders in the period 1949-61. Men born before 1900 and those with only an ordinary driving licence were excluded. According to the occupation registered on their licence, the 6630 drivers were distributed into three groups: (1) professional drivers (n = 1726), (2) non-professional drivers "more exposed" to exhaust gas and fumes (this group included occupations such as vehicle mechanic, policeman, road sweeper; n = 712), and (3) non-professional drivers "less exposed," composed of all other occupations (n = 4192). The cohort was followed up from 1949 to December 1986 and the trace of 197 men (3%) was lost. Compared with the general population of the Canton of Geneva, professional drivers experienced significant excess risks, taking into account 15 years of latency, for all causes of death (standardised mortality ratio (SMR) 115, 90% confidence interval (90% CI) 107-123) and for all malignant neoplasms (SMR 125, 90% CI 112-140; standardised incidence ratio (SIR) 128, 90% CI 115-142). Cause specific analysis showed significant excesses for lung cancer (SMR 150, 90% CI 123-181; SIR 161, 90% CI 129-198), oesophageal cancer (SMR 183, 90% CI 108-291), stomach cancer (SMR 179, 90% CI 117-263; SIR233, 90% CI 156-336), rectal cancer (SMR 258, 90% CIU 162-392; SIR 200, 90% CI 127-300), and cirrhosis of the liver (SMR 145, 90% CI 104-198). Risk of lung cancer increased significantly with time from first exposure. Among non-professional drivers no significant excess risk was found except for lung cancer mortality among the "less exposed" group (SMR 121, 90% CI 103-140), and for incidence of lung cancer among the "more exposed" group (SIR 161, 90% CI 111-227). The possible casual relation between exposure to engine exhaust emissions and the increased risk for lung cancer and for cancer of the gastrointestinal tract found among professional drivers is discussed.


Molecular cloning of a differentiation-related mRNA in the adipogenic cell line 1246.

The 1246 cell line is a C3H mouse teratoma-derived adipogenic cell line that can proliferate and differentiate in defined medium. We have constructed a recombinant phage library containing complementary DNAs (cDNAs) prepared from mRNA of differentiated 1246 cells. This library was screened using a differential hybridization technique. We have isolated five different cDNA clones corresponding to mRNAs that are induced during adipogenesis of 1246 cells and one cDNA clone corresponding to mRNA that is decreased during adipogenesis. Among the mRNAs expressed during adipose differentiation, some are not expressed in undifferentiated cells, whereas some are expressed at very low levels under these conditions. Moreover, the level of induction during differentiation and the temporal expression of the mRNAs corresponding to these cDNAs varied. Our results indicate that one of the cDNA clones isolated, called 154, which selects a 2.2-kilobase mRNA, was induced 100-fold at a very early time during the onset of the differentiation program in 1246 cells and also in adipocyte precursors in primary culture. Direct sequencing of 154 cDNA insert revealed no homology with sequences in GenBank and PIR protein databases. The expression of 154 mRNA was stimulated by accelerators of differentiation such as dexamethasone and isobutylmethylxanthine and inhibited by tumor necrosis factor alpha, transforming growth factor beta, and epidermal growth factor, which are known inhibitors of 1246 cell differentiation. In addition, 154 mRNA level in adipocytes was down-regulated by tumor necrosis factor alpha, but not by transforming growth factor beta or epidermal growth factor. These results suggest that the increase in 154 mRNA expression is related to the onset of adipose differentiation. Further analysis of this clone should allow characterization of a novel protein induced early during the process of differentiation.


The int-2 gene product acts as a growth factor and substitutes for basic fibroblast growth factor in promoting the differentiation of a normal mouse mammary epithelial cell line.

We have investigated the effect of basic fibroblast growth factor (bFGF) and the related int-2 gene on the growth, transformation, and differentiation of HC11 mouse mammary epithelial cells. We show that in HC11 cells infected with int-2 retroviral expression vectors, the int-2 protein can function as a bFGF-like growth factor in stimulating: (a) HC11 cell proliferation in monolayer, (b) anchorage-independent growth in soft agar, and (c) soft agar growth of the bFGF-responsive SW13 tumor cell line. These effects are observed irrespective of whether the int-2 protein is expressed in its wild-type form or is linked to a signal peptide. A candidate bFGF receptor, which is the product of the flg gene and which may recognize the int-2 protein, is expressed at high levels in HC11 cells. Following epidermal growth factor or bFGF priming and subsequent treatment with lactogenic hormones, all of the int-2 infected and the parental HC11 cells synthesize similar levels of beta-casein. However, the autocrine expression of int-2 in HC11 cells abrogates their requirement for either exogenous epidermal growth factor or bFGF priming. These data suggest that, in HC11 cells, the growth factor activity of the int-2 gene is indistinguishable from that of bFGF and does not interfere with the mammary cell differentiation program associated with lactogenesis.


Changes of epidermal cell morphology and keratin expression induced by inhibitors of protein kinase C.

Several lines of evidence show protein kinase C as being involved in various regulatory processes in keratinocyte biology, e.g. proliferation and differentiation. In the present study, we investigated the effects of three different inhibitors of protein kinase C, staurosporine, CP 46'665-1, and tiflucarbine, on cell morphology and keratin expression in a non-tumorigenic human keratinocyte cell line (HaCaT cells). Staurosporine, being the most potent inhibitor of protein kinase C activity in vitro, and CP 46'665-1 induced morphological transformation to a fibroblast-like cell shape. In contrast, no changes in cell morphology were observed after exposure to tiflucarbine. The investigation of keratin expression in HaCaT cells grown in the presence of the different compounds revealed the following changes: After 72 h of cultivation, keratins 8 and 18 were still expressed in treated cells, whereas expression of keratin 13 was decreased as compared to control cells. Immunoblotting to detect vimentin demonstrated its absence in treated and control cells. Since tiflucarbine is known as a dual protein kinase C/calmodulin inhibitor whereas staurosporine and CP 46'665-1 do not antagonize calmodulin function, it might be possible that not only protein kinase C but also calmodulin is involved in the process leading to the morphological changes.


Perfluorocarbon liquid effects on corneal endothelial permeability.

We investigated the effects of perfluoro-n-octane liquid on the rabbit corneal endothelial permeability to inulin and dextran. Permeability measurements were made either after 10 minute in vitro exposure of the endothelium to 50 microliters of the test liquid or after one week exposure in vivo following injection of 50 microliters of the test fluid into the anterior chamber. Retention of the perfluorocarbon on the in vitro cornea and in the anterior chamber during the appropriate exposure time was visually verified. The corneal endothelial permeability was unchanged after either short or sub-chronic treatment with perfluoro-n-octane indicating an absence of toxicity for this substance should it reach the cornea either intraoperatively or postoperatively.


An unusual coloured compound in digests of senile nuclear cataract lens proteins.

An unusual pink compound has been isolated from the proteolytic digests of senile nuclear cataract proteins by adsorption onto Sephadex G-10. The tiny amounts obtained precluded definitive structural elucidation, however TLC comparisons show that it resembles triphenodioxazine-1,8-dicarboxylic acid, a pink heterocyclic chemical formed by oxidative condensation of 3-hydroxyanthranilic acid.


Helical epitope of the group B meningococcal alpha(2-8)-linked sialic acid polysaccharide.

The immunological properties of the group B meningococcal alpha(2-8)-linked sialic acid polysaccharide have been rationalized in terms of a model where the random coil nature of the polymer can be described by the presence of local helices. The conformational versatility of the alpha NeuAc(2-8)alpha NeuAc linkage has been explored by NMR studies at 600 MHz in conjunction with potential energy calculations for colominic acid, an alpha(2-8)NeuAc polymer, and the trisaccharide alpha NeuAc(2-8)alpha NeuAc(2-8)beta NeuAc. Potential energy calculations were used to estimate the energetically favorable conformers and to describe the wide range of helices which the polymer can adopt. No unique conformer was found to satisfy all NMR constraints, and only ensemble averaged nuclear Overhauser enhancements could correctly simulate the experimental data. Conformational differences between the polymer and the trisaccharide could be best explained in terms of slight changes in the relative distribution of conformers in solution. Similar helical parameters for the alpha(2-8)NeuAc polymer and poly(A) were proposed as the basis for their cross-reactivity to a monoclonal antibody IgMNOV. The unusual length dependency for binding of oligosaccharide to group B specific antibodies was postulated to arise from the recognition of a high-order local helix with an extended conformation which was not highly populated in solution.


Activation of the collagen-binding of endogenous serum vitronectin by heating, urea and glycosaminoglycans.

The present study describes that the collagen-binding activity of vitronectin in human serum increases by treatment with heparin, heating and urea. Vitronectin purified from human serum was bound to native collagen, whereas endogenous vitronectin in the serum was not. We have examined the conditions to change the collagen-binding activity of endogenous vitronectin. Endogenous vitronectin in human serum became considerably bound to collagen when the serum was boiled in 4-8 M urea for 5 min and mixed with heparin (0.5-5 micrograms/ml). Each treatment of heating, urea or heparin alone, and any combination of the two factors, inefficiently activated the binding. Dextran sulfate could substitute for heparin, but dermatan sulfate, keratan sulfate, chondroitin sulfate A and C, heparan sulfate and hyaluronan could not. Possible explanations for the activation of endogenous vitronectin are discussed.


Molecular cloning and characterization of mouse mast cell chymases.

Mouse mast cell chymases are granule-associated serine proteinases with chymotrypsin-like substrate specificities. cDNAs for two new chymases were isolated from a cDNA library constructed using mRNA from ABFTL-6 mouse mast cells by screening with a rat mast cell proteinase cDNA. The deduced amino acid sequence of mouse chymase 1 consists of a 226 amino acid catalytic portion and a 21 amino acid preprosequence. Chymase 1 is unusual in that an Asn occurs in the substrate binding pocket, a feature that has not been observed in any other serine proteinase. Also, chymase 1 is expected to have a large positive charge (+13) at physiological pH. A partial cDNA for chymase 2 encodes 177 residues of the carboxy terminal portion of a second proteinase distinct from chymase 1. Chymase 2 cDNA contains a highly conserved intron/exon junction, a high positive charge (+17) and a novel, second potential N-glycosylation site. Transcripts for both chymases are found in ABFTL-6 mast cells, but only chymase 2 mRNA is in mouse connective tissue mast cells. These data suggest that these chymases have distinct enzymatic properties and tissue-specific patterns of gene expression.


Sequential and combined G-banding for identifying breakpoints in sister chromatid exchanges.

A simple new method is described for obtaining sequential and a combination of differential sister chromatid staining and G-banding in the same metaphase. Using this method the sister chromatid exchanges and chromosome lesion breakpoints can be precisely localized in particular bands of individual chromosomes.


Quality assurance and standardization in immunohistochemistry. A proposal for the annual meeting of the Biological Stain Commission, June, 1991.

Quality assurance, quality control, proficiency testing, reagent documentation and validation are standard parts of everyday practice in clinical laboratories throughout the United States. Immunohistochemical stains employ reagents and principles in common with immunoenzyme methods utilized in the clinical laboratory. However, immunohistochemistry has not routinely been subjected to similar standardization and quality assurance procedures that manufacturers and pathologists alike have applied to essentially the same techniques in the clinical laboratory environment. The current proposal was invited by the Biological Stain Commission with the charge of incorporating the findings of previous workshops on quality control in immunohistochemistry into a practical design for implementation. The status of quality assurance, quality control and standardization in immunohistochemistry is reviewed and a phased strategy for implementation is proposed.


A two step stain for normal and leukemic monocytes using two different dyes applied in sequence.

A staining procedure for monocytes in specimens of blood and bone marrow was developed. The technique was a two step procedure in which unfixed cells were exposed first to a methanolic solution of C.I. basic blue 54. Next, an aqueous alkaline buffered solution of C.I. basic blue 141 was added to the first staining solution. After staining for 10 min in the solution with two stains, slides or coverslips were washed for 5 sec in pH 5.6 phosphate buffer and drained dry. The cytoplasm of monocytes stained intensely deep purple and frequently nuclei were stained red. Similar staining was not found in other types of normal or abnormal blood and bone marrow cells.


Simultaneous measurement of DAPI-sulforhodamine 101 stained nuclear DNA and protein in higher plants by flow cytometry.

A 2-step staining procedure is presented for simultaneous measurement of nuclear DNA and protein content in higher plants by flow cytometry. To release nuclei, plant tissues were chopped and stirred in the presence of the DNA specific fluorochrome 4',6-diamidino-2-phenylindole (DAPI) and the nonionic detergent Triton X-100. Plant protoplasts were stirred in the DAPI dye solution with the detergent. After a short incubation period a second dye solution containing DAPI and the protein fluorochrome sulforhodamine 101 (SR 101) without detergent was added. Following another incubation, and after filtration through nylon gauze, the highly fluorescent nuclei were analyzed with an impulse cytophotometer. Accurate bivariate DNA-protein histograms were obtained with CV-values of about 2% or less for the 2C-peak of the univariate DNA parameter. The method presented here can be used for basic and applied cytogenetic studies of higher plants, for characterization of subcompartments of the cell cycle phases, or for examination of heterogeneity in plant tissues.


Unknown G0/G1 cell subpopulation revealed by hydrochloric acid/acridine orange staining.

An unknown cell subpopulation was observed in mouse and rat thymus, spleen and bone marrow cells, as well as in human peripheral blood mononuclear cells (resting and stimulated by PHA) using equilibrium HCl/acridine orange staining. This subpopulation includes cells with decreased green and unchanged red fluorescence. The staining does not affect cells in S- and G2/M-phases. The mechanism and biological meaning of the effect await further investigation.


A modified Hortega-Globus stain is superior to Bielschowsky and Bodian stains for demonstrating neuritic plaques.

The quantitative assessment of the age-dependent number of neuritic plaques is essential for the diagnosis of Alzheimer type dementia. This study reports the superiority of a modified Hortega-Globus stain compared to Bielschowsky and Bodian stains applied to samples obtained from ten brains of patients with a clinical history of progressive dementia. In two of ten cases only the modified Hortega-Globus stain allowed confirmation of the diagnosis of senile dementia of the Alzheimer type (SDAT). The counts of neuritic plaques in sections stained by other methods were not sufficient to establish the histological diagnosis of SDAT. These results indicate that the choice of the most sensitive staining method is critical for the correct histopathologic diagnosis of the Alzheimer type dementia.


Block-surface staining for differentiation of starch and cell walls in wheat endosperm.

A staining technique for differentiating starch granules and cell walls was developed for computer-assisted studies of starch granule distribution in cells of wheat (Triticum aestivum L.) caryopses. Blocks of embedded caryopses were sectioned, exposing the endosperm tissue, and stained with iodine potassium iodide (IKI) and Calcofluor White. Excessive tissue hydration during staining was avoided by using stains prepared in 80% ethanol and using short staining times. The IKI quenched background fluorescence which facilitated the use of higher concentrations of Calcofluor White. Cell wall definition was improved with the IKI-Calcofluor staining combination compared to Calcofluor alone. The high contrast between darkly stained starch granules and fluorescent cell walls permitted computer assisted analysis of data from selected hard and soft wheat varieties. The ratio of starch granule area to cell area was similar for both wheat classes. The starch granule sizes ranged from 2.1 microns 3 to 22,000 microns 3 with approximately 90% of the granules measuring less than 752 microns 3 (ca. 11 microns in diameter). Hard wheat samples had a greater number of small starch granules and a lower mean starch granule area compared to the soft wheat varieties tested. The starch size distribution curve was bimodal for both the hard and soft wheat varieties. Three-dimensional starch size distribution was measured for four cells near the central cheek region of a single caryopsis. The percentage of small granules was higher at the ends than at the mid-section of the cells.


Pre-analytical and biological variability of prostatic acid phosphatase and prostate-specific antigen in serum from patients with prostatic pathology.

We determined the pre-analytical and biological variation of prostatic acid phosphatase and prostate-specific antigen in the same patient samples. Prostatic acid phosphatase and prostate-specific antigen were both stable when stored for at least 3 weeks with acidification (acetate buffer) or without acidification, except for prostate-specific antigen in samples stored unacidified at 4 degrees C. A significant elevation of prostate-specific antigen was noted in four patients with benign prostatic hyperplasia between 1/2 and 6 hours after prostatic massage. No significant effect was shown of changes in the glomerular filtration rate on prostate-specific antigen concentration, in spite of its low molecular mass. The estimate of within-subject biological variation showed a coefficient of variation of 33.8% for prostatic acid phosphatase and 14% for prostate-specific antigen. Desirable analytical imprecisions based on these findings were about 17% for prostatic acid phosphatase and 7% for prostate-specific antigen, these goals being achieved in practice for marker values higher than or equal to the upper reference limit.


Infections and pseudoinfections due to povidone-iodine solution contaminated with Pseudomonas cepacia.

In 1989 we investigated the first instance of Pseudomonas cepacia infections due to intrinsic contamination of a povidone-iodine product. Six patients in a Texas pediatric facility had P. cepacia infection or pseudoinfection (three, peritonitis; one, pseudoperitonitis; and two, pseudobacteremia). Epidemiological studies showed one risk factor for infection of peritoneal fluid with P. cepacia: performance of peritoneal dialysis in the dialysis unit with use of one lot of povidone-iodine later found to be intrinsically contaminated (4/5 vs. 0/16, P = .001). Blood cultures yielded P. cepacia after nurses wiped the tops of blood culture bottles with the povidone-iodine solution before inoculation. P. cepacia was cultured from three povidone-iodine containers used at the hospital and from four containers of the same lot obtained from other health-care facilities in Texas and California. Isolates from patients and the povidone-iodine had similar antibiograms, identical plasmid profiles, and identical DNA banding patterns on the basis of results of ribonucleotide typing. This investigation demonstrates that intrinsic contamination of povidone-iodine solution with P. cepacia can result in infections in addition to colonization and/or pseudoinfection.


Influence of ion occupancy and membrane deformation on gramicidin A channel stability in lipid membranes.

The average lifetime of gramicidin A channels in monoolein/decane bilayer membranes was measured. The results support the hypothesis of channel stabilization by ion occupancy. The effects of electric field and salt concentration are consistent with the expected effects on both occupancy and membrane compression. The lifetime in asymmetric solutions with divalent cation blockers on one side of the membrane shows a voltage dependence such that the lifetime decreases for positive voltages applied from the blocking side and increases for negative voltages. This result strongly supports the occupancy hypothesis. The lifetime increases with permeant ion concentration, and at the one molar level it also increases with voltage. The voltage dependence of lifetime for a low concentration of permeant ion depends on the total salt level. The results for these conditions are consistent with the assumption that membrane compression also influences the lifetime, even for the "soft" solvent-containing membrane considered here. It is proposed that the channel nearest neighbor lipids need not be fixed in a plane at the channel end. Using a liquid crystal model it may then be shown that surface tension is the major component of the membrane deformation free energy, which may explain the significant effects of the membrane compression on the lifetime.


Dynamic ion-ion and water-ion interactions in ion channels.

The dynamic interactions among ions and water molecules in ion channels are treated based on an assumption that ions at binding sites can be knocked off by both transient entering ions and local water molecules. The theory, when applied to a single-site model K+ channel, provides solutions for super- and subsaturations, flux-ratio exponent (n') greater than 1, osmotic streaming current, activity-dependent reversal potentials, and anomalous mole-fraction behavior. The analysis predicts that: (a) the saturation may but, in general, does not follow the Michaelis-Menten relation; (b) streaming current results from imbalanced water-ion knock-off interactions; (c) n' greater than 1 even for single-site channels, but it is unlikely to exceed 1.4 unless the pore is occupied by one or more ion(s); (d) in the calculation involving two permeant ion species with similar radii, the heavier ions show higher affinity; the ion-ion knock-off dissociation from the site is more effective when two interacting ions are identical. Therefore, the "multi-ion behaviors" found in most ion channels are the consequences of dynamic ion-ion and water-ion interactions. The presence of these interactions does not require two or more binding sites in channels.


Constant fields and constant gradients in open ionic channels.

Ions enter cells through pores in proteins that are holes in dielectrics. The energy of interaction between ion and charge induced on the dielectric is many kT, and so the dielectric properties of channel and pore are important. We describe ionic movement by (three-dimensional) Nemst-Planck equations (including flux and net charge). Potential is described by Poisson's equation in the pore and Laplace's equation in the channel wall, allowing induced but not permanent charge. Asymptotic expansions are constructed exploiting the long narrow shape of the pore and the relatively high dielectric constant of the pore's contents. The resulting one-dimensional equations can be integrated numerically; they can be analyzed when channels are short or long (compared with the Debye length). Traditional constant field equations are derived if the induced charge is small, e.g., if the channel is short or if the total concentration gradient is zero. A constant gradient of concentration is derived if the channel is long. Plots directly comparable to experiments are given of current vs voltage, reversal potential vs. concentration, and slope conductance vs. concentration. This dielectric theory can easily be tested: its parameters can be determined by traditional constant field measurements. The dielectric theory then predicts current-voltage relations quite different from constant field, usually more linear, when gradients of total concentration are imposed. Numerical analysis shows that the interaction of ion and channel can be described by a mean potential if, but only if, the induced charge is negligible, that is to say, the electric field is spatially constant.


Large scale rearrangement of protein domains is associated with voltage gating of the VDAC channel.

The VDAC channel of the mitochondrial outer membrane is voltage-gated like the larger, more complex voltage-gated channels of the plasma membrane. However, VDAC is a low molecular weight (30 kDa), abundant protein, which is readily purified and reconstituted, making it an ideal system for analyzing the molecular basis for ion selectivity and voltage-gating. We have probed the VDAC channel by subjecting the cloned yeast (S. cerevisiae) VDAC gene to site-directed mutagenesis and introducing the resulting mutant channels into planar bilayers to detect the effects of specific sequence changes on channel properties. This approach has allowed us to formulate and test a model of the open state structure of the VDAC channel. Now we have applied the same approach to analyzing the structure of the channel's low-conducting "closed state" (essentially closed to important metabolites). We have identified protein domains forming the wall of the closed conformation and domains that seem to be removed from the wall of the pore during channel closure. The latter can explain the reduction in pore diameter and volume and the dramatically altered channel selectivity resulting from the channel closure. This process would make a natural coupling between motion of the sensor and channel gating.


Formation of non-beta 6.3-helical gramicidin channels between sequence-substituted gramicidin analogues.

Using the linear gramicidins as an example, we have previously shown how the statistical properties of heterodimeric (hybrid) channels (formed between the parent [Val1]gramicidin A (gA) and a sequence-altered analogue) can be used to assess whether the analogue forms channels that are structurally equivalent to the parent channels (Durkin, J. T., R. E. Koeppe II, and O. S. Andersen. 1990. J. Mol. Biol. 211:221-234). Generally, the gramicidins are tolerant of amino acid sequence alterations. We report here an exception. The optically reversed analogue, gramicidin M- (gM-) (Heitz, F., G. Spach, and Y. Trudelle. 1982. Biophys. J. 40:87-89), forms channels that are the mirror-image of [Val1]gA channels; gM- should thus form no hybrid channels with analogues having the same helix sense as [Val1]gA. Surprisingly, however, gM- forms hybrid channels with the shortened analogues des-Val1-[Ala2]gA and des-Val1-gC, but these channels differ fundamentally from the parent channels: (a) the appearance rate of these heterodimers is only approximately 1/10 of that predicted from the random assortment of monomers into conducting dimers, indicating the existence of an energy barrier to their formation (e.g., monomer refolding into a new channel-forming conformation); and (b), once formed, the hybrid channels are stabilized approximately 1,000-fold relative to the parent channels. The increased stability suggests a structure that is joined by many hydrogen bonds, such as one of the double-stranded helical dimers shown to be adopted by gramicidins in organic solvents (Veatch, W. R., E. T. Fossel, and E. R. Blout. 1974. Biochemistry. 13:5249-5256).


Mutational analysis of gap junction formation.

The paired oocyte cell-cell channel assay was used to investigate the mechanisms involved in the process of formation of gap junction channels. Single oocytes, injected with connexin-specific mRNAs, accumulate a pool of precursors from which cell-cell channels can form rapidly upon pairing. Several lines of evidence, including immunohistochemistry and surface labeling, indicate that part of this precursor pool is located in the cell membrane, probably in the form of closed hemichannels. The homophilic binding of hemichannels to each other can be mimicked by synthetic peptides representing the extracellular loop sequences of connexin32. The peptides specifically suppress channel formation. A crucial role is established for the six cysteines in the extracellular domains that are conserved in all vertebrate gap junction proteins. Change of any of these cysteines into serines results in absolute loss of function of the mutant connexin. The effects of thiol-specific reagents on channel formation suggest that docking and/or opening of channels involves disulfide exchange. Several of the variable amino acids in the extracellular loop sequences were found to determine specificity of connexin-connexin interactions.


Molecular analysis of voltage dependence of heterotypic gap junctions formed by connexins 26 and 32.

Heterotypic gap junctions formed by pairing Xenopus oocytes expressing hemichannels formed of Cx32 with those expressing hemichannels formed of Cx26 displayed novel transjunctional voltage (Vj) dependence not predicted by the behavior of these connexins in homotypic configurations. Rectification of initial and steady-state currents was observed. Relative positivity and negativity on the Cx26 side of the junction resulted in increased and decreased initial conductance (gj0), respectively. Only relative positivity on the Cx26 decreased steady-state conductance (gj infinity). This behavior suggested that interactions between hemichannels influences gap junction gating. The role of the first extracellular loop (E1) in these interactions was examined by pairing Cx32 and Cx26 with a chimeric connexin in which Cx32 E1 was replaced with Cx26 E1 (Cx32*26E1). Both junctions rectified with gj0/Vj relations that were less steep than that observed for Cx32/Cx26. Decreases in gj infinity occurred for either polarity Vj in the Cx32/Cx32*26E1 junction. Mutation of two amino acids in Cx26 E1 increased the steepness of both the gj0/Vj and gj infinity/Vj relations. These data demonstrate that fast rectification can arise from mismatched E1 domains and that E1 may contribute to the voltage sensing mechanisms underlying both fast and slow Vj-dependent processes.


Threonine in the selectivity filter of the acetylcholine receptor channel.

The acetylcholine receptor (AChR) is a cation selective channel whose biophysical properties as well as its molecular composition are fairly well characterized. Previous studies on the rat muscle alpha-subunit indicate that a threonine residue located near the cytoplasmic side of the M2 segment is a determinant of ion flow. We have studied the role of this threonine in ionic selectivity by measuring conductance sequences for monovalent alkali cations and bionic reversal potentials of the wild type (alpha beta gamma delta channel) and two mutant channels in which this threonine was replaced by either valine (alpha T264V) or glycine (alpha T264G). For the wild type channel we found the selectivity sequence Rb greater than Cs greater than K greater than Na. The alpha T264V mutant channel had the sequence Rb greater than K greater than Cs greater than Na. The alpha T264G mutant channel on the other hand had the same selectivity sequence as the wild type, but larger permeability ratios Px/PNa for the larger cations. Conductance concentration curves indicate that the effect of both mutations is to change both the maximum conductance as well as the apparent binding constant of the ions to the channel. A difference in Mg2+ sensitivity between wild-type and mutant channels, which is a consequence of the differences in ion binding, was also found. The present results suggest that alpha T264 form part of the selectivity filter of the AChR channel were large ions are selected according to their dehydrated size.


Locating a residue in the diphtheria toxin channel.

We are studying structure-function relationships in the Diphtheria Toxin (DT) channel using a combination of site-directed mutagenesis and electrophysiology in artificial lipid bilayers. We made site-directed mutations of charged residues in the toxin's channel-forming B fragment, and after expressing the mutant proteins in Escherichia coli, we analyzed the single channels they formed in lipid bilayers. Changing aspartate 352, which is located in a short hydrophilic loop separating two hydrophobic stretches, to asparagine or lysine dramatically reduces the single-channel conductance of the pore at pH 5.3 cis, 7.2 trans (5.3/7.2). Lowering the pH on both sides of the membrane essentially eliminates the difference between wild-type and D352N; this finding is consistent with the idea that an aspartate with a (protonated) neutral side-chain and the always neutral asparagine have similar electrostatic influences on permeant ions. Using a high concentration of permeant buffer to clamp the pH of the cis compartment and the pore, and varying the pH on the trans side, we have located D352 at or near the trans compartment. We further find that D352N channels, in contrast to wild-type, display conductances independent of trans pH. This observation allows us to determine the titration curve of aspartate 352 in the wild-type toxin, establishing its pKa at approximately 5.5.


The effect of polyimmune gammaglobulin for prophylaxis against reactivation cytomegalovirus infection in kidney and kidney/pancreas transplant recipients.

Cytomegalovirus (CMV) remains the most important infection in the renal transplant recipient. Few data are available that provide guidance for approaches that seek to reduce the reactivation of latent disease after transplantation. To test the efficacy of polyimmune gammaglobulin in kidney and kidney/pancreas transplantation, consenting recipients with serologic evidence of previous CMV disease were randomized to receive i.v. polyimmune gammaglobulin (500 mg/kg) within 3 days of transplant with 250 mg/kg at weeks 1, 2, 4, and 6 or no prophylaxis. Both groups received identical induction and rejection immunosuppressive therapy. Polyimmune gammaglobulin prophylaxis reduced CMV reactivation infections. The incidence of reactivation infections was half in patients receiving Nashville/rabbit antithymocyte serum (N/R-ATS) compared with those receiving monoclonal anti-CD-3 therapy. Patients receiving polyimmune gammaglobulin along with N/R-ATS had an incidence of infection of only 10%. Reactivation infections were twice as common in patients who had primary nonfunction and nearly three times as common in patients with acute rejection. Both risk factors were associated with longer anti-T-cell therapy. Polyimmune gammaglobulin prophylaxis should be considered in transplant patients with previous CMV exposure who will be receiving prolonged anti-T-cell therapy because of acute rejection or primary nonfunction.


Regulation of cultured human mesangial cell growth by ionized macromolecules.

We evaluated the importance of the net charge of polyionic macromolecules in the regulation of cultured human mesangial cell growth. Structurally unrelated polyanionic compounds, i.e., heparin, suramin, poly-L-aspartic acid, and poly-L-glutamic acid, strongly inhibited 10% fetal bovine serum-stimulated cell proliferation. On the other hand, two polycations, protamin sulfate and poly-L-lysine, were equally effective in inhibiting cell growth. The antiproliferative activity of each compound was neutralized by molecules with opposite net charge. These data indicate that both anionic and cationic macromolecules exert an antimitogenic effect on cultured human mesangial cells. This inhibitory effect is dependent upon charge density rather than on the net electric charge of each compound.


Ovarian and hepatic metastases of gastric carcinoma associated with high serum levels of human chorionic gonadotropin (hCG), alpha-fetoprotein (AFP), and carcinoembryonic antigen (CEA): a case report.

A 45-year-old woman who underwent gastrectomy for gastric carcinoma which had metastasized to the liver and ovaries, showed high serum levels of hCG, AFP and CEA. To locate the source, an immunohistochemical technique was utilized. HCG-producing cells were detected in poorly differentiated adenocarcinoma of a primary tumor and an ovarian metastatic site, and AFP-producing cells in poorly differentiated adenocarcinoma forming a medullary pattern of primary site and metastatic foci. CEA-producing cells were found diffused in primary tumor and metastatic foci. From the viewpoint of oncodevelopmental gene expression (Cancer Res 36:3423, 1976), it is interesting that the serum levels of these three tumor markers (hCG, AFP, CEA) were elevated simultaneously.


Granulocyte colony-stimulating factor-producing large cell undifferentiated carcinoma of the lung.

We report a case of granulocyte colony-stimulating factor (G-CSF) producing lung cancer. A 38-year-old Japanese woman had a large cell undifferentiated carcinoma of the left lung with severe granulocytosis without any evidence of infection. A specimen was taken from a metastatic cervical lymph node and a tumor cell line was established. The culture supernatant of the line as well as patient's serum exhibited a high level of G-CSF by sandwich enzyme immunoassay. Immunohistochemical analysis demonstrated that tumor cells from transplanted nude mice were stained granularly in the cytoplasma by anti-human G-CSF monoclonal antibody, 4A6.


Carnitine deficiency: a possible cause of gastrointestinal dysmotility.

An infant with delayed development and peripheral myopathy, nourished on a soy-based liquid diet deficient in carnitine, had gastrointestinal dysmotility manifested by postprandial vomiting, oral drooling, delayed gastric emptying and infrequent bowel movements. Oesophageal manometry showed a reduced lower oesophageal sphincter pressure for age with abnormal distal motility. Serum carnitine concentration was 9.9 mumol l-1. After dietary supplementation of carnitine the gastrointestinal symptoms resolved, oesophageal manometry returned to normal, and serum carnitine increased to 37.2 mumol l-1. Dietary carnitine deficiency in infancy may be a cause of smooth muscle dysmotility of the gastrointestinal tract.


Mild course of cystic fibrosis associated with heterozygosity for infrequent mutations in the first nucleotide-binding fold of CFTR.

The mild clinical course of a patient with cystic fibrosis is presented who inherited the two mutations Gly551----Asp and Arg553----Stop in the cystic fibrosis transmembrane conductance regulator gene. The missense mutation Arg553----Stop discovered in American Blacks is also present on cystic fibrosis chromosomes of Caucasian ancestry.


Cardiac rhythm in healthy elderly subjects.

To study the normal cardiac rhythm in elderly subjects we performed 24-h Holter monitoring on 94 subjects aged over 70 years. We had previously discarded those with cardiac disease by using history, physical examination, electrocardiography (ECG), chest X-radiography and Doppler echocardiography. The maximum, average and minimum heart rates were 113, 79 and 62, respectively, during the day, and 90, 64 and 53 during the night. Supraventricular and ventricular arrhythmias were frequent (91% and 89.4%, respectively). Some 50% of the subjects had complex ventricular arrhythmias. Two subjects presented with sinus pauses of more than 2 s, and 4 had Wenckebach second-degree atrioventricular (AV) block. During a follow-up averaging 20.8 months, there were no deaths or symptoms of an arrhythmic origin.


[Prevalence of hepatitis C virus in poly-transfused patients with hematologic and oncologic diseases].

Patients with hematological and oncological diseases often require intensive, supportive hematotherapy due to the underlying disease or chemo-/radiotherapy. As a consequence, they had an increased transfusion-associated risk of hepatitis C virus infections (non-A, non-B-posttransfusion hepatitis) until 1989-1990, when specific and sensitive HCV antibody tests became available. This is confirmed by our study of 'first' and 'second' generation (1.0 and 2.0) anti-HCV EIAs against structural and non-structural (NS) antigen-determinants. Ten of 101 patients (10.9%) were anti-HCV positive in 2.0 tests. HCV antibodies were detected more often by 2.0 EIAs and the new HCV-immunoblot (4-RIBA), than by 1.0 EIAs. In this respect, the patients' serological HCV profile differs from that of healthy blood donors, which display a prevalence of NS-antibodies.


Comparison of hematopoietic and immune recovery after autologous bone marrow or blood stem cell transplants.

We studied hematopoietic and immune recovery in 40 subjects receiving autologous bone marrow (ABMT) or blood stem cell transplants (ABSCT). Supportive care, transplant-related morbidity, duration of hospitalization and cost were also considered. ABSCT was associated with more rapid recovery of all hematopoietic lineages than was ABMT. However, kinetics of immune recovery were similar between the groups. In the ABSCT group, there was a correlation between numbers of blood progenitor cells infused and the rate of hematopoietic recovery. The accelerated hematopoietic recovery following ABSCT correlated with less morbidity, fewer transfusions, briefer hospitalization and lower cost than ABMT.


[RP 67580, a potent and selective substance P non-peptide antagonist].

The pharmacological properties of 7,7-Diphenyl-2 [1-imino-2 (2-methoxy-phenyl)-ethyl] perhydroisoindol-4-one (3 aR, 7 aR) or RP67580 are described. This compound, derived from a novel chemical family, is a potent and selective substance P (SP) antagonist, in vitro and in vivo. In vitro, it inhibited in a competitive manner (IC50 = 10 nM) 3H-SP binding in rat brain (NK1 receptors). It did not interact with the two other tachykinin receptor sites (NK2 and NK3) nor the other receptor sites tested. Moreover, RP67580 competitively antagonized the contractile activity of SP on guinea-pig ileum (pA2 = 7.16); in contrast, it was inactive in rabbit pulmonary artery and in rat portal vein tissues which contain NK2 and NK3 receptors, respectively. In vivo, in the rat, RP67580 inhibited the plasmatic extravasation induced by administration of SP (ED50 = 0.04 mg/kg i.v.) as well as that induced by antidromic stimulation of a peripheral sensory nerve (ED50 = 0.15 mg/kg i.v.). In mice and rats, RP67580, like morphine, potently blocked the nociceptive effects of phenylbenzoquinone and formalin; its antinociceptive effect does not involve opiate receptors since it was not reversed by naloxone. These results indicate that RP67580 is a particularly valuable tool for investigating the physiological and pathological role of SP.


Acute exposure to human interferon-alpha affects ion currents in human natural killer cells.

Interferon-alpha (IFN-alpha) is a particularly potent stimulator of human natural killer (NK) cell activity. The initial trigger for IFN action is not known, but there is indirect evidence from a number of cell types that changes in ion channel activity are among the earliest responses. Previous evidence includes changes in Ca2+ fluxes and intracellular activity, membrane potential changes, and effects of ion-channel blockers. Killing by human NK cells is dependent on external Ca2+ and on K+ channel activity. In the present study we have confirmed this dependence and the augmentation by human IFN-alpha. Then we directly studied the effects of IFN-alpha on ion currents in human NK cells using the patch-clamp electrophysiological techniques. We find that IFN-alpha can increase the predominant K+ current near the resting potential but suppresses it at higher voltages. Within 1 min after acute IFN-alpha treatment a new current is induced. This small current appears to be through nonselective cation channels that allow monovalent and divalent cations, including Ca2+ to permeate. This current presents a possible early triggering mechanism whereby acute exposure to IFN-alpha augments NK cytotoxicity.


Predicted prostate specific antigen results using transrectal ultrasound gland volume. Differentiation of benign prostatic hyperplasia and prostate cancer.

The diagnostic performance of transrectal ultrasound (TRUS) gland volume and prostate specific antigen (PSA) results were evaluated in 204 men consecutively scheduled to undergo transurethral prostatic resection (TUR). Nonpalpable prostate cancer was detected by TRUS alone in 18% (29 of 161) and by TUR alone in 9% (14/161), for an overall cancer incidence of 27%. A predicted PSA value (TRUS gland volume x 0.20 ng/ml/g = polyclonal PSA) was used for comparison with serum PSA for each patient. TRUS positive predictive value improved from 52% to 86% when serum PSA exceeded the predicted value. The specificity and positive predictive value of PSA at 2.5 ng/ml were 23% and 37%, respectively, which increased to 88% and 72%, respectively, when serum PSA exceeded the predicted value. Predicted PSA values produce decision levels near the 95th percentile for each patient and assist individual biopsy decisions better than grouped gland volume ranges. Wider application of TRUS and PSA in any clinical setting or early detection program is now possible.


Clinical diagnosis of prostate cancer.

The clinical diagnosis of localized prostate carcinoma in the asymptomatic male has been based on a careful digital rectal examination (DRE). The DRE, prostate specific antigen (PSA), transrectal ultrasonography (TRUS), prostate needle biopsy (PNB), and other modalities are examined for their role in prostate cancer diagnosis. Up to 20% of localized prostatic cancer is still diagnosed "retrospectively" on transurethral resection (TURP) for clinically benign disease and prostatism. The role of fine-needle aspiration (FNA), flow cytometric study (FCM), and magnetic resonance imaging (MRI) in the diagnosis of prostate cancer is limited. Those men older than 50 years of age who have lower tract symptoms, either obstructive or irritative, or who have abnormal serum levels of PSA, regardless of DRE findings, are advised to undergo TRUS with ultrasound-guided PNB.


Tumor markers in prostate cancer.

Prostatic specific antigen (PSA) is a tissue specific marker that is now the most widely used biochemical test for the assessment and follow-up of prostate cancer. The levels of PSA rise with tumor stage, but there is considerable overlap of their distribution between stages. PSA measurement now forms a part of the workup of a suspected carcinoma of the prostate, with a level of more than 4 ng/ml being an indication for further investigation. The sensitivity of PSA makes it an essential test for the postoperative assessment of radical prostatectomy and curative radiation therapy. The rates of change of PSA levels in locally advanced and metastatic disease treated by hormone manipulation can provide prognostic information. Low levels of PSA (less than 10 ng/ml) 6 months after treatment are a sign that the response will be prolonged. However, the sensitivity of PSA often results in a rising level preceding clinical evidence of progression by several months and is not necessarily an indication to change treatment. Alkaline phosphatase and prostatic acid phosphatase provide a less sensitive test for the bone response to skeletal metastases and tumor activity in advanced disease, respectively.


Prostate specific antigen predominantly forms a complex with alpha 1-antichymotrypsin in blood. Implications for procedures to measure prostate specific antigen in serum.

Prostate specific antigen (PSA) is a zymogen of a 33-kilodalton (kD) serine proteinase with extensive similarity to glandular kallikreins. The mechanism responsible for converting the zymogen into active proteinase has not been defined, but active PSA may be irreversibly inactivated in vitro by two of the major proteinase inhibitors in blood: alpha 1-antichymotrypsin and alpha 2-macroglobulin. Procedures have been designed to characterize the different molecular forms of PSA in serum. One assay detects PSA epitopes available on both PSA binding to serine proteinase inhibitors and PSA not binding to a proteinase inhibitor. A second assay only detects PSA in complex with alpha 1-antichymotrypsin. A third assay mainly detects PSA not binding to a proteinase inhibitor. In serum samples, an 80-kD to 90-kD species of PSA in complex with alpha 1-antichymotrypsin is the predominant molecular form and a minor molecular form of serum PSA was an approximately 30-kD fraction not binding to a proteinase inhibitor. The benefits of detecting different molecular forms of serum PSA should be investigated regarding possibilities to facilitate differential diagnosis of carcinoma of the prostate (CAP) and benign prostatic hyperplasia (BPH).


Pathology of carcinoma of the prostate.

In this presentation the authors review the pathology of prostatic carcinoma (PCa), and discuss criteria for pathologic diagnosis, premalignant lesions, lesions that simulate PCa, immunopathology, special types of PCa, effects of therapy on the prostate, and recent efforts to improve diagnostic and prognostic capabilities. The possible role of study of nucleolar organizing regions is reported. A new method for demonstration of chromosome in formalin-fixed, paraffin-embedded tissue is presented. The need for research in all aspects of pathology is emphasized.


Critical assessment of prostate cancer staging.

Staging of prostatic adenocarcinoma is a systematic classification of the extent of disease based on clinical and pathologic criteria. This classification determines treatment and reflects ultimate expected clinical outcome. The technologic changes in diagnostic modalities need to be incorporated into the staging classification and a better assessment of biologic hazard of each individual tumor needs to be developed to further refine current treatment of prostate cancer.


Benign prostatic hyperplasia. The scope of the problem.

The prevalence and incidence of clinical problems secondary to and associated with benign prostatic hyperplasia (BPH) are of increasing concern as the population ages. Selected published reports using anatomical and clinical criteria to identify BPH and its clinical sequelae were reviewed. The following observations seem to reflect the current state of knowledge: (1) BPH develops with increasing frequency as men age; (2) BPH causes significant pathologic changes in the urinary tract of some patients and symptoms in others; and (3) other identifiable or cryptic etiologic factors may be the predominant cause of identical voiding dysfunction in patients with BPH. Essential information about factors initiating and promoting development of BPH, the exact mechanisms by which BPH alters voiding mechanisms, and definitive diagnostic criteria to establish the role of BPH in clinical changes are lacking. Progress in these problem areas is essential to guide appropriate clinical management.