Intercellular adhesion molecule-2, a second counter-receptor for CD11a/CD18 (leukocyte function-associated antigen-1), provides a costimulatory signal for T-cell receptor-initiated activation of human T cells. Activation of T cells often requires both activation signals delivered by ligation of the TCR and those resulting from costimulatory interactions between certain T cell surface accessory molecules and their respective counter-receptors on APC. CD11a/CD18 complex on T cells modulate the activation of T cells by interacting with its counter-receptors intracellular adhesion molecule (ICAM-1) (CD54) and/or ICAM-2 on the surface of APC. The costimulatory ability of ICAM-1 has been demonstrated. Using a soluble ICAM-2 Ig fusion protein (receptor globulin, Rg) we demonstrate the costimulatory effect of ICAM-2 during the activation of CD4+ T cells. When coimmobilized with anti-TCR-1 mAb ICAM-2 Rg induced vigorous proliferative response of CD4+ T cells. This costimulatory effect of ICAM-2 was dependent on its coimmobilization with mAb directed at the CD3/TCR complex but not those directed at CD2 or CD28. Both resting as well as Ag-primed CD4+ T cells responded to the costimulatory effects of ICAM-2. The addition of mAb directed at the CD11a or CD18 molecules almost completely inhibited the responses to ICAM-2 Rg. These results are consistent with the role of CD11a/CD18 complex as a receptor for ICAM-2 mediating its costimulatory effects. Stimulation of T cells with coimmobilized anti-TCR-1 and ICAM-2 resulted in the induction of IL-2R (CD25), and anti-Tac (CD25) mAb inhibited this response suggesting the contribution of endogenously synthesized IL-2 during this stimulation. These results demonstrate that like its homologue ICAM-1, ICAM-2 also exerts a strong costimulatory effect during the TCR-initiated activation of T cells. The costimulatory effects generated by the CD11a/CD18:ICAM-2 interaction may be critical during the initiation of T cell activation by ICAM-1low APC.


Autoreactivity of low but not of high CD4 variants of an antigen-specific, I-A-restricted mouse T cell clone. Variant lines expressing high and low surface densities of the accessory molecule CD4 have been developed by repeated preparative flow cytometric cell sortings from the murine Th cell clone D10.G4.1 (D10). The high CD4 variant line (D10H) fully maintained the original I-Ak restricted specificity for conalbumin of wild-type D10 cells. In contrast, the low CD4 variant line (D10L) showed a strong autoreactivity to I-Ak carrying stimulator cells alone which was only slightly augmented by addition of conalbumin. Cell surface molecules other than CD4, including TCR, CD3, CD11a, CD2, CD45, CD44, and MHC class I, remained identical on D10H and D10L sublines as on D10 wild-type cells. The possibility that D10L cells had suffered alterations of their TCR-alpha beta was excluded by demonstrating their reactivity with a panel of eight different anti-clonotypic mAb specific for various epitopes of the D10 TCR. By limiting dilution analysis we show that the majority of responding cells of D10L sublines were autoreactive. Although the reactivity for allogeneic I-A also increased as compared with D10H cells, a clear preference for self-I-Ak was maintained so that a true autoreactive phenotype was evident. The results indicate that the surface concentration of CD4 has a decisive influence on self-non-self discrimination of MHC class II-restricted Th cells.


CD4+ lymphocyte cell enumeration for prediction of clinical course of human immunodeficiency virus disease: a review. Over the last 10 years the appreciation of the full breadth of the spectrum of human immunodeficiency virus (HIV) infection has gradually increased; it is now known that AIDS represents merely the end stage of this progressive infectious process. The surrogate marker that most closely correlates with the stage of HIV infection is the CD4+, or T helper, cell count. Because this count is relied on in making important therapeutic decisions, it is of paramount importance that clinicians be cognizant of and fully understand the multiple factors that can influence this parameter: the variability of the count from day to day and from morning to night, the influence of intercurrent viral infections, the influence of drugs. In this AIDS Commentary, Sten H. Vermund and his colleagues at the National Institutes of Health discuss these issues and put the CD4+ cell count into a lucid and practical clinical perspective.


Signaling by lymphocyte function-associated antigen 1 (LFA-1) in B cells: enhanced antigen presentation after stimulation through LFA-1. To examine the role of lymphocyte function-associated antigen 1 (LFA-1) expression on murine B cells as it pertains to their function in T cell activation, we carried out antigen-presentation assays in tissue culture wells coated with a purified, secreted form of the murine intercellular adhesion molecule 1 (ICAM-1). We observed a significant decrease in the concentration of antigen required to activate a T cell hybridoma and primary T cells in wells coated with ICAM-1. This effect was dependent on the amount of ICAM-1 used to coat the wells and was also observed in wells coated with anti-LFA-1-monoclonal antibodies and was blocked by soluble anti-LFA-1 antibodies. The effect on antigen dose was most pronounced in assays carried out with an ICAM-1-deficient mutant B lymphoma cell line, small resting primary B cells, and unfractionated primary B cells at low concentrations. No decrease in the antigen dose was observed if the B cells were chemically fixed or treated with ricin, or when antigen was presented by a HeLa cell line transfected with murine class II major histocompatibility complex (MHC) genes, indicating that the immobilized ICAM-1 was mediating its effect through B cell LFA-1, and that B cell protein synthesis was required. The enhancing effect was also observed if the B cells were prepulsed with antigen, indicating that improved uptake or processing of antigen, or increased class II MHC expression were unlikely mechanisms.


Searching for hematopoietic stem cells: evidence that Thy-1.1lo Lin- Sca-1+ cells are the only stem cells in C57BL/Ka-Thy-1.1 bone marrow. Hematopoietic stem cells (HSCs) are defined in mice by three activities: they must rescue lethally irradiated mice (radioprotection), they must self-renew, and they must restore all blood cell lineages permanently. We initially demonstrated that HSCs were contained in a rare (approximately 0.05%) subset of bone marrow cells with the following surface marker profile: Thy-1.1lo Lin- Sca-1+. These cells were capable of long-term, multi-lineage reconstitution and radioprotection of lethally irradiated mice with an enrichment that mirrors their representation in bone marrow, namely, 1,000-2,000-fold. However, the experiments reported did not exclude the possibility that stem cell activity may also reside in populations that are Thy-1.1-, Sca-1-, or Lin+. In this article stem cell activity was determined by measuring: (a) radioprotection provided by sorted cells; (b) long-term, multi-lineage reconstitution of these surviving mice; and (c) long-term, multi-lineage reconstitution by donor cells when radioprotection is provided by coinjection of congenic host bone marrow cells. Here we demonstrate that HSC activity was detected in Thy-1.1+, Sca-1+, and Lin- fractions, but not Thy-1.1-, Sca-1-, or Lin+ bone marrow cells. We conclude that Thy-1.1lo Lin- Sca-1+ cells comprise the only adult C57BL/Ka-Thy-1.1 mouse bone marrow subset that contains pluripotent HSCs.


Development of humanized bispecific antibodies reactive with cytotoxic lymphocytes and tumor cells overexpressing the HER2 protooncogene. The HER2 protooncogene encodes a 185-kD transmembrane phosphoglycoproteins, human epidermal growth factor receptor 2 (p185HER2), whose amplified expression on the cell surface can lead to malignant transformation. Overexpression of HER2/p185HER2 is strongly correlated with progression of human ovarian and breast carcinomas. Recent studies have shown that human T cells can be targeted with bispecific antibody to react against human tumor cells in vitro. We have developed a bispecific F(ab')2 antibody molecule consisting of a humanized arm with a specificity to p185HER2 linked to another arm derived from a murine anti-CD3 monoclonal antibody that we have cloned from UCHT1 hybridoma. The antigen-binding loops for the anti-CD3 were installed in the context of human variable region framework residues, thus forming a fully humanized BsF(ab')2 fragment. Additional variants were produced by replacement of amino acid residues located in light chain complementarity determining region 2 and heavy chain framework region 3 of the humanized anti-CD3 arm. Flow cytometry analysis showed that the bispecific F(ab')2 molecules can bind specifically to cells overexpressing p185HER2 and to normal human peripheral blood mononuclear cells bearing the CD3 surface marker. In additional experiments, the presence of bispecific F(ab')2 caused up to fourfold enhancement in the cytotoxic activities of human T cells against tumor cells overexpressing p185HER2 as determined by a 51Cr release assay. These bispecific molecules have a potential use as therapeutic agents for the treatment of cancer.


Defective RNAs in mosquito cells persistently infected with Bunyamwera virus. Viral protein and RNA synthesis were compared in BHK and Aedes albopictus C6/36 (mosquito) cells infected with Bunyamwera virus. In BHK cells host protein synthesis was inhibited and viral proteins were detected until the cells died; in C6/36 cells there was little inhibition of host proteins and viral proteins could not be detected after 36 h post-infection. Relatively more S segment RNA than L or M segment RNA was produced in infected C6/36 cells compared to BHK cells. A persistent infection of C6/36 cells was established and the cells were passaged at weekly intervals for over a year. The titre of virus released from the cells and the level of viral RNA in the cells at different passages fluctuated markedly, but there was no simple relationship between virus titre and the amount of viral RNA. Northern blot analysis of viral RNA extracted from persistently infected cells revealed the presence of subgenomic RNAs derived from the L RNA segment. These defective RNAs were not packaged into nucleocapsids. The presence of the defective RNAs did not correlate with resistance of cells cloned from the persistently infected population to superinfection with homologous virus. Hence the role of these defective RNAs in the maintenance of the persistent state remains to be elucidated.


Spinal subdural abscess. Case report.

Spinal subdural abscess. Case report. Only 44 cases of spinal subdural abscess have been reported to date. The authors present another case and review the relevant literature. The findings of intraspinal gassification on computerized tomography scans and Escherichia coli as the causative organism have not previously been described in relation to spinal subdural abscess. Most frequently, Staphylococcus aureus is the responsible organism. Hematogenous spread of infection from a distant source often takes place. In a surprising number of incidences, iatrogenic causes are the primary foci of spinal subdural abscess. Spinal subdural abscess is an unpredictable disease, with an unfavorable outcome if left untreated. If there is suspicion of a spinal subdural abscess, urgent radiological examination followed by immediate surgical drainage and appropriate antibiotic therapy is warranted.


A proposed mechanism of action for the antinociceptive effect of intrathecally administered calcium in the mouse. Administration of i.t. calcium has been shown to produce effects which are opposite to those observed when calcium is injected into the brain. The purpose of this study was to elucidate the mechanism of the antinociceptive action of calcium (i.t.). Injection of calcium (i.t.) produced antinociceptive effects in the tail-flick and p-phenylquinone (PPQ) stretching tests. The ED50 value for calcium (i.t.) in the PPQ test was 4.8 (4.2-5.5) nmol per mouse vs. 344 (251-469) nmol per mouse for calcium (i.t.) in the tail-flick test. The antinociceptive effects of calcium (i.t.) were attenuated significantly in the tail-flick test by pretreatment with naloxone (i.t.) (AD50 value = 200 pmol/mouse) and ICI-174,864 (i.t.) (AD50 value = 20 nmol/mouse), but not by the kappa receptor-selective antagonist nor-BNI. The antinociceptive effects of calcium (i.t.) were attenuated significantly in the PPQ test by pretreatment with naloxone (i.t.) (AD50 value = 50 pmol/mouse) and norbinaltorphimine (i.t.) (AD50 value = 110 pmol/mouse), but not by the delta receptor-selective antagonists naltrindole and ICI-174, 864. Administration of calcium (i.t.) significantly enhanced the antinociceptive effects of mu [D-Ala2,N-Me-Phe4,Gly-ol]enkephalin, delta [D-Pen2,D-Pen5]enkephalin and kappa (U50,488H) opioid receptor-selective peptides. The injection of the dibutyryl derivative of cyclic AMP (i.t.), as well as forskolin (i.t.), blocked the antinociceptive effects of calcium (i.t.) (AD50 values = 39 nmol and 1.7 nmol/mouse, respectively). Injection of apamin (AD50 value = 2.9 pmol/mouse) and charybodotoxin (58 fmol/mouse), blockers of calcium-gated potassium channels, significantly blocked calcium (i.t.). The antinociceptive effects of calcium (i.t.) were also blocked by verapamil (30 and 60 nmol/mouse), theophylline (275 nmol/mouse) and substance P (7.4 nmol/mouse, i.t.). Thus, the data indicate that the mechanism underlying the antinociceptive effect of calcium (i.t.) involves mediation, at least in part, by opioid peptides, alterations in intraneuronal cyclic AMP and/or neuronal hyperpolarization, and decreased release of substance P. The administration of calcium (i.t.) may also enhance the release of adenosine as a significant factor in the antinociceptive effects of the calcium.


Characterization of receptors involved in dopamine-induced activation of phospholipase-C in rat renal cortex. Dopamine (DA) is reported to stimulate phospholipase-C (PL-C) in rat renal cortex. Inasmuch as DA activates alpha adrenoceptors and DA receptors, the relative contribution of these receptors to DA-induced activation of PL-C is not yet established. We examined the effect of DA on PL-C activity in rat renal cortical slices prelabeled with myo-2-[3H]inositol in the presence of Li+. PL-C activity was expressed as fractional release (FR) of combined [3H]inositol phosphates expressed as dpm inositol phosphates accumulated/total dpm incorporated X 100. DA (1 mM) produced time-dependent increases in FR up to 60 min. DA (1, 3 and 10 mM) produced 61%, 88% and 110% increases in FR over control. When DA was given in the presence of SCH 23390, a selective DA-1 receptor antagonist, the increase in FR was significantly reduced to 33%, 51% and 62%, respectively, but the increase in FR remained unaffected in the presence of a DA-2 receptor antagonist, domperidone (30 microM). Phentolamine (10 microM) also inhibited the response to DA to 41%, 47% and 43% at the respective concentrations. DA-induced stimulation of PL-C was completely abolished in the combined presence of both SCH 23390 (30 microM) and phentolamine (10 microM). SCH 23390, domperidone or phentolamine alone did not significantly change the basal PL-C activity in renal cortical slices. These results demonstrate that 1) DA stimulates PL-C in rat renal cortex via activation of both DA-1 receptors and alpha adrenoceptors and DA-2 receptors are not involved in this response; and 2) during normal sodium intake, intrarenal DA does not modulate the PL-C activity in rat renal cortex.


Evidence for a noradrenergic innervation to "atypical" beta adrenoceptors (or putative beta-3 adrenoceptors) in the ileum of guinea pig. Indirect evidence suggests that beta-1 adrenoceptors in the guinea pig ileum are innervated but it has not been determined whether "atypical" beta adrenoceptors also receive a postganglionic sympathetic innervation. To answer this question, experiments were undertaken using electrical stimulation of para-arterial sympathetic neurons to evoke relaxation in isolated segments of guinea pig ileum. Tension was developed in the ileal segments by either transmural electrical field stimulation to evoke the cholinergic "twitch" response, or by histamine to produce a steady-state contracture. Para-arterial sympathetic nerve stimulation evoked a frequency-dependent inhibition of the twitch response which was blocked by guanethidine and restored by dexamphetamine, indicating typical noradrenergic transmission. In preparations contracted with histamine and pretreated with benextramine to block alpha adrenoceptors, para-arterial sympathetic nerve stimulation evoked frequency-dependent relaxations which were reduced in magnitude but not abolished by the following beta adrenoceptor antagonists: bromoacetylalprenololmenthane (1 microM) or a combination of ICI 118,551 (0.3 microM) and CGP 20712A (0.1 microM). Remaining responses were blocked by compounds exhibiting affinity for atypical beta adrenoceptors, (-)-alprenolol (3 microM) and nadolol (300 microM), as well as the agonist (-)-isoproterenol (10 microM; to saturate the atypical beta adrenoceptor). However, relaxations to papaverine were unaffected by these treatments. Experiments revealed that potential cotransmitters (ATP, neuropeptide Y and somatostatin) do not appear to play a detectable role in relaxations produced by para-arterial sympathetic nerve stimulation. The results demonstrate, for the first time, that atypical beta adrenoceptors in guinea pig ileum receive a noradrenergic innervation.


Effect of fenoldopam on the acute and subacute nephrotoxicity produced by amphotericin B in the dog. The effect of the selective dopamine DA1 receptor agonist fenoldopam (1 microgram/kg/min i.v.) on the acute nephrotoxic response to amphotericin B (2 mg/kg i.v.) has been studied in the anesthetized dog. Animals were prepared for the measurement of blood pressure, renal blood flow, urine flow, glomerular filtration rate and sodium and potassium excretion. Amphotericin B was given over 20 min and the animals were followed for an additional 160 min. The fenoldopam infusion was started 20 min before amphotericin B and was continued for the duration of the experiment. In control animals, amphotericin B markedly increased renal vascular resistance without affecting blood pressure and thus produced a significant reduction in renal blood flow. The renal vasoconstrictor response to amphotericin B was not attenuated by fenoldopam. Concomitant with the renal vasoconstriction produced by amphotericin B was a marked reduction in glomerular filtration rate, sodium excretion and urine flow rate, which lasted for at least 160 min after amphotericin B treatment. Fenoldopam did not have any effect on the initial reductions in glomerular filtration rate, sodium excretion and urine flow rate but did produce a significant return of these parameters toward control levels by 160 min, despite the continued renal vasoconstriction. The effect of fenoldopam (0.5 microgram/kg/min) given continuously by i.v. infusion on the subacute nephrotoxic response produced by amphotericin B given every other day for 8 days was also investigated. One day after the start of the fenoldopam infusion, venous samples were drawn for the analysis of serum creatinine and blood urea nitrogen.(ABSTRACT TRUNCATED AT 250 WORDS)


Differential inhibition of cholinergic and noncholinergic neurogenic contractions by 5-hydroxytryptamine1A receptor agonists in guinea pig ileum. Previous studies have shown that 5-hydroxytryptamine1A (5-HT1A) receptors are localized only to a subset of myenteric neurons in guinea pig ileum; the purpose of this study was to determine the possible functional significance of this differential receptor localization. Nerve-mediated contractions of the longitudinal muscle, myenteric plexus of guinea pig ileum were studied in vitro. Contractions were evoked by single transmural electrical stimuli (0.5-msec duration, at 0.1 Hz; cholinergic) or trains of stimuli (10 or 20 Hz for 1 sec; noncholinergic; scopolamine, 1 microM present). The 5-HT1A agonist, 8-hydroxy-2-(n-dipropylamino)tetralin (DPAT), reduced cholinergic contractions by no more than 14% in the concentration range of 0.001 to 0.3 microM. At high concentrations (1 to 100 microM), DPAT inhibited longitudinal muscle, myenteric plexus contractions; the EC50 was 6 microM. 5-Hydroxytryptamine (5-HT) and 5-carboxamidotryptamine (5-CT) reduced longitudinal muscle, myenteric plexus contractions by a maximum of 35% and 24% at 100 and 30 microM, respectively. Spiperone and 1-(2-methoxyphenyl)-4-[4-(2-phthalimidobutyl]piperazine (NAN-190), two 5-HT1A receptor antagonists, did not block DPAT-induced inhibition of cholinergic contractions. DPAT (3, 10 and 30 microM) shifted histamine concentration-response curves to the right in an apparently competitive manner; Schild analysis yielded a pA2 of 5.2. DPAT (3, 10, 30 and 100 microM) also shifted bethanechol concentration-response curves to the right in an apparently competitive manner; Schild analysis yielded a pA2 of 5.5. These data indicate that DPAT blocks histamine and muscarinic receptors on longitudinal muscle.(ABSTRACT TRUNCATED AT 250 WORDS)


Cholecystokinin release from the rat caudate-putamen, cortex and hippocampus is increased by activation of the D1 dopamine receptor. Studies were undertaken to examine the role of dopamine in the regulation of cholecystokinin (CCK) release in slices of the rat caudate-putamen (CP), a region where both neuroactive substances are abundant yet not extensively colocalized. It was found that dopamine, acting through a D1 dopamine receptor, is a major factor regulating CCK release in this tissue. Haloperidol and the selective D1 antagonist, SKF 83566, but not the D2 antagonist, sulpiride, inhibited potassium-evoked release from CP slices. Apomorphine enhanced both basal and potassium-evoked release, an effect which was reversed by (+/-)SKF 83566, but not by sulpiride. The D1 agonist, (+)SKF 38393, displayed agonist and antagonist activity in this system, whereas the D2 agonist, quinpirole, had no effect. Depletion of endogenous dopamine by pretreatment of animals with alpha-methyl-para-tyrosine decreased potassium-induced release by 45%, and in these animals, haloperidol no longer inhibited release. These dopaminergic agents altered CCK release in cortex and hippocampus in a similar fashion, although those tissues were less sensitive to their effects. These results suggest that dopamine, acting through D1 receptors, increases CCK release in CP, cortex and hippocampus. The results of previous studies showing that phorbol esters increase CCK release and that occupation of D1 receptors increases phosphoinositol turnover make it possible to hypothesize that dopamine, acting through D1 receptors, is increasing CCK release by increasing phosphoinositol turnover. Further studies will be required to verify this hypothesis and to determine whether dopamine is acting directly or indirectly to modulate CCK release.


Captopril and ANP: changes in renal hemodynamics, glomerular-ANP receptors and guanylate cyclase activity in rats with heart failure. To define the renal effects of atrial natriuretic peptide (ANP) in heart failure, we studied rats with heart failure after coronary artery ligation. The rats received either captopril (2 milligrams drinking water) or placebo for 4 weeks. Glomerular filtration rate, renal plasma flow, filtration fraction, urine volume, urinary sodium excretion and the percent fractional excretion of sodium were measured before and after an infusion of ANP (0.3 microgram/kg/min). To determine whether changes in ANP receptor binding and responsiveness occur in heart failure and after captopril treatment, we performed radioreceptor binding studies and measured guanylate cyclase activity. Atrial natriuretic peptide in sham-operated rats decreased mean arterial pressure from 118 +/- 5 to 95 +/- 5 mm Hg (P less than .001), increased urine volume from 0.06 +/- 0.02 to 0.16 +/- 0.05 ml/min/kg (P less than .05), urinary sodium excretion, 14.2 +/- 3.1 to 41.4 +/- 8.9 mu eq/min/kg (P less than .02), filtration fraction from 0.30 +/- 0.03 to 0.40 +/- 0.4 (P less than .05), and the percent fractional excretion of sodium from 0.84 +/- 0.19 to 2.85 +/- 0.61 (P less than .02). Atrial natriuretic peptide in untreated rats with heart failure produced no significant systemic or renal hemodynamic effects. In rats with heart failure treated with captopril, ANP decreased mean arterial pressure from 93 +/- 4 to 86 +/- 4 mm Hg (P less than .05) and increased hematocrit from 50 +/- 2 to 52 +/- 1 (P less than .001).(ABSTRACT TRUNCATED AT 250 WORDS)


Opposite effects of NMDA receptor blockade on dopaminergic D1- and D2-mediated behavior in the 6-hydroxydopamine model of turning: relationship with c-fos expression. In rats with unilateral 6-hydroxydopamine (6-OHDA) lesions of the nigrostriatal dopaminergic pathway, I-dihydroxyphenylalanine (L-DOPA) induces contralateral turning through activation of denervated D1 and D2 receptors. Blockade of N-methyl-D-aspartate (NMDA) receptors by the noncompetitive antagonist (+)MK-801 (0.1 mg/kg, i.p.), potentiated L-DOPA-induced contralateral turning. In 6-OHDA lesioned rats, selective agonists of D1 (SKF 38393, CY 208-243) or D2 (LY 171555) receptors also induce contralateral turning; however, (+)MK-801 pretreatment, although markedly potentiating D1, almost completely inhibited D2-mediated turning. The potentiation of SKF 38393-induced contralateral turning by MK-801 was stereospecific and was observed also with the noncompetitive NMDA antagonist phencyclidine, and with the competitive antagonist CPP. Administration of the D1 antagonist SCH 23390 (0.1 mg/kg s.c.) blocked (+)MK-801-induced potentiation of L-DOPA contralateral turning, confirming the D1 nature of the effects observed. Expression of the early gene c-fos in the caudate-putamen (CPu) is known to be activated by stimulation of supersensitive D1 receptors. Immunohistochemical studies on c-fos revealed sparse c-fos positive nuclei in the lesioned CPu after 1.5 mg/kg of SKF 38393, whereas after combined administration of (+)MK-801 and SKF 38393, dense labeling of nuclei was obtained in the dorso-lateral aspect of the CPu. Therefore, blockade of NMDA receptors acts synergistically with D1 and antagonistically with D2 receptor stimulation in the 6-OHDA model of turning, suggesting that different neuronal pathways are involved in the mediation of D1 and D2 responses.


The insulin-secreting cell line, RINm5F, expresses an alpha-2D adrenoceptor and nonadrenergic idazoxan-binding sites. The pharmacological properties of alpha-2 adrenoceptors and the existence of nonadrenergic idazoxan-binding sites (NAIBS) were investigated in the insulin-secreting cell-line, RINm5F, using [3H]RX821002 and [3H]idazoxan. Analysis of [3H]RX821002 saturation isotherms revealed the presence of a single class of binding sites (Bmax = 47.5 +/- 3.5 fmol/mg protein) having high affinity (Kd = 1.26 +/- 0.18 nM). Inhibition of [3H]RX821002 binding by adrenergic compounds showed that the labeled sites displayed the properties expected for an alpha-2 adrenoceptor. Based on competition data with drugs having alpha-2 adrenoceptor subtype selectivity, the receptor from RINm5F is neither an alpha-2B nor an alpha-2C. It resembles the alpha-2A, but deviates from this subtype because of a weak affinity for yohimbine and rauwolscine. In this respect, RINm5F alpha-2 adrenoceptor is identical to the receptor previously described in rat intestinal mucosa and corresponds to a fourth subtype: alpha-2D. Agonist inhibition curves were better fitted by a two-site model and indicated that about half of the receptor population was under a high-affinity state corresponding to G protein-coupled receptors. [32P]ADP-ribosylation with pertussis toxin and immunodetection with specific antibodies permitted the identification of three distinct G proteins: Gi2, Gi3 and G0. Binding experiments with [3H]idazoxan showed that this imidazoline labeled two types of sites corresponding to alpha-2 adrenoceptors and NAIBS. Analysis of saturation isotherms under binding conditions allowing to discriminate between the two site populations indicated that the density of NAIBS (44 +/- 2 fmol/mg protein) was fairly identical to that of alpha-2 adrenoceptors. The pharmacological properties of NAIBS, as assessed by determining the relative affinity of imidazolinic and nonimidazolinic compounds, reasonably matched that reported in other tissues. Taken together, these data make the RINm5F cell-line 1) the first model in permanent culture known as expressing an alpha-2 adrenoceptor of the alpha-2D subtype; 2) a good system for studying in vitro the respective role of alpha-2 adrenoceptors and NAIBS in the regulation of insulin secretion by beta cells.


Electrophysiological actions of the pimobendan metabolite, UD-CG 212 Cl, in guinea pig myocardium. Pimobendan (UD-CG 115 BS), an inotropic agent and inhibitor of type III phosphodiesterase activity, is demethylated in vivo to form UD-CG 212 Cl, which is a more potent type III phosphodiesterase inhibitor. This study examined cyclic AMP (cAMP)-mediated actions of UD-CG 212 Cl. In guinea pig papillary muscles, UD-CG 212 Cl increased cAMP and stimulated Ca(++)-dependent slow action potentials (APs) in a dose-dependent manner. When compared to previous studies using pimobendan, UD-CG 212 Cl was approximately 100-fold more potent. UD-CG 212 Cl had no additional effects on slow APs in the presence of a maximal dose of isoproterenol (1 microM). Propranolol had little effect on UD-CG 212 Cl-induced slow APs. These results, along with previous studies, indicate that slow AP induction by UD-CG 212 Cl was cAMP-dependent, and the increase in cAMP levels was most likely due to phosphodiesterase inhibition and not beta receptor stimulation. Experiments with tetraethylammonium.Cl suggested that UD-CG 212 Cl probably did not induce slow APs by blocking K+ channels. In voltage-clamped ventricular myocytes UD-CG 212 Cl (100 microM) could stimulate Ca++ current (+21 +/- 5%) when basal cAMP levels were enhanced with a submaximal dose of isoproterenol (10(-9)-10(-8) M). Isoproterenol was not required to observe the stimulating effect of UD-CG 212 Cl on Ca++ current in intact, nondialyzed cells prepared using the nystatin-perforated patch method. Studies with the stereoisomers of UD-CG 212 Cl showed that the D-isomer was more potent than the L-isomer.(ABSTRACT TRUNCATED AT 250 WORDS)


Comparison of mu opioid receptor binding on intact neuroblastoma cells with guinea pig brain and neuroblastoma cell membranes. To better understand opioid binding parameters found in situ, binding studies were conducted to mu-opioid receptors on intact SH-SY5Y neuroblastoma cells and compared with binding to SH-SY5Y membrane and guinea pig brain membrane preparations. The mu-selective peptide antagonist [3H]D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 (CTOP) was used for the binding studies. The fact that CTOP is an antagonist and hydrophilic is important for binding to be achieved using intact cells. In intact cells, using a physiological buffer, there appears to be only a low affinity "agonist" conformation of the receptor. This is in contrast to binding in either brain or SH-SY5Y membranes in Tris buffer, in which high-affinity agonist binding was prevalent. As expected from the binding profiles, pertussis toxin treatment of cells has no effect on binding to intact cells, but significantly decreases affinity of agonists to cell membranes. In intact cells, binding appears to be to a single site and a single state of the mu receptor. Although in membrane preparations inhibition curves are shallow, with slope factors less than 1.0 for many agonists, on intact cells agonist inhibition curves are very steep, with slope factors slightly greater than 1.0.


Restriction fragment length polymorphism of polymerase chain reaction products from vaccine and wild-type varicella-zoster virus isolates. The nucleotide changes that result in two restriction endonuclease polymorphisms that differentiate wild-type varicella-zoster virus (VZV) from the vaccine strain were determined. Oligonucleotide primers that flank these sites were used to amplify the intervening sequences with the polymerase chain reaction to identify VZV DNA in clinical isolates. Restriction enzyme digestion of the amplification products distinguished vaccine and wild-type genomes from one another. This study confirms the feasibility of amplifying VZV sequences so that they may be detected in clinical specimens and provides a molecular epidemiological approach to strain identification of VZV in vesicular lesions.


Gastric intramucosal pH as a therapeutic index of tissue oxygenation in critically ill patients. Falls in gastric intramucosal pH (pHi) are associated with morbidity and mortality in patients admitted to intensive-care units (ICU). We tested the hypothesis that ICU outcome can be improved by therapy guided by changes in pHi and aimed at improving systemic oxygen availability. We studied 260 patients admitted to ICUs with APACHE II scores of 15-25. After insertion of a gastric tonometer, each patient was randomly assigned to a control or protocol group within the admission pHi category (normal = 7.35 or higher; low = below 7.35). The control groups were treated according to standard ICU practices. The protocol groups received, in addition, treatment to increase systemic oxygen transport or to reduce oxygen demand, whenever the pHi fell below 7.35 or by more than 0.10 units from the previous measurement. The protocol was used, because pHi fell, in 67 (85%) of the protocol group with normal pHi on admission. There were no significant differences between protocol and control groups in demographic characteristics, admission blood gases or haemoglobin concentration, number or type of organ system failures, or the intensity of ICU care. For patients admitted with low pHi, survival was similar in the protocol and control groups (37% vs 36%), whereas for those admitted with normal pHi, survival was significantly greater in the protocol than in the control group (58% vs 42%; p less than 0.01). Therapy guided by pHi measurements improved survival in patients whose pHi on admission to ICU was normal. pHi-guided resuscitation may help improve outcome in such patients by preventing splanchnic organ hypoxia and the development of a systemic oxygen deficit.


Combined chemotherapy with ABCM versus melphalan for treatment of myelomatosis. The Medical Research Council Working Party for Leukaemia in Adults. Both melphalan and cyclophosphamide increase life expectancy in patients with myelomatosis, but few large randomised studies have compared combination chemotherapy regimens with these single agents. In the Vth MRC myelomatosis trial, the survival of 314 patients randomised to receive ABCM (adriamycin, BCNU, cyclophosphamide, and melphalan) as first-line treatment was significantly longer than that of 316 patients given intermittent melphalan (M7) (p = 0.0003). The 75%, median, and 25% survivals were 7, 24, and 42 months, respectively, with M7 and 10, 32, and 56 months, respectively, with ABCM. Stable disease with few symptoms (plateau) was achieved by 61% of patients given ABCM and 49% of those given M7 (p = 0.004). Myelotoxicity was comparable between regimens. Cross-trial analysis suggests that M7 is comparable to melphalan and prednisone or melphalan, prednisone, and vincristine; that the efficacy of ABCM in the Vth trial and VIth MRC trials is comparable; and that ABCM gave better survival than intermittent melphalan regimens in the prognostic groups analysed. The results indicate that ABCM is an acceptable regimen that is more effective than melphalan, with or without prednisone, for first-line treatment of myelomatosis.


Multicentre study of gestrinone in cyclical breast pain. Although the aetiology of cyclical mastalgia is poorly understood, the consistent finding of an increased prolactin stimulation response probably due to oestrogen dominance has led to the use of treatment with prolactin-lowering drugs and antioestrogens. The efficacy and safety in cyclical mastalgia of gestrinone, which has androgenic, anti-oestrogenic, and antiprogestagenic properties, were investigated in a multicentre study. In a double-blind randomisation procedure, 72 patients were allocated placebo and 73 treatment with gestrinone (2.5 mg twice a week) for 3 months. The patients recorded the severity of breast pain on a visual analogue scale before and during treatment and scored other breast symptoms as not present (0), mild (1), moderate (2), or severe (3). The gestrinone group had significantly greater reductions than the placebo group in breast pain score at months 1, 2, and 3 of treatment (mean reduction 59.5 [SD 22.6] to 11.7 [17.0] vs 58.2 [17.6] to 36.7 [23.0] at month 3; p less than 0.0001). All six breast symptoms had lower scores in the gestrinone than in the placebo group by the end of treatment. In a subset of 30 participants (15 from each group), serum concentrations of oestradiol, progesterone, and tri-iodothyronine were significantly lower than baseline after 3 months of gestrinone, but concentrations of luteinising hormone, follicle-stimulating hormone, prolactin, thyroid-stimulating hormone, and thyroxine did not change. 41% of gestrinone-treated and 14% of placebo-treated patients reported at least one side-effect; most of these were androgen-mediated. 11 placebo-treated patients and 4 on gestrinone discontinued treatment. Thus, gestrinone was very effective in the treatment of cyclical mastalgia and was well tolerated.


Cassava cyanogens and konzo, an upper motoneuron disease found in Africa. Konzo is a distinct form of tropical myelopathy characterised by abrupt onset of spastic paraparesis. Epidemics in East Africa have been attributed to dietary cyanide exposure from insufficiently processed cassava but a study done in Zaire disputed such an aetiology. We investigated a konzo-affected population in rural Zaire and measured the cyanogen content of cassava flour, determined urinary thiocyanate as an indicator of cyanide intake, and compared blood cyanide concentrations in cases and controls. The affected population consumed flour made from short-soaked (one day) cassava roots and thus had high dietary cyanide exposure (urinary thiocyanate in 31 children = 757 mumol/l) compared with the unaffected population (urinary thiocyanate in 46 children = 50 mumol/l) that ate cassava that had been soaked for three days before consumption. 3 konzo patients, but only 2 of 23 controls, had blood cyanide concentrations above 4 mumol/l (p less than 0.01), although serum thiocyanate concentrations were similar. Our findings indicate a causal role in konzo of sustained high blood cyanide concentrations maintained by a deficient sulphur intake impairing cyanide to thiocyanate conversion. The underlying causes of konzo are poverty and food shortage, but a minor improvement of food processing may, as in beri-beri, be preventive.


Ventricular arrhythmias and four-year mortality in haemodialysis patients. Gruppo Emodialisi e Patologie Cardiovascolari. 127 randomly selected patients on haemodialysis showed a high prevalence of ventricular arrhythmias, the frequency of which rose significantly during and after dialysis. These patients have now been followed up for 4 years. Only age and ischaemic heart disease correlated independently with mortality. Although ventricular arrhythmias are often associated with cardiac disease in patients on chronic haemodialysis, they do not seem to predict overall mortality.


Control of scarring in adult wounds by neutralising antibody to transforming growth factor beta. Adult wounds heal with scar-tissue formation, whereas fetal wounds heal without scarring and with a lesser inflammatory and cytokine response. We injected the margins of healing dermal wounds in adult rats with neutralising antibody (NA) to transforming growth factor-beta (TGF-beta). All control wounds (irrelevant antibody, or TGF-beta, or no injection) healed with scarring, whereas the NA-treated wounds healed without scar-tissue formation; NA-treated wounds had fewer macrophages and blood vessels, lower collagen and fibronectin contents, but identical tensile strength and more normal dermal architecture than the other wounds. Early manipulation of the concentrations of selected cytokines may be a new approach to the control of scarring.


Randomised controlled trial of day care for hypertension in pregnancy. Our aim was to assess the effect of the introduction of a day-care unit on the care of women with non-proteinuric hypertension in pregnancy. A randomised controlled trial was carried out on 54 women who presented at 26 weeks of pregnancy or later with non-proteinuric hypertension (systolic blood pressure 150-170 mm Hg and/or diastolic pressure 90-105 mm Hg on two occasions at least 15 min apart). 30 women were allocated to care by the day unit and 24 were managed according to the established practice of their clinicians without access to the day unit (control group). Women in the control group spent on average 4.6 times longer as inpatients (difference in mean stay 4.0 days [95% confidence interval 2.1-5.9 days]) than the day-unit group and were 8.8 times (95% CI 3.0-25.8) more likely to be admitted to hospital. Induction of labour was 4.9 times (95% CI 1.6-13.8) more likely in the control than in the day-unit group and the development of proteinuria 11.4 times (95% CI 1.8-71.4) more likely. The control group had a mean of 1.5 fewer hospital outpatient visits (95% CI 0.36-2.64). The groups did not differ in their use of antihypertensive drugs. Day-unit care for hypertension in pregnancy significantly reduced the need for and the length of antenatal inpatient admissions and the number of medical interventions, at the cost of an increase in outpatient attendances. Our results are further evidence that inpatient care does not improve outcomes or prevent the development of proteinuria in this disorder.


Contraceptive efficacy of lactational amenorrhoea. Pregnancy is rare among breastfeeding women with lactational amenorrhoea. The lactational amenorrhoea method (LAM) is the informed use of breastfeeding as a contraceptive method by a woman who is still amenorrhoeic, and who is not feeding her baby with supplements, for up to 6 months after delivery. Under these three conditions, LAM users are thought to have 98% protection from pregnancy. It can be difficult, however, to determine when supplementation of the baby's diet begins. We have analysed data from nine studies of the recovery of fertility in breastfeeding women to assess the effectiveness of lactational amenorrhoea alone, irrespective of whether supplements have been introduced, as a fertility regulation method post partum. Cumulative probabilities of ovulation during lactational amenorrhoea were 30.9 and 67.3 per 100 women at 6 and 12 months, respectively, compared with 27.2 at 6 months when all three criteria of the LAM were met. Cumulative pregnancy rates during lactational amenorrhoea were 2.9 and 5.9 per 100 women at 6 and 12 months, compared with 0.7 at 6 months for the LAM. The probability of pregnancy during lactational amenorrhoea calculated from these studies is similar to that of other modern contraceptive methods, and it seems reasonable for a woman to rely on lactational amenorrhoea without regard to whether she is fully or partly breastfeeding. So that amenorrhoea and fertility suppression can be maintained, counselling about good breastfeeding and weaning practices remains important.


Renal effects of dopamine and dopexamine in the newborn anesthetized rabbit. The renal effects of dopexamine, a new dopaminergic agonist with marked beta 2-adrenergic agonist properties, but no alpha-adrenergic effect, has been studied in 8 newborn New Zealand rabbits, whose renal functional characteristics show close similarities with those of premature infants. Six animals were used as controls. After a control period, dopexamine was infused intravenously at a rate of 4 micrograms/kg per min and after a wash-out period, at 10 micrograms/kg per min. The renal effects of dopamine were studied in similar conditions. Glomerular filtration rate (GFR) and renal plasma flow (RPF) were determined by inulin and para-aminohippuric acid clearances, respectively. Dopexamine, 4 micrograms/kg per min, did not induce changes in cardiovascular and renal hemodynamics or in renal functions. At 10 micrograms/kg per min, a significant increase in urine flow rate (25 +/- 5%; p less than 0.01), urine sodium excretion (77 +/- 17%; p less than 0.01) and fractional sodium excretion (69 +/- 25%; p less than 0.05) was observed. The GFR, RPF and renal vascular resistance (RVR) were not affected. Heart rate increased slightly but significantly (8 +/- 3%; p less than 0.05), without change in mean blood pressure (MBP). Dopamine, 4 micrograms/kg per min, decreased slightly albeit significantly MBP (3 +/- 1%; p less than 0.05). At 10 micrograms/kg per min the only renal effect was a significant increase in RVR (19 +/- 6%; p less than 0.02). The different actions of these two dopaminergic agonists in this immature model could be explained by their respective ability to activate electively the adrenergic and dopaminergic peripheral receptors. The natriuretic and diuretic effect of dopexamine in normal immature rabbits, in the absence of changes in RPF or GFR is probably mediated by a direct action of this agent on dopaminergic tubular receptors. Failure of these two drugs to increase RPF may be related to an immaturity of the dopaminergic vascular receptors.


Postsynaptic contribution to long-term potentiation revealed by the analysis of miniature synaptic currents. Miniature excitatory synaptic currents were recorded from CA1 pyramidal cells in hippocampal slices to study the site of the persistent change in synaptic efficacy during long-term potentiation. Induction of long-term potentiation produced a large increase in the amplitude of these currents. Such a change in amplitude suggests an increase in postsynaptic transmitter sensitivity.


Activation of a C. elegans Antennapedia homologue in migrating cells controls their direction of migration. Anterior-posterior patterning in insects, vertebrates and nematodes involves members of conserved Antennapedia-class homeobox gene clusters (HOM-C) that are thought to give specific body regions their identities. The effects of these genes on region-specific body structures have been described extensively, particularly in Drosophila, but little is known about how HOM-C genes affect the behaviours of cells that migrate into their domains of function. In Caenorhabditis elegans, the Antennapedia-like HOM-C gene mab-5 not only specifies postembryonic fates of cells in a posterior body region, but also influences the migration of mesodermal and neural cells that move through this region. Here we show that as one neuroblast migrates into this posterior region, it switches on mab-5 gene expression; mab-5 then acts as a developmental switch to control the migratory behaviour of the neuroblast descendants. HOM-C genes can therefore not only direct region-specific patterns of cell division and differentiation, but can also act within migrating cells to programme region-specific migratory behaviour.


The eukaryotic initiation factor 2-associated 67-kDa polypeptide (p67) plays a critical role in regulation of protein synthesis initiation in animal cells. The eukaryotic initiation factor 2 (eIF-2)-associated 67-kDa polypeptide (p67) isolated from reticulocyte lysate protects the eIF-2 alpha subunit from eIF-2 kinase-catalyzed phosphorylation and promotes protein synthesis in the presence of active eIF-2 kinases. We have now studied the roles of p67 and eIF-2 kinases in regulation of protein synthesis using several animal cell lysates and an animal cell line (KRC-7) in culture under various growth conditions. The results are as follows. (i) Both p67 and eIF-2 kinase(s) are present in active forms in all animal cells under normal growth conditions and p67 protects the eIF-2 alpha subunit from eIF-2 kinase-catalyzed phosphorylation, thus promoting protein synthesis in the presence of active eIF-2 kinases. (ii) In heme-deficient reticulocyte lysates and in serum-starved KRC-7 cells in culture, p67 is deglycosylated and subsequently degraded. This leads to eIF-2 kinase-catalyzed eIF-2 alpha-subunit phosphorylation and thus to protein synthesis inhibition. (iii) Addition of a mitogen (namely, phorbol 12-myristate 13-acetate) to serum-starved KRC-7 cells in culture induces an increase of p67 and thus increases protein synthesis. These results suggest the following conclusions. (i) Protein synthesis inhibition in a heme-deficient reticulocyte lysate is not due to the activation of an eIF-2 kinase (heme-regulated inhibitor), as is generally believed, but is due to degradation of p67. The heme-regulated inhibitor is present in an active form and possibly in equal amounts in both heme-deficient and heme-supplemented reticulocyte lysates but cannot phosphorylate eIF-2 alpha subunit because of the presence of p67. (ii) p67 is essential for protein synthesis as it protects the eIF-2 alpha subunit from eIF-2 kinase-catalyzed phosphorylation and promotes protein synthesis in the presence of one or more active eIF-2 kinases present in all animal cells. (iii) p67 is both degradable and inducible. Only the p67 level correlates directly with the protein synthesis activity of the cell, indicating that p67 is a critical factor in protein synthesis regulation in animal cells.


Synaptic localization of kappa opioid receptors in guinea pig neostriatum. Distribution of kappa opioid receptors was examined by EM radioautography in sections of guinea pig neostriatum with the selective 125I-labeled dynorphin analog [D-Pro10]dynorphin-(1-11). Most specifically labeled binding sites were found by probability circle analysis to be associated with neuronal membrane appositions. Because of limitations in resolution of the method, the radioactive sources could not be ascribed directly to either one of the apposed plasma membranes. Nevertheless, three lines of evidence favored a predominant association of ligand with dendrites of intrinsic striatal neurons: (i) the high frequency with which labeled interfaces implicated a dendrite, (ii) the enrichment of dendro-dendritic interfaces, and (iii) the occurrence of dendritic profiles labeled at several contact points along their plasma membranes. A small proportion of labeled sites was associated with axo-axonic interfaces, which may subserve the kappa opioid-induced regulation of presynaptic dopamine and acetylcholine release documented in guinea pig neostriatum. Although most membrane-associated kappa sites were found at extrasynaptic locations, approximately 23% were associated with synaptic specializations. This proportion is markedly higher than that previously reported for either mu or delta sites in rat neostriatum. Whether labeled synapses represent preferential sites of action for kappa ligands remains to be established. In any event, these results support the hypothesis that in mammalian brain kappa opioid receptors are conformationally and functionally distinct from mu and delta types.


Most gamma delta T cells develop normally in beta 2-microglobulin-deficient mice. The specificity of T cells bearing gamma delta T-cell receptors (gamma delta+ T cells) is poorly characterized. Earlier studies suggest that like alpha beta+CD8+ T cells, some gamma delta+ T cells may recognize antigens associated with class I major histocompatibility complex molecules. alpha beta+CD8+ T cells are nearly absent in class I-deficient mice (mutant for beta 2-microglobulin), reflecting a requirement for intrathymic "positive selection" of these cells by class I molecules. Here, we examine whether the development of gamma delta+ T cells is altered in the beta 2-microglobulin mutant mice. We show that the cellularity, marker expression, repertoire, and functional competence of gamma delta+ T cells are not detectably deficient in beta 2-microglobulin mutant mice. We conclude that class I expression is unnecessary for the development of most gamma delta+ T cells.


Hyperthyroidism due to inappropriate secretion of thyrotropin in 10 patients. The syndrome of inappropriate thyroid-stimulating hormone (TSH) secretion, characterized by elevated serum free thyroxine and triiodothyronine levels in association with measurable serum TSH concentrations, remains an uncommon cause of hyperthyroidism that is being recognized with increasing frequency. The hyperthyroidism may be due to either neoplastic pituitary TSH secretion or selective pituitary resistance to thyroid hormone. In an effort to better understand this rare cause of hyperthyroidism, we undertook a retrospective analysis of our institution's experience with this condition. We reviewed our cumulative experience (10 patients) with hyperthyroidism due to the syndrome of inappropriate secretion of TSH. Six patients were diagnosed with TSH-secreting pituitary adenomas and four were found to have selective pituitary resistance to thyroid hormone. One patient with tumor had a TSH-secreting pituitary adenoma in the setting of multiple endocrine neoplasia syndrome. In all patients with tumor, hyperthyroidism was successfully treated with transsphenoidal adenomectomy with or without pituitary radiotherapy. All four patients with pituitary resistance had thyroid ablation or resection prior to their correct diagnosis. Therefore, therapy for this group of patients involved thyroid hormone replacement and efforts to suppress TSH hypersecretion. All 10 patients have done well clinically, with follow-up ranging from 2 weeks to 13 years. Adequate treatment exists for the two primary causes of TSH hypersecretion. TSH-secreting pituitary adenomas are treated with surgery and, if necessary, adjuvant pituitary radiotherapy. The results are generally good if the tumor is diagnosed and treated at an early stage. Primary therapy for hyperthyroidism due to selective pituitary resistance to thyroid hormone is aimed at suppression of pituitary TSH hypersecretion. The evaluation of any patient with hyperthyroidism must be thorough and, in some cases, should include measurement of TSH to determine the presence of inappropriate secretion. Eliminating this diagnosis will help avoid improper and potentially harmful treatment of hyperthyroid patients.


Neu proto-oncogene amplification and expression in ovarian adenocarcinoma cell lines. In this communication, the authors summarize their characterization of eight ovarian adenocarcinoma-derived cell lines for level of neu gene amplification, expression of neu transcripts and protein, and intraperitoneal tumorigenicity in nude mice. Two of the eight cell lines in our study (SKOV3 and YAOVBIX1) exhibited five- to ninefold neu DNA sequence amplification, accompanied by up to 200-fold overexpression of transcripts and protein (p185). Both of these cell lines expressed a major approximately 7.5 kb neu-complementary transcript not previously reported in other neu-positive tumor cell lines. One pair of cell lines (YAOVBIX1 and YAOVBIX3), isolated from a single ovarian carcinoma patient's ascites sample differed dramatically in regard to level of neu gene amplification and expression. Immunohistochemical staining of the primary ovarian tumor from which these two lines were derived demonstrated populations of both neu-positive and neu-negative malignant epithelial cells. Seven of the eight ovarian carcinoma lines produced intra-abdominal tumors after intraperitoneal injection into nude mice, irrespective of level of neu gene expression. This study demonstrates tumor cell heterogeneity with regard to neu gene amplification and expression in an ovarian adenocarcinoma, reveals the overexpression of novel neu-complementary transcripts in two independently isolated ovarian adenocarcinoma cell lines, and suggests that neu gene expression is not required for intraperitoneal tumorigenicity of ovarian carcinoma xenografts in a nude mouse model system.


Relative role of the glutaminase, glutamate dehydrogenase, and AMP-deaminase pathways in hepatic ureagenesis: studies with 15N. We have studied the relative roles of the glutaminase versus glutamate dehydrogenase (GLDH) and purine nucleotide cycle (PNC) pathways in furnishing ammonia for urea synthesis. Isolated rat hepatocytes were incubated at pH 7.4 and 37 degrees C in Krebs buffer supplemented with 0.1 mM L-ornithine and 1 mM [2-15N]glutamine, [5-15N]glutamine, [15N]aspartate, or [15N]glutamate as the sole labeled nitrogen source in the presence and absence of 1 mM amino-oxyacetate (AOA). A separate series of incubations was carried out in a medium containing either 15N-labeled precursor together with an additional 19 unlabeled amino acids at concentrations similar to those of rat plasma. GC-MS was utilized to determine the precursor product relationship and the flux of 15N-labeled substrate toward 15NH3, the 6-amino group of adenine nucleotides ([6-15NH2]adenine), 15N-amino acids, and [15N]urea. Following 40 min incubation with [15N]aspartate the isotopic enrichment of singly and doubly labeled urea was 70 and 20 atom % excess, respectively; with [15N]glutamate these values were approximately 65 and approximately 30 atom % excess for singly and doubly labeled urea, respectively. In experiments with [15N]aspartate as a sole substrate 15NH3 enrichment exceeded that in [6-NH2]adenine, indicating that [6-15NH2]adenine could not be a major precursor to 15NH3. Addition of AOA inhibited the formation of [15N]glutamate, 15NH3 and doubly labeled urea from [15N]aspartate. However, AOA had little effect on [6-15NH2]adenine production. In experiments with [15N]glutamate, AOA inhibited the formation of [15N]aspartate and doubly labeled urea, whereas 15NH3 formation was increased. In the presence of a physiologic amino acid mixture, [15N]glutamate contributed less than 5% to urea-N. In contrast, the amide and the amino nitrogen of glutamine contributed approximately 65% of total urea-N regardless of the incubation medium. The current data indicate that when glutamate is a sole substrate the flux through GLDH is more prominent in furnishing NH3 for urea synthesis than the flux through the PNC. However, in experiments with medium containing a mixture of amino acids utilized by the rat liver in vivo, the fraction of NH3 derived via GLDH or PNC was negligible compared with the amount of ammonia derived via the glutaminase pathway. Therefore, the current data suggest that ammonia derived from 5-N of glutamine via glutaminase is the major source of nitrogen for hepatic urea-genesis.


Isolation of two novel sialyl-Lewis X-active oligosaccharides by high-performance liquid affinity chromatography using monoclonal antibody Onc-M26. A monoclonal antibody, Onc-M26, that recognizes a cancer-associated antigen expressed by most human adenocarcinomas of the breast was shown previously to recognize a carbohydrate epitope carried on a hexaglycosyl ganglioside carrying the sialyl-Lewis X (SLex) antigen (P.S. Linsley et al., 1988, Cancer Res. 48, 2138-2148). Evidence that the antibody binds even more avidly to minor gangliosides containing more complex carbohydrate chains prompted us to search for a higher affinity epitope among sialylated oligosaccharides from pooled human milk. Affinity chromatography of a partially purified fraction of monosialylated milk oligosaccharides on a column containing monoclonal antibody Onc-M26 bound to a macroporous silica matrix gave a peak with a retention volume significantly greater than that of a standard SLex-active hexasaccharide. The retained material consisted of two nonasaccharides, each containing the SLex tetrasaccharide sequence, Neu5Ac alpha 2-3Gal beta 1-4(Fuc alpha 1-3) GlcNAc, linked beta 1-6 to a 3,6-disubstituted galactosyl residue.


Changing profiles of failed coronary angioplasty patients: impact on surgical results. As more high-risk patients undergo percutaneous transluminal coronary angioplasty (PTCA), the changing profiles of PTCA patients who may require emergent coronary artery bypass grafting may alter operative morbidity and mortality. This study compared profiles of recent patients undergoing emergent coronary artery bypass grafting after a failed PTCA with earlier patients to determine their impact on operative results. From 1980 to 1988, 53 patients underwent emergent coronary artery bypass grafting after a failed PTCA at the Boston University Medical Center. These patients were divided into two groups based on the year of the PTCA: group I, 1980 to 1985 (n = 18); and group II, 1986 to 1988 (n = 35). Group II patients tended to be older (age greater than or equal to 65 years, 47% group II versus 11% group I), were more likely to have unstable angina before PTCA (74% versus 33%), and had lower ejection fractions (0.53 +/- 0.02 versus 0.63 +/- 0.05) and more vessels with 50% or greater stenosis (2.1 +/- 0.2 versus 1.6 +/- 0.2). Nevertheless, there was no significant difference in the incidence of perioperative myocardial infarcts using enzyme and electrocardiographic criteria (37% in group II versus 39% in group I), 30-day operative mortality (11% in group II versus 11% in group I), or major postoperative complications (14% in group II versus 22% in group I). We conclude that despite the changing profiles of patients undergoing PTCA, which include older patients with more extensive coronary artery disease and lower ejection fractions, operative results after emergent coronary artery bypass grafting for failed PTCAs remain unchanged.


A novel variant of Sindbis virus is both neurovirulent and neuroinvasive in adult mice. A strain of Sindbis virus (SV), recently isolated from mosquitoes in Israel, was used as a source for variants which differ in neuroinvasiveness and virulence that were generated by serial passage of SV in suckling and weanling mouse brain. At the 15th passage a neurovirulent variant was observed and designated SVN (neurovirulent). After 7 more passages in weanling mouse brains, another variant was observed and designated SVNI (neuroinvasive) and both were isolated and purified. All strains caused similar viremia after intraperitoneal (I.P.) injection of weanling mice, but whereas SV was neuroinvasive but nonvirulent, SVN was neurovirulent but noninvasive and SVNI was both virulent and invasive. SVNI is the first SV variant which is both neurovirulent and neuroinvasive in weanling mice. Co-injection I.P. of SV + SVN resulted in presence of SV alone in the mouse brain; co-injection of SVNI + SVN resulted in full-titered replication of both strains in the brain. We assume that this is achieved through a breach of the blood brain barrier effected by SVNI replication and used by SVN for co-invasion. SV probably invades the brain by a different mechanism. I.P. infection with SVNI of inbred BALB/c mice gave rise to clinical signs only in a few mice even though substantial viremia was demonstrated.


A cDNA construct of tissue inhibitor of metalloproteinases (TIMP) linked to the last exon of Thy-1 confers glycophospholipid anchorage on this naturally secreted protein. A naturally secreted protein, tissue inhibitor of metalloproteinases (TIMP), has been transiently expressed on the surface of transfected COS cells and stably on transfected murine BW 5147 thymoma cells, by linkage of the entire coding sequence of the cDNA to the last exon of Thy-1. Thy-1 is a glycophospholipid-linked protein. In COS cells the chimaeric protein can be labelled by [3H]ethanolamine, which is a component of glycophospholipid anchors. Ltk- cells cannot anchor proteins by glycan phosphatidylinositol linkage and were found to be unable to express the engineered protein extracellularly on their plasma membranes. Phosphatidylinositol-specific phospholipase C treatment released 90% of the protein from all BW 5147 cells, but very little from the COS-1 cells. It is concluded that the last exon of Thy-1 has conferred the property of glycophospholipid anchorage on the normally secreted protein TIMP.


Induction of peroxisomal beta-oxidation genes by retinoic acid in cultured rat hepatocytes. Retinoic acid is reported here to induce peroxisomal beta-oxidation activities in cultured rat hepatocytes, with a concomitant increase in respective peroxisomal mRNAs. The concentrations of retinoic acid required for inducing liver peroxisomal acyl-CoA oxidase were similar to those required for inducing liver transglutaminase. A putative 5'-flanking response element for retinoic acid may be found within the enhancer region involved in the induction of peroxisomal genes by xenobiotic amphipathic carboxylates.


Differential expression of two msh-related homeobox genes Chox-7 and Chox-8 during chick limb development. We have isolated two closely related cDNAs, Chox-7 and Chox-8, encoding homeodomain-containing proteins homologous to Drosophila msh. The Chox-7 and Chox-8 genes are chicken cognates of mouse Hox-7.1 and Hox-8.1, respectively. In situ hybridization using 3' regions of the cDNAs as probes revealed that the Chox-7 gene is highly expressed in the mesenchyme subjacent to the apical ectodermal ridge whereas Chox-8 expression is localized in the anterodistal mesenchymal region at early stages of limb formation, suggesting different roles during limb development. At later stages, both genes are expressed in the anterior and posterior mesenchymes and in the interdigital mesenchyme where programmed cell death occurs.


High-density lipoprotein antagonizes the inhibitory effects of oxidized low-density lipoprotein and lysolecithin on soluble guanylyl cyclase. Oxidatively modified LDL (LDLox) reduces the response of soluble guanylyl cyclase to nitrovasodilators. We now demonstrate that this desensitization can be antagonized by HDL. Similar to its protective effect against LDLox, HDL also inhibited the lysolecithin-induced desensitization of soluble guanylyl cyclase. Since the lysolecithin content of LDLox correlated with the amount of lysolecithin necessary to diminish stimulation of soluble guanylyl cyclase, our data support the hypothesis that lysolecithin may be responsible for the inhibitory effect of LDLox on smooth muscle relaxation and provide evidence that the antagonistic effect of HDL against desensitization of soluble guanylyl cyclase by atherogenic compounds could be responsible for the protective role of HDL in atherosclerosis.


Structure of the mouse tyrosine hydroxylase gene. The mouse tyrosine hydroxylase (TH) gene was isolated from a genomic library by cross-hybridization with human TH cDNA probe. Nucleotide sequence analysis of two overlapping genomic clones showed that this gene is split into 13 exons distributed about 7.5 kb in length. The transcription initiation site was determined by primer extension analysis with mouse adrenal gland poly(A)+RNA. The structure of the mouse TH gene was similar to that of the human TH gene, but it contained neither the alternative splice donor site around the 3'-end of the first exon nor an independent exon corresponding to the second exon of the human TH gene. There were the canonical TATA and GC boxes, cyclic AMP responsive element (CRE), and AP1 binding site in the 5'-flanking region of the mouse TH gene.


Lipoproteins and their genetic variation in subjects with and without angiographically verified coronary artery disease. To examine the concentration of serum lipoproteins and the association of their genetic variation with the occurrence of coronary artery disease (CAD), composite serum lipoprotein profiles including lipoprotein(a) (Lp[a]), apolipoprotein (apo) E phenotypes, and apo B Xba I genotypes were determined in patients with angiographically verified CAD (CAD+ group, n = 111) and in subjects with no angiographic evidence of CAD (CAD- group, n = 46). In addition, we determined the concentrations of serum lipids, lipoproteins, and apolipoproteins in 96 healthy controls. Both CAD- and CAD+ groups had lower concentrations of apos A-I and A-II but higher concentrations of serum total and very low density lipoprotein triglyceride and very low density lipoprotein cholesterol than did healthy controls. The mean concentrations of serum total and low density lipoprotein cholesterol and the median values of Lp(a) were similar in the CAD+ and CAD- groups, both having higher concentrations of low density lipoprotein cholesterol and apo B than the healthy controls. Irrespective of gender, patients with CAD had significantly lower serum high density lipoprotein cholesterol than did those without CAD (1.48 +/- 0.40 versus 1.16 +/- 0.29 mmol/l, p less than 0.001). In women, the mean serum total and very low density lipoprotein triglyceride concentration was also higher in the CAD+ than in the CAD- group. The frequency of the apo E4 allele (epsilon 4) was significantly higher in the CAD+ group (0.293) than in the CAD- group (0.174; p less than 0.001). The frequencies of the two apo B alleles, X1 (Xba I restriction site absent) and X2 (Xba I restriction site present), were similar in the two groups. Stepwise discriminant analysis revealed that in men, serum high density lipoprotein cholesterol had the highest power to discriminate for CAD. In addition, the concentration of plasma apo B levels and the occurrence of apo E phenotypes were independently associated with CAD in men. In women, the only independent factor associated with CAD after adjustment for beta-blocker and diuretics usage was the concentration of serum triglycerides.


Heat shock protein synthesis of the cyanobacterium Synechocystis PCC 6803: purification of the GroEL-related chaperonin. Synechocystis PCC 6803 cells could be induced to synthesize four major HSPs with apparent molecular sizes of 70, 64, 15 and 14 kDa. Heat stress at 42.5 degrees C appeared to be the optimum temperature for HSP formation in cells grown at 30 degrees C. The relative rate of synthesis of HSP70 and HSP15 reached a maximum at 30 min after the temperature shift-up whereas the capability of cells to accumulate HSP64 and HSP14 continued through 2 h. The two most abundant HSPs, HSP70 and HSP64, were recognized on western blots by antibodies raised against authentic DnaK and GroEL from Escherichia coli. To furnish sufficient evidence for the assumption that HSP64 is a GroEL-related chaperonin, this protein was purified to homogeneity. There was a 76% sequence identity between the amino acid sequence of HSP64 and the corresponding protein in Synechococcus PCC 7942. Moreover, the purified HSP64 cross-reacted to anti-E. coli GroEL antibody. To our knowledge, this is the first report about the purification and partial protein sequencing of a cyanobacterial chaperonin.


Identification and sequence analysis of the promoter for the leukocyte integrin beta-subunit (CD18): a retinoic acid-inducible gene. Leukocyte adhesion receptors (LFA-1; Mac-1; p150,95) are a family of heterodimeric cell-surface adhesion molecules expressed exclusively in granulocytes, lymphocytes, and macrophages. Expression of these proteins is under complex regulatory control, but to date promoters for these genes have not been identified. The CD18 gene codes for the common beta-subunit of the leukocyte adhesion receptors. Transcription of CD18 is highly tissue-specific, hormonally inducible (by retinoic acid [RA]), and coordinately regulated with leukocyte integrin alpha-chains. To identify the CD18 promoter, we screened a human genomic phage library with a human CD18 cDNA probe and obtained a clone that contains an exon coding for the 5' untranslated region (UTR). Using rapid amplification of cDNA ends (RACE), RNAse protection, S1 nuclease, and primer extension assays, we demonstrated the existence of multiple transcription start sites clustered in a 45-nt region. We investigated the transcription-promoting activity of the genomic sequences 5' to the CD18 gene by performing transient expression assays with a growth hormone reporter gene in various hematopoietic cell lines. The CD18 promoter was active in Jurkat cells, a lineage that normally expresses CD18 but was considerably less active in K562, an early erythroid line that does not normally express CD18. The genomic sequences upstream of the start site cluster lack CAAT and TATA boxes, but have two Sp1 binding sites and 10 T(G/C)AC(C/A) boxes, which may represent binding sites for RA receptors (RAR). These features distinguish the CD18 promoter from the promoters of other tissue-specific, hormone-inducible genes, and may be representative of leukocyte integrin promoters in general.


DNA sequence variation in a negative control region 5' to the beta-globin gene correlates with the phenotypic expression of the beta s mutation. The clinical diversity of sickle cell anemia is strongly related to the degree of intracellular hemoglobin S (Hb S) polymerization, which in turn is dependent on the intracellular concentration of Hb S. We have recently defined a region of DNA approximately 500 bp 5' to the human beta-globin gene that acts as a silencer for the transcription of this gene and have shown that a polymorphism in this sequence is associated with a thalassemic phenotype of the beta-globin gene. In this work we have examined the correlation of DNA sequence polymorphisms in this silencer with binding of a previously identified putative repressor protein, BP1, and with the expression of Hb S in individuals heterozygous for the beta s allele. It was found that specific configurations of the motif, (AT)x(T)y, are homogeneous for the major haplotypes of the beta-globin gene cluster described on beta s chromosomes. Binding of BP1 was measured to DNA of three haplotypes: Indian, Benin, and Bantu. BP1 binds most tightly to DNA of the Indian haplotype, and these patients produce less beta s protein than Benin patients, whose DNA exhibits weaker affinity for BP1. Binding of BP1 is the weakest to DNA of the Bantu haplotype, which is associated with clinically more severe sickle cell symptoms. These data are consistent with the hypothesis that these polymorphisms may not be neutral and that the DNA sequence at this site may affect the expression of the beta s gene. Such an effect may be synergistic with other genetic variables, such as fetal hemoglobin levels, F-cell numbers, and the number of alpha-globin genes, in determining intracellular polymerization and, thus, the severity of the sickle cell syndromes.


Control of pain.

Control of pain. Pain in critically ill and injured pediatric patients may go unrecognized and undertreated since children often suffer silently and caretakers are often fearful to intervene aggressively to alleviate pain. Methods are now readily available to relieve pain in the vast majority of ICU patients. Administration of inadequate doses of opioids at infrequent intervals or via a noxious route (intramuscularly) can be supplanted by far superior pain management methods. Provision of nearly constant therapeutic levels of opioid via a painless route is recommended and will usually be well tolerated even by very sick children. This can be achieved by the use of continuous intravenous infusions of opioids, PCA, or epidural administration of local anesthetics or opioids. Flexibility is essential so that the appropriate technique or agent can be selected for a particular pediatric ICU patient.


p53 mutation and loss of heterozygosity on chromosomes 17 and 10 during human astrocytoma progression. The human brain tumor, astrocytoma, typically progresses through three histopathologically defined stages with the passage of time: one premalignant stage, low-grade astrocytoma; and two malignant stages, anaplastic astrocytoma and glioblastoma multiforme. We correlated the results of a sequence analysis of the tumor suppressor gene, p53, and a restriction fragment length polymorphism analysis of chromosomes 17 and 10 in 45 patients with cerebral astrocytomas at different stages. To detect p53 mutations in tumor DNA, we analyzed polymerase chain reaction products corresponding to every p53-coding exon for single-strand conformation polymorphisms and confirmed the mutations by sequencing. Loss of heterozygosity (LOH) was determined by Southern transfer analysis of somatic and tumor DNA from these same patients using polymorphic markers for various loci on chromosomes 10 and 17. p53 mutations were found in 7 of 25 glioblastomas (28%), in 5 of 14 anaplastic astrocytomas (36%) but in 0 of 6 low-grade astrocytomas. p53 mutations were found in 62% of patients with LOH on chromosome 17p. These results indicated that p53 inactivation is a common genetic event in astrocytoma progression that may signal the transition from benign to malignant tumor stages. LOH on chromosome 10 was found in 61% of glioblastomas, in 23% of anaplastic astrocytomas, but in 0% of low-grade astrocytomas. LOH on chromosome 10 and p53 mutation were found together only in patients with glioblastoma multiforme (22%), suggesting that these genetic changes may accumulate during astrocytoma progression.


Loss of heterozygosity affecting the p53, Rb, and mcc/apc tumor suppressor gene loci in dysplastic and cancerous ulcerative colitis. Allelic deletions of tumor suppressor genes have been observed frequently in a variety of human tumors. These losses are believed to contribute to the development of human cancer. Three of the most frequently deleted chromosomal loci contain the tumor suppressor genes p53, retinoblastoma (Rb), and mcc/apc. In order to detect loss of heterozygosity (LOH) within these genes in dysplastic and cancerous ulcerative colitis, we used an application of the polymerase chain reaction. LOH affecting p53 was observed in 8 of 17 (47%) of heterozygous patients, while LOH of Rb and the mcc/apc locus was observed in 9 of 27 (33%) and 13 of 39 (33%) of heterozygotes, respectively. Among 35 patients heterozygous at 2 or more loci, LOH of p53, Rb, and/or mcc/apc was observed in 18 (51%). LOH was more common in left-sided neoplasms. These data suggest that allelic deletion of p53, Rb, mcc, and/or apc is involved in the pathogenesis and/or progression of at least a subset of colonic dysplasias and carcinomas occurring in the setting of ulcerative colitis.


Plasmodium falciparum-infected erythrocytes bind ICAM-1 at a site distinct from LFA-1, Mac-1, and human rhinovirus. The attachment of erythrocytes infected with P. falciparum to human venular endothelium is the primary step leading to complications from severe and cerebral malaria. Intercellular adhesion molecule-1 (ICAM-1, CD54) has been implicated as a cytoadhesion receptor for P. falciparum-infected erythrocytes. Characterization of domain deletion, human/murine chimeric ICAM-1 molecules, and amino acid substitution mutants localized the primary binding site for parasitized erythrocytes to the first amino-terminal immunoglobulin-like domain of ICAM-1. The ICAM-1 binding site is distinct from those recognized by LFA-1, Mac-1, and the human major-type rhinoviruses. Synthetic peptides encompassing the binding site on ICAM-1 inhibited malaria-infected erythrocyte adhesion to ICAM-1-coated surfaces with a Ki of 0.1-0.3 mM, whereas the Ki for soluble ICAM-1 is 0.15 microM. These findings have implications for the therapeutic reversal of malaria-infected erythrocyte sequestration in the host microvasculature.


Analysis of the CYP2D6 gene in relation to debrisoquin and desipramine hydroxylation in a Swedish population. The molecular basis of polymorphic debrisoquin hydroxylation was studied in 223 Swedish white subjects, 187 extensive metabolizers and 36 poor metabolizers phenotyped with debrisoquin and desipramine. Restriction fragment length polymorphism (RFLP) analysis of the CYP2D6 gene revealed that 52% of unrelated poor metabolizers were homozygous for Xba I 29 kb fragment, and only 8% had two mutant alleles detected with RFLP. Allele-specific polymerase chain reaction (PCR)-based DNA amplification, however, revealed that all but one of the poor metabolizers had two mutant alleles of the CYP2D6A or CYP2D6B type or both. Extensive metabolizers who were heterozygous for wild-type and CYP2D6B genes had metabolic ratios for debrisoquin and desipramine that were higher than those of subjects who were homozygous for the wild-type gene. The 16 + 9 kb Xba I RFLP pattern was associated with the poor metabolizer phenotype and CYP2D6B mutations. Three extremely rapid metabolizers of debrisoquin had a 44 kb Xba I fragment that did not carry either CYP2D6A or CYP2D6B mutations. In conclusion, in the Swedish population studied, allele-specific PCR amplification allowed prediction of the debrisoquin hydroxylation phenotype with 99% accuracy.


Southeast Asian mitochondrial DNA analysis reveals genetic continuity of ancient mongoloid migrations. Human mitochondrial DNAs (mtDNAs) from 153 independent samples encompassing seven Asian populations were surveyed for sequence variation using the polymerase chain reaction (PCR), restriction endonuclease analysis and oligonucleotide hybridization. All Asian populations were found to share two ancient AluI/DdeI polymorphisms at nps 10394 and 10397 and to be genetically similar indicating that they share a common ancestry. The greatest mtDNA diversity and the highest frequency of mtDNAs with HpaI/HincII morph 1 were observed in the Vietnamese suggesting a Southern Mongoloid origin of Asians. Remnants of the founding populations of Papua New Guinea (PNG) were found in Malaysia, and a marked frequency cline for the COII/tRNA(Lys) intergenic deletion was observed along coastal Asia. Phylogenetic analysis indicates that both insertion and deletion mutations in the COII/tRNA(Lys) region have occurred more than once.


Native American mitochondrial DNA analysis indicates that the Amerind and the Nadene populations were founded by two independent migrations. Mitochondrial DNAs (mtDNAs) from 167 American Indians including 87 Amerind-speakers (Amerinds) and 80 Nadene-speakers (Nadene) were surveyed for sequence variation by detailed restriction analysis. All Native American mtDNAs clustered into one of four distinct lineages, defined by the restriction site variants: HincII site loss at np 13,259, AluI site loss at np 5,176, 9-base pair (9-bp) COII-tRNA(Lys) intergenic deletion and HaeIII site gain at np 663. The HincII np 13,259 and AluI np 5,176 lineages were observed exclusively in Amerinds and were shared by all such tribal groups analyzed, thus demonstrating that North, Central and South American Amerinds originated from a common ancestral genetic stock. The 9-bp deletion and HaeIII np 663 lineages were found in both the Amerinds and Nadene but the Nadene HaeIII np 663 lineage had a unique sublineage defined by an RsaI site loss at np 16,329. The amount of sequence variation accumulated in the Amerind HincII np 13,259 and AluI np 5,176 lineages and that in the Amerind portion of the HaeIII np 663 lineage all gave divergence times in the order of 20,000 years before present. The divergence time for the Nadene portion of the HaeIII np 663 lineage was about 6,000-10,000 years. Hence, the ancestral Nadene migrated from Asia independently and considerably more recently than the progenitors of the Amerinds. The divergence times of both the Amerind and Nadene branches of the COII-tRNA(Lys) deletion lineage were intermediate between the Amerind and Nadene specific lineages, raising the possibility of a third source of mtDNA in American Indians.


A restriction fragment length polymorphism map and electrophoretic karyotype of the fungal maize pathogen Cochliobolus heterostrophus. A restriction fragment length polymorphism (RFLP) map has been constructed of the nuclear genome of the plant pathogenic ascomycete Cochliobolus heterostrophus. The segregation of 128 RFLP and 4 phenotypic markers was analyzed among 91 random progeny of a single cross; linkages were detected among 126 of the markers. The intact chromosomal DNAs of the parents and certain progeny were separated using pulsed field gel electrophoresis and hybridized with probes used to detect the RFLPs. In this way, 125 markers were assigned to specific chromosomes and linkages among 120 of the markers were confirmed. These linkages totalled 941 centimorgans (cM). Several RFLPs and a reciprocal translocation were identified tightly linked to Tox1, a locus controlling host-specific virulence. Other differences in chromosome arrangement between the parents were also detected. Fourteen gaps of at least 40 cM were identified between linkage groups on the same chromosomes; the total map length was therefore estimated to be, at a minimum, 1501 cM. Fifteen A chromosomes ranging from about 1.3 megabases (Mb) to about 3.7 Mb were identified; one of the strains also has an apparent B chromosome. This chromosome appears to be completely dispensable; in some progeny, all of 15 markers that mapped to this chromosome were absent. The total genome size was estimated to be roughly 35 Mb. Based on these estimates of map length and physical genome size, the average kb/cM ratio in this cross was calculated to be approximately 23. This low ratio of physical length to map distance should make this RFLP map a useful tool for cloning genes.


Identification of a Serratia entomophila genetic locus encoding amber disease in New Zealand grass grub (Costelytra zealandica). Serratia entomophila UC9 (A1MO2), which causes amber disease in the New Zealand grass grub Costelytra zealandica, was subjected to transposon (TnphoA)-induced mutagenesis. A mutant (UC21) was found to be nonpathogenic (Path-) to grass grub larvae in bioassays and was shown, by Southern hybridization, to contain a single TnphoA insertion. This mutant failed to adhere to the gut wall (Adn-) of the larvae and also failed to produce pili (Pil-). A comparative study of the total protein profiles of wild-type S. entomophila UC9 and mutant UC21 revealed that the mutant lacked an approximately 44-kDa protein and overexpressed an approximately 20-kDa protein. Transfer of cosmids containing homologous wild-type sequences into mutant strain UC21 restored wild-type phenotypes (Path+, Pil+, and Adn+). One of the complementing cosmids (pSER107) conferred piliation on Pil- Escherichia coli HB101. The TnphoA insertion in UC21 was mapped within an 8.6-kb BamHI fragment common to the complementing cosmids, and we designated this gene locus amb-1. Six gene products with molecular masses of 44, 36, 34, 33, 20, and 18 kDa were detected in E. coli minicells exclusive to the cloned 8.6-kb fragment (pSER201A). The 44-kDa gene product was not detected in E. coli minicells containing the cloned mutant fragment. Saturation mutagenesis of this fragment produced four unlinked insertional mutations with active fusions to TnphoA. These active fusions disrupted the expression of one or more gene products encoded by amb-1. The 8.6-kb fragment cloned in the opposite orientation (pSER201B) expressed only a 20-kDa protein. We propose that these are the products of structural and/or regulatory genes involved in adhesion and/or piliation which are prerequisites in the S. entomophila-grass grub interaction leading to amber disease.


Interaction of the Bacillus subtilis glnRA repressor with operator and promoter sequences in vivo. In vivo dimethyl sulfate footprinting of the Bacillus subtilis glnRA regulatory region under repressing and derepressing conditions demonstrated that the GlnR protein, encoded by glnR, interacts with two sites situated within and adjacent to the glnRA promoter. One site, glnRAo1, between positions -40 and -60 relative to the start point of transcription, is a 21-bp symmetrical element that has been identified as essential for glnRA regulation (H. J. Schreier, C. A. Rostkowski, J. F. Nomellini, and K. D. Hirschi, J. Mol. Biol. 220:241-253, 1991). The second site, glnRAo2, is a quasisymmetrical element having partial homology to glnRAo1 and is located within the promoter between positions -17 and -37. The symmetry and extent of modifications observed for each site during repression and derepression indicated that GlnR interacts with the glnRA regulatory region by binding to both sites in approximately the same manner. Experiments using potassium permanganate to probe open complex formation by RNA polymerase demonstrated that transcriptional initiation is inhibited by GlnR. Furthermore, distortion of the DNA helix within glnRAo2 occurred upon GlnR binding. While glutamine synthetase, encoded by glnA, has been implicated in controlling glnRA expression, analyses with dimethyl sulfate and potassium permanganate ruled out a role for glutamine synthetase in directly influencing transcription by binding to operator and promoter regions. Our results suggested that inhibition of transcription from the glnRA promoter involves GlnR occupancy at both glnRAo1 and glnRAo2. In addition, modification of bases within the glnRAo2 operator indicated that control of glnRA expression under nitrogen-limiting (derepressing) conditions included the involvement of a factor(s) other than GlnR.


Increased muscarinic cholinergic receptor density on CD4+ lymphocytes in progressive multiple sclerosis. We measured the density and affinity of muscarinic cholinergic receptors (MR) in 29 chronic progressive and ten stable multiple sclerosis (MS) patients and 27 control subjects using [3H]N-methyl-scopolamine. The density of MR on CD4+ lymphocytes was significantly higher in chronic progressive MS (CPMS) than in controls (7.9 +/- 0.7 vs. 4.5 +/- 0.4 fmol/10(6) cells, p less than 0.001). Stable patients did not differ significantly from control subjects. Receptors of the M1 subtype were measured on CD4+ lymphocytes of nine patients and seven controls with the selective antagonist [3H]methylpirenzepine: M1/total receptor ratio was 64.1% in CPMS and 81.2% in controls, suggesting a selective increase of M2-type MR in CPMS. The findings may relate to parasympathetic denervation hypersensitivity of lymphocytes or to lymphocyte activation which is known to be associated with increased MR number.


Putative periodontopathogens in "diseased" and "non-diseased" persons exhibiting poor oral hygiene. The aim of the study was to assess the occurrence of some putative periodonto-pathogens in "test" and "control" sites in "diseased" and "non-diseased" persons, respectively, from an adult rural Kenyan population exhibiting poor oral hygiene and widespread loss of attachment (LA). 14 persons (less than 35 years) were assigned to a "diseased" category on the basis of at least 4 sites with LA greater than or equal to 4 mm; at least 5 mm LA and a pocket greater than or equal to 4 mm interproximally in a lower incisor ("test" site): and less than 2 mm LA and no pocket greater than or equal to 4 mm distal to a lower canine or mesial to a lower first premolar ("control" site). Age-matched "non-diseased" persons were identified on the basis of no sites with LA greater than 2 mm and no pockets greater than or equal to 4 mm associated with LA. Paperpoint samples from test and control sites as well as a scraping sample from the dorsum of tongue were examined for presence of Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Bacteroides intermedius, B. melaninogenicus group, Capnocytophaga, Selenomonas spp., and Wolinella recta. P. gingivalis was found in 79% of test sites and 36% of control sites in "diseased" persons, and in 18% and 35% of test and control sites, respectively, in "non-diseased" persons. "No other bacterial group discriminated significantly between test and control sites or between diseased and non-diseased subjects. The surprisingly high occurrence of P. gingivalis in non-diseased subjects, both subgingivally and on tongue, indicates that deep periodontal pockets are not prerequisite ecological environments for P. gingivalis establishment.


Use of intravenous esmolol to predict efficacy of oral beta-adrenergic blocker therapy in patients with neurocardiogenic syncope. The usefulness of esmolol in predicting the efficacy of treatment with an oral beta-adrenergic blocking agent was evaluated in 27 consecutive patients with neurocardiogenic syncope. Seventeen patients had a positive head-up tilt test response at baseline and 10 patients required intravenous isoproterenol for provocation of hypotension. All patients were then given a continuous esmolol infusion (500 micrograms/kg per min loading dose for 3 min followed by 300 micrograms/kg per min maintenance dose) and rechallenged with a head-up tilt test at baseline or with isoproterenol. Of the 17 patients with a positive baseline tilt test response, 11 continued to have a positive response to esmolol challenge. Sixteen patients (including all 10 patients with a positive tilt test response with isoproterenol) exhibited a negative response to upright tilt during esmolol infusion. Irrespective of their response to esmolol infusion, all patients had a follow-up tilt test with oral metoprolol after an interval of greater than or equal to 5 half-lives of the drug. All 16 patients (100%) with a negative tilt test response during esmolol infusion had a negative tilt test response with oral metoprolol. Of the 11 patients with a positive tilt test response during esmolol infusion, 10 (90%) continued to have a positive response with oral metoprolol. It is concluded that in the electrophysiology laboratory, esmolol can accurately predict the outcome of a head-up tilt response to oral metoprolol. This information may be helpful in formulating a therapeutic strategy at the initial head-up tilt test in patients with neurocardiogenic syncope.


Cytokine regulation of proliferation and ICAM-1 expression of human dermal microvascular endothelial cells in vitro. The effects of recombinant human interleukin 1 alpha (IL-1 alpha), interleukin 1 beta (IL-1 beta), interleukin 6 (IL-6), granulocyte/macrophage colony-stimulating factor (GM-CSF) and tumor necrosis factor-alpha (TNF) on the cell proliferation and the expression of intercellular adhesion molecule-1 (ICAM-1) were assessed in cultured human dermal microvascular endothelial cells (HDMEC). IL-1 alpha and IL-1 beta stimulated the proliferation of HDMEC in a dose-dependent manner, whereas in control experiments using human umbilical vein endothelial cells (HUVEC), IL-1 alpha and IL-1 beta did not stimulate HUVEC growth. Also GM-CSF stimulated the proliferation of HDMEC, whereas IL-6 did not affect endothelial cell growth in vitro. Treatment with IL-1 alpha, IL-1 beta, and TNF markedly increased the expression of ICAM-1 on HDMEC in a time- and dose-dependent manner, in contrast to IL-6 and GM-CSF. By pre-embedding immunoelectron microscopy, membrane-bound expression of ICAM-1 was visualized with pronounced labeling in areas of microvillous cell protrusions. The TNF-induced expression of ICAM-1 on HDMEC was blocked by co-incubation with a neutralizing antibody against TNF, but not with neutralizing antibodies against IL-1 alpha, IL-1 beta, or IL-6. In addition, co-incubation of HDMEC with TNF and the retinoid compound acitretin, dexamethasone, or indomethacin did not abrogate the TNF-induced ICAM-1 expression. These results disclose IL-1 as a major, multifunctional endothelial cell-targeted cytokine and further confirm the concept that pro-inflammatory cytokines exert differential regulatory effects on dermal microvascular endothelial cell proliferation and expression of cell-adhesion molecules.


Efficacy of epanolol versus metoprolol in angina pectoris: report from a Swedish multicentre study of exercise tolerance. The efficacy of epanolol vs. metoprolol in stable angina pectoris was compared in 114 patients recruited to a randomized double-blind cross-over study, consisting of a 4-week period on each drug. Epanolol (200 mg) or metoprolol (200 mg) was administered daily. Bicycle ergometry was performed at the end of each treatment period. The maximum workload was 134 +/- 18 W on epanolol and 133 +/- 37 W on metoprolol (NS). Values for resting heart rate (epanolol, 72 +/- 11 beats min-1; metoprolol, 64 +/- 12 beats min-1; P less than 0.001), systolic blood pressure (epanolol, 143 +/- 21 mmHg; metoprolol, 137 +/- 21 mmHg; P less than 0.05) and diastolic blood pressure (epanolol, 88 +/- 10 mmHg; metoprolol, 84 +/- 11 mmHg; P less than 0.01) were all higher on epanolol treatment. During exercise, the increase in heart rate and blood pressure was of similar magnitude during the two treatment periods, and these parameters did not differ significantly at the last identical workload. The rating of chest pain, fatigue and dyspnoea did not differ between the two drugs during submaximal or maximal exercise. In conclusion, 200 mg of epanolol and metoprolol have similar efficacy with regard to exercise tolerance. As expected from the partial agonist activity present in epanolol but not in metoprolol, the former drug resulted in a higher heart rate and blood pressure at rest. The observed increase in these parameters during exercise was similar for both drugs.


Activation-induced death by apoptosis in CD4+ T cells from human immunodeficiency virus-infected asymptomatic individuals. In immature thymocytes, T cell receptor for antigen (TCR) mobilization leads to an active T cell suicide process, apoptosis, which is involved in the selection of the T cell repertoire. We have proposed that inappropriate induction of such a cell death program in the mature CD4+ T cell population could account for both early qualitative and late quantitative CD4+ T lymphocyte defects of human immunodeficiency virus (HIV)-infected individuals (Ameisen, J.C., and A. Capron. 1991. Immunol. Today. 4:102). Here, we report that the selective failure of CD4+ T cells from 59 clinically asymptomatic HIV-infected individuals to proliferate in vitro to TCR mobilization by major histocompatibility complex class II-dependent superantigens and to pokeweed mitogen (PWM) is due to an active CD4+ T cell death process, with the biochemical and ultrastructural features of apoptosis. Activation-induced cell death occurred only in the CD4+ T cell population from HIV-infected asymptomatic individuals and was not observed in T cells from any of 58 HIV-seronegative controls, including nine patients with other acute or chronic infectious diseases. Activation-induced CD4+ T cell death was prevented by cycloheximide, cyclosporin A, and a CD28 monoclonal antibody (mAb). The CD28 mAb not only prevented apoptosis but also restored T cell proliferation to stimuli, including PWM, superantigens, and the tetanus and influenza recall antigens. These findings may have implications for the understanding of the pathogenesis of acquired immune deficiency syndrome and for the design of specific therapeutic strategies.