Pulmonary function and stress response after laparoscopic cholecystectomy: comparison with subcostal incision and influence of thoracic epidural analgesia. Laparoscopic cholecystectomy (LPC) is increasingly used to treat symptomatic cholelithiasis. We compared the effects of cholecystectomy by subcostal incision to those of LPC on lung function and endocrine metabolic response. The effects of thoracic epidural analgesia for LPC were studied as well. Thirty patients undergoing elective cholecystectomy under general anesthesia were allocated to three study groups: group I, cholecystectomy by subcostal incision; group II, LPC; group III, LPC and epidural analgesia with 0.5% bupivacaine with epinephrine, followed by continuous epidural infusion of 6 mL of 0.5% bupivacaine. Forced vital capacity (FVC), peak expiratory flow, and forced expiratory volume in 1 s were measured with the patients in a half-sitting position. In all groups, sustained decreases in FVC, forced expiratory volume in 1 s, and peak expiratory flow were observed up to 24 h after surgery. Reduction of FVC was significantly more in group I compared with groups II and III (P less than 0.05). The FVC in group I decreased from 3.8 +/- 0.42 (SD) to 1.1 +/- 0.27 L (P less than 0.01), in group II from 3.6 +/- 1.46 to 2.1 +/- 0.94 L (P less than 0.05), and in group III from 3.8 +/- 0.92 to 2.8 +/- 0.90 L (P less than 0.05). In all groups, plasma glucose and cortisol increased after surgery compared with baseline levels (P less than 0.05). At 240 min after surgery, a small but significant decrease of cortisol was measured in group III (P less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)


Histochemical and immunohistochemical study of the mucosal lymphoid system in swine. Enzyme histochemical and immunohistochemical techniques were used to examine palatine tonsils and aggregated lymphoid follicles (Peyer's patches) of the ileum in 6- to 9-day-old and in 6-month-old pigs. Histochemical techniques were used to detect alpha-naphthyl-acetate esterase (ANAE), alpha-naphthyl-butyrate esterase (ANBE), beta-glucuronidase, adenosine triphosphatase (ATPase), and acid phosphatase (AcP). Nonspecific esterases (ANAE, ANBE) were detected in macrophages, T-cell area lymphocytes, eosinophils, fibroblastic reticular cells (FRC), follicular dendritic cells (FDC), and interdigitating cells (IDC). beta-Glucuronidase reactivity was strong in macrophages, eosinophils, FDC, and IDC, and weaker in FRC. Adenosine triphosphatase reactivity was detected in B-cell area lymphocytes, FDC, FRC, and IDC. Cell types with acid phosphatase reactivity were macrophages, FDC, FRC, and IDC. Nonepithelial cells of tonsils and aggregated lymphoid follicles of the ileum had similar enzymatic reactions. In Peyer's patches, however, epithelial cells were positive for all enzymes studied; in tonsils, only nonspecific esterases were detected. Immunoperoxidase techniques were used to detect S-100 protein and cytoplasmic immunoglobulins (IgG, IgM, and IgA). The S-100 protein was detected in lymphocytes, FDC, and FRC of tonsils and Peyer's patches; in tonsillar epithelial and endothelial cells; and in IDC of Peyer's patches.(ABSTRACT TRUNCATED AT 250 WORDS)


Characterization and pathogenicity for pigs of a hog cholera virus strain isolated from wild boars. One hog cholera virus strain isolated from an outbreak of the disease in a wild boar breeding herd in Brittany (France) in 1990 has been characterized with a panel of monoclonal antibodies to hog cholera virus and ruminant pestiviruses: the strain was found to be indistinguishable from that of other domestic pig isolates. The pathogenicity of the strain to domestic pigs was evaluated by infecting intranasally, intramuscularly and by contact 17 specific pathogen-free 6-week- and 12-week-old pigs. Sixteen of the 17 pigs showed symptoms of hog cholera. The virus was detected in the blood of the 16 pigs during all phases of hyperthermia which persisted up to death or the terminal phase, ie between 16 and 29 days post-infection. One animal recovered after presenting a mild form of the disease. This pig was the only one which raised antibodies to the virus. Typical hog cholera lesions were observed in 2 pigs only; the other animal showed very few pathological changes. No relationship between intensity or duration of the disease and pathological changes could be established.


Serological alpha 1-antichymotrypsin in Down's syndrome and Alzheimer's disease. alpha 1-Antichymotrypsin (ACT) is a serine protease inhibitor that is markedly elevated in the serum and cerebrospinal fluid of patients with Alzheimer's disease (AD). Patients with Down's syndrome are known to develop neuropathological changes of AD by age 40 years and many become demented. Therefore, in the present study, we obtained serum ACT levels from patients with Down's syndrome and AD, diagnosed by autopsy or clinically, and healthy control subjects. Newman-Keuls' multiple range test revealed a significantly greater (p less than 0.01) mean ACT level in the Autopsy AD (906.4 +/- 94.64 mg/L) and Clinical AD (745.00 +/- 59.95 mg/L) groups in contrast to the Old Control group (531.00 +/- 23.05 mg/L). The mean ACT level of the Down's Syndrome group (513.33 +/- 14.73 mg/L) was not significantly different from that of the Young Control subjects. Furthermore, we did not observe a positive correlation of ACT levels with age in the Down's Syndrome group, in spite of the age-dependent premature increase in neuropathological changes of AD that are known to occur in patients with Down's syndrome. A positive correlation between serum ACT levels and the density of plaques or tangles, neuropathological hallmarks of AD, in brains of patients with AD also did not exist. Thus, our results suggest that ACT levels may not parallel the development of the classical neuropathological hallmarks of AD.


Absolute bioavailability of clarithromycin after oral administration in humans. The absolute bioavailability of clarithromycin, a new macrolide antimicrobial agent, was assessed in a three-way, randomized, single-dose, crossover study conducted with 22 healthy volunteers, 19 of whom provided analyzable study data. The bioavailability parameters of two 250-mg oral tablet formulations were calculated with reference to an identical dose administered by intravenous infusion of the lactobionate salt. After adjustment for formulation potency, the mean absolute bioavailabilities of the two oral formulations were 52 and 55%, on the basis of the appearance of parent compound in the systemic circulation. Metabolite peak concentration and area under the plasma concentration-time curve data after oral dosing were generally greater than those after intravenous infusion, suggesting that marked first-pass metabolism of clarithromycin occurs after oral administration. Pharmacokinetic analysis of the parent drug and the active 14-hydroxy metabolite data suggests complete (or nearly complete) absorption of the drug after oral administration.


Comparison of the intracellular activities of clarithromycin and erythromycin against Mycobacterium avium complex strains in J774 cells and in alveolar macrophages from human immunodeficiency virus type 1-infected individuals. The intracellular activities of clarithromycin and erythromycin, alone and in combination with other antimicrobial agents, were tested against Mycobacterium avium complex (MAC) strains inside mouse J774 cells and inside alveolar macrophages obtained from human immunodeficiency type 1-infected individuals. Clarithromycin alone had greater intracellular activity than erythromycin alone, and drug combinations that included clarithromycin were usually more active than combinations that included erythromycin.


Clarithromycin-ciprofloxacin-amikacin for therapy of Mycobacterium avium-Mycobacterium intracellulare bacteremia in patients with AIDS. A combination of clarithromycin, ciprofloxacin, and amikacin for the treatment of Mycobacterium avium-Mycobacterium intracellulare bacteremia was evaluated in 12 AIDS patients. Mycobacteremia cleared in all patients by 2 to 8 weeks of treatment, and symptoms resolved. Four patients died; all had negative blood cultures until death, and disseminated M. avium-M. intracellulare complex infection was not considered the primary cause of death.


Replacement of the valvular prosthesis in a patient with a Bentall procedure. Patients who have had the Bentall-DeBono procedure using a composite conduit with a tissue valvular prosthesis pose a great challenge when problems develop with the tissue prosthesis. We herein report the surgical management of one such case, in which the valvular prosthesis was removed and replaced without replacement of the conduit.


Siderophore and organic acid production in root nodule bacteria. Nineteen strains of root nodule bacteria were grown under various iron regimes (0.1, 1.0 and 20 microM added iron) and tested for catechol and hydroxamate siderophore production and the excretion of malate and citrate. The growth response of the strains to iron differed markedly. For 12 strains (Bradyrhizobium strains NC92B and 32H1, B. japonicum USDA110 and CB1809, B. lupini WU8, cowpea Rhizobium NGR234, Rhizobium meliloti strains U45 and CC169, Rhizobium leguminosarum bv viciae WU235 and Rhizobium leguminosarum bv trifolii strains TA1, T1 and WU95) the mean generation time showed no variation with the 200-fold increase in iron concentration. In contrast, in Bradyrhizobium strains NC921, CB756 and TAL1000, B. japonicum strain 61A76 and R. leguminosarum bv viciae MNF300 there was a 2-5 fold decrease in growth rate at low iron. R. meliloti strains WSM419 and WSM540 showed decreased growth at high iron. All strains of root nodule bacteria tested gave a positive CAS (chrome azurol S) assay for siderophore production. No catechol-type siderophores were found in any strain, and only R. leguminosarum bv trifolii T1 and bv viciae WU235 produced hydroxamate under low iron (0.1 and 1.0 microM added iron). Malate was excreted by all strains grown under all iron regimes. Citrate was excreted by B. japonicum USDA110 and B. lupini WU8 in all iron concentrations, while Bradyrhizobium TAL1000, R. leguminosarum bv viciae MNF300 and B. japonicum 61A76 only produced citrate under low iron (0.1 and/or 1.0 microM added iron) during the stationary phase of growth.


The cell cycle regulator, human p50weel, is a tyrosine kinase and not a serine/tyrosine kinase. The human weel protein, a homologue of the yeast weel protein, was expressed in E. coli and purified to homogeneity. The purified weel protein phosphorylated the tyrosine residue of cdc2 kinase in HeLa cell extracts in the presence of human cyclin B1. It also phosphorylated the tyrosine but not the threonine residue in the peptide of the amino-terminal of cdc2 kinase, although both these residues have been shown to be phosphorylated in higher eukaryotes in vivo. Furthermore, serine and tyrosine residues of the yeast weel protein are reportedly autophosphorylated in vitro, however the tyrosine residue of the human weel protein was autophosphorylated whereas the serine and threonine residues were not. These data indicate that human p50weel is tyrosine kinase and that it phosphorylated the tyrosine residue of the amino-terminal of cdc2 kinase in the presence of cyclin B1 and that the threonine residue is phosphorylated by another, unknown kinase.


Internalization of V2-vasopressin receptors in LLC-PK1-cells: evidence for receptor-mediated endocytosis. The mechanism of internalization of the vasopressin-receptor (V2-subtype) of LLC-PK1-cells, a pig renal tubular cell line, is unknown. We studied internalization utilizing a novel, highly specific vasopressin analogue ((125I)-[8-p(OH)-phenylpropionyl]-LVP, 2000 Ci/mmol). Scatchard analysis performed with membranes of LLC-PK1-cells revealed a Kd of 0.8 +/- 0.2 nM and a Bmax of 366 +/- 41 fmol/mg of protein. Degradation of the ligand was excluded by RP-HPLC-analysis. Internalization was proven by the acid-wash technique, quantitative light-microscopic autoradiography and electron microscopy. The ligand was internalized in a time- and temperature-dependent manner. At 4 degrees C, no uptake was found; at 22 degrees C, after 30 min of incubation, more than 50% of the radioligand was found inside the cell. Electron microscopy demonstrated that plasma-membrane bound vasopressin receptors are internalized by receptor-mediated endocytosis via coated pits.


Regulatory role of GM3 ganglioside in integrin function, as evidenced by its effect on function of alpha 5 beta 1-liposomes: a preliminary note. Mouse mammary carcinoma mutant cell line FUA169, characterized by high GM3 ganglioside content, was established from parent cell line FM3A/F28-7, which has high LacCer content but no GM3. Although both cell lines showed the same quantity and quality of integrin receptors, FUA169 showed much stronger adhesion to fibronectin (FN)-coated plates than did F28-7. Liposomes containing phosphatidylcholine, cholesterol, alpha 5 beta 1, and a moderate amount of GM3 showed greatly enhanced adhesion to FN-coated plates, but adhesion of similar liposomes containing a large amount of GM3, or no GM3, was much lower. Our results suggest that GM3 regulates integrin receptor function essential for cell adhesion to FN.


Electrophoretic profiles of mitochondrial plasmids in Neurospora suggest they replicate by a rolling circle mechanism. Migratory behaviour of mitochondrial plasmids from Neurospora crassa Mauriceville-1c and N. intermediate LaBelle has been studied by pulsed field gel electrophoresis (PFGE). Electrophoretic profiles demonstrate that long, linear molecules of a heterogeneous size are the prevailing form of plasmid DNA in vivo. Circular forms represent less than 8-9% of plasmid DNA. Single stranded DNA regions are abundant and lead to electrophoretic inertia of a significant amount of plasmid DNA. These profiles indicate that both plasmids replicate by the recombination dependent rolling circle mechanism.


Binding characteristics of the new thromboxane A2/prostaglandin H2 receptor antagonist [3H]BAY U 3405 to washed human platelets and platelet membranes. The new thromboxane A2 antagonist [3H]BAY U 3405 was characterized for its binding to washed human platelets and platelet membranes. In washed platelets the specific binding was reversible, selective and stereospecific, but not saturable. The dissociation constant (Kd) was 6 +/- 2.5 nM, the number of specific binding sites 1177 +/- 306 per platelet. Three structurally different thromboxane A2 (TXA2)/prostaglandin H2 (prostaglandin endoperoxide) (PGH2) receptor ligands completely inhibited the specific binding of [3H]BAY U 3405 in a concentration-dependent manner, indicating that the observed high affinity binding site is the TXA2/PGH2 receptor. In platelet membranes, however, specific [3H]BAY U 3405 binding showed saturability in addition to reversibility, selectivity, and stereospecifity. The Kd of the binding was 9.6 +/- 2.3 nM in kinetic studies and 8.7 +/- 3.7 nM in saturation studies, the inhibition constant (Ki) was 10 +/- 1.1 nM in displacement studies. The TXA2/PGH2 receptor agonists U 46619 and CTA2, and the antagonists Daltroban (BM 13505), I-PTA-OH and SQ 29548 all completely inhibited the specific binding of [3H]BAY U 3405 thus defining the observed binding site as the TXA2/PGH2 receptor. In conclusion, the data suggest that the previously reported TXA2 antagonism of BAY U 3405 is mediated by binding to a specific high affinity binding site of human platelets and platelet membranes that represents the TXA2/PGH2 receptor.


Antiflammins. Anti-inflammatory activity and effect on human phospholipase A2. Two anti-inflammatory peptides (antiflammins) corresponding to a high amino acid similarity region between lipocortin I and uteroglobin were tested for their ability to inhibit purified human synovial fluid phospholipase A2 (HSF-PLA2). No inhibitory activity was observed, even at such high concentrations of peptides as 50 microM. When antiflammins were preincubated with the enzyme and/or the substrate, no HSF-PLA2 inhibition was detected. In vivo anti-inflammatory activity of these peptides was evaluated in several experimental models of inflammation induced by carrageenan, croton-oil, oxazolone and Naja naja naja venom phospholipase A2 (PLA2). In contrast to the in vitro results, anti-inflammatory activity was observed in all tests, except when inflammation was induced by snake venom PLA2. Taken together, our results do not support the hypothesis that the in vivo anti-inflammatory effect of antiflammins is directly related to inhibition of PLA2 activity.


Metabolism of NAD(P)H by blood components. Relevance to bioreductively activated prodrugs in a targeted enzyme therapy system. NADH was metabolized both by serum components and at the cell surface. The metabolism by serum was either oxidation to NAD+, or hydrolysis of the pyrophosphate to yield nicotinamide mononucleotide (reduced) (NMNH) and AMP. NMNH was further hydrolysed to yield nicotinamide riboside (reduced) (NRH), which was stable. NAD+ was hydrolysed (although at a slower rate than was NADH), but was also reduced to yield NADH. The reduction of NAD+ was catalysed by the enzyme serum L(+)lactate dehydrogenase (EC 1.1.1.27) and was dependent on the concentration of L(+)lactate in the serum. NADPH was hydrolysed in a similar manner to NADH but not oxidized by serum. NADH generated from NAD+ by serum derived from human, foetal calf and horse sources was capable of driving the bioreductive activation of CB 1954 by the enzyme DT diaphorase. Cell surfaces oxidized NADH to NAD+, but did not oxidize NADPH or NRH. These observations suggest that NAD(P)H would be unsuitable as a source of reducing equivalents for the bioreductive activation of prodrugs by a reductase enzyme in Antibody Directed Enzyme Prodrug Therapy (ADEPT). In contrast, NAD+ (which could act as a source of NADH) and NRH could avoid the shortcomings of NAD(P)H, and act as suitable cofactors for an enzyme in an ADEPT system.


Regulation of inflammation by lipocortin 1. The mechanisms that regulate inflammatory responses, and their dysfunction in chronic inflammatory diseases, are poorly understood. Lipocortin 1 is a potential mediator of the anti-inflammatory effects of glucocorticoid hormones. Following the discovery of putative receptors on phagocytes for lipocortin 1, Nick Goulding and Paul Guyre here offer one explanation for the self-limitation of inflammation and the impairment of the mechanism in chronic inflammatory disease.


Is there a role for gamma delta T cells in malaria? Peripheral blood lymphocytes of the V gamma 9+ family of gamma delta T cells proliferate vigorously in response to Plasmodium falciparum. In this brief article, Jean Langhorne and colleagues discuss this response and assess the possible role of gamma delta T cells in the pathogenesis of malaria.


The effects on toxicity of circadian patterning of continuous hepatic artery infusion. Long term continuous hepatic artery infusion of FUDR was carried out in 34 rats. In the animals who received a constant infusion schedule of 15 mg/kg/day all died of toxicity with a mean survival of 9.3 days. If the pattern of the continuous infusion was changed so that over 60% of the infusion was given during the hours of 3pm to 9pm than all of the animals survived the 14 day infusion. If the maximum dose of infusion was changed so that 60% of the infusion was given at night from 3am to 9am the infusion became more toxic and all the animals died in a mean of 5.5 days. Pathologic sectioning of all the livers reflected the above outcomes with the greatest amount of hepatic necrosis in the animals on the night cycles. This study underscores the recent advances in chronobiology demonstrating that for continuous hepatic arterial infusions the timing of delivery is crucial in determining the toxicity.


18O isotopic 13C NMR shift as proof that bifunctional peptidylglycine alpha-amidating enzyme is a monooxygenase. The biosynthesis of C-terminal alpha-amidated peptides from their corresponding C-terminal glycine-extended precursors is catalyzed by peptidylglycine alpha-amidating enzyme (alpha-AE) in a reaction that requires copper, ascorbate, and molecular oxygen. Using bifunctional type A rat alpha-AE, we have shown that O2 is the source of the alpha-carbonyl oxygen of pyruvate produced during the amidation of dansyl-Tyr-Val-[alpha-13C]-D-Ala, as demonstrated by the 18O isotopic shift in the 13C NMR spectrum of [alpha-13C]lactate generated from [alpha-13C]pyruvate in the presence of lactate dehydrogenase and NADH. In addition, one-to-one stoichiometries have been determined for glyoxylate formed/dansyl-Tyr-Val-Gly consumed, pyruvate formed/dansyl-Tyr-Val-D-Ala consumed, dansyl-Tyr-Val-NH2 formed/ascorbate oxidized, and dansyl-Tyr-Val-NH2 formed/O2 consumed. Quantitative coupling of NADH oxidation to dansyl-Tyr-Val-NH2 production using Neurospora crassa semidehydroascorbate reductase showed that two one-electron reductions by ascorbate occurred per alpha-AE turnover. The stoichiometry of approximately 1.0 dansyl-Tyr-Val-NH2 produced/ascorbate oxidized observed in the absence of a semidehydroascorbate trap resulted from the disproportionation of two semidehydroascorbate molecules to ascorbate and dehydroascorbate.


Dynamic conformations compared for IgE and IgG1 in solution and bound to receptors. Dynamic conformations of two distinct immunoglobulin (Ig) isotypes, murine IgE and human IgG1, were examined with fluorescence resonance energy transfer measurements. The IgE mutant epsilon/C gamma 3* and the IgG1 mutant gamma/C gamma 3* each bind [5-(dimethylamino)naphthalen-1-yl]sulfonyl (DNS) in two identical antigen binding sites at the amino (N)-terminal ends of the Ig in the Fab segments. Eosin-DNS bound in these Fab sites served as the acceptor probe in these studies. Both Ig have a carboxy (C)-terminal domain (C gamma 3*) which contains genetically introduced cysteine residues. Modification of these cysteine sulfhydryls with fluorescein maleimide provided donor probes near the C-terminal ends of the Ig in the Fc segment. Energy transfer between the C-terminal and N-terminal ends was compared for these two Ig in solution and when they were found to their respective high-affinity receptors on plasma membranes: IgE-Fc epsilon RI on RBL cell membranes and IgG1-Fc gamma RI on U937 cell membranes. Previous energy-transfer measurements with these probes yielded an average end-to-end distance of 71 A for IgE in solution and 69 A for IgE bound to Fc epsilon RI, indicating that in both situations IgE is bent such that the axes of the Fab segments and the axis of the Fc segment do not form a planar Y-shape [Zheng, Shopes, Holowka, & Baird (1991) Biochemistry 30, 9125]. In the current study we found the average end-to-end distance for IgG1 in solution is 75 A and greater than or equal to 85 A for IgG1 bound to Fc gamma RI, suggesting an average bend conformation for IgG1 as well. The contributions of segmental flexibility to the average distances were assessed directly by measuring the efficiency of energy transfer as a function of variations in donor quantum yield caused by a collisional quencher and using these data to extract a Gaussian distribution of end-to-end distances. The distribution average (rho) and half-width (hw) were determined to be as follows: rho = 75 A, hw = 24 A for IgE in solution; rho = 71 A, hw = 12 A for IgE bound to Fc epsilon RI; and rho = 100 A, hw = 88 A for IgG in solution.(ABSTRACT TRUNCATED AT 400 WORDS)


Effect of heart rate on centerline velocities of pulsatile intracardiac jets: an in vitro study with laser Doppler anemometry and pulsed Doppler ultrasound. This article confirms a recently developed distal jet centerline technique for noninvasively quantifying regurgitant cardiac valve flows. The basic principle that allows flow rate to be calculated is conservation of momentum. As the jet entrains more mass, its centerline velocity decays inversely with distance from the orifice. Under pulsatile flow conditions, it has been shown that this technique remains applicable at peak flow for a normal resting adult heart rate of 60 to 70 beats/min. It is, however, conceivable that at higher heart rates, the same inverse relation between centerline velocity and distance is never fully established, because there is a finite time interval required for the jet to penetrate the receiving chamber. Therefore, the purpose of this study was to determine whether the inverse relationship of centerline velocity to distance develops sufficiently rapidly so that quantitative techniques based on that decay would be applicable over a wide range of heart rates. Two different techniques, an engineering tool, laser Doppler anemometry, and a clinical tool, Doppler ultrasound, were used for measuring jet centerline velocities (averaged over multiple beats). Physiologic pulsatile flows were pumped through two circular orifices, 4 and 6 mm in diameter, at 60 to 150 beats/min; peak orifice velocities ranged from 2 to 5 m/sec. Steady flow experiments were also performed with the same orifice diameters and over the same velocity range. Peak centerline velocities in the fully developed turbulent jet region decayed inversely with distance at all heart rates studied. With laser Doppler anemometry, the proportionality constant of the decay curve was found to be in the range 6.4 +/- 0.5. The pulsed Doppler results provided a jet constant in the range 6.7 +/- 0.3 with the 4-mm orifice diameter, whereas the constant was 6.5 +/- 0.3 with the 6-mm orifice diameter. In steady flow, the proportionality constant was found to be 6.1 +/- 0.2. Therefore, within a wide range of physiologic heart rates, full jet development occurs with sufficient speed so that the expected centerline velocity decay is established (jet empirical constant of 6.3). The conservation of momentum technique for calculating orifice flow rate on the basis of these centerline velocities is thus applicable under physiologic conditions.


Roles of Mg2+ in the mechanism of formation and dissociation of open complexes between Escherichia coli RNA polymerase and the lambda PR promoter: kinetic evidence for a second open complex requiring Mg2+. Comparative studies of the effects of Mg2+ vs Na+ and of acetate (OAc-) vs Cl- on the kinetics of formation and dissociation of E. coli RNA polymerase (E sigma 70)-lambda PR promoter open complexes have been used to probe the mechanism of this interaction. Composite second-order association rate constants ka and first-order dissociation rate constants kd, and their power dependences on salt concentration SKa (SKa identical to d log ka/d log [salt]) and Skd (Skd identical to d log kd/d log [salt]), were determined in MgCl2 and NaOAc to compare with the results of Roe and Record (1985) in NaCl. Replacement of NaCl by MgCl2 reduces the magnitude of Ska 2-fold (Ska = -11.9 +/- 1.1 in NaCl; Ska = -5.2 +/- 0.3 in MgCl2) and (by extrapolation) drastically reduces the magnitude of ka at any specified salt concentration (e.g., approximately 10(6)-fold at 0.2 M). Replacement of NaCl by NaOAc does not significantly affect Ska (Ska = -12.0 +/- 0.7 in NaOAc) and (by extrapolation) increased ka by approximately 80-fold at any fixed [Na+]. In the absence of Mg2+, replacement of NaCl by NaOAc is found to increase the half-life of the open complex by approximately 560-fold at fixed [Na+] without affecting Skd [Skd = 7.6 +/- 0.1 in NaOAc; in NaCl, Skd = 7.7 +/- 0.2 (Roe & Record, 1985)]. Replacement of NaCl by MgCl2 drastically reduces both Skd and the half-life of the open complex at any salt concentration below approximately 0.2 M. Strikingly, Skd = 0.4 +/- 0.1 in MgCl2, indicating that the net uptake of Mg2+ ions in the kinetically significant steps in dissociation of the open complex is much smaller than that expected by analogy with the uptake of approximately 8 Na+ ions in the corresponding steps in NaCl. In NaCl/MgCl2 mixtures, at a constant [NaCl] in the range 0.1-0.2 M, initial addition of MgCl2 (0.5 mM less than or equal to [MgCl2] less than or equal to 1 mM) increases the half-life of the open complex; further addition of MgCl2 causes the half-life to decrease, though the effect of [MgCl2] on kd is always less than that predicted by a simple competitive model. The observed effects of MgCl2 on Skd and kd differ profoundly from those expected from the behavior of kd and Skd in NaCl and NaOAc and indicate that the role of Mg2+ in dissociation is not merely that of a nonspecific divalent competitor with RNAP for interactions with DNA phosphates and of a DNA helix-stabilizer, both of which should cause kd to increase monotonically with increasing [Mg2+].(ABSTRACT TRUNCATED AT 400 WORDS)


Two-dimensional NMR, circular dichroism, and fluorescence studies of PP-50, a synthetic ATP-binding peptide from the beta-subunit of mitochondrial ATP synthase. PP-50, a peptide based on residues 141-190 of the beta-subunit of mitochondrial F1-ATPase, contains the GX4GKT consensus region for nucleoside triphosphate binding and has been shown to bind ATP [Garboczi, D.N., Shenbagamurthi, W.K., Hullihen, J., & Pedersen, P.L. (1988) J. Biol. Chem. 263, 812-816]. At pH 4.0, appropriate for NMR studies, PP-50 retains the ability to bind ATP tightly (KD = 17.5 microM) with a 1:1 stoichiometry as shown by titrations measuring the partial quenching of ATP fluorescence by PP-50. CD spectra of PP-50 at pH 4.0 and at low ionic strength show 5.8% helix, 30.2% beta-structure, and 64% coil. ATP binding increases the structure of PP-50, changing the CD to 7.5% helix, 44.5% beta-structure, and 48% coil. Increasing the ionic strength to 50 mM KCl also increases the structure, changing the CD to 7.4% helix, 64.4% beta-structure, and 28.2% coil. The 600-MHz proton NMR spectrum of PP-50, at pH 4.0 and low ionic strength, has been assigned by 2D methods (TOCSY, DQF-COSY, and NOESY with jump-return water suppression). Based on strong d alpha N NOEs, J alpha N values, and NH chemical shifts differing from random coil values, regions of extended structure are detected from residues 1-7 and 43-48. Based on dNN, dNN(i,i+2), and d alpha N(i,i+2) NOEs and 3J alpha N values, possible type I' and type I turns are found from residues 11-14 and 31-34, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)


Early steps of the Mg(2+)-ATPase of relaxed myofibrils. A comparison with Ca(2+)-activated myofibrils and myosin subfragment 1. The early steps of the Mg(2+)-ATPase activity of relaxed rabbit psoas myofibrils were studied in a buffer of near-physiological ionic strength at 4 degrees C by the rapid flow quench technique. The initial ATP binding steps were studied by the ATP chase, and the cleavage and release of product steps by the Pi burst method. The data obtained were interpreted by [formula: see text] where M represents the myosin heads with or without actin interaction. This work is a continuation of our study on Ca(2+)-activated myofibrils [Houadjeto, M., Travers, F., & Barman, T. (1992) Biochemistry 31, 1564-1569]. Here the constants obtained with relaxed myofibrils were compared with those with activated myofibrils and myosin subfragment 1 (S1). We find that whereas Ca2+ increases 80X the release of products (k4), it has little effect upon the kinetics of the initial binding and cleavage steps. As with activated myofibrils and S1, the second-order binding constant for ATP (k2/K1) was about 1 microM-1 s-1 and the ATP was bound very tightly. With activated myofibrils, it was difficult to obtain an estimate for the koff for ATP(k-2) but it is much less than kcat. Here with relaxed myofibrils we estimate k-2 less than 8 x 10(-4) s-1, which is considerably smaller than kcat (0.019 s-1) and also previous estimates for this constant. The overall Kd for ATP to relaxed myofibrils is less than 8 x 10(-10) M. With S1 this Kd is about 10(-11) M.(ABSTRACT TRUNCATED AT 250 WORDS)


Gestational hyperlipidemia in the rat is characterized by accumulation of n - 6 and n - 3 fatty acids, especially docosahexaenoic acid. We have evaluated the relative and quantitative changes in long-chain fatty acids in maternal liver, serum, carcass and conceptus (fetuses plus placentae) during pregnancy in the rat, to ascertain whether previous concern over lower proportions of n - 6 and n - 3 fatty acids in maternal serum could be indicative of suboptimal n - 6 or n - 3 fatty acid status. Gestational hyperlipidemia was characterized by proportional decreases in linoleic, stearic and arachidonic acids but increases in palmitic and docosahexaenoic acids. However, the quantitative amount (microgram/ml) of linoleic, arachidonic and docosahexaenoic acids in serum lipids actually increased 2-5-fold from mid-pregnancy to term. Compared to non-pregnant rats, gestational hyperlipidemia was also associated with a lower proportion but similar quantity of linoleic acid in maternal carcass and adipose stores. We conclude that gestational hyperlipidemia in the rat is characterized by a relative but not quantitative decrease in whole-body stores of n - 6 fatty acids and a marked proportional and quantitative increase in docosahexaenoic acid in maternal organs and in the conceptus.


Limitations of phosphatidylcholine/deoxycholate mixtures for the analysis of phospholipase A2 inhibition and activation: illustration with annexins. The effects of human recombinant lipocortin I (annexin I) and bovine lung calpactin I (annexin II) on porcine pancreatic phospholipase A2 (PLA2) activity in phosphatidylcholine (PC)/deoxycholate (DOC) mixtures were investigated. Annexin-associated decreases in PLA2 activity were observed under some conditions, for example, at high DOC/PC molar ratios; however, activation was observed under other conditions. NaCl, which lowers the non-critical micellar concentration (NCMC) of deoxycholate, caused significant decreases in control PLA2 activity in the absence of annexins, and greater decreases in PLA2 activity in annexin-containing samples, resulting in an apparent increase in inhibition. The PC/DOC substrate mixtures themselves appeared unstable. Despite a large excess of detergent, precipitates were, at times, observed upon incubation of some PC/DOC mixtures at 37 degrees C. Such behavior is of interest in view of the numerous reports of PLA2 inhibition by annexins and annexin-derived peptides in the PC/DOC system. The influence of the annexins on activity in this system is consistent with effects on the phase behavior of the PC/DOC mixture and/or competition with the enzyme for available Ca2+. These results caution against use of the PC/DOC system for analysis of potential PLA2 inhibitors unless the phase behavior of the system is more fully delineated.


Primary structure of V-ATPase subunit B from Manduca sexta midgut. The amino acid sequence of a vacuolar-type ATPase (V-ATPase) subunit B has been deduced from a cDNA clone isolated from a Manduca sexta larval midgut library. The library was screened by hybridization with a labeled cDNA encoding subunit B of Arabidopsis thaliana tonoplast V-ATPase. The M. sexta V-ATPase subunit B consists of 494 amino acids with a calculated M(r) of 54,902. The amino acid sequence deduced for V-ATPase subunit B of M. sexta is between 98% and 76% identical with that of seven other V-ATPase subunits B and greater than 52% identical with three archaebacterial ATPase subunits B.


Antiemetic agents.

Antiemetic agents. Despite major progress in the treatment of chemotherapy-induced emesis, nearly one third of patients undergoing cisplatin-based regimens still experience emesis within the first 24 hours of chemotherapy. An adequate treatment of delayed and anticipatory emesis remains to be determined. For highly emetogenic chemotherapy, the combination of ondansetron and dexamethasone is superior to dexamethasone alone and protects most patients. For moderately emetogenic regimens, the high level of complete control that can be achieved with the use of standard antiemetics is comparable to that obtained with ondansetron. This would suggest that, to reduce the cost of antiemetic therapy, ondansetron can be limited in case of failure of standard therapy. Delayed emesis remains poorly controlled with no difference between metoclopramide, dexamethasone, ondansetron, and placebo. Although some data suggest an improved efficacy when combining ondansetron with dexamethasone, convincing confirmatory studies are needed.


Pea dehydrins: identification, characterisation and expression. An antiserum raised against dehydrin from maize (Zea mays) recognised several polypeptides in extracts of pea (Pisum sativum) cotyledons. A cDNA expression library was prepared from mRNA of developing cotyledons, screened with the antiserum and positive clones were purified and characterised. The nucleotide sequence of one such clone, pPsB12, contained an open reading frame which would encode a polypeptide with regions of significant amino acid sequence similarity to dehydrins from other plant species. The deduced amino acid sequence of the pea dehydrin encoded by B12 is 197 amino acids in length, has a high glycine content (25.9%), lacks tryptophan and is highly hydrophilic. The polypeptide has an estimated molecular mass of 20.4 kDa and pI = 6.4. An in vitro synthesised product from the clone comigrates with one of the in vivo proteins recognised by the antiserum. A comparison of the pea dehydrin sequence with sequences from other species revealed conserved amino acid regions: an N-terminal DEYGNP and a lysine-rich block (KIKEKLPG), both of which are present in two copies. Unexpectedly, pea dehydrin lacks a stretch of serine residues which is conserved in other dehydrins. B12 mRNA and dehydrin proteins accumulated in dehydration-stressed seedlings, associated with elevated levels of endogenous abscisic acid (ABA). Applied ABA induced expression of dehydrins in unstressed seedlings. Dehydrin expression was rapidly reversed when seedlings were removed from the stress or from treatment with ABA and placed in water. During pea cotyledon development, dehydrin mRNA and proteins accumulated in mid to late embryogenesis. Dehydrin proteins were some of the most actively synthesised at about the time of maximum fresh weight and represent about 2% of protein in mature cotyledons.


Alterations in thrombin-induced protein tyrosine phosphorylation of platelets from patients with chronic myelogenous leukemia. Thrombin is known to stimulate platelet protein tyrosine kinase (PTK). We studied thrombin-induced tyrosine-specific protein phosphorylation in normal platelets and those from patients with chronic myelogenous leukemia (CML) and other myeloproliferative disorders (MPD) using immunoblotting with antiphosphotyrosine (anti-P-Tyr) antibody. In resting platelets, two major phosphotyrosyl (P-Tyr) proteins with molecular masses of 120 kDa (p120) and 60 kDa (p60) were consistently detected both in normal subjects and in CML and other MPD patients. In addition to these P-Tyr proteins, a 36 kDa protein (p36) was predominantly phosphorylated only in CML platelets, using antilipocortin II antibody, we identified this p36 protein as lipocortin. Thrombin enhanced the tyrosine phosphorylation of p120 and p60, not only in normal platelets, but also in CML platelets, although the response was more delayed and the duration was shorter in CML platelets than those in normal platelets. Interestingly, decreased thrombin-induced aggregation was associated with a transient stimulation of p36 phosphorylation in CML platelets. These results suggest that the tyrosine phosphorylation of p36, which was probably identical to lipocortin, inhibits thrombin-induced platelet aggregation through anti-phospholipase A2 (anti-PLA2) activity.


Comparison of antithrombin III, protein C and protein S levels in capillary and venous blood of newborn infants. The plasma concentrations of antithrombin III, protein C and protein S in capillary and venous blood samples obtained simultaneously from 30 neonates were compared in order to determine the suitability of using capillary blood for estimation of these proteins with anticoagulant action. Our findings showed that while capillary and venous blood did not differ significantly in antithrombin III functional activity and protein C antigen levels, the capillary samples had significantly lower protein C functional activity and higher antithrombin III antigen level. Protein S antigen level was also significantly higher in the capillary samples although the difference was relatively small. The capillary and venous concentrations of the binding protein of protein S, C4b binding protein, were almost identical.


The incidence of DPB1 differences between serological and mixed lymphocyte culture matched unrelated individuals: implications for selection of bone marrow donors. Bone marrow transplantation from unrelated donors is being used increasingly for the treatment of patients with leukaemia and other disorders of lymphohaemopoiesis. Selection of histocompatible unrelated bone marrow donors currently relies on serological HLA identity and negative mixed lymphocyte cultures (MLC) between potential donor-recipient pairs. Since serological HLA-DP typing is not feasible, we used the HLA-DPB1 oligonucleotide typing method to test whether the current selection procedure can also guarantee identity for HLA-DP. In 40 consecutive patients, one-third (62/193) of the serologically HLA-A, -B, -C, -DR and -DQ identical donors were judged as MLC negative (relative response below 5%) with the presumptive recipient. HLA-DPB1 oligonucleotide typing of the MLC negative donors revealed that only one-third of these (20/62) were also identical for DP. In the majority of the pairs, we found a DPB1 disparity. A difference in the graft-versus-host direction was seen in 25/62 cases in the host-versus-graft direction in 28/62 cases and in both directions in 29/62 cases. These data indicate that, in spite of the strict MLC criteria used, the current procedure did not guarantee complete MHC class II identity. Therefore oligotyping for DPB1 can improve matching for DP and should be introduced for typing of volunteers. We suspect that DP differences may contribute to the higher incidence of graft-versus-host disease or graft rejection in unrelated donor transplants.


Mixed allogeneic chimeras prepared by a non-myeloablative regimen: requirement for chimerism to maintain tolerance. We have recently described a non-myeloablative conditioning regimen permitting engraftment of allogeneic bone marrow in mice which involves administration of anti-CD4 (GK1.5) plus anti-CD8 (2.43) monoclonal antibodies in vivo, 3 Gy whole body irradiation, plus 7 Gy thymic irradiation. B10 (H-2b) mice prepared by this regimen and infused with unmanipulated B10.D2 (H-2d) bone marrow develop permanent mixed lymphohematopoietic chimerism and specific tolerance to donor skin grafts. We now demonstrate that mixed chimerism persists longer than 170 days in the lymphoid tissues including spleen, thymus and bone marrow of such animals, and that equivalent levels of donor chimerism are observed in both T and B cell compartments. In addition stable mixed chimeras were found to be unresponsive to host (B10) and donor (B10.D2) stimulator cells in mixed lymphocyte reaction and in cell mediated lympholysis assays, while responses to a third party (B10.BR, H-2k) were intact. Persistent chimerism was found to be necessary for the maintenance of skin graft tolerance in these animals, since in vivo depletion of donor cells by treatment with an anti-H-2d (34-2-12) monoclonal antibody resulted in the subsequent rejection of donor skin grafts. These studies demonstrate that mixed allogeneic chimeras produced using this regimen are specifically tolerant to donor in vitro and in vivo, and that persistence of donor chimerism is critical for the maintenance of tolerance.


N-methyl-D-aspartic acid (NMDA) receptor antagonist MK-801 blocks non-opioid stress-induced analgesia. II. Comparison across three swim-stress paradigms in selectively bred mice. The effects of the specific N-methyl-D-aspartic acid (NMDA) receptor antagonist MK-801 (dizocilpine, 0.075 mg/kg, i.p.) on swim-stress-induced analgesia (SSIA) were studied in control (C) mice and in mice selectively bred for high (HA) or low (LA) SSIA. In three consecutive experiments, animals were subjected to forced swimming at water temperature of 20 degrees C, 32 degrees C and 15 degrees C and the resulting analgesia (hot-plate test) was found to be mixed opioid/non-opioid, opioid and non-opioid, respectively, as a function of the degree of antagonism by naloxone (10 mg/kg, i.p.). The major finding of this study is that MK-801 attenuated 15 degrees C SSIA, against which naloxone was ineffective, but had no effect on 32 degrees C SSIA, which naloxone blocked completely. A combination of naloxone and MK-801 significantly attenuated 20 degrees C SSIA in C and HA mice and in HA mice this attenuation was significantly larger than that produced by either drug alone. Morphine analgesia (10 mg/kg, i.p.) was unaffected by MK-801. It is concluded that low doses of MK-801 selectively block non-opioid mechanisms of SSIA.


Localization of dopamine D2 receptor protein in rat brain using polyclonal antibody. The precise distribution of the dopamine type D2 receptor has been mapped for the first time in rat brain using an antibody to D2 receptor protein. Polyclonal antisera were collected from rabbits inoculated with an undecapeptide identical to residues 24-34 of the D2 protein sequence. Rat brain slices, 40 microns in thickness, were incubated with either primary antiserum, the antiserum plus free peptide antigen, or pre-immune serum. Antibody binding was visualized by peroxidase-antiperoxidase (PAP) reaction followed by light microscopy. PAP complex bound moderately-to-densely throughout the medial forebrain bundle, and was seen in more discrete regions in the midbrain, consistent with the binding of D2 radioligands. There were some unexpected results, namely in the cerebral cortex and nucleus accumbens, there were unexpectedly steep gradients in binding density, decreasing caudally; no binding was detected in the hippocampus or the substantia nigra pars reticulata. In all positive-staining regions examined, the antibody was highly localized to neuronal cell bodies, except in the frontal cortex where antibody was also evident on basilar dendrites. These data confirm that the polyclonal antibody recognized dopamine D2 receptor protein throughout the rat brain, and suggest that the D2 receptor is distributed more abundantly on somata than on cellular processes.


Lipocortin-1 inhibits NMDA receptor-mediated neuronal damage in the striatum of the rat. Lipocortin-1 (annexin-1), an endogenous phospholipid and calcium binding protein, has been shown to significantly attenuate the damage produced by focal cerebral ischaemia in the rat. In the present study we have therefore investigated its effect on N-methyl-D-aspartate (NMDA) induced neuronal damage. Unilateral intrastriatal infusion of a potent and selective NMDA agonist, cis-2,4-methanoglutamate (MGlu), induced an extensive lesion of the striatum in the rat, which was inhibited (greater than 80%) by prior injection of MK801 (4 mg/kg, i.p.). Infusion of 1.2 micrograms of an active fragment of lipocortin-1 (N-terminal 1-188 aa) immediately after MGlu significantly reduced the extent of damage by 44.2 +/- 8.0%. In contrast, infusion of 3 microliters of neutralizing anti-lipocortin-1 antibody with MGlu increased lesion size by 158.9 +/- 22.0%. These findings indicate that the damage produced by intrastriatal infusion of MGlu is mediated by the NMDA receptor. Lipocortin-1 fragment markedly attenuated, and the neutralizing antibody increased, this NMDA mediated neuronal damage. These observations may explain the neuroprotective action of lipocortin following cerebral ischaemia.


The loss of beta II-protein kinase C in the striatum from patients with Huntington's disease. We have examined the levels of protein kinase C (PKC) in autopsied brains of patients with Huntington's disease (HD), using [3H]4-beta-phorbol-12,13-dibutyrate ([3H] PDBu) and antisera against the PKC subspecies. In the caudate nucleus and putamen from patients with HD, the specific binding for [3H]PDBu was significantly decreased by 74 and 68%, respectively, as compared to findings in controls. The beta II-PKC immunoreactivities were significantly reduced by 65%, whereas the alpha-PKC immunoreactivities increased by 146%, in the putamen. There were no differences in the beta I- or gamma-PKC immunoreactivities in the putamen between HD and controls. These results suggest the differential localization of four PKC subspecies in human striatum and the involvement of four subspecies in different aspects of HD pathophysiology.


The 'initial burst' of muscle spindle afferents with or without terminals on the bag1 intrafusal muscle fibre. The initial burst has been re-examined following conditioning of the muscle to investigate its intrafusal origins. The burst was present in the response to stretch of spindle afferents that innervated the bag1 fibre and those that did not. There was, however, a difference in amplitude of the burst. These findings are discussed in terms of intrafusal mechanisms.


Antagonists of the NMDA receptor and allopurinol protect the olfactory cortex but not the striatum after intra-cerebral injection of kainic acid. Overstimulation of the NMDA receptor, as well as generation of excessive amounts of free radicals, has been implicated in excitotoxic brain injuries. We report here that two antagonists of the NMDA receptor and an inhibitor of the free radical-generating enzyme, xanthine oxidase, protect the olfactory cortex but not the striatum after intrastriatal injection of kainic acid. Our results suggest the existence of a precise link between excitotoxic activation of the NMDA receptor and neuropathology related to excessive amounts of free radicals. The focal point of this link may be the entry of Ca2+ through the NMDA receptor and the consequent activation of proteases and free radical-generating systems.


Effects of intra-amygdala injections of NMDA receptor antagonists on acquisition and retention of inhibitory avoidance. These experiments examined the effects of intra-amygdala injections of NMDA receptor antagonists on the acquisition and retention of inhibitory avoidance. In Expt. I, rats received bilateral intra-amygdala injections of the NMDA antagonists D,L-AP5 (1-10 micrograms), D-AP5 (0.03-1 micrograms), CPP (0.125 or 0.375 microgram), or MK-801 (0.2 or 0.5 microgram) prior to training in a continuous multiple-trial inhibitory avoidance (CMIA) task. Acquisition of the task was not significantly affected by any of the drug injections. In contrast, all three competitive antagonists, D,L-AP5, D-AP5 and CPP, produced dose-dependent impairment of 48 h retention performance. Although the MK-801 injections did not significantly impair retention performance, the retention scores of the 0.5 microgram MK-801 group were bimodally distributed, indicating retention impairment in a subgroup of the animals given that dose. Intra-amygdala injections of 3 or 10 micrograms D,L-AP5 did not affect footshock sensitivity (Expt. II) or locomotor activity (Expt. III) and their retention-impairing effects were not due to induction of state dependency (Expt. IV). The retention-impairing effects of intra-amygdala injections of NMDA antagonists were not due to diffusion of the drugs dorsally: injections of 1 microgram D-AP5 into the striatal area directly above the amygdala impaired acquisition but not retention performance (Expt. V). The retention-impairing effects of 1 microgram D-AP5 or 0.5 microgram MK-801 were attenuated by giving additional training to the animals shortly after receiving intra-amygdala injections (Expt. VI). The implications of these findings for hypotheses concerning amygdala function in learning and memory are discussed.


A 1992 update on hepatitis B vaccination. Surveillance studies of people at high risk for hepatitis B, combined with almost 10 years of experience with the vaccine, have allowed cost effective vaccination programs to be devised. It would make good sense to incorporate the hepatitis B vaccination into the standard childhood immunization protocols. Present teenagers, who will not benefit from such a program, would be well advised to be vaccinated. All clinical dental staff should also be immunized. These three actions would be very effective in curbing the spread of this preventable disease.


Estimates of costs and effects of screening for colorectal cancer in the United States. In many ways, colorectal cancer might be an excellent candidate for mass screening because of the following: (1) it is the second leading cause of cancer mortality in the United States; (2) it develops slowly from a precursor lesion; and (3) methods of early detection are available. Barriers to screening include unproven efficacy of the procedure and high costs. Cost analyses are derived from two mathematic models that estimate screening costs and effects based on expert opinion and data from uncontrolled screening studies. One screening option that follows the guidelines of the American Cancer Society and the National Cancer Institute (annual testing for occult fecal blood and sigmoidoscopy every 5 years) could result in a 40% decrease in colon cancer mortality for American adults between the ages of 50 and 75 years if they comply with screening. This model, developed by David Eddy, projects an average of 44 days of extra life per person screened, at a net cost of $57 per day of life gained. Using assumptions much less favorable to screening, the Office of Technology Assessment modeled this same screening strategy for those aged 65 years and older. This model predicted a similar benefit of extra life per person at a cost of $118 per day of life gained. This doubling of the predicted cost was caused by the inclusion of subsequent colonoscopic surveillance costs for those found to have polyps. Direct costs of screening annually for fecal occult blood and every 5 years by sigmoidoscopy would cost an average of approximately $48 per person per year for screening and follow-up testing of all positive results. Fecal occult blood testing alone, although less effective, costs only $20 per person per year, including follow-up testing of all positive findings. The results from randomized trials of fecal occult blood screening will be known in the next 5 years, but trials of screening with sigmoidoscopy will not be complete for 10-15 years. Because mass screening programs will be difficult to fund without better data on their efficacy, colorectal cancer screening will continue to be a matter of individual decision making in the clinical setting for years to come. Clearer presentations of costs and benefits that can be understood by both patients and physicians are needed.


Inhibition of spontaneous testicular Leydig cell tumor development in F-344 rats by dehydroepiandrosterone. The incidence of spontaneous Leydig cell tumors of testis is very high in old F-344 rats. We have examined the effect of dehydroepiandrosterone (DHEA), a steroid hormone with antimitotic and anticarcinogenic properties, on spontaneous Leydig cell tumorigenesis. Fifteen-week-old male F-344 rats were fed a diet containing DHEA (0.45% w/w) for 84 weeks. At the termination of experiment none of the 13 rats had Leydig cell hyperplasia or Leydig cell tumors. All the eight control rats of comparable age had Leydig cell tumors. These findings suggest that DHEA is a potent inhibitor of spontaneous Leydig cell tumors of testis in aged rats.


In search of specific cytotoxic T lymphocytes infiltrating or accompanying human ovarian carcinoma. Lymphocytes infiltrating human ovarian carcinoma obtained directly from the tumour mass (tumour-infiltrating lymphocytes, TIL) or from the carcinomatous ascites (tumour-associated lymphocytes, TAL) were expanded in vitro in long-term cultures with interleukin-2 and tested for their specific cytolytic activity. Killing of the autologous tumour was detected only in a proportion of the patients, less frequently in TIL compared to TAL. In fact two out of ten TIL and four out of nine TAL cultures tested showed significant levels of lysis against the autologous tumour. This cytotoxic activity was not restricted to the autologous tumour, as other tumour cell lines, including non-ovarian ones, were lysed as well. The cultures that were not cytotoxic against the autologous tumour were in most cases able to lyse other tumour cell lines of ovarian or other histology. Cloning of TIL from one patient was performed: of 22 clones tested, 4 displayed higher cytotoxicity against the autologous tumour compared to the uncloned population and 3 out of these 4 did not kill an irrelevant carcinoma cell line. In order to stimulate the expansion of putative specific effectors we performed mixed lymphocyte/tumour cultures (MLTC) with autologous or allogeneic tumour cells. No stimulation of cytotoxicity against the autologous tumour was detected after MLTC in nine different TAL populations, using autologous or allogeneic tumours as stimulators. On the contrary, peripheral blood lymphocytes from two patients after MLTC with the autologous tumour showed increased killing of the autologous and decreased killing of an allogeneic target. In conclusion TIL and TAL from ovarian carcinoma expanded in vitro with interleukin-2 usually have non-MHC-restricted cytotoxicity and variable degrees of reactivity against the autologous tumour. A preferential killing for the autologous tumour was not observed even after MLTC. These results do not exclude the existence of tumour-specific cytotoxic T lymphocytes in ovarian carcinoma; nevertheless they suggest that putative specific effectors have very low frequency and that culture techniques for expanding their growth more selectively are still to be optimized.


Role of arterial hypertension in left ventricle hypertrophy in hemodialysis patients: an echocardiographic study. To assess the role of arterial hypertension in left ventricle (LV) hypertrophy among hemodialysis patients, echocardiographic evaluation was performed in 10 hypertensive and 13 normotensive hemodialysis subjects matched for age, sex, race, duration of dialysis treatment and degree of interdialytic volume expansion. We excluded from the latter group patients with previous hypertension since hypertensive heart disease may persist after adequate blood pressure control. We also studied 17 normal controls and 10 non-uremic patients with essential hypertension. Comparisons between the two uremic groups showed that the hypertensive patients had a higher mass index (222 +/- 74 x 108 +/- 26, p = 0.0001) and posterior wall thickness (12 +/- 2 x 9 +/- 2, p = 0.0001) and a reduced LV radius/wall thickness ratio (4.4 +/- 0.7 x 5.8 +/- 1, p = 0.0001). There were no significant echocardiographic differences between normal controls and normotensive uremics. In contrast, compared to controls, hypertensive uremic patients showed an increased LV mass index (222 +/- 74 x 83 +/- 21, p = 0.0001) and posterior wall thickness (12 +/- 2 x 7 +/- 1, p = 0.0001) and a reduced LV radius/wall thickness ratio (4.4 +/- 0.7 x 6.5 +/- 1.1, p = 0.001), characterizing concentric hypertrophy. They also had ventricular dilation with larger LV dimensions than in controls (53 +/- 5 x 47 +/- 4, p = 0.004). In patients with essential hypertension, the mass index (135 +/- 22), wall thickness (11 +/- 1) and LV radius/wall thickness ratio (4.3 +/- 0.7) significantly differed (p = 0.0001) from those in the controls.(ABSTRACT TRUNCATED AT 250 WORDS)


Augmented plasma protein C activity after coronary thrombolysis with urokinase in patients with acute myocardial infarction. Coronary thrombolysis reduces morbidity and mortality in patients with acute myocardial infarction, however, the exact effects of thrombolytic agents on the status of intrinsic hemostases are not fully understood. In the present study, we examined serial changes in plasma thrombin and protein C activities of 6 patients with acute myocardial infarction treated with urokinase. Fibrinolysis occurred immediately after urokinase injection with an increase in the plasma thrombin-antithrombin III complex, suggesting a subsequent procoagulant state due to thrombin generation. Correspondent increases in plasma protein C activity were observed, however, protein S levels did not change at all. Our findings suggest that urokinase administration for coronary thrombolysis not only causes fibrinolysis, but also induces thrombin activity, which may be antagonized by augmented intrinsic protein C activity.


MHC-unrestricted recognition of bacteria-infected target cells by human CD8+ cytotoxic T lymphocytes. A CD8+ alpha beta TCR+ T cell clone (A35) was isolated from the synovial fluid of a patient with post-enteric reactive arthritis caused by Yersinia enterocolitica. This clone efficiently killed autologous and allogeneic target cells that had been preincubated with live but not with heat-killed bacteria. There was no restriction by polymorphic parts of HLA-A, -B, or -C molecules and a HLA class II-deficient mutant cell line was lysed as efficiently as its normal counterpart, whereas infected HLA class I-deficient cells (Daudi cells) were not. The clone showed crossreaction between Yersinia enterocolitica, Escherichia coli, Pseudomonas aeruginosa, and Streptococcus pyogenes, but did not lyse target cells preincubated with Staphylococcus epidermidis. MAb to CD2, CD3, and CD8 efficiently blocked A35, whereas the addition of mAb to HLA class II or to HLA class I did not. This clone apparently represents a novel effector mechanism against bacteria-infected or -modified cells that could be involved in the immunopathology of reactive arthritis.


Nonspecific regulatory mechanism of contact sensitivity: nonspecific suppressor factor (NSF)-treated intermediate cells produce a second nonspecific suppressor factor (NSFint). Nonspecific suppressor factor (NSF), which inhibits the passive transfer of contact sensitivity (CS), is produced spontaneously from macrophage-like suppressor cells induced by intravenous administration of oxazolone (Ox)-conjugated spleen cells. NSF binds selectively to Ia-positive, cyclophosphamide (CY)-sensitive, and plastic-adherent cells (named intermediate cells) present in the normal spleen. NSF-treated intermediate cells acquire the ability to suppress the passive transfer of CS nonspecifically. In this study, NSF-treated intermediate cells were found to release a second nonspecific suppressor factor (NSFint) during a 2-hr culture, while retaining their suppressor activity. Investigation of the relationship between these two factors showed that both NSF and NSFint were trypsin-sensitive, nondialyzable proteins. However, gel chromatography revealed that NSF was about 43 kDa, while NSFint was about 20 kDa. NSF was released from macrophage-like suppressor cells after RNA-dependent protein synthesis. In contrast, production of NSFint was energy dependent but did not require protein synthesis. Intermediate cells pretreated with lysosomotropic agents, such as ammonium chloride or chloroquine, did not acquire suppressor activity nor release suppressor factors due to NSF treatment. These observations suggest that NSFint is an altered form of NSF released by the intermediate after having undergone some modification; the biochemical mechanism is not known. This study showed that the intermediate cells play an active role in the suppressor cascade of NSF.


Antigen concentration determines helper T cell subset participation in IgE antibody responses. A recently developed in vitro system for antigen-stimulated primary and secondary murine IgE antibody responses has been used to define (a) the relative participation of the Th1 and Th2 cell-derived lymphokines IFN-gamma and IL-4, respectively, in such responses, and (b) the role of antigen concentration in determining functional helper T cell activity. These studies confirm that IL-4 and IFN-gamma exert regulatory effects on IgE synthesis, but the nature and extent of their respective effects on primary and secondary IgE responses differ. Thus, primary IgE responses are considerably more sensitive to and dependent on IL-4 than are secondary IgE responses since (1) anti-IL-4 monoclonal antibody totally inhibited primary IgE responses, but only partially affected secondary responses; and (2) exogenously added IL-4 could stimulate primary IgE responses to optimal antigen concentrations, but had no effect on secondary IgE production. Likewise, antigen-stimulated primary IgE responses are about eightfold more sensitive than are secondary responses to the inhibitory effects of IFN-gamma. Studying the effect of antigen dose on the quantity of IgE antibody produced revealed that although IFN-gamma could be detected by ELISA in cultures exhibiting high-dose antigen-dependent diminution of IgE production, anti-IFN-gamma monoclonal antibody could not reverse this phenomenon. Thus, IFN-gamma is not solely responsible for decreased IgE synthesis associated with high-dose antigen exposure. IL-4 activity was detected in the fluid from cultures stimulated with low, but not high, levels of antigen. Moreover, addition of exogenous IL-4 restored IgE production to normal levels in cultures exposed to high antigen concentrations. Therefore, it appears that high levels of antigen result in selective stimulation of Th1 cells which produce IFN-gamma, and diminished activation of IL-4-producing Th2 cells. These results help explain observations regarding the influence of antigen dose on the generation of experimental and clinical IgE antibody responses in vivo.


Alloreactivity against IE-encoded antigens: evidence of the discrepancy between graft rejection and reactivity of IE-reactive T cells. Participation of IE antigens (Ag) in immune response as the transplantation Ag was examined. IE- B10.A(4R)(4R; Kk, IAk, IE-, Db) mice could not reject skin graft from IE Ag alone-disparate B10.A(2R) (2R; Kk, IAk, IEk, Db) mice despite intravenous (iv) injection of 2R spleen cells (SC) before or after skin grafting, indicating that graft rejection could not be caused across IE Ag-barrier alone. Furthermore, 4R SC could not induce lethal graft-versus-host disease (GVHD) in supralethally (950 rad) irradiated 2R mice. On the other hand, infiltration of lymphoid cells was observed at the site of transplanted 2R skin in 4R mice. SC of 4R mice unprimed or primed with 2R skin or 2R SC showed the capability to proliferate in vitro in response to 2R Ag. In immunofluorescence analysis of lymph node cells (LNC) of 4R mice injected iv with 2R SC 7 days earlier, IE-reactive CD4+Vbeta 11+ T cells did not change in number, but slightly increased the expression of interleukin-2 receptor (IL-2R). In 2R mice irradiated with 670 rad and injected iv with 4R SC 7 days earlier, 4R-derived CD4+V beta 11+ T cells proliferated, changed to blastoid form, and showed a markedly increased expression of IL-2R. To further investigate the influence of IE alloantigens on transplantation immunity, IL-2 production and anti-class I CTL activity were assayed. The 4R SC capable of recognizing IEk and Dk Ag of B10.BR (Kk, IAk, IEk, Dk) generated levels of both IL-2 and CTL activities higher than those of 2R SC capable of recognizing Dk Ag alone. These results strongly suggest that IE alloantigens indirectly act as the transplantation Ag by the stimulation of IE-reactive CD4+ helper T cells resulting in the differentiation of class I-restricted CD8+ T cells.


Comparative absorption and distribution pharmacokinetics of intravenous and epidural sufentanil for major abdominal surgery. The pharmacokinetics of absorption and distribution of a single bolus dose of sufentanil 150 micrograms for major abdominal surgery were compared in 20 patients after random intravenous or epidural administration. Samples of plasma and cerebrospinal fluid were taken at regular intervals from time zero to 180 min after injection and at the time of tracheal extubation (3.43 to 12.66h). Sufentanil was analysed by radioimmunoassay. The area under the concentration-time curve (AUC) from zero to 1h, 2h, 3h, tracheal extubation and infinity, the absorption and distribution half-lives, maximum plasma and CSF concentrations, time to the peak concentration of sufentanil, and the fraction of sufentanil that reached the central circulation after epidural administration were assessed. Except in the first sample, plasma concentrations of sufentanil were comparable between the 2 groups. The initial transfer of sufentanil from the epidural space to the systemic circulation appeared to be very rapid. Explanations for this phenomenon are given. In only 3 patients could an uptake of sufentanil from the systemic circulation into the CSF be demonstrated. The transfer of sufentanil from the epidural space into the CSF is slower than the transfer into the plasma and it varied interindividually.


Advances in occupational asthma.

Advances in occupational asthma. Clinical and research interests in occupational asthma increased dramatically in the 1980s. Advances in our knowledge base have led to improved recognition, management, and methods for preventing this disorder. An accelerated pace of basic and clinical research is anticipated in the 1990s. These efforts will likely lead to a more complete understanding of the disease (and pay dividends in understanding asthma itself). Occupational asthma is predicted to be the preeminent occupational lung disease in the next decade.


Can midazolam diminish sufentanil analgesia in patients with major trauma? A retrospective study with 43 patients. Benzodiazepine agonists in combination with opioid analgesics are commonly used for combined analgesia and sedation in intensive care patients as well as for anesthesia. In animals, studies indicate either agonistic or antagonistic interactions of benzodiazepine agonists and opioids. This retrospective study of 43 patients evaluated the possible clinical relevance of benzodiazepine-opioid interactions related to pain management. We observed an increase of greater than 50% of the maximal sufentanil infusion rate in significantly more patients in group 2 (13 patients vs 6 patients; chi 2: p = 0.04) and a decrease of the sufentanil infusion rate in eight group 1 patients, but only in one patient in group 2 (chi 2: p = 0.03). We believe that an interaction between midazolam and sufentanil on nociceptive transmission and/or a rapid development of tolerance to sufentanil may be responsible for the observed difference. Contrary to the common clinical impression that midazolam potentiates opioid analgesia, these results indicate that systemic co-administration of midazolam over a period of more than three days can diminish sufentanil efficacy.


Effects of anabolic-androgenic steroids on the pilosebaceous unit. Examination of skin biopsy specimens from persons using anabolic-androgenic steroids demonstrates dramatic hypertrophy of the sebaceous glands. High dosages of testosterone and anabolic-androgenic steroids, often self-administered by athletes, increase skin surface lipids, the cutaneous population of Propionibacteria acnes and the cholesterol and free fatty acids of the skin surface lipids. Acne, oily hair and skin, sebaceous cysts, hirsutism, androgenic alopecia, striae atrophicae, seborrheic dermatitis, and secondary infections including furunculosis may occur in persons using these drugs.


Acne mechanica in athletes.

Acne mechanica in athletes. Acne mechanica is a papulopustular eruption caused by the physical factors of pressure, occlusion, friction, and heat acting upon the skin, either separately or in unison. Athletes in certain sports are at particular risk for this condition. Dermatologists, if familiar with its presentation, can play a critical role in relieving its distracting symptoms during a competitive season, and eliminating it completely at the season's end.


Pelvic peritoneal reconstruction to prevent radiation enteritis in rectal carcinoma. Some patients with rectal cancer who undergo exenterative surgery may require radiation therapy as an adjuvant treatment for recurrent or residual disease. A common devastating side effect of this treatment modality is radiation enteritis, a radiation-induced small bowel injury. Hence, the prevention of such a complication is essential for both the surgeon and the radiation oncologist. A new surgical method using the posterior rectus sheath and peritoneum to partition the abdominal cavity at the level of the umbilicus to the sacral promontory seems to accomplish this purpose, keeping the small bowel away from the pelvic cavity. After removal of the rectal lesion [eight abdominoperineal resections (APRs), nine Hartmann's procedures, and one low anterior resection (LAR)] in 18 patients with rectal cancer, this new surgical procedure was performed. One of the patients had an early postoperative intestinal obstruction, and all but one of the patients received postoperative adjuvant radiation therapy. In addition, a small bowel series was performed before the radiation therapy and six months and one year after surgery. Upon examination, most of these patients still had their small bowel kept intact in the abdominal cavity. During the follow-up period of 10 months to 2 years with an average of 18 months, two late complications of intestinal obstruction were noted. Exploratory laparotomy of these two patients revealed radiation enteritis of the small bowel. Therefore, the failure rate of the following procedure is 12 percent, since 2 of the 17 patients received small bowel injury. Although the follow-up period for this surgical method is short, the results have encouraged us to continue the use of this procedure on advanced rectal cancer patients who require postoperative radiation therapy.


The effect of phospholipids, calcium ions and protein S on rate constants of human factor Va inactivation by activated human protein C. Rate constants for human factor Va inactivation by activated human protein C (APC) were determined in the absence and presence of Ca2+ ions, protein S and varying concentrations of phospholipid vesicles of different lipid composition. APC-catalyzed factor Va inactivation in free solution (in the presence of 2 mM Ca2+) was studied under first-order reaction conditions with respect to both APC and factor Va and was characterized by an apparent second-order rate constant of 6.1 x 10(5) M-1 s-1. Stimulation of APC-catalyzed factor Va inactivation by phospholipids was dependent on the concentration and composition of the phospholipid vesicles. Optimal acceleration (230-fold) of factor Va inactivation was observed with 10 microM phospholipid vesicles composed of 20 mol% dioleoylglycerophosphoserine (Ole2GroPSer) and 80 mol% dioleoylglycerophosphocholine (Ole2GroPCho). At higher vesicle concentrations and at higher molar fractions of Ole2GroPSer some inhibition of APC-catalyzed factor Va inactivation was observed. Membranes that contained anionic phospholipids other than phosphatidylserine also promoted factor Va inactivation. The ability of different anionic lipids to enhance factor Va inactivation increased in the order phosphatidylethanolamine less than oleic acid less than phosphatidic acid less than phosphatidylglycerol less than phosphatidylmethanol less than phosphatidylserine. APC-catalyzed factor Va inactivation in the presence of phospholipid vesicles could be saturated with respect to factor Va and the reaction obeyed Michaelis-Menten kinetics. Both the Km for factor Va and the Vmax of factor Va inactivation were a function of the phospholipid concentration. The Km increased from 1 nM at 2.5 microM phospholipid (Ole2GroPSer/Ole2GroPCho 20:80, mol/mol) to 65 nM at 250 microM phospholipid. The Vmax increased from 20 mol factor Va inactivated.min-1.mol APC-1 at 2.5 microM phospholipid to 62 mol factor Va inactivated.min-1.mol APC-1 at 10 microM phospholipid and remained constant at higher phospholipid concentrations. Protein S appeared to be a rather poor stimulator of APC-catalyzed factor Va inactivation. Protein-S-dependent rate enhancements were only observed in reaction mixtures that contained negatively charged phospholipid vesicles. Independent of the concentration and the lipid composition of the vesicles, protein S caused a twofold stimulation of APC-catalyzed factor Va inactivation. This suggests that, in the human system, enhancement of APC binding to phospholipid vesicles by protein S is of minor importance. Considering that protein S is a physiologically essential antithrombotic agent, it is likely that other factors or phenomena contribute to the in vivo antithrombotic action of protein S.


Modulation of cell-surface transferrin receptor by the imino sugar N-butyldeoxynojirimycin. The imino sugar, N-butyldeoxynojirimycin, is an inhibitor of the glycoprotein-processing enzyme glucosidase I and exhibits anti-(human immunodeficiency virus) activity in vitro. We have investigated the effect(s) of this compound on cell-surface glycoproteins by flow cytometry. We observed selective modulation of the transferrin receptor in response to treatment with 0.5 mM N-butyldeoxynojirimycin resulting in reduced cell-surface transferrin-receptor expression. The receptor modulation was dose dependent, resulted in reduced 59Fe uptake by treated cells and was fully reversible within 24 h of culture in the absence of the compound. Pulse/chase analysis in conjunction with endoglycosidase-H digestion demonstrated that transferrin-receptor glycosylation was altered following N-butyldeoxynojirimycin treatment, which is compatible with glucosidase inhibition. In addition, modulation of transferrin receptor in response to N-butyldeoxynojirimycin was not confined to a single cell line, but was also observed with certain human lymphoid and myeloid cell lines. Mechanism(s) of action of the imino sugar resulting in reduced cell-surface transferrin-receptor expression are discussed.


Role of F0 and F1 subunits in the gating and coupling function of mitochondrial H(+)-ATP synthase. The effect of dithiol reagents. A study is presented on the role of F0 and F1 subunits in oligomycin-sensitive H+ conduction and energy transfer reactions of bovine heart mitochondrial F0F1 H(+)-ATP synthase. Mild treatment with azodicarboxylic acid bis(dimethylamide) (diamide) enhanced oligomycin-sensitive H+ conduction in submitochondrial particles containing F1 attached to F0. This effect was associated with stimulation of the ATPase activity, with no effect on its inhibition by oligomycin, and depression of the 32Pi-ATP exchange. The stimulatory effect of diamide on H+ conduction decreased in particles from which F1 subunits were partially removed by urea. The stimulatory effect exerted by diamide in the submitochondrial particles with F1 attached to F0 was directly correlated with a decrease of the original electrophoretic bands of a subunit of F0 (F0I-PVP protein) and the gamma subunit of F1, with corresponding formation of their cross-linking product. In F0 liposomes, devoid of gamma subunit, diamide failed to stimulate H+ conduction and to cause disappearance of F0I-PVP protein, unless purified gamma subunit was added back. The addition to F0 liposomes of gamma subunit, but not that of alpha and beta subunits, caused per se inhibition of H+ conduction. It is concluded that F0I-PVP and gamma subunits are directly involved in the gate of the F0F1 H(+)-ATP synthase. Data are also presented indicating contribution to the gate of oligomycin-sensitivity conferral protein and of another protein subunit of F0, F6.


Interaction of membranes of the Golgi complex with microtubules in vitro. We have developed an in vitro assay for characterizing the binding of elements of the Golgi complex to microtubules. The binding assay comprises three distinct components, Golgi elements purified from Vero cells by subcellular fractionation, taxol-polymerized tubulin from bovine brain coupled to magnetic beads and cytosol from HeLa cells. Binding of Golgi elements to microtubules is quantitated by measuring the activity of the Golgi marker enzyme, galactosyltransferase, associated with the microtubule-coated beads retrieved with a magnet. In the presence of cytosol, 35 to 45% of the total input of galactosyltransferase activity (Golgi elements) bind to microtubules; only 3% of the Golgi elements bind to microtubules, however, in the absence of cytosolic factors. This binding is saturable at a cytosol concentration of approximately 5 mg/ml or at a high input of Golgi elements. Cytosol-stimulated binding of Golgi elements to microtubules is decreased to less than 15% when cytosol is pretreated with 2 mM N-ethylmaleimide (NEM) and it is abolished when cytosolic proteins are inactivated by heat or when microtubules have been coated with heat-stable microtubule-associated proteins (MAPs). Trypsinization of the membranes of the Golgi elements abolishes their ability to bind to microtubules. Furthermore, inactivation of cytoplasmic dynein by UV/vanadate treatment does not affect the binding. This suggests that the interaction of Golgi elements with microtubules depends on NEM-sensitive cytosolic factors and membrane-associated receptors, but not on the microtubule-based motor protein cytoplasmic dynein.


Stimulation of the biosynthesis of lactosamine repeats in glycoproteins in differentiating U937 cells and its suppression in the presence of NH4Cl. We prepared a mouse monoclonal antibody, 2D5, which recognized a highly glycosylated human lysosomal membrane antigen. The apparent molecular mass of this antigen was cell type dependent and ranged between 100 kDa and 130 kDa. The difference was due to a variation in the carbohydrate moiety, since upon removal of the N-linked oligosaccharides the size of the glycoprotein was reduced to approximately 50 kDa in all cases. The high carbohydrate contents, subcellular localization and N-terminal sequence indicated a high similarity or identity of this antigen with the lamp-2 protein. In U937 cells several agents known to elicit differentiation induced synthesis of a larger form of the lamp antigen. Thus, treatment of cells with calcitriol resulted in a shift in its average molecular mass from 115 kDa to 130 kDa. The difference was due to an increase in the contents of lactosamine repeats. In subcellular membranes from calcitriol-treated cells the specific activity of the UDP-N-acetylglucosamine: N-acetyllactosamine N-acetylglucosaminyltransferase was enhanced 3-fold. The enhancement was accompanied with an elongation of lactosamine repeats in N-linked oligosaccharides in the 46 kDa mannose 6-phosphate receptor and the homing receptor, the leucocyte antigen CD44. In contrast, the apparent size of the leucocyte antigen CD43 which bears numerous O-linked oligosaccharides was not changed indicating a selectivity in the modulation of the formation of lactosamine repeats in N- and O-linked carbohydrates. It is shown further that the synthesis of lactosamine repeats in U937 cells is impeded in the presence of NH4Cl.


Selective screening on hormonedependent tumours of women's reproductive system organs. This work is devoted to research on the selective screening possibilities of hormonodependent tumours of the female reproductive system organs (endometrial cancer, ovarian cancer, breast cancer). After morbidity analysis using case-control study elucidation of risk factors affecting cancer of the uterine cervix, endometrium, ovary and breast were carried out on of female population the Ashkhabad (311 cancer patients and 14872 healthy women). Using established risk factors and mathematical methods optimized computer programmes were developed processing individual risk and models forming risk groups or hormonedependent tumours which were used in practice. As a result in the risk group were found 0.9% subclinical states and hormonedependent tumours (control -0.16%). The research accomplished has shown the practical importance of hormonedependent selective screening and necessity of elucidating new disease-epidemiological and laboratory risk factors in cancer of the reproductive system's organs.


Synergistic release of calcium in sea urchin eggs by caffeine and ryanodine. A transient rise in intracellular Ca2+ during fertilization is necessary for activation of the quiescent sea urchin egg. Several mechanisms contribute to the rise in Ca2+ including influx across the egg plasma membrane and release from intracellular stores. The egg contains both IP3-sensitive and -insensitive Ca2+ release mechanisms and in this study we have used single-cell spectrofluorimetry to examine the effects of caffeine and ryanodine on Ca2+ release in eggs preloaded with fura 2. Caffeine induced a small Ca2+ release that was insensitive to heparin or ruthenium red. Ca2+ liberation by caffeine could be augmented by prior treatment with thapsigargin, an inhibitor of endoplasmic reticulum Ca2+ ATPase. Variable Ca2+ releases were observed in response to microinjection of ryanodine. The action of ryanodine appeared to be enhanced by prior injection of heparin and partially inhibited by ruthenium red. The release of Ca2+ by caffeine or ryanodine was generally insufficient to trigger cortical granule exocytosis, thus these eggs could be fertilized and a second Ca2+ release during fertilization was measured. Unlike the caffeine- and ryanodine-sensitive Ca(2+)-induced Ca2+ release mechanism in somatic cells, the graded responses in eggs suggested this caffeine- and ryanodine-sensitive release mechanism is not sensitive to sudden changes in Ca2+. Thus we could examine the combined actions of caffeine and ryanodine on Ca2+ release, which were synergistic. Caffeine treatment of ryanodine-injected eggs or ryanodine injection of caffeine-treated eggs stimulated a Ca2+ release significantly larger than the release by either drug independently. The experiments presented here suggest that sea urchin eggs liberate Ca2+ in response to caffeine and ryanodine; however, the regulation of this release differs from that described for caffeine- and ryanodine-sensitive Ca(2+)-induced Ca2+ release of somatic cells.


Lys-373 of actin is involved in binding to caldesmon. Limited proteolysis of actin with trypsin removes its two or three C-terminal amino acid residues [Proc. Natl. Acad. Sci. USA 81 (1984) 3680-3684]. Carboxypeptidase B-treatment of G- and F-actin previously digested with trypsin revealed that in the first case preferential release of three and in the second two C-terminal amino acid residues takes place. Tryptic removal of three but not two C-terminal amino acid residues of actin causes weakening of its interaction with caldesmon and lowering of the caldesmon-induced inhibitory effect on actomyosin ATPase activity. Therefore, it is concluded that the third amino acid residue from the C terminus of actin, Lys-373, is important for the interaction with caldesmon.


Aromatase inhibitors: clinical pharmacology and therapeutic implications in breast cancer. Aminoglutethimide was the first aromatase inhibitor to be used in breast cancer therapy but, since it interacts with the synthetic glucocorticoids, hydrocortisone must also be given as a replacement. The most important side-effects of aminoglutethimide are at the level of the central nervous system. Other aromatase inhibitors with greater potency and selectivity are being developed. Pyridoglutethimide, a compound resulting from modifications to the structure of aminoglutethimide, seems to be devoid of sedative properties according to preliminary tests on the central nervous system. 4-Hydroxyandrostenedione is significantly more potent and better tolerated than aminoglutethimide. Fadrozole (CGS 16,949 A) is 200-400 times more potent than aminoglutethimide and is now in phase II of its clinical development. CGS 20,267 has no effect on adrenal steroidogenesis and is currently in phase I of its clinical development. Availability of newer aromatase inhibitors could make a worthwhile contribution to endocrine therapy in breast cancer.


Isradipine in the treatment of peripheral occlusive vascular disease of the lower limbs: a pilot study. The long-term effects of isradipine on peripheral occlusive vascular disease of the lower limbs were investigated in 23 normotensive patients with stable Fontaine stage IIa disease and with an absolute pain-free interval (treadmill speed 4 km/h, no incline) of 300 - 700 m, and Doppler ankle - arm arterial pressure index of less than 0.80 in at least one leg. Using a double-blind, parallel-group design, patients received either 2.5 mg isradipine twice daily or placebo for 12 months. Both isradipine (n = 11) and placebo (n = 12) increased the absolute pain-free interval mean values; the increases were not significantly different. Similar trends were observed in the mean values for relative pain-free interval and ankle--arm arterial pressure index. In a subgroup of patients with a baseline absolute pain-free interval of greater than 500 m, isradipine (n = 6) significantly (P less than 0.001) increased both the absolute and the relative pain-free intervals and increased the ankle--arm arterial pressure index compared with placebo (n = 7). The favourable effects of long-term isradipine treatment suggest that isradipine could positively interfere with factors involved in the progression of atherosclerotic lesions or improve collateral vessel flow.


Expression of natriuretic peptide genes in cardiac tissues of hypertensive rats. A quantitative assay using a reverse transcriptase-linked polymerase chain reaction has been developed for measuring the levels of rANP and iso-rANP mRNA. A linear correlation between total RNA template and amplified cDNA was obtained for the amplification of cDNA from both iso-rANP and rANP mRNAs even when both cDNAs were amplified in the same assay. Application of the assay showed that in contrast to rANP levels of iso-rANP transcript in hypertensive rats remained approximately the same in atrium but were increased 10-fold in ventricle compared to normal rats. Given the relative size of the ventricle the increase of iso-rANP in this tissue in SHR may be a major response to the hypertensive state.


Postnatal ontogenesis of vasopressin receptors in the rat collecting duct. The ontogenesis of vasopressin receptors in the rat collecting duct was studied by measuring the binding of [1-(beta-mercapto-beta,beta-cyclopentamethylenepropionic acid),2-O-methyltyrosine,4-threonine,8-ornithine,9-125I-tyrosylamide+ ++]-vasotocin (125I-d(CH2)5[Tyr(Me)2,Thr4,Tyr-NH(9)2]-OVT) to isolated cortical collecting ducts (CCD), outer medullary collecting ducts (OMCD) and inner medullary collecting ducts (IMCD) microdissected from collagenase-treated kidneys of 2- to 34-day-old rats and adult animals. The stereospecificity for recognition of a series of seven vasopressin structural analogues by CCD and OMCD receptors reveals that the labeled binding sites identified in 11- to 16-day-old and adult rats are homologous respectively and contain a major population of V2 type and a minor population of V1a type of vasopressin receptors. At all postnatal stages examined, the receptor density (expressed as 10(-18) mol radioligand bound per square millimeter tubular outer surface area) decreases gradually from the CCD to the IMCD. For the three segments, the numbers of receptors detected remained constant during the first 2 weeks after birth and increased sharply after 20 days to reach the corresponding adult levels during the fifth week.


Fractures caused by falling from a wheelchair in patients with neuromuscular disease. This report discusses the occurrence of fractures caused by falling from a wheelchair. 13 distal femur, two proximal humerus and three distal tibia fractures were seen in 13 patients, of whom 11 had Duchenne muscular dystrophy, one had Becker muscular dystrophy and one had spinal muscular atrophy. All were non-ambulant and had been wheelchair-dependent for at least one year before the fall occurred. Six patients sustained fractures between 1976 and 1982, and five more between 1983 and 1985. Eight subsequent fractures from wheelchair falls have occurred since 1986. Although wheelchair design, controls and adaptations have continued to improve, wheelchair safety has not.


Suppression of sympathetic nervous system activity by nicorandil during exercise. 1. Treadmill testing was done and plasma epinephrine and norepinephrine were measured during exercise and recovery in 21 patients with coronary artery disease given nicorandil. 2. Epinephrine levels during exercise did not change significantly with nicorandil, but the percent change in epinephrine with nicorandil tended to be lower during exercise. 3. Norepinephrine levels after exercise were suppressed with nicorandil (with, 424 +/- 15 pg/ml; without, 760 +/- 16, P less than 0.05). 4. Also, the percent change in norepinephrine with nicorandil was significantly decreased during exercise and recovery (with, 259 +/- 11%; without, 555 +/- 19, P less than 0.01). 5. Therefore, nicorandil suppressed the sympathetic nervous system hyper-response to exercise.


Long-term effects of imipramine on striatal dopamine autoreceptor function: involvement of both noradrenergic and serotonergic systems. 1. The effects of apomorphine (APO) administration on DA system activity were assessed by measuring dopamine metabolite levels (HVA) in several circumstances. 2. Pretreatment with IMI reduced the effect of APO on HVA levels. 3. Pretreatments with either IDE or DMI did not reduce the effect of APO on HVA levels. 4. Reductions of either NE and 5-HT levels after DSP4 and pCPA restored the effect of APO after IMI pretreatment.


Characterization of dopamine receptors involved in apomorphine-induced pecking in pigeons. 1. The possible involvement of subtypes of dopamine-receptors in apomorphine induced pecking was studied in pigeons. Different doses of apomorphine induced pecking in pigeons which was dose-dependent. 2. The response was decreased by SCH 23390 (D-1 antagonist) or high doses of sulpiride (D-2 antagonist) pretreatment, but increased by lower doses of sulpiride. 3. Combination of SCH 23390 with sulpiride completely antagonized the apomorphine effect. 4. Single dose administration of SKF 38393 (D-1 agonist) or bromocriptine (D-2 agonist) and combination of these drugs did not induce pecking, although either SK 23390 or bromocriptine increased the apomorphine-induced pecking which was decreased by SCH 23390 or sulpiride pretreatment. 5. It may be concluded that pecking, induced by apomorphine in pigeons, is elicited through activation of both D-1 and D-2 dopamine-receptors.


Sequence of the Bacillus subtilis homolog of the Escherichia coli cell-division gene murG. The Bacillus subtilis homology of the Escherichia coli murG gene [encoding UDP-N-acetylglucosamine:N-acetylmuramyl-(pentapeptide) pyrophosphoryl-undecaprenol N-acetylglucosamine transferase] was cloned in E. coli K-12 and sequenced. The murG homolog encodes a protein of M(r) 39,936 [363 amino acid (aa) residues] of which 108 aa residues (29.8%) are identical with the E. coli murG product.


Chimeric HU-IHF proteins that alter DNA-binding ability. Chimeric proteins between Escherichia coli histone-like HU and IHF were constructed by genetic engineering, in which part of the arm region was replaced by the corresponding region of IHF alpha (designated as HupANhimA) or IHF beta (HupANhimD); alternatively, an alpha-helix 2-beta 1 region was replaced by the corresponding region of IHF alpha (HupAXhimA) or IHF beta (HupAXhimD) (symbols N and X indicate NotI and XhoI junctions). These proteins were synthesized in a hupA-hupB double-deletion mutant. HupANhimA exhibited marked reduction in nonspecific DNA binding in vitro, and a drastic loss of HU activity in replicative transposition of Mu phage in vivo. HupANhimD also showed a significant reduction in the ability for DNA binding, though this protein supported Mu phage development. In contrast, the other two chimeric HU proteins showed only slight changes in nonspecific DNA-binding ability: they retained activities for transposition of Mu phage in vivo. These observations confirm that the flexible arm of HU-2, a domain proposed for DNA binding [Tanaka et al., Nature 310 (1984) 376-381; Goshima et al., Gene 96 (1990) 141-145], plays an important role in the physiological function of this protein. The results indicate that a unique conformation of the arm structure of HU protein, particularly the N-terminal half of a two-strand antiparallel beta-ribbon of the structure, is important for the DNA-binding ability of this protein.


Overproduction of a human snRNP-associated Sm-D autoantigen in Escherichia coli and Saccharomyces cerevisiae. To conduct functional and autoimmunity studies, we overproduced human Sm-D1 (hSm-D1), a small nuclear ribonucleoprotein 'core' protein and autoantigen, in Escherichia coli and Saccharomyces cerevisiae. Optimal expression in these organisms was achieved by designing vectors that synthesized abundant hSm-D1 mRNA under the control of the strong, regulatable promoters: T7 phi 10 (E. coli) and GAL1 (yeast). In addition, efficient translation initiation of the hSm-D1 coding sequence was effected in E. coli by utilizing a two-cistron approach; for expression in yeast, we created a 5' untranslated leader whose sequence was based on the consensus of highly expressed genes in S. cerevisiae. The hSm-D1 protein accumulated at high levels in both bacteria and yeast, representing, respectively, approx. 10% and 7% of the total protein. However, in comparison with the authentic protein, the recombinant hSm-D1 displayed different immunoreactive determinants as assessed by Western blot. We thus conclude that certain hSm-D1 immunologic properties are most likely dependent on posttranslational modifications that take place in the cells of higher eukaryotes.