Helicase Hmi1 stimulates the synthesis of concatemeric mitochondrial DNA molecules in yeast Saccharomyces cerevisiae

Hmi1p is a helicase in the yeast Saccharomyces cerevisiae required for maintenance of the wild-type mitochondrial genome. Disruption of the HMI1 ORF generates ^– and ⁰ cells. Here we demonstrate that, in ^– yeast strains, Hmi1p stimulates the synthesis of long concatemeric mitochondrial DNA molecules associated with a reduction in the number of nucleoids used for mitochondrial DNA packaging. Surprisingly, the ATPase negative mutants of Hmi1p can also stimulate the synthesis of long concatemeric ^– mitochondrial DNA molecules and support the maintenance of the wild-type mitochondrial genome, albeit with reduced efficiency. We show that, in the mutant hmi1–5 background, the wild-type mitochondrial DNA is fragmented; and we propose that, in hmi1 yeast cells, the loss of the wild-type mitochondrial genome is caused by this fragmentation of the mitochondrial DNA.     

Temporospatial Heterogeneity of Myocardial Perfusion and Blood Volume in the Porcine Heart Wall

Spatial heterogeneity of myocardial perfusion has been recognized for many years. Whether this is primarily the result of heterogeneity of parameters such as myocardial metabolism, of intramyocardial mechanical forces, or of vasomotor function within the myocardial microcirculation, is not clear. A practical problem is that it has been almost impossible to measure any two of these parameters simultaneously in the same piece of myocardium so that an unambiguous correlation, much less a cause-and-effect relationship, has been difficult to establish. In this study of six anesthetized pigs, we propose that whole-body computed tomography is a method for providing the simultaneous measurement of heterogeneity of myocardial perfusion (F) and myocardial blood volume (). The first finding was that the empirical relationship =AF+BF^0.5 between myocardial blood flow (F) and intramyocardial blood volume () is maintained over a range of sizes of regions of interest (approximately 1 to 0.125 cm³) within the myocardium of each individual animal despite the spatial heterogeneity of the F and the values. The value of A ranges from 0.014 to 0.021 min and of B ranges from 0.061 to 0.076 ml^0.5 g^–0.5 min^0.5. A second finding was that the pattern of spatial heterogeneity of F and of remained reasonably stable over at least a 1 h period. © 1998 Biomedical Engineering Society. PAC98: 8745Ft, 8759Fm     

Cytokine messenger RNA expression and proliferation status of intestinal mononuclear cells in noninflamed gut and Crohn's disease

T-cell activation and local cytokine production probably contribute to the pathogenesis of Crohn's disease. This study investigates the proliferative status of intestinal mononuclear cells (MNC) and cytokine messenger RNA (mRNA) production in gut tissue sections from patients with Crohn's disease and noninflamed controls. mRNA in situ hybridization was performed using ³³P-labelled riboprobes for human interleukin (IL)-1, IL-2, IL-4, IL-5, IL-6, tumour necrosis factor- and interferon-. The expression of the proliferation-associated antigen Ki-67 was analysed by immunohistochemical single and double staining. Compared with controls, where proliferation of MNC and cytokine expression was restricted to mucosal lymphoid follicles, inflamed gut tissue contained increased numbers of cells expressing cytokine mRNA, most prominently IL-1 and IL-6, but also interferon- and tumour necrosis factor-. Proliferating T-cells were increased in number, and small amounts of IL-2-expressing cells were detected. IL-4 was expressed by a few cells exclusively in follicular germinal centres. IL-5 was negative. Proinflammatory cytokines are strongly expressed in situ in Crohn's disease and largely predominate over lymphokine mRNA. Our results provide in situ evidence of a local lymphocyte response in Crohn's disease with characteristics of a delayed-type hypersensitivity reaction.     

Queensland fruit fly virus,a probable member of the Picornaviridae

Summary A picornavirus was isolated from various life stages of the Queensland fruit fly,Dacus tryoni. This virus, Queensland fruit fly virus (QFFV) has virions with a diameter of 30 nm and a sedimentation coefficient of 178 S. One third of the particles in preparations were empty capsids or natural top component (NTC) with a sedimentation coefficient of 95 S. The buoyant density () of virions and NTC in CsCl was 1.34 and 1.30 g/ml respectively; small amounts of a dense component (=1.45 g/ml) were also detected. The capsid contained three major protein species of molecular weight (mol.wt.) 41,700, 36,500, and 31,300, in approximately equimolar proportions. NTC contained three major species of mol. wt. 44,700, 41,700, and 31,300. The nucleic acid present only in the bottom component virions was RNA and comprised about 30% of the particle weight and had a mol. wt of 2.88 kd, contained a poly(A) tract, and had a base ratio: G=20; A=32; C=15; U=33. The mol. wt. of the virion was estimated to be 9.5 kd. When virions were heated at 56°C and above, they converted into artificial top component (ATC), which had the same protein composition as the virion when analysed by SDS-PAGE. In immunodiffusion tests the virions and NTC were indistinguishable, but a minor difference in antigenicity was detected between the virions and ATC. Virions were stable between pH 3 and 9 inclusive, and between 5 and 7 in the presence of 0.14 M NaCl. Immunodiffusion tests showed that QFFV was serologically unrelated to a range of picornaviruses as well as an unclassified virus isolated from the Mediterranean fruit fly,Ceratitis capitata. The data show that QFFV is probably a member of thePicornaviridae, genus Enterovirus.     

Über die „thermo-dilution“-Methode zur Bestimmung des Herzzeitvolumens am narkotisierten und unnarkotisierten Hund

Zusammenfassung 1. Vergleichende Messungen des Herzzeitvolumens mit der thermodilution-Methode, nach dem Fickschen Prinzip und mit dem Farbstoff-Verdünnungs-Verfahren zeigten gute Übereinstimmung und bewiesen die Brauchbarkeit der thermo-dilution-Methode.2. Die Versuche ergaben weiterhin, daß die Herzzeitvolumen-Bestimmung mit der thermo-dilution-Methode auch am unnarkotisierten Tier möglich ist.Mit 4 Textabbildungen     

Comparison of the properties of five pox virus strains isolated from monkeys

Summary Five strains of monkey pox viruses were compared with respect to their cultural characteristics in primary and continuous cell cultures and the lesions developed in embryonated eggs and in rabbit skin as well as to their hemagglutinating activity.Four strains (Copenhagen 65-31 65-32 and 7-61) appeared to be similar in their properties. The cytopathogenic effect (CPE) was identical to that induced by vaccinia virus. There was no detectable virus multiplication in an pig kidney cell line (PEK). All four strains produced small, white, compact, hemorrhagic pock-like lesions on the chorioallantoic membrane.The strain 64–7275, isolated from healthy monkeys kidneys, had all properties of variola virus. It multiplied in the PEK cell line with a CPE. The lesions on the CAM were more compact without hemorrhage. In rabbit skin no detectable reaction occurred after infection with this strain.     

Ultrastructure and permeability of the Schwann cell layer surrounding the giant axon of the squid

Summary The ultrastructure of the Schwann cell layer surrounding the giant axon of the squidAlloteuthis subulata is described, and the permeability of extracellular compartments assessed by exposure to electron-dense tracers. Morphometric analysis is used to deduce the number, size and shape of the Schwann cells, and the routes for ion flux across the Schwann cell layer. Axons (mean diameter 233 m) were surrounded by a 1–2 m thick layer of Schwann cells which were 1 m thick, 70 m long and 23 m wide. There were around 62 000 Schwann cells per cm² axon surface. The outer (abaxonal) surface of the Schwann cells was invaginated, with evidence for a covering of fine Schwann cell processes; the inner (adaxonal) surface of the Schwann cells was less folded. The percentage area occupied by mesaxonal cleft openings to the axon and to the basal lamina was 0.02% and 1.09% respectively. A system of tubules, the glial tubular system, occupied 3.9% of the Schwann cell volume, and opened to both axonal and basal lamina surfaces, with more elaborate lattice-like clusters towards the basal side of the cell. Tubule openings accounted for 0.26% of the surface area facing the axon and 0.37% of the area facing the basal lamina (where there was greater clustering of openings). The electron dense tracers horseradish peroxidase, ionic lanthanum and tannic acid filled mesaxon clefts, glial tubular system and periaxonal space. If ion flux occurred via the mesaxonal clefts, a theoretical series resistance (R_sth) of > 20 cm², would be predicted, whereas if it occurred via the tubular system, the figure would be < 2 cm², closer to physiological estimates. The results presented show that the glial tubular system is likely to be the major route for ion flux into and across the Schwann cell layer, and for clearance of K⁺ from the periaxonal space during periods of axonal stimulation. The implications for K⁺ homeostasis in the axonal microenvironment are discussed.     

Identification and quantitation of cortisol metabolites in rat liver homogenate and -slices

Summary Cortisol-1, 2-H³ was incubated with rat liver homogenate and/or rat liver slices in the presence of a NADPH-generating system. The following metabolites could be identified in adult male rats: -cortol, allo--cortol, 3-allo--cortol, 20-hydroxy-cortisol, 11, 17, 20, 21-tetrahydroxy-5-pregnan-3-one, 3-allotetrahydrocortisol, tetrahydrocortisol, trace amounts of allotetrahydrocortisol and two highly polar metabolites only partly identified. In female rats only tetrahydrocortisol, allotetrahydrocortisol and allodihydrocortisol could be detected in significant amounts.The radioactive metabolites mentioned above were localized and quantitated on paper chromatograms by a 4-radiochromatogram scanner. A nearly perfect correlation was found between these results so obtained and those given by liquid-scintillation counting of each metabolite after its elution from the paper.Part of this work was supported by grant n° 695 of the National Fonds voor Wetenschappelijk Geneeskundig Onderzoek.Stagiair of the Nationaal Fonds voor Wetenschappelijk Onderzoek.     

Comparative activity of selected antiviral compounds against clinical isolates of varicella-zoster virus

Sixteen freshly isolated varicella-zoster virus (VZV) strains were evaluated in vitro, in parallel with two reference strains expressing a functional thymidine kinase (TK⁺) (Oka and YS) and two thymidine kinase-deficient mutants (TK^–) (07–1 and YS-R), for their susceptibility to a broad range of antiviral compounds. The following compounds were included: acyclovir (ACV), brivudine (BVDU), sorivudine (BVaraU), other BVDU congeners such as BTDU, CTDU, CVDC and CVDU, ganciclovir (GCV), FIAC, araT, araA, araC, foscarnet (PFA), phosphonoacetic acid (PAA), the acyclic nucleoside phosphonates HPMPC, cHPMPC, HPMPA, cHPMPA, HPMPc³A, PMEA and PMEDAP, the N⁷-isomeric acyclic nucleoside analogue N⁷ AP, penciclovir (PCV), compounds 882C87 and H2G and two oxetanocin derivatives OXT-A and OXT-G. Fourteen of the 16 clinical isolates displayed the following order of decreasing selectivity against VZV: BVaraU > BVDU > CVDU CVDC > H2G > N⁷AP } CTDU BTDU OXT-G 882C87 > ACV > FIAC araT > HPMPC cHPMPC HPMPA HPMPc³A cHPMPA > PCV GCV OXT-A > PMEDAP PMEA > PFA PAA araA > araC. Two VZV strains (isolated from the cerebrospinal fluid of an AIDS patient) that were shown to have a truncated TK were clearly resistant to all the compounds that need the viral TK for their phosphorylation, while sensitivity to the acyclic nucleoside phosphonates, PFA, PAA, OXT-A and araA, remained unchanged. A slight (5- and 10-fold) increase was noted in the 50 % inhibitory concentration of N⁷AP and OXT-G, respectively, for the TK^– VZV strains as compared to the TK⁺ VZV strains. Ganciclovir and FIAC also showed a marked decrease in their activity against these two strains, but this was not as pronounced as for the other viral TK-dependent drugs. From our results, it appears that although acyclic nucleoside phosphonates may not have as favourable a therapeutic index as drugs requiring the viral TK, they should be considered for the treatment of TK^– VZV life-threatening infections that are resistant to the viral TK-dependent drugs     

Prostate specific antigen and prostate specific acid phosphatase in adenocarcinoma of Skene's paraurethral glands and ducts

An autopsy case of adenocarcinoma of Skene's paraurethral gland co-incident with renal cell carcinoma is described. The adenocarcinoma showed distinct prostate specific antigen and prostate specific acid phosphatase pointing to the equivalence between the male prostate and Skene's paraurethral glands and ducts. Skene's gland are the homologue of the prostate in females and tumours arising from them are immunohistochemically similar to male prostate carcinoma.In the title and text the authors used the official term of Nomina Anatomica paraurethral (Skene's) glands and ducts. Nevertheless recently published data on cross-antigenicity between the male prostate and Skene's glands and the newly discovered exocrine and neuroendocrine parameters of the prostate homologue in the female, comparable with the male prostate (Zaviai 1987), support the use of the same term — the prostate — for prostatic tissue in both sexes (Zaviai 1987, Zaviai et al. 1985). The designations female prostate homologue or female prostate equivalent are a compromise between terms the female prostate and Skene's paraurethral glands.     

Nachweis von 6 neuen Steroiden im Plasma von menschlichem Placentablut (Nabelsehnurblut)

Zusammenfassung 16-Hydroxyprogesteron, 17-Hydroxyprogesteron, ⁴-Pregnen-20-ol-3-on, ⁴-Pregnen-17, 20-diol-3-on, Adrenosteron und 11-Hydroxyandrostendion wurden in Extrakten von Plasma des menschlichen Placentablutes (Nabelschnurblut) nachgewiesen.     

Cervical Concentrations of Interleukin-10 and Interleukin-12 Do Not Correlate with Plasma Levels

Human papillomavirus (HPV) infects the transformation zone of the cervix and is the primary cause of cervical cancer. The infection is localized to the cervix and mucosal immunity is likely to be an important determinant for viral clearance. Previous studies of immunity to HPV have measured immune markers in the blood, but the relationship of systemic immunity to cervical immunity is poorly understood. In this study of 70 women enrolled in the ASCUS-LSIL Triage Study (ALTS), a clinical trial for management of low-grade cytologic abnormalities of the cervix, we collected paired plasma and cervical secretions to investigate the relationship between cervical concentrations of interleukin-10 (IL-10) and interleukin-12 (IL-12) and plasma levels. Neither IL-10 ( = 0.11), or IL-12 ( = –0.04) nor the ratio of IL-12 to IL-10 ( = 0.06) were correlated between blood and cervical secretions. Except for weak correlations of IL-10 among nonsmokers ( = 0.35, P = 0.019) and those in day 18–27 of their menstrual cycle ( = 0.51, P = 0.015), this lack of correlation persisted in all subgroups defined by genital inflammation or infection, current oral contraceptive use, heme contamination and volume of collected secretions, HPV16 seropositivity, and repeat HPV infection and/or cytologic abnormalities. The lack of correlation and high concentrations in cervical secretions indicate that the cervical IL-10 and IL-12 concentrations exceed what could be expected from blood as a principle source of IL-10 and IL-12 and suggest that cytokine concentrations in cervical secretions are predominantly the result of local cytokine production.     

Genetic analysis of the products of a cross involving a suppressive ‘petite’ mutant of S. cerevisiae

Summary A genetically defined highly suppressive petite yeast strain ( ^–cob⁺A^sE^oC^oO^oP^o) was crossed with a grande strain carrying a multiply marked mitochondrial genome ( ⁺A^rE^rC^rO rp^r). Petite diploid progeny, isolated from individual zygotic clones consisting either of wholly petite or mixtures of grande and petite cells, were characterised genetically by crossing to grande haploids. The diploid petites were found to closely resemble the petite parent and in general not to carry mitochondrial markers from the grande parent. In the petites from the mixed clones recombination was detected, but only within the region of homology between the genomes. These observations are inconsistent with models of suppressiveness based on destructive recombination and suggest that the petite genome eliminates the grande genome from zygotic progeny through being preferentially replicated. The most plausible model to explain the observed pattern of zygotic clones postulates a limited number of mDNA replication sites in zygotes, competition for sites between input mDNA molecules and an advantage in this competition for suppressive ^– mDNA.     

Cationic proteins of human granulocytes Effects on human platelet aggregation and serotonin release

Immune-aggregate and thrombin-mediated [³H]serotonin release from human platelets are shown to be enhanced when platelets are preincubated with the antibacterial chymotrypsin-like cationic protein isolated from human granulocytes. The enhancement is dose dependent and inhibited by heating of the cationic protein. Release with chymotrypsin-like cationic protein alone was not observed, although the protein was shown to micro-aggregate platelets irreversibly by an ADP-dependent reaction. Platelet macro-aggregation induced by immune-aggregate was also enhanced by chymotrypsin-like cationic protein whereas platelet macro-aggregation induced by thrombin was inhibited competitively. Platelet micro-aggregation induced by chymotrypsin-like cationic protein was inhibited when preincubated for more than 5 min with a 2-fold molar excess of-1-antitrypsin. Chymotrypsin-like cationic protein interaction with several platelet reactions suggests a close relationship between neutrophils and platelets in the inflammatory process.     

A monovalent ion-selective cation current activated by noradrenaline in smooth muscle cells of rabbit ear artery

We have studied chloride influx and efflux in a highly purified preparation of type n cells freshly isolated from adult guinea-pig lung using ³⁶Cl^–. Chloride uptake was time-dependent, saturable (K_m<10 mM) and was inhibited by 4,4-diisothiocyanatostilbene-2,2-disulphonic acid (DIDS; K_i80 M). In the absence of external chloride (substituted by gluconate), ³⁶Cl^– uptake exhibited an overshoot above equilibrium. The rate of ³⁶Cl^– entry was strongly inhibited by addition of external nitrate; sulphate was a weaker inhibitor. ³⁶Cl^– efflux was stimulated by external bromide > bicarbonate chloride citrate; and was inhibited by proprionate > acetate > oxalate. Although the chloride channel blocker 4-nitro-2-(3-phenylpropylamino)benzoate (0.14 mM) caused an inhibition, ³⁶Cl^– influx did not appear to be electrogenic. These data are compatible with the existence of a substantial electroneutral anionexchange pathway for chloride transport in freshly isolated adult type II pneumocytes.     

Neutralizing monoclonal antibodies directed to infectious bovine rhinotracheitis virus

Summary Infectious bovine rhinotracheitis virus (IBRV) has been shown in this report to have thirty-three polypeptides. Ten of the eleven polypeptides which can be labeled with (³H)-glucosamine are located on the surface of the virus since they can be surface labeled with sodium boro(³H)hydride. In order to define the immunologically important viral proteins, monoclonal antibodies were prepared against the virus and selected for their ability to neutralize infectivity. Four such hybridoma lines were obtained for characterization of the antigens that elicit neutralizing antibodies. The viral polypeptides were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and the specificity of each monoclonal antibody was determined by Western blot analysis and/or by immunoprecipitation of (³⁵S)-methionine and (³H)-glucosamine labeled infected cell lysates by the monoclonal antibodies. One monoclonal antibody reacted with two glycoproteins, gp135 and gp78a, on the Western blot but immunoprecipitated three glycoproteins, gp135, gp78a, and gp54 from labeled infected cell lysates. The other three monoclonal antibodies immunoprecipitated a single glycoprotein, gp78b, from (³H)-glucosamine labeled infected cell lysates but not from (³⁵S)-methionine labeled infected cell lysates.With 5 Figures     

Single channel Ca2+ currents inHelix pomatia neurons

Unitary Ca²⁺ currents of TEA injected Helix neurons were recorded in the Giga seal situation (6, 7) from microscopic membrane patches exposed to 50 mM [Ca²⁺]_o, O [Na⁺]_o, 20 mM [TEA⁺]_o and 2.5 M [TTX]_o. Constant field assumptions yield a channel permeability of 2.9±1.0×10^–14 cm³s^–1 corresponding to slope conductances of 5 to 15 pS between 0 and –30 mV. Frequency of occurrence of the units strongly increased with depolarization. Mean open time of the Ca²⁺ channels was about 3 ms without obvious dependence on voltage. A similar open time was seen with [Ba²⁺]_o, yielding about double the current strength when compared with [Ca²⁺]_o.     

Nuclease treatment results in high specific purification of Creutzfeldt-Jakob disease infectivity with a density characteristic of nucleic acid-protein complexes

Summary Representative preparations of partially purified Creutzfeldt-Jakob disease (CJD), including disaggregated density gradient fractions, were treated with a variety of nucleases. RNases as well as exhaustive digestions with micrococcal nuclease did not significantly diminish infectivity, but resulted in an 7,000-fold specific purification of infectivity with respect of nucleic acid. Protected nucleic acids included species of up to 2,000 bases in length. After nuclease treatment, infectivity co-migrated with nucleic acid-protein complexes at a density of 1.27 g/cm³ in sucrose. Substantial specific protein purification were also achieved in the gradient step ( 11,000-fold), where 70% the host Gp34 (prion protein) as well as other free proteins separated from infectivity. These CJD purifications are better than those previously attained in scrapie, and may be useful for further studies of non-host protein and nucleic acid species. The data are consistent with the hypothesis that CJD-like agents are composed of nucleic acid-protein complexes.     

Quantitative determination of lactic acid in cell culture media and biological fluids: Optimization by automatic rate analysis

Summary An automated assay for the enzymatic determination of L(+)-lactic acid by rate analysis is described. It is shown that the initial rate (1 min) of reduction of-nicotinamide adenine dinucleotide by the enzyme lactate dehydrogenase is proportional to the amount of lactic acid originally present in the sample. The reliability of the assay (C.V. 5%) as well as the sensitivity ( 10 g/ml) and the recovery of lactic acid from samples (95%) are such that the assay may be used for the quantitation of the metabolite in virtually any cell culture media or biological fluids. Because the automated procedure is shorter and requires less reagent than established end-point assays, it is considered that these factors represent major advantages in cell culture systems where numerous samples have often to be processed.To whom requests for reprints should be addressed.     

The sensitivity to Zn2+ discriminates between typical and atypical mast cells

The effects of zinc gluconate have been studied on rat peritoneal mast cells and rat basophilic leukemia cells (RBL 2H3) stimulated by various secretagogues. The IC₅₀'s of zinc gluconate on peritoneal cells were (M)1.6, 1.9, 5.4 and 18 for ionophore A23187, phorbol 12-myristate 13-acetate, substance P and immunoglobulin E-antigen, respectively. Higher concentrations of zinc gluconate were required to inhibit histamine secretion from RBL 2H3 cells, i.e. 12 M (ionophore A23187) and 140 M (immunoglobulin E-antigen). Zinc gluconate (10^–4 to 10^–3 M) also inhibited the IgE-dependent contraction of guinea pig trachea but was unable to affect that induced by exogenous histamine. These results suggest that zinc gluconate acts intracellularly and is selective of typical or connective tissue mast cells.