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.     

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.     

Key word: Chromosome

The word chromosome has survived for over 100 years, because it succinctly defines what early cytologists were able to see with the most modern instrument of their time, a light microscope. It was introduced in a review that became widely known and was published almost simultaneously in German, English and French (Waldeyer 1888, 1889, 1890a, 1890b). In the late 19th century, these three languages were in strong competition for international status as the idiom of science. At the same time, Greek was also considered as a candidate for a nationalistically neutral language of science, and it seems more than coincidence that the word ó matches well the coherent Greek terminology used to describe the cell cycle in mitosis as well as meiosis. Emil Heitz (1935) maintained – in the face of reactionary German efforts to replace the term – that in using the ineradicable word chromosome we think last of all that it indicates a body that stains intensely. Significantly, the key word is no longer restricted to eukaryotes, but has been readily adopted by microbial geneticists (Heidelberg et al. 2000) and acknowledged as defining the elementary unit of genomic partition.     

Activation of atrial muscarinic K+ channels by low concentrations ofβγ subunits of rat brain G protein

Effects of G protein subunits from rat brain on cardiac K⁺ channel was examined in single atrial cells of guinea-pig, using patch clamp techniques. We found that 10 pM concentration of rat brain subunits preparation could activate the atrial muscarine receptor-gated K⁺ channel (I_K.ACh). Neither the detergent, CHAPS, used to suspend nor the boiled preparation activated I_K.ACh. Furthermore, preincubation of subunits preparation in Mg²⁺-free solution, which easily inactivated -GTP-S, did not affect -activation of I_K.ACh. We concluded, therefore, that subunits themselves can activate I_K.ACh.Supported by the grants from the Ministry of Education, Culture and Science of Japan and from the Calcium Signal Workshop on Cardiovascular Systems     

A Theoretical Model for the Margination of Particles within Blood Vessels

The margination of a particle circulating in the blood stream has been analyzed. The contribution of buoyancy, hemodynamic forces, van der Waals, electrostatic and steric interactions between the circulating particle and the endothelium lining the vasculature has been considered. For practical applications, the contribution of buoyancy, hemodynamic forces and van der Waals interactions should be only taken into account, whilst the effect of electrostatic and steric repulsion becomes important only at very short distances from the endothelium (1–10 nm). The margination speed and the time for margination t_s have been estimated as a function of the density of the particle relative to blood , the Hamaker constant A and radius R of the particle. A critical radius R_c exists for which the margination time t_s has a maximum, which is influenced by both and A: the critical radius decreases as the relative density increases and the Hamaker constant decreases. Therefore, particles used for drug delivery should have a radius smaller than the critical value (in the range of 100 nm) to facilitate margination and interaction with the endothelium. While particles used as nanoharvesting agents in proteomics or genomics analysis should have a radius close to the critical value to minimize margination and increase their circulation time.     

Isolation of mutants lacking branched-chain amino acid transaminase

Variants of the Chinese hamster ovary cell have been isolated which can no longer grow when valine, leucine, or isoleucine is replaced in the culture medium by its respective -keto acid: -ketoisovaleric acid, -ketoisocaproic acid, or -keto--methylvaleric acid. These variants lack branched-chain amino acid transaminase activity. Evidence is presented indicating these variants to be single gene mutants. Genetic evidence is also presented confirming previous biochemical evidence that a single enzyme carries out transaminase functions on valine, leucine, and isoleucine. The branched-chain transaminase-deficient (trans^–)mutants can be reverted to wild-type behavior by treatment with mutagenic agents. These mutants promise to be useful in exploring regulatory mechanisms in biochemical, genetic, and cancer research.Trainee, U.S. Public Health Service Grant No. GM00781-17.     

The role ofγδ T cells in the normal and disordered immune system

Summary A small population of T cells does not express the conventional T cell receptor characterized by the and polypeptide chains (TCR) but instead, two polypeptides termed and (TCR). This alternative receptor is able to recognize antigen. It appears early in T cell ontogeny, but its role in the thymus prior to the availability of TCR remains unclear. In selected sites such as skin or gut TCR predominates in mice which might suggest a role of T cells in the first line of defense against infection, T cells secrete lymphokines and display cytotoxic activity. However, their activation requirements may differ from what is known for T cells since MHC-nonrestricted and also CD4 and CD8 negative T cells have been described. Preferential activation by mycobacterial antigens possibly indicates a special repertoire of the T cells. In various diseases slightly increased numbers of T cells were found, but these preliminary studies have not yet provided evidence for a major pathogenetic role of T cells.List of abbreviations C constant region (immunoglobulin or TCR gene segment) - CD4 cluster of differentiation 4 (mainly on helper cells) - CD8 cluster of differentiation 8 (mainly on cytotoxic cells) - D diversity region (immunoglobulin or TCR gene segment) - DNA desoxyribonucleic acid - IL2 interleukin 2 - J joining region (immunoglobulin or TCR gene segment) - kD kiloDalton - MHC major histocompatibility complex - NK natural killer (cells) - RA rheumatoid arthritis - TCR T cell receptor - V variable region (immunoglobulin or TCR gene segment)     

Phenotypic characteristics of lewis lung carcinoma cells

We studied adhesive properties and physiological activity in vivo of cells from Lewis lung carcinoma and its metastases. These cells differed in tumorogenic activity and metastatic potential in the syngeneic system. In vivo non-metastasizing cells are characterized by a lower content of surface lectins to tetrasaccharides SiaLe^x [Neu5Ac2-3Gal1-4(Fuc1-3) GlcNAc] and SiaLe^a [Neu5Ac2-3Gal1-3(Fuc1-4)GlcNAc] and trisaccharide HSO₃Le^x [HSO₃2-3Gal1-4(Fuc1-3)GlcNAc] compared to cells forming metastases in the syngeneic system. Metastatic cells with low tumorogenic activity weakly expressed lectins to disaccharide ligands 6-SiaLac [Neu5Ac2-6Gal1-4Glc], 6-HSO₃LacNAc, and A-di [GalNAc 1-3Gal] and trisaccharides H-type 1 [Fuc1-2Gal1-3GlcNAc and Le^x [Fuc1-3(Gal 1-4)GlcNAc] compared to cells that initiated tumor formation in the syngeneic system (similarly to transplanted tumors). We hypothesized that cell receptors to these carbohydrate determinates are involved in the development and growth of primary tumors, while lectins to SiaLe^x, SiaLe^a, and HSO₃Le^x play a role in the progress of tumor process and metastasizing.     

Structural polypeptides of California encephalitis virus: BFS-283

Summary The polypeptides of California encephalitis virus (BFS-283) were analyzed by polyacrylamide gel electrophoresis (PAGE). Four polypeptides were detected in virions grown in both BHK-21 and LLC-MK₂ cell cultures with molecular weights of 17,500, 30,000, 38,000, and 82,000 (VP-1, VP-2, VP-3, and VP-4, respectively). Viral proteins 2, 3, and 4 were glycoproteins and appeared to be associated with the envelope of the virus. Treatment of virions (=1.18 g/cm³) with then non-ionic detergent, NP-40, allowed detection of a RNA-rich fraction (=1.26/cm³) with contained the smallest polypeptide (VP-1).With 8 Figures     

Partitioning of Cortical and Deep Cytoskeleton Responses from Transient Magnetic Bead Twisting

We attempted to estimate in living adherent epithelial alveolar cells, the degree of structural and mechanical heterogeneity by considering two individualized cytoskeleton components, i.e., a submembranous cortical cytoskeleton and a deep cytoskeleton (CSK). F-actin structure characterizing each CSK component was visualized from spatial reconstructions at low and high density, respectively, especially in a 10-m-cubic neighborhood including the bead. Specific mechanical properties (Young elastic and viscous modulus E and ) were revealed after partitioning the magnetic twisting cytometry response using a double viscoelastic solid model with asymmetric plastic relaxation. Results show that the cortical CSK response is a faster ( ₁ 0.7s), softer (E₁: 63-109 Pa), moderately viscous (₁: 7-18 Pa s), slightly tensed, and easily damaged structure compared to the deep CSK structure which appears slower (₂ min), stiffer (E₂: 95-204 Pa), highly viscous (₂: 760-1967 Pa s), more tensed, and fully elastic, while exhibiting a larger stress hardening behavior. Adding drug depolymerizing actin filaments decreased predominantly the deep CSK stiffness. By contrast, an agent altering cell–matrix interactions affected essentially the cortical CSK stiffness. We concluded that partitioning the CSK within cortical and deep structures is largely consistent with their respective functional activities. © 2003 Biomedical Engineering Society. PAC2003: 8716Ka, 8716Ac, 8380Lz     

Pseudogenes and short repeated sequences in the rice chloroplast genome

Summary The rice chloroplast genome has been derived from a tobacco-like ancestral form by three major inversions. In the rice genome we have found six pseudogenes, trnG, trnI, 3-rps 12a, trnT, trnE and trnfM/G, all located near inversion endpoints, as well as four short repeated sequences. A comparison of rice, wheat and tobacco sequences indicated that similar pseudogenes are present in wheat but not in tobacco, suggesting that the creation of these pseudogenes occurred before the divergence of rice and wheat. The region downstream of rbcL is a variable region and contains rpl23 in rice and wheat and another 3-rps 12b further downstream in rice. This 3-rps 12b shows a higher homology to the functional rps 12 than 3-rps 12a, which suggests that it appeared more recently. The involvement of these pseudogenes in genome inversions and the creation of the pseudogenes and short repeated sequences are discussed.     

Effects of serine protease inhibitor,tame, on IL-1β in LPS-stimulated human monocytes: Relationship between synthesis and release of a 33-kDa precursor and the 17-kDa biologically active species

LPS stimulation of human monocytes in vitro induced release of the 17-kDa mature IL-1 (mIL-1) but did not result in release of precursor IL-1 (pIL-1). In contrast, the presence of a serine protease inhibitor, N-(p-toluene sulfonyl)-L-arginine methyl ester (TAME; 10 mM) for 6 or 18 h was associated with the LPS-stimulated release of the 33-kDa pIL-1 as well. These effects were initially discerned from observations that the fraction of the total IL-1 produced (as detected by ELISA) that was released from monocytes increased in the presence of TAME, and immunoblot assays confirmed that this fraction was predominantly 33-kDa IL-1. A global decrease in monocyte protein synthesis was also observed after prolonged (18-h) exposure to TAME and was associated with a decrease in IL-1 synthesis, predominantly affecting 31-kDa pIL-1, and a dose-dependent inhibition of TNF- production. Parallel examination of lactate dehydrogenase (LDH) release indicated thatpIL-1 release was unrelated to cell lysis. These results demonstrate that TAME-inhibitable serine proteases are probably involved in the production and eventual proteolysis of the 33-kDa pIL-1 in situ but are probably not mechanistically related to either maturation of the IL-1 molecule or signaling of IL-1 release. IL-1 release appears to be dependent on the amount of total IL-1 synthesized. Serine proteolysis may constitute a degradative pathway for excess precursor, which, if interfered with, could result in release of the higher-molecular-weight forms of IL-1.     

Effects of anti-inflammatory drugs on cartilage recovery from catabolin-induced degradation

Effects of aspirin (200 g/ml), hydrocortisone (10 g/ml), sodium aurothiomalate (100 g/ml), and indomethacin (10 g/ml) on recovery of cartilage from interleukin 1 or catabolin-induced degradation were examined in this initialin vitro study. The experimental protocol involved a degradative phase of eight days during which cartilage plugs were incubated in the presence or absence of spent human rheumatoid synovial culture media. A recovery period of six days followed during which the effects of the aforementioned drugs on treated cartilage were analyzed. Incorporation of [³⁵S]sulfate and [³H]proline precursors, and total contents of hydroxyproline and glycosaminoglycan in cartilage were determined two, four, and six days after insult. Aspirin treatment caused a rise in total proteoglycan content over degraded controls (p<0.002), however, this increase was not associated with increased [³⁵S]sulfate incorporation into glycosaminoglycans. Hydrocortisone resulted in a delayed rise in proteoglycan content concommitant with increased [³⁵S]sulfate uptake, whereas sodium aurothiomalate treatment was without effect on proteoglycans. Indomethacin treatment was associated with an increased release of newly synthesized macromolecules by cartilage into the media (p<0.01). These results suggest that common anti-inflammatory drugs may exhibit distinctly different effects on thein vitro synthesis and retention of proteoglycans by cartilage explants previously exposed to a degradative phase. Further work is necessary to assess the influence of drug concentration in this experimental system.     

Inhibition of L-type calcium channels by internal GTP [γS] in mouse pancreatic β cells

Pretreatment of pancreatic cells with pertussis toxin resulted in a 30% increase in peak whole-cell Ca²⁺ currents recorded in the absence of exogenous intracellular guanine nucleotides. Intracellular application of 90 M GTP[S], by liberation from a caged precursor, resulted in 40% reduction of the peak Ca²⁺ current irrespective of whether the current was carried by Ca²⁺ or Ba²⁺. Effects on the delayed outward K⁺ current were small and restricted to a transient Ca²⁺-dependent K⁺ current component. Inhibition by GTP[S] of the Ca²⁺ current was not mimicked by standard GTP and could not be prevented either by pretreatment with pertussis toxin or by inclusion of GDP[S] or cyclic AMP in the intracellular medium. The inhibitory effect of GTP[S] could be counteracted by a prepulse to a large depolarizing voltage. A similar effect of a depolarizing prepulse was observed in control cells with no exogenous guanine nucleotides. These observations indicate that inhibition of cell Ca²⁺ current by G protein activation results from direct interaction with the channel and does not involve second-messenger systems. Our findings also suggest that the cell Ca²⁺ current is subject to resting inhibition by G proteins.     

Characteristics of c-fos Gene Expression in the Brains of Rats with Different Investigative and Defensive Behaviors

Differences were found in the expression of the c-fos gene in brain structures of rats with active and passive types of behavior in the open field, hole exploration, and step-down tests. The most marked and intense expression of the c-fosgene was seen in passive rats. After electrocutaneous stimulation in the step-down test, these animals showed maximum expression of the c-fos gene in most areas of the cerebral cortex, the amygdala, in olfactory structures, the hypothalamus, and the brainstem. In active rats in the same conditions, expression of the c-fos gene was seen only in the infralimbic cortex and olfactory nuclei. These results, along with existing data on neurological mapping of the c-fos gene, demonstrate a relationship between the level and topography of c-fosexpression, and with typological and, perhaps, individual characteristics of rats.     

Klinische,morphologische und biochemische Untersuchungen bei einem androgenbildenden Hodentumor

Zusammenfassung Bei einem Knaben mit einer Pseudopubertas praecox wurde ein androgenproduzierender Hodentumor entfernt. Präoperativ war die Harnausscheidung der gesamten 17-Ketosteroide, von Androsteron und Ätiocholanolon, der 11-Oxy-17-Ketosteroide, insbesondere von 11-Hydroxy-androsteron, von Pregnandiol, Pregnantriol sowie der Oestrogene abnorm erhöht und fiel nach der Operation auf Normwerte ab. Nach Inkubation von Glucose allein mit Tumorschnitten wurden 11-Hydroxy- ⁴-androstendion und ⁴-Androstendion gefunden, nach Inkubation von ⁴-Androstendion: 11-Hydroxy- ⁴-androstendion, Testoseron und Adrenosteron, jedoch keine Oestrogene, nach Inkubation von Desoxycorticosteron: Corticosteron und 17-Hydroxy-desoxy-corticosteron. Im V. spermatica-Blut wurden große Mengen ⁴-Androstendion, ferner 17-Hydroxy-progesteron, 11-Hydroxy- ⁴-androstendion und Testosteron nachgewiesen.Die biochemischen und morphologischen Gesichtspunkte sowie die klinischen Auswirkungen des Tumors werden diskutiert. Morphologisch hatte der Tumor den Aspekt eines Leydigzell-Adenoms. In Übereinstimmung hiermit wurde eine hohe Sekretion von Androgenen festgestellt. Durch den Nachweis einer 11-Hydroxylase-Aktivität zeigte der Tumor jedoch gleichzeitig eine Eigenschaft, die sonst nur NNR-Zellen zukommt. Der Tumor wird vermutungsweise von atypischen Leydigzellen abgeleitet.Diese Art von Leydigzell-Adenom wird der beidseitigen tumorartigen Hodenvergrößerung, die gelegentlich beim angeb. adrenogenitalen Syndrom beobachtet wird, gegenübergestellt. Die gemeinsamen und unterschiedlichen Eigenschaften der beiden Wucherungen werden besprochen.

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Summary An androgen producing tumour of the testis was removed from a boy with pseudopubertas praecox. Urinary excretion of total 17-oxosteroids, androsterone and aetiocholanolone, 11-oxy-17-oxosteroids (particularly 11-hydroxy-androsterone), pregnandiol, pregnantriol and oestrogens were abnormally high before operation, and had decreased to normal after operation. After incubation of tumour slices with glucose alone androst-4-en-3,17-dione and 11-hydroxyandrost-4-en-3,17-dione were found; after incubation with androst-4-en-3,17-dione: 11-hydroxyandrost-4-en-3,17-dione, testosterone and adrenosterone, but no oestrogens were found; after incubation with desoxycorticosterone: corticosterone and 17-hydroxycorticosterone. A large amount of androst-4-en-3,17-dione was found in Vena spermatica blood, as well as 17-hydroxyprogesterone, 11-hydroxyandrost-4-en-3,17-dione and testosterone.The biochemical and morphological aspects are discussed in relation to the clinical manifestations of the tumour. The tumour had the morphological appearance of a Leydig cell adenoma and also showed a high rate of androgen secretion. The finding of 11-hydroxylase activity in the tumour shows that it possessed a characteristic usually found only in adrenocortical cells. The tumour was conceivably derived from atypical Leydig cells.This type of Leydig cell adenoma is compared with the bilateral tumour-like enlargement of the testis, which is ocassionally observed in congenital adrenogenital syndrome. The common and contrasting characteristics of the two growths are discussed.

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Herrn Prof. Dr.E. Rehn zum 85. Geburtstag gewidmet.     

Causal dimensions of college students' perceptions of physical symptoms

According to attribution theory, controllability, locus, and stability are important dimensions underlying causal explanations. The extent to which these theoretical dimensions underlie lay explanations for physical symptoms is unclear. Accordingly, in this study, attributes relevant to the lay public were empirically derived using a multidimensional scaling (MDS) procedure. Undergraduates (N=194) provided similarity judgments for 18 potential causes of physical discomfort. The MDS analysis yielded a three-dimensional solution. The first dimension captured the distinction between physical and nonphysical causes. The second dimension distinguished either variable versus stable causes or those that are controllable versus uncontrollable by health care professionals. The third dimension differentiated causes under low versus high personal control. These findings empirically confirm the theoretically proposed dimensions of personal control and stability and suggest the utility of considering the physical/nonphysical and controllability by health care professional distinctions in future work on attributions in the health domain.     

Monocyte chemoattractant protein (MCP)-1 production via functionally reconstituted Fcα receptor (CD89) on glomerular mesangial cells

Background: Fc alpha receptor (FcR; CD89) is the receptor for Fc portion of IgA in various cells, and displays various immunological responses on binding. It is important to analyze the mesangial functions via FcR in the pathogenesis of IgA nephropathy. However, it is still controversial whether FcR is expressed on mesangial cells. To assess biological functions of FcR on the mesangial cells, we established mesangial transfectants that expressed FcR with or without FcR chain that is a common signaling molecule of FcRs. The production of monocyte chemoattractant protein-1 (MCP-1) by mesangial cells is known to contribute to cellular infiltration into glomeruli and subsequent glomerular injuries. Methods: Murine mesangial cell lines (SV40 MES 13) were transfected with cDNA of the human FcR. Furthermore, we co-transfected some of the FcR transfectants with cDNA of human FcR chain. The tyrosine phosphorylation of the intra-mesangial proteins after FcR cross-linking was examined by immunoprecipitation. MCP-1 production from each transfectant stimulated with heat aggregated IgA was determined by sandwich ELISA. Results: Two kinds of mesangial transfectants stably expressed human FcR with or without FcR chain (FcR⁺, FcR⁺/⁺). Phosphorylation of FcR chain and syk kinase was detected in FcR⁺ and FcR⁺/⁺ cells, but not in untransfected cells. Aggregated IgA induced significantly higher MCP-1 production in FcR⁺/⁺ than those in FcR⁺ or untransfected control. Conclusions: Present study demonstrated that FcR and FcR chain could be reconstituted in mesangial cells and mediated MCP-1 production by aggregated IgA in a dose-dependent manner. Current data would argue that FcR can be activated in mesangial cells through their own machinery, although underlying mechanisms for FcR induction in mesangial cells remain unclear.Received 10 February 2003; returned for revision 14 May 2003; accepted by M. J. Parnham 16 June 2003     

Modulation of Ca2+-activated K+ channels by Mg2+ and ATP in frog oxyntic cells

Ca²⁺-activated K⁺ channels in the basolateral plasma membrane of bullfrog oxynticopeptic cells are intimately involved in the regulation of acid secretion. Patch-clamp techniques were applied to study the regulating mechanism of these channels. In the excised inside-out configuration, intracellular Mg²⁺ decreased channel activity in a dose-dependent manner. In the absence of Mg²⁺, administration of adenosine 5 triphosphate (ATP) to the cytoplasmic side also inhibited channel activity. On the other hand, in the presence of Mg²⁺, addition of ATP markedly increased channel activity. At a fixed concentration of free Mg²⁺ the Mg-ATP complex caused channel activation and shifted the dose response relationship between channel activity and the intracellular Ca²⁺ concentration to the left. A nonhydrolysable ATP analogue, adenosine 5-[,-imido]triphosphate (AMP-PNP) adenylyl [,-methylene]diphosphate (AMP-PCP), could not substitute for ATP in channel activation, but a hydrolysable ATP analogue, adenosine 5-O-(3-thiotriphosphate) (ATP[S]) could do so. Furthermore, application of alkaline phosphatase to the cytoplasmic side inhibited channel activity. These results demonstrate that Ca²⁺-activated K⁺ channels are regulated by Mg²⁺ and ATP, and suggest that a phosphorylation reaction may be involved in the regulation mechanism of these channels.