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PubMed1984

[Modulation of coronary vessel tonus: molecular and cellular mechanisms].

The cyclic interactions between myosin cross bridges and the actin filament in the presence of Ca++ with a sliding of both filaments passed each other, is considered also in vascular smooth muscle as the basic contractile mechanism. While in the striated muscle the regulation of the actin-myosin interaction occurs at the level of the actin filaments, there is a growing body of evidence that the contractile activation of the vascular smooth muscle is primarily regulated by phosphorylation of the 20,000-Dalton myosin light chain. This reaction is catalyzed by a calcium-calmodulin-dependent myosin light chain kinase. Additionally, dephosphorylated myosin cross bridges which remain attached to actin filaments over prolonged periods of time ("latch bridges") at low myoplasmic Ca2+-concentrations seem to be involved in the vascular smooth muscle in maintaining tonic active stress at a very low energy expenditure. In most arterial smooth muscle cells, the initiation of contraction (electromechanical coupling) is not associated with action potentials, but is coupled with graded membrane depolarization. During the process of excitation-contraction coupling, two mechanisms lead to increased myoplasmic calcium: a) Calcium influx through voltage-dependent channels along an electro-chemical gradient. b) Release of calcium from the sarcoplasmic reticulum or from the inside of the cell membrane, triggered either by calcium influx or directly by membrane depolarization. The pharmaco-mechanical coupling, i.e., the contractile activation by drugs without depolarization as initiating step, seems to be realized only in a few specific vessels. The stimulation of the phosphatidyl-inositol turnover (PI-cycle) in the plasma membrane by activation of alpha 1-adrenergic receptors can also be demonstrated in vascular smooth muscle cells. However, whether or not this PI-response plays a primary role in the increase of myoplasmic Ca2+ remains to be settled. The activation of alpha 2-adrenergic receptors seems to involve the action of an inhibitory guanine nucleotide-binding protein on the catalytic moiety of the adenylate cyclase. Thus, the contractile response observed may be attributed to the decrease of cyclic AMP (which is responsible for dilating effects via phosphorylation of various regulatory proteins). The decrease in the myoplasmic concentration of free-ionized calcium as a basic principle of relaxation comes about by different mechanisms, which can be classified as follows: a) Inhibition of transmembrane calcium influx into vascular smooth muscle cells by Ca-antagonists, which specifically interfere with plasmalemmal Ca2+-channels.(ABSTRACT TRUNCATED AT 400 WORDS)

Actins↗

PubMed1980

Sperm semi-autoanalysis by a combination of multiple exposure photography (MEP) and computer techniques.

A semiautomatic method for sperm analysis is described. The method combines a previously developed multiple exposure photography system with a digitizer connected to a computer. An undiluted specimen is inserted into a 10-mic chamber and the sperms are microscopically photographed for 1 second while illuminated by six short light pulses. On the developed film nonmotile sperms appear very bright, while motile sperms are seen as six-ringed chains. In addition, high magnification photographs of immobilized preheated sperms are taken for morphology analysis. The photographed images are projected onto sheets of paper where the traces of the nonmotile and motile sperms as well as the normal and abnormal forms are marked by a pen. For motility determination, sheets containing the sperm tracks are placed on the digitizer tablet. By means of a sonic pen the coordinates of the ends of the tracks are automatically recorded and the number of motile and nonmotile sperms are counted. Then the percent of abnormal forms are entered at the keyboard. Within a few seconds the computer supplies a printout of the results, including sperm concentration, percent of motile sperms, average velocity and frequency distribution of velocities of moving sperms, percent of abnormal forms, and total number of analyzed sperms.

Autoanalysis↗

PubMed1984

Phospholipids as acyl donors to membrane proteins of Mycoplasma capricolum.

Mycoplasma capricolum, a procaryotic sterol and fatty acid auxotroph, contains a large number of membrane proteins covalently modified by both saturated and unsaturated fatty acids (Dahl, C.E., Dahl, J.S., and Bloch, K. (1983) J. Biol. Chem. 258, 11814-11818). Pulse-chase experiments show that the radioactivity in the fatty acid moieties of the acyl proteins increases rather than decreases during a 4.5-h incubation period suggesting that a large intracellular pool of metabolites such as phospholipid serves as the donor for protein acylation. We find that cells incubated for 4 h in a growth medium containing [3H]palmitate-labeled phosphatidylglycerol or 2-[3H]palmitoyl dipalmitoylphosphatidylcholine show the same labeling pattern as cells incubated for 4 h in a complete growth medium with [3H]palmitate. Exogenously added phospholipids are not hydrolyzed to free fatty acid during the labeling period. Acylation of proteins is inhibited in cells treated with chloramphenicol showing that there is no pool of proacyl protein in the cell. Labeling of membrane proteins also occurs with [3H]glycerol. Glycerol is incorporated primarily into the same proteins as oleate suggesting that acylation by unsaturated fatty acid may involve a protein bound diglyceride moiety. Palmitate, on the other hand, appears to bind to other sites along the polypeptide chain in addition to the diglyceride moiety.

Acylation↗

PubMed1983

[24,25-3H]Cholesterol: presence of tritium at additional sites in the side chain.

In order to measure the distribution of radioactivity present in the side chain of [24,25-3H]cholesterol prepared by a sequence involving catalytic tritiation of 3 alpha, 5 alpha-cyclocholest-24-en-6 beta-ol 6-methyl ether, the cholesterol was oxidized to 4-cholesten-3-one, which was then cleaved between C-24 and C-25 to afford the C24 alcohol. Oxidation to the corresponding cholenoic acid, followed by alkali equilibration and esterification completed the sequence. It was found that about 20% of the tritium in the labeled cholesterol is not lost when this tracer is physiologically converted to bile acids. Consequently, measurements of bile acid formation using this tracer must be corrected upward by this amount.

Cholesterol↗

PubMed1982

Effects of histone hyperacetylation and hypoacetylation on RNA synthesis in HTC cells.

We have analyzed the RNA synthetic activity in intact HTC cells which have artificially high or low levels of histone acetylation. Rates of total RNA synthesis and the number of nascent transcription complexes have been measured for 20-h butyrate-treated cells and for butyrate-released cells which contain hypoacetylated histones. Exposure of HTC cells to butyrate for 20 h leads to a approximately 40% decrease in the number of nascent RNA molecules and a corresponding approximately 40% decrease in the rate of total RNA synthesis. During the induction of hypoacetylation following the release of cells from butyrate, rates of RNA synthesis and the number of nascent transcripts remain unchanged. Between the fifth and eighth h of release from butyrate, the number of nascent transcripts and the rate of total RNA synthesis increase to control cell levels. This increase in the number of nascent RNA polymerase complexes, while occurring when bulk histone is hypoacetylated, is temporally correlated with an increase in the rate of acetylation for a subset of histones. In contrast, changes in levels and rates of histone acetylation were found to be without effect on the average rate of RNA chain elongation. Additionally, we have found that the complexity of nuclear RNA is unchanged by a 20-h exposure of HTC cells to butyrate, suggesting that the approximately 40% decrease in frequency of nascent polymerase complexes produced by butyrate does not result from the complete loss of transcription of a large number of genes.

Acetylation↗

PubMedDate not supplied

[Possibilities of forming a hydrogen-bonded cytosine-adenine pair in the structure of transfer ribonucleic acid and at the wobble-position of the codon-anticodon complex].

220 nucleotide sequences of tRNAs were investigated to elucidate the frequency of appearance of C-A pairs in their main two-stranded regions, in the positions 26--44 and 15--48. It was supposed, that in the formation of C-A pairs on antiparallel polynucleotide chains the atomic groups--N4-H and -N3 of cytosine make up H-bonds with the groups N7- and H-N6--of adenine. On parallel chains, H-bonds, probably, form -N6-H and --N1 groups of adenine with N3- and H-N4--of cytosine. The calculation results predicted a significant energy of ineraction between cytosine and adenine. By the investigation of the molecular models it was shown that the formation of H-bonded C-A pairs requires considerable changes of conformation in rebosephosphate chains. In addition a theoretical analysis revealed the possibility of formation of C-A pairs at the wobble-position of codon-anticodon complex. The significance of this nucleotide pair in the processes of genetic coding proved to depend on the stability of the codon-anticodon complex, modification of cytosine 34 and structural features of the distant regions of the tRNA.

Adenine↗

PubMed1981

Changes in plasma amino acid profiles following abdominal operations.

Plasma amino acid profiles along with hemoglobin, hematocrit, albumin, protein, blood urea nitrogen and serum creatinine values for ten patients undergoing abdominal operations were studied before operation and for 16 days there-after at different intervals. Six patients in the control group were studied in a similar manner. From the observations obtained, we concluded that total amino acid valued are a more sensitive reflection of patient nutrition in both the preoperative and postoperative periods. In future, total amino acid levels may become part of the nutritional assessment of a patient undergoing an operation. The histidine levels in plasma remain low for the longest period of time, an indication of a great need for histidine. Hence, greater attention should be paid to the histidine content of a diet or solution administered parenterally, or both. In addition, branched chain amino acids, alanine, glycine, cystine, arginine, lysine, tryptophan and threonine are required in greater quantity than the other amino acids as a result of the increased catabolism and partial starvation of the patients postoperatively. In formulation hyperalimentation solutions, an increased need for these amino acids should be kept in mind.

Abdomen↗

PubMed1995

Antiviral cytotoxic T lymphocytes in vaginal mucosa of simian immunodeficiency virus-infected rhesus macaques.

The mucosal immune system of the female reproductive tract is of central importance for protection against sexually transmitted diseases, including HIV; however, this arm of the immune system remains poorly understood. Antiviral CTL responses never have been documented in the genital tract and the role of CTL in this anatomic site is unknown. In this study, CD8+ intraepithelial lymphocytes (IEL) in the vaginas of six simian immunodeficiency virus (SIV)-infected female rhesus macaques were identified by immunohistochemistry to be CD2+ and TCR beta-chain+. In addition, the majority of CD8+ IEL contained TIA-1+ cytoplasmic granules that are associated with CTL activity. CD8+ T cells were isolated from the vaginal epithelium and submucosa and amplified by limiting dilution in the presence of feeder cells. SIV p55gag and/or gp160env-specific lysis was detected in cultures of vaginal epithelial but not submucosal CD8+ T lymphocytes. Estimated SIV-specific precursor CTL frequencies were higher in the vaginal CD8+ IEL population of chronically infected monkeys than in the same cells from acutely infected monkeys or a naive control monkey. These results provide the first demonstration that antiviral CTL are present in the vaginal epithelium, and suggest that a vaccine may be able to generate anti-HIV CTL in the genital mucosa.

Animals↗

PubMed1993

Sugar chains of human cord serum alpha-fetoprotein: characteristics of N-linked sugar chains of glycoproteins produced in human liver and hepatocellular carcinomas.

Human serum alpha-fetoprotein (AFP) is elevated in not only hepatocellular carcinoma (HCC) but also benign liver diseases. AFP produced in HCC and benign liver diseases was separated into several isoforms corresponding to different sugar chain structures by several types of lectin affinity electrophoresis, and the HCC-specific AFP isoform was discriminated from those of benign liver diseases. Because a small amount of HCC-specific AFP isoform was detected in cord serum AFP, the whole sugar chain structures of human cord serum AFP were determined, as follows: Neu5Ac alpha 2-->Gal beta 1-->4GlcNAc beta 1-->2Man alpha 1-->6(Neu5Ac alpha 2 -->6Gal beta 1-->4GlcNAc beta 1-->2Man alpha 1-->3)Man beta 1-->4R1 and R2, Gal beta 1-->4GlcNAc beta 1-->2Man alpha 1-->6(Neu5Ac alpha 2-->6Gal beta 1 -->4GlcNAc beta 1-->2Man alpha 1-->3)Man beta 1-->4R1 and R2, and Neu5Ac alpha 2-->3Gal beta 1-->4GlcNAc beta 1-->2Man alpha 1-->6(Neu5Ac alpha 2 -->6Gal beta 1-->4GlcNAc beta 1-->2Man alpha 1-->3)Man beta 1-->4R1 and R2 in the ratio of 81.6:8.9:9.5. R1 and R2 denote GlcNAc beta 1-->4GlcNAcOT (subscript OT represents an NaB3H4-reduced oligosaccharide) and GlcNAc beta 1-->4(Fuc alpha 1-->6)GlcNAcOT, respectively, and the ratio between R1 and R2 in the respective fractions was approximately 19:1. In contrast, the sugar chain structure of HCC highly specific AFP isoform was found to comprise a monosialyl-biantennary sugar chain with additional fucosylation of the proximal N-acetylglucosamine. Fucosylation of AFP produced in fetal liver increased in inverse proportion to the gestation period, in weeks, indicating that fucosylation of AFP in HCC may be related to the dedifferentiation of human hepatocytes through malignant transformation.

Biomarkers, Tumor↗

PubMed1993

Engineering Fab' fragments for efficient F(ab)2 formation in Escherichia coli and for improved in vivo stability.

We previously developed an efficient route to humanized F(ab')2 fragments by high level secretion of the Fab' arms from Escherichia coli followed by directed chemical coupling in vitro. Here the number and type of interchain linkages in F(ab')2 molecules has been modified to simplify their production and improve their serum stability. All F(ab')2 variants had comparable binding affinity for the p185HER2 Ag and antiproliferative activity against p185HER2-overexpressing tumor cells. This was anticipated since the modifications are distant from the Ag-binding loops. Replacement of a single disulfide bridge between Fab' arms with a more stable thioether bridge increased the serum permanence time in normal mice by threefold to 2.1 h. Removal of the disulfide bond between L and H chains in the thioether-bridged F(ab')2 did not affect the pharmacokinetics, suggesting that the L chain remains associated with the H chain. An additional Fab' variant containing three repeats of the motif, CysProPro, was constructed with the aim of promoting efficient formation of F(ab')2 in E. coli. This Fab' (CPP)3 variant was recovered predominantly (up to 70%) as F(ab')2 directly from fermentation cell pastes, thus circumventing the need for in vitro coupling. The F(ab')2 (CPP)3 variant has a similar serum pharmacokinetics to the thioether-bridged molecules. The improvements described here for deriving F(ab')2 fragments from E. coli should enhance the clinical potential of these molecules.

Amino Acid Sequence↗

PubMed1994

Clonal composition of human adrenocortical neoplasms.

The mechanisms of tumorigenesis of adrenocortical neoplasms are still not understood. Tumor formation may be the result of spontaneous transformation of adrenocortical cells by somatic mutations. Another factor stimulating adrenocortical cell growth and potentially associated with formation of adrenal adenomas and, less frequently, carcinomas is the chronic elevation of proopiomelanocortin-derived peptides in diseases like ACTH-dependent Cushing's syndrome and congenital adrenal hyperplasia. To further investigate the pathogenesis of adrenocortical neoplasms, we studied the clonal composition of such tumors using X-chromosome inactivation analysis of the highly polymorphic region Xcen-Xp11.4 with the hybridization probe M27 beta, which maps to a variable number of tandem repeats on the X-chromosome. In addition, polymerase chain reaction amplification of a phosphoglycerokinase gene polymorphism was performed. After DNA extraction from tumorous adrenal tissue and normal leukocytes in parallel, the active X-chromosome of each sample was digested with the methylation-sensitive restriction enzyme HpaII. A second digestion with an appropriate restriction enzyme revealed the polymorphism of the region Xcen-Xp11.4 and the phosphoglycerokinase locus. Whereas in normal polyclonal tissue both the paternal and maternal alleles are detected, a monoclonal tumor shows only one of the parental alleles. A total of 21 female patients with adrenal lesions were analyzed; 17 turned out to be heterozygous for at least one of the loci. Our results were as follows: diffuse (n = 4) and nodular (n = 1) adrenal hyperplasia in patients with ACTH-dependent Cushing's syndrome, polyclonal pattern; adrenocortical adenomas (n = 8), monoclonal (n = 7), as well as polyclonal (n = 1); adrenal carcinomas (n = 3), monoclonal pattern. One metastasis of an adrenocortical carcinoma showed a pattern most likely due to tumor-associated loss of methylation. In the special case of a patient with bilateral ACTH-independent macronodular hyperplasia, diffuse hyperplastic areas and a small nodule showed a polyclonal pattern, whereas a large nodule was monoclonal. We conclude that most adrenal adenomas and carcinomas are monoclonal, whereas diffuse and nodular adrenal hyperplasias are polyclonal. The clonal composition of ACTH-independent massive macronodular hyperplasia seems to be heterogeneous, consisting of polyclonal and monoclonal areas.

Adenoma↗

PubMed1994

Immunodeficiency-associated malignant lymphoma. Three cases showing genotypic evidence of both T- and B-cell lineages.

Dual rearrangement of both immunoglobulin and T-cell receptor (TCR) genes has been described in up to 30% of precursor lymphoid neoplasms, but this phenomenon occurs rarely in lymphomas with a mature phenotype. We describe three cases of immunodeficiency-associated lymphomas, two in patients with acquired immunodeficiency syndrome (AIDS) and one in a patient with a renal transplant, in which both immunoglobulin and TCR gene rearrangements were identified. Two cases exhibited clonal JH, clonal JK, and clonal TCR beta gene rearrangements; the other case had clonal JK and TCR beta gene rearrangements with deletion of JH. Two of these bigenotypic lesions exhibited a B-cell phenotype, and the other exhibited evidence of both a B- and T-cell phenotype, containing cytoplasmic CD3 as well as monoclonal kappa light chain. In addition, two of the cases exhibited the presence of clonal Epstein-Barr viral DNA. These observations suggest that lymphomas arising in immunodeficiency states may be more prone to disordered differentiation than those arising in an intact immune system.

Adult↗

PubMed1994

Review of investigative diagnostic techniques for cutaneous lymphoma.

This article reviews the contribution of modern investigative techniques to the diagnosis of cutaneous lymphoproliferative diseases. Special attention is given to the significance of immunoglobulin and T-cell receptor rearrangements; as detected by Southern blotting and/or the polymerase chain reaction. Additional topics discussed include immunohistochemistry, cytogenetics, onco- and tumour suppressor genes and in-situ hybridization. It is recommended that the results of these techniques are interpreted in the form of a multifaceted diagnostic profile.

Blotting, Southern↗

PubMed1994

Effects of adenosine receptor and muscarinic cholinergic receptor agonists on cardiac protein phosphorylation. Influence of pertussis toxin.

In isolated 32P-labeled guinea pig ventricular cardiomyocytes phospholamban (PLB), myosin light chain 2 and the inhibitory subunit of troponin (Tnl) were identified by antibodies. In isolated 32P-labeled guinea pig ventricular cardiomyocytes isoproterenol (0.1 mumol/l) increased phosphorylation of PLB and of the inhibitory subunit of Tnl to 159 and 119% of control, respectively, without effect on the phosphorylation state of myosin light chain 2. Additionally applied carbachol or (-)-N6-phenylisopropyladenosine reduced phosphorylation of PLB and Tnl without reducing cyclic AMP content. After pretreatment of guinea pigs with pertussis toxin (18 micrograms/100 g b.wt.), carbachol or (-)-N6-phenylisopropyladenosine failed to reduce isoproterenol-stimulated phosphorylation of both PLB and Tnl. It is concluded that the reductions by carbachol and (-)-N6-phenylisopropyladenosine of isoproterenol-stimulated PLB phosphorylation and Tnl phosphorylation are pertussis toxin-sensitive.

Animals↗

PubMed1995

Properties of iridoviruses from ornamental fish.

An iridovirus from 2 species of freshwater tropical imported fish was examined for its antigenic and biochemical properties and virulence for 3 species of food fish. The virus was found in both guppy (Poecilia reticulata) and doctor fish (Labroides dimidatus). The virus replicated in numerous fish cell lines including BF-2, CCO and EPC. Virus titers reached 10(8) TCID50/ml in each cell line. The virions had similar size and morphology to those previously reported to cause systemic infections among catfish in Europe and from redfin perch and rainbow trout in Australia. A comparison of the ornamental fish iridovirus with these systemic agents indicated that all share common antigens detected by indirect fluorescent antibody tests and Western blots. Additionally, polymerase chain reactions using primer sets developed for the red fin perch virus, amplified a segment of genomic DNA from the ornamental fish isolate. An examination of the virulence of the ornamental fish isolate for 2 important food fish species, rainbow trout (Oncorhynchus mykiss) and channel catfish (Ictalurus punctatus), indicated the former species was susceptible although mortality was low. Dead fish had significant lesions in the liver and kidney and had titers of virus greater than 10(8) TCID50/g. We speculate that these iridoviruses, first detected in Australia in redfin perch, are all related strains in the Ranavirus genus that may have reached these geographically distant sites by movements of ornamental fish.

Animals↗

PubMed1997

Lower levels of 1H MRS-visible mobile lipids in H-ras transformed tumorigenic fibroblasts with respect to their untransformed parental cells.

High resolution 1H MRS studies report increased mobile neutral lipid (MNL) signals in transformed and malignant as well as in some in vitro cultured embryonic cells. Nature, subcellular localization and biological function of MNL are still under debate. This work was aimed at assessing alterations induced in MNL signals of NIH-3T3 mouse embryo fibroblasts by transformation with human HJ-ras oncogene. Lower MRS-visible MNL levels were unexpectedly detected in ras-transformed, in vivo tumorigenic fibroblasts, with respect to their untransformed and non-tumorigenic parental cells. MRS, gas chromatography and chemical analysis on cells and their lipid extracts indicated that these spectral differences could hardly be attributed to different triacylglycerol, free fatty acids and total cholesterol levels or to changes in the fatty acyl degree of unsaturation and average chain length. Additional, possibly more relevant mechanisms of regulation of MNL mobility may implicate the extensive morphogenetic changes and reorganization of cytoskeleton components (notably actin) associated with ras-transformation.

Animals↗

PubMed1998

Advance in the Rh blood group system.

The Rh blood group antigens are associated with non-glycosylated red blood cell membrane proteins encoded by two closely related genes, RHD and RHCE. Both RH genes have 10 exons and the open reading frames of their transcripts are composed of 1251 nucleotides. The sequence analyses of RHCE transcripts revealed that RhC/c and RhE/e are related to six nucleotide substitutions and a single substitution, respectively. Molecular analyses have elucidated the genetic background of various RhD and RhCE variants including the Rhnull phenotype with neither RhD nor RhCE antigen. The Rh polypeptides require some co-molecules for their assembly in the plasma membranes. In fact, several unrelated proteins other than those of the Rh polypeptides are reduced or absent on Rhnull red blood cells. This group includes the Rh50 glycoprotein, LW glycoprotein, glycophorin B, Duffy glycoprotein, and CD47 glycoprotein. The mutation of the RH50 gene is inducible of the Rhnull phenotypes. Although the Rh50 glycoprotein is a critical co-expressing factor, one or more factors seem to be required to express RhD or RhC(c)E(e) antigens on the plasma membranes. Additionally, polymerase chain reaction-based tests for Rh genotyping were developed and some have been put in practice. Much information obtained from the molecular approach to the Rh blood group system has shed considerable light on the Rh polypeptides. In the near future, the mechanism of expression, the function, and the evolution of Rh polypeptides will be clarified further.

Anemia, Hemolytic, Autoimmune↗

PubMed1998

Mitochondrial function as a determinant of recovery or death in cell response to injury.

Many pathological conditions can be the cause or the consequence of mitochondrial dysfunction. For instance anoxia, which is initiated by a critical reduction of oxygen availability for mitochondrial oxidations, is followed by a wide variety of mitochondrial alterations. A crucial role in the evolution of cell injury is to be attributed to the direction of operation of the F0F1 ATPase, which may turn mitochondria into the major consumers of cellular ATP in the futile attempt to restore the proton electrochemical gradient. On the other hand, functional mitochondria can paradoxically accelerate or exacerbate cell damage. This concept is particularly relevant for the ischemic myocardium. Indeed, inhibition of the respiratory chain or addition of uncouplers of oxidative phosphorylation can both limit the extent of enzyme release in the intact heart and prevent the onset of irreversible morphological changes in isolated myocytes. From studies on different tissues in a variety of pathological conditions a general consensus emerges on the role of intracellular Ca2+ overload as a pivotal link between cellular alterations and mitochondrial dysfunction. Oxidative phosphorylation is reduced by a massive mitochondrial uptake of Ca2+, resulting in a vicious cycle whereby the reduced ATP availability is followed by a failure of the mechanisms which extrude Ca2+ from the sarcoplasm. In addition, the rise in [Ca2+]i could promote opening of the cyclosporin-sensitive mitochondrial permeability transition pore, leading to a sudden deltapsi(m) dissipation. Here, we review the changes in intracellular and intramitochondrial ionic homeostasis occurring during ischemia and reperfusion. In particular, we evaluate the potential contribution of the permeability transition pore to cellular damage and discuss the mechanisms which can determine the cellular fate from a mitochondrial point of view.

Adenine Nucleotides↗