Actin cytoskeletal isoforms in human endothelial cellsin vitro: Alteration with cell passage

Summary The microfilamentous actin component of the cytoskeleton is crucial to endothelial angiogenesis and vascular permeability. Differences in actin cytoskeletal profiles in cultured human endothelial cells were explored: when first isolated, both primary human umbilical vein endothelial cells (HUVEC) and primary human placental microvascular endothelial cells (HPMEC) expressed F-actin, but notβ-actin orα-smooth muscle actin. A similar endothelial actin profile was observed in cryo-sections of freshly delivered term umbilical cord and placenta. In subsequent cell culture, although the actin cytoskeleton of HUVEC remained unchanged, the actin profiles of HPMEC altered after the second passage with the induction ofα-smooth muscle actin expression, which was intercellularly heterogeneous and increased to 20% at P4. This behavior occurred in HPMEC monolayers cultured on a variety of extracellular matrices. Comparisons with a spontaneously immortalized human microvascular cell-line, HGTEN 21, revealed that inprolonged passage, bothα-smooth muscle actin andβ-actin were expressed, whereas HPMEC at P4 showed a lower level ofβ-actin expression. Therefore, in comparison with large vessels, microvascular cells are more likely to dedifferentiate. This may reflect the ability of microvascular cells to remodel according to changing requirement for new vessel formation. In conclusion, passage of human microvascular endothelial cells, but not of larger vessel endothelial cells, alters the expression of actin isoforms. This may be important in relation to comparisons ofin vitro andin vivo vascular permeability; higher passage microvascular endothelial cells should thus be used with caution in such studies.     

Purification and characterization of tubulin from the catfishHeteropneustes fossilis

Tubulin was purified from the brain of the catfishHeteropneustes fossilis by cycles of temperature-dependent assembly and disassembly. Fish tubulin assembles into microtubules in the absence of high molecular weight microtubule associated proteins. Its subunits comigrate with goat brain α andβ tubulin subunits and is composed of 4 major α andβ tubulins each as analyzed by isoelectric focusing and two dimensional gel electrophoresis. Peptide mapping showed it to be very similar to goat brain tubulin. Polymerization of catfish brain tubulin occurs optimally between 18–37°C and the critical protein concentrations of assembly at 18°C and 37°C are the same, as opposed to mammalian brain tubulins.     

S-100 protein subunits in bovine oviduct epithelium:In situ distribution and changes during primary cell culture

Summary Cultures of bovine oviduct epithelial cells are widely used in co-culture systems to improve the results ofin vitro fertilization. The aim of the present study was to evaluate the suitability of S-100 protein as a differentiation marker for bovine oviduct epithelial cellsin vitro. The distribution of S-100α and S-100β was examined immunohistochemically in bovine oviduct epitheliumin situ and in primary cell cultures derived from it. Three segments of the Fallopian tube (isthmus, ampulla and fimbriae) were compared and analysed during different stages of the oestrus cycle (luteal phase and follicular phase). Ciliated and non-ciliated cells of the epithelium reacted with anti-S-100α, S-100a(αβ) and S-100β antibodies, except for isthmic non-ciliated cells, which did not bind anti-S-100β or anti-S-100a(αβ). In addition, basal cells never showed immunoreactivity for S-100. In confluent monolayers of cultured oviduct epithelial cells, disappearance of reactivity for S-100 paralleled morphological signs of dedifferentiation (loss of cilia, cytoplasmic vacuolization). Free-floating oviduct epithelial cells, in contrast, retained morphological differentiation and still expressed S-100 antigen even after seven daysin vitro. The immunohistochemical findings were confirmed by polyacrylamide gel electrophoresis and Western blotting. The results indicate that the presence of S-100 is closely connected to morphological differentiation and to the specific functional condition of bovine oviduct epithelial cells.     

Mapping and nucleotide sequence of a newHLA class II light chain gene,DQB3

A genomic clone specifying a new HLA class II antigen β chain,DQB3, was isolated from a human genomic phage library using aDQB1 cDNA probe under low stringency conditions. Southern hybridization and nucleotide sequence analyses identified the β2 domain exon (exon 3) with several deleterious mutations and the CP-TM-CY exon [connecting peptide, transmembrane, and cytoplasmic regions, (exon 4)], but the first, second, and fifth exons encoding the 5′ UT-leader, the β1 domain, and the 3′ UT domain of normal β chains, respectively, were entirely missing. The nucleotide sequences of these two exons were distinct from those of other class II β chain genes, but slightly more related to theDQB1 andDQB2 genes than to other class II genes. TheDQB3 sequence mapped betweenDQA2 andDQB1, 15 kb upstream fromDQA2, by analysis of overlapping cosmid clones. This mapping was supported by the fact thatTaq I,Msp I, andBam HIDQB3 polymorphisms were perfectly correlated with theDQA2 polymorphism and not with any polymorphisms in theDR orDQ subregion, suggesting the presence of a hot spot for recombination betweenDQB3 andDQB1. The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession number M26577.     

Interaction of the monoaminergic and opioidergic brain systems in the course of nociceptive and antinociceptive reactions in cats

Nociceptive responses were evoked in cats by electrical transcutaneous stimulation of the forepaw or electrical stimulation of respective brain structures; these responses could be modulated (intensified or suppressed) by combined electrical stimulation of different brain structures or by neurochemical influences upon these structures. Intensification of nociceptive responses was observed after stimulation of the noradrenergic orP-ergic systems localized in the ventral zone of the central gray (vl SGC) and the structures monosynaptically connected with the latter: the posterior and lateral hypothalamic nuclei (Hp andHl) and preoptic region (RPO). Similar effects were induced by suppression of the serotoninergic system concentrated within the dorsolateral central gray (dl SGC), dorsal raphe nucleus (Rd), and closely related structures: the ventromedial, dorsomedial, and paraventricular hypothalamic nuclei (Hvm, Hdm, andHpv), septum (Sep), basolateral amygdalar nucleus (Am bl), fields 3–4 of the hippocampus (CA3–4), and cingular cortex (GC). Suppression of the serotoninergic system resulted in a decrease in the levels of functioning of the met-enkephalin- and β-endorphinergic systems and facilitation of theP-ergic system. Moderation of nociceptive responses, i.e., an analgesic effect, was observed after either stimulation of the serotonin-, met-enkephalin-, and β-endorphinergic systems localized in thedl SGC, Rd, Hvm, Hdm, Sep, Am bl, CA3–4, andGC, or suppression of the noradrenergic system. The latter influence resulted in inhibition of theP-ergic system and a rise in the functional activity of the met-enkephalin- and β-endorphinergic systems. The composition of two antagonistic brain systems, nociceptive and antinociceptive, is considered. The antinociceptive system includes serotonin-, met-enkephalin-, and β-endorphinergic elements. Leu-enkephalin is a nonspecific activator of the met-enkephalin-, β-endorphin-, andP-ergic systems. The nociceptive system consists of thevl SGC, Hp, Hl, andRPO, while the antinociceptive system includes thedl SGC, Rd, Hvm, Hdm, Hpv, Sep, Am dl, CA3–4, andGC.     

Preliminary study of taxonomy ofAzotobacter andAzomonas by using rocket line immunoelectrophoresis

Rocket line immunoelectrophoresis was used to study the taxonomy ofAzotobacter andAzomonas assessed by reaction with antiserum to the AVO₂ strain ofAzotobacter. Forty-five cultures, comprising seventeen species in five genera, showed that antigen “β”, like high-titer somatic agglutination, was restricted to all 11 strains ofAzotobacter vinelandii and to one strain which has been namedAzotobacter macrocytogenes (10EM). A thermoresistant antigen (“γ”) was found to be shared by all strains and species ofAzotobacter andAzomonas investigated.     

Cytochrome P4501A induced differentially in endothelial cells cultured from different organs of Anguilia rostrata

Summary Endothelial cells are a structural barrier and an active regulator of many bodily processes. Cytochrome P4501A (CYPIA) activity is induced in the endothelium of teleosts and mammals exposed to lipophilic xenobiotics, such as polycyclic aromatic hydrocarbons, and can have significant consequences for endothelial functions. We exposed cultures of characterized endothelial cells from the heart, kidney, and rete mirabile of the eel, Anguilla rostrata, to aryl hydrocarbon receptor (AhR) agonists. In heart endothelial cells, the maximum response (based on O-deethylation of 7-ethoxyresorufin to resorufin [EROD] activity) to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), 113 pmol/mg/min, was at 1 nM TCDD and the peak response to β-napthoflavone (βNF), 135 pmol/mg/min, was at 3 μM βNF. The maximum response to TCDD in the kidney endothelial cells is 12 pmol/mg/min at 0.3 nM TCDD. The rete mirabile capillary endothelial cells responded minimally or not at all to exposure to TCDD and βNF. Both the heart and kidney endothelial cells (but not the rete mirabile capillary cells) have a low level of EROD activity (12.7 and 5.2 pmol/mg/min, respectively) in untreated or dimethylsulfoxide-treated cells. The robust response of the heart endothelial cells to induction and the lack of response in the rete mirabile capillary endothelial cells indicate that these cells are a good resource to use to investigate the physiological consequences of AhR agonist exposure and CYP1A induction in different areas of the vasculature.     

Natural killer (NK) activity of cultured S100β-positive T-leukemia cells

In order to clarify the function of human S100β- positive T-cells, S100β-positive T-leukemia cells (S100β TLC) were examined in vitro. S100β TLC were obtained from the peripheral blood of a patient with S100β-positive T-cell leukemia and enriched by an E-rosetting method. Two dimensional flow cytometric analysis indicated that the vast majority of the E-positive fraction were S100β TLC expressing CD3 and CD8 antigens. Although S100β TLC expressed CD3 antigen, they were negative for the α/β and γ/δ T-cell antigen receptor (TCR) defined by monoclonal antibodies (mabs) WT-31 and δ TCS-1, respectively. It was speculated that S100β TLC initially expressed α/β TCR but lost it during malignant transformation. When S100β TLC were cultured for 24 h, they acquired cytotoxic activity towards various NK-sensitive cell lines including K-562, Molt-3 and CEM-CCLF, but did not exhibit lysing activity towards NK-resistant cell lines including Raji, Daudi and MT-1. Despite the NK-activity of cultured S100β TLC, they lacked the morphological features of large granular lymphocytes (LGL). S100β TLC did not exhibit lymphokine-activated killer (LAK) activity. When S100β TLC were cocultivated with NK-sensitive cells or NK-resistant cells, they selectively bound to NK-sensitive cells, indicating that they lysed target cells by cell-to-cell contact. The finding that S100 β TLC lacked TCR molecules and their NK activity was not inhibited by mabs reactive with the CD3-TCR complex indicated that the CD3-TCR complex was not involved in their target recognition. These findings suggest that S100 β-positive T-cells are functionally similar to NK cells. We discuss the roles of S100 β-positive T-cells in the human immune system.     

The helper T-cell repertoire of mice expressing class II major histocompatibility complex β chains in the absence of α chains

 Mutant mice generated by disrupting the H2-Aa ^b major histocompatibility complex (Mhc) gene are demonstrated here to express Aβ^b chains in the absence of α chains. These mice possess a CD4⁺ helper T cell (Th) repertoire that uses predominantly the Vβ7 T-cell antigen receptor (Tcr) segment for recognition of any protein antigen presented by the α-free Aβ molecule. As an alloantigen, the Aα-free Aβ molecule is recognized very poorly by T cells from a series of class II disparate mouse strains, indicating that it is grossly different from normal α/β heterodimers. Indeed, molecular modeling suggests a β/β homodimer arrangement with an altered geometry of the Tcr contact area. Interestingly, the mutant mice exhibit normal alloreactivity, without a restricted Vβ usage, toward a series of foreign α/β class II heterodimers, although their T cells developed in the absence of such heterodimers. Thus, the complementarity of Tcr to normal α/β heterodimers, and thereby also alloreactivity, appears to be an ontogeny independent (i. e., germline-encoded) feature. Received: 30 September 1996 / Revised: 18 October 1996     

Polymorphism of T-cell receptor genes among laboratory and wild mice: Diverse origins of laboratory mice

Southern blots of genomic DNA from 23 strains of laboratory mice and 19 individual wild mice were examined for restriction fragment length polymorphisms in their loci encoding the T-cell receptors (Tcr): the constant regions of the α, β, and γ chains (C _α,C _β, andC _γ) and a variable region family of the β chain (V _β8). Only a few polymorphisms were observed for each locus in the laboratory mice after using three restriction enzymes,Bam HI,Eco RI, andHind III. All the laboratory mice examined fall into one of two types for theC _α,C _β andV _β8 loci and one of three types for theC _γ. These types are found in some of the wild mice studied, indicating that they were already present in the founder mice of laboratory mouse strains. In contrast, theTcr genes are highly polymorphic among wild mice. Analysis of the polymorphisms in these loci suggests that laboratory mice have inherited their genes not only fromMus musculus domesticus, but also from other subspecies, and much more than previously believed from Asian subspecies.     

Culture and characterization of bovine mesenteric lymphatic endothelium

Summary Lymphatic endothelial cells isolated from bovine mesenteric lymphatic vessels were cultured and characterized. Lymphatic endothelial cells grew as a monolayer displaying an elongated morphology in preconfluent primary cultures. When confluent, the cells exhibited a polygonal morphology to form a “cobblestone” pattern previously described for cultured vascular endothelium. All culture lymphatic endothelial cells expressed Factor VIII-related antigen and boundUlex europaeus I lectin. Ultrastructurally, cultured lymphatic endothelium was characterized by the presence of Weibel-Palade bodies as well as the usual cytoplasmic organelles.     

Guggulsterone induced changes in the levels of biogenic monoamines and dopamineβ-hydroxylase activity of rat tissues

The effect of lipid lowering agent, guggulsterone, the purified fraction of guggulipid, on biogenic monoamine levels and dopamineβ-hydroxylase activity of rat brain and heart has been studied. Administration of guggulsterone caused inhibition of brain dopamineβ-hydroxylase activity with marked stimulation in heart bothin vivo andin vitro. The levels of catecholamines were similarily affected. The contents of serotonin and histamine were found to be enhanced in brain and decreased in heart. Alterations in biogenic amines and dopamineβ-hydroxylase activity may be one of the possible mechanisms for the antilipaemic effect of the compound C.D.R.I. communication No. 3683. Part of this work presented at 52nd Annual Meeting of the Society of Biological Chemists (India), held at National Chemical Laboratory, Pune, November 1983.     

Biosynthesis of medium-chain-length poly(hydroxyalkanoates) with altered composition by mutant hybrid PHA synthases

Pseudomonas resinovorans harbors two isogenic poly(hydroxyalkanoates) (PHAs) synthase genes (phaC1 _Pre , phaC2 _Pre ) responsible for the production of intracellular medium-chain-length (mcl-)PHAs. Sequence analysis showed that the putative gene-products of these genes contain a conserved α/β-hydrolase fold in the carboxy-terminal half of the proteins. Hybrid genes pha7 and pha8 were constructed by exchanging the α/β-hydrolase-fold coding portions of phaC1 _Pre and phaC2 _Pre at the 3′ terminal. When grown with decanoate as carbon source, the pha7- or pha8-transformed Escherichia coli LS1298 produced PHAs containing 73–75% β-hydroxydecanoate (β-HD) and 25–27% β-hydroxyoctanoate (β-HO). Deletion mutants, Δpha7 and Δpha8, were isolated during the PCR-based construction of pha7 and pha8, respectively. Cells harboring these mutants produced PHAs containing 55–60 mol% β-HD and 40–45 mol% β-HO. These results demonstrate the feasibility of generating active hybrid mcl-PHA synthase genes and their mutants with the potential of producing polymers having a varied repeat-unit composition.     

Origin and evolution of the sulawesi macaques 1. Electrophoretic analysis of hemoglobins

The monkeys on the island of Sulawesi (Celebes), Indonesia, comprise seven species ofMacaca, that isM. maura, M. tonkeana, M. hecki, M. nigrescens, M. nigra, M. ochreata, andM. brunnescens. Hemoglobins from 248 individuals of these seven species were analyzed by isoelectric focusing electrophoresis (IEFE) and by starch gel electrophoresis in the presence of urea (USGE). Eighteen phenotypes consisting of eight molecular types were identified by IEFE analysis. The speciestonkeana inhabiting the central part of the island revealed 11 phenotypes, while peripheral species such asnigrescens andbrunnescens carried only 3 and 2 phenotypes, respectively. On USGE, three α chains and three β chains were identified and named α1, α2, and α6, and β1, β3, and β5, respectively. The α1 chain has the same mobility as the α chains of other macaques, while the α2 chain is less positively charged than α1, and α6 is the least positive among these α chains. The α2 chain is widely distributed in the Sulawesi macaques as the major component. Four species,ochreata, tonkeana, maura, andnigrescens, carried the α1 and α6 chains as minor components. The electrophoretic mobility of β1 was the same as that of other macaques, while β3 and β5 were more positively charged and less positively charged than β1, respectively. All of the Sulawesi species had β3 in high or low gene frequencies and inmaura, tonkeana, andbrunnescens, this type was most abundant. β5 chain existed in the species of the northern peninsula, as the major type. The subordinate type was β3 innigra andnigrescens and β1 inhecki. On the other hand, β1 was most frequently observed inochreata.     

Reversible change in the fibroblast lysosomal enzyme dipeptidyl aminopeptidase-1 (cathepsin c) related to the commercial source of fetal bovine serum in the culture medium

Summary The commercial source of fetal bovine serum used to supplement the growth medium of human skin fibroblasts alters the activity of the lysosomal enzyme dipeptidyl aminopeptidase-1 (DAP-1). Cells grown with one serum were found to have a threefold higher level of DAP-1 than those grown with serum from another source (P<0.001). The effect on DAP-1 activity was specific inasmuch as no differences were found in the activities of a variety of other lysosomal and nonlysosomal hydrolases: DAP-II, DAP-III, DAP-IV, β-glucosidase, β-glucuronidase, andN-acetyl-β-galactosaminidase. The effect is reversible and is observed over a wide range of cell population doublings. Cell growth kinetics were not significantly different with the different sera. This work was supported in part by grants from the National Institutes of Health, Bethesda, MD (NS 16287).     

Overexpression of proline oxidase induces proline-dependent and mitochondria-mediated apoptosis

The neuropathogenesis of influenza-associated encephalopathy in children and Reye's syndrome remains unclear. A surveillance effort conducted during 2000-2003 in South-West Japan reveals that almost all fatal and handicapped influenza-associated encephalopathy patients exhibit a disorder of mitochondrial β-oxidation with elevated serum acylcarnitine ratios (C_16:0+C_18:1)/C₂. Here we show invasion by a non-neurotropic epidemic influenza A H3N2 virus in cerebral capillaries with progressive brain edema after intranasal infection of mice having impaired mitochondrial β-oxidation congenitally or posteriorly in the newborn/ suckling periods. Mice genetically lacking of carnitine transporter OCTN2, resulting in carnitine deficiency and impaired β-oxidation, exhibited significant higher virus-genome numbers in the brain, accumulation of virus antigen exclusively in the cerebral capillaries and increased brain vascular permeability compared to in wild type mice. Mini-plasmin, which proteolytically potentiates influenza virus multiplication in vivo and destroys the blood-brain barrier, accumulated with virus antigen in the brain capillaries of OCTN2-deficient mice but only a little in wild-type mice. These results suggest that the impaired mitochondrial β-oxidation changes the susceptibility to a non-neurotropic influenza A virus as to multiplication in the brain capillaries and to cause brain edema. These pathological findings in the brain of mice having impaired mitochondrial β-oxidation after influenza virus infection may have implications for human influenza-associated encephalopathy.     

Biological characteristics of the peptides α and β isolated from bovine seminal plasma

The bull seminal plasma peptides α andβ have been examined for their biological properties. While both the peptides were able to inhibit the human chorionic gonadotropin-dependent uterine response in the mouse, α alone exhibits the property of suppressing post-castrational rise in gonadotropin in appropriate animal models. This suggests that the peptideβ must be acting directly on the ovary to suppress estrogen production and, consequently, the uterine weight increase. Such a possibility was confirmed when α andβ were examined by the coupled bioassay which is capable of discriminating between pituitary feedback factors and those acting directly on the gonad. In a test system designed to examine chronic effects, both α andβ showed evidence of acting directly on the ovary to inhibit human menopausal gonadotropin-induced estrogen production. Such a direct action could not be correlated with the relative potencies of these peptides when examined for their follicle stimulating hormone-receptor binding inhibitor and lutinizing hormone-receptor binding inhibitor activities.     

Ontogenetic correlations between noradrenaline level and the density of adrenergic receptors in the rat brain

We studied the level of noradrenaline and the density of α2-and β-adrenoreceptors in the brain stem and cerebral cortex of 12-day- and 21-day-old rat fetuses, as well as of rats at the ages of 1, 2, 5, 7, 9, 16, 21, 35, and 70 days. We found a positive correlation between the level of noradrenaline in the brain stem and the density of β-receptors in the cerebral cortex, and between the amount of α2- and β-receptors in the cerebral cortex, as well as between the values of each of these indices of the neurochemical system and body weight. Significant negative correlations (r=−0.72 andr=−0.88, respectively) were found between the amount of α2-adrenoreceptors in the brain stem and the content of noradrenaline in this brain region, as well as in the cerebral cortex. Explanations of these positive and negative correlations between the level of noradrenaline and the amount of adrenergic receptors in the rat brain during ontogenesis are discussed.     

Cloning,production and secretion of β-galactosidase inAcetobacter pasteurianus

The gene coding for β-galactosidase fromEscherichia coli was cloned into plasmid pACT71 containing the replicon from plasmid pAC1 fromAcetobacter pasteurianus. E. coli MC4100,E. coli JM105,E. coli LE392.23 andA. pasteurianus 3614 were transformed with the recombinant plasmid pACB815. Cells were cultivated in LB, YPG and M media supplemented with glucose, glycerol, lactose or ethanol and β-galactosidase activity was detected in the cells and in the cultivation medium. The best substrate for production of β-galactosidase was lactose. To release β-galactosidase fromA. pasteurianus cells amino acids were added to the cultivation medium. The highest secretory activity was achieved using 1.5% glycine after 36 h of cultivation in the M medium.