Glucosephosphate isomerase polymorphism in sheep

Two types of glucosephosphate isomerase (GPI) are described for the first time in sheep. Type T produced a 3-band pattern on polyacrylamide gel electrophoresis of serum samples, whereas type M produced a pattern of 5-7 bands. When 23 Scottish Halfbred ewes showing type T were mated to 3 Suffolk rams showing type M, their 32 offspring had frequencies of 0.72 and 0.28 for T and M respectively. There was no conclusive evidence that the types were controlled by codominant alleles or by simple Mendelian inheritance and neither was sex-linked. The frequency of the rarer type (M) was sufficiently high to provide conclusive evidence of true enzyme polymorphism.     

Two types of murine helper T cell clone. I. Definition according to profiles of lymphokine activities and secreted proteins

A panel of antigen-specific mouse helper T cell clones was characterized according to patterns of lymphokine activity production, and two types of T cell were distinguished. Type 1 T helper cells (TH1) produced IL 2, interferon-gamma, GM-CSF, and IL 3 in response to antigen + presenting cells or to Con A, whereas type 2 helper T cells (TH2) produced IL 3, BSF1, and two other activities unique to the TH2 subset, a mast cell growth factor distinct from IL 3 and a T cell growth factor distinct from IL 2. Clones representing each type of T cell were characterized, and the pattern of lymphokine activities was consistent within each set. The secreted proteins induced by Con A were analyzed by biosynthetic labeling and SDS gel electrophoresis, and significant differences were seen between the two groups of T cell line. Both types of T cell grew in response to alternating cycles of antigen stimulation, followed by growth in IL 2-containing medium. Examples of both types of T cell were also specific for or restricted by the I region of the MHC, and the surface marker phenotype of the majority of both types was Ly-1+, Lyt-2-, L3T4+, Both types of helper T cell could provide help for B cells, but the nature of the help differed. TH1 cells were found among examples of T cell clones specific for chicken RBC and mouse alloantigens. TH2 cells were found among clones specific for mouse alloantigens, fowl gamma-globulin, and KLH. The relationship between these two types of T cells and previously described subsets of T helper cells is discussed.     

Mimicry in recognition of cardiac myosin peptides by heart-intralesional T cell clones from rheumatic heart disease

Molecular mimicry between Streptococcus pyogenes Ags and human proteins has been considered as a mechanism leading to autoimmune reactions in rheumatic fever and rheumatic heart disease (RHD). Cardiac myosin has been shown as a putative autoantigen recognized by autoantibodies of rheumatic fever patients. We assessed the human heart-intralesional T cell response against human light meromyosin (LMM) and streptococcal M5 peptides and mitral-valve-derived proteins by proliferation assay. Cytokines induced by LMM peptides were also evaluated. The frequency of intralesional T cell clones that recognized LMM peptides was 63.2%. Thirty-four percent of T cell clones presented cross-reactivity with different patterns: 1) myosin and valve-derived proteins; 2) myosin and streptococcal M5 peptides; and 3) myosin, valve-derived proteins and M5 peptides. In addition, several LMM peptides were recognized simultaneously showing a multiple reactivity pattern of heart-infiltrating T cells. Inflammatory cytokines (IFN-gamma and TNF-alpha) were predominantly produced by heart-infiltrating T cells upon stimulation with LMM peptides. The alignment of LMM and streptococcal M5 peptides showed frequent homology among conserved amino acid substitutions. This is the first study showing the cellular response by human heart-infiltrating T cells against cardiac myosin epitopes in RHD patients. The high percentage of reactivity against cardiac myosin strengthens its role as one of the major autoantigens involved in rheumatic heart lesions. T cell reactivity toward myosin epitopes in RHD patients may also trigger the broad recognition of valvular proteins with structural or functional similarities.     

Identification of a botulinum C3-like enzyme in bovine brain that catalyzes ADP-ribosylation of GTP-binding proteins.

A novel enzyme activity was found in bovine brain cytosol that transfers the ADP-ribosyl moiety of NAD to proteins with Mr values of 22,000 and 25,000. The substrates were the same GTP-binding proteins serving as the substrate of an ADP-ribosyltransferase C3 which was produced by a type C strain of Clostridium botulinum. The brain enzyme was partially purified from the cytosol and had a molecular mass of approximately 20,000 on a gel filtration column. The brain endogenous enzyme displayed unique properties similar to those observed with botulinum C3 enzyme. The enzyme activity was markedly stimulated by a protein factor that had been initially found in the cytosol as an activator for botulinum C3-catalyzed ADP-ribosylation (Ohtsuka, T., Nagata, K., Iiri, T., Nozawa, Y., Ueno, K., Ui, M., and Katada, T. (1989) J. Biol. Chem. 264, 15000-15005). The activity of the brain enzyme was also affected by certain types of detergents or phospholipids. The substrate of the brain enzyme was specific for GTP-binding proteins serving as the substrate of botulinum C3 enzyme; the alpha-subunits of trimeric GTP-binding proteins which served as the substrate of cholera or pertussis toxin were not ADP-ribosylated by the endogenous enzyme. Thus, this is the first report showing an endogenous enzyme in mammalian cells that catalyzes ADP-ribosylation of small molecular weight GTP-binding proteins.     

Application of Enzyme Production Properties in Subtyping of Group A Streptococci According to T Type

The production of extracellular nicotinamide adenine dinucleotide glycohydrolase (NADG) and the cell-bound lipoproteinase (serum opacity reaction, SOR) by strains of different serological types of group A streptococci, in relation to the T typing, was studied. The production of both NADG and SOR, or only one of them, was found to be characteristic of serotypes, as determined by M and T antigen. No difference in the production of these enzymes was found in relation to M-positive and M-negative variants. Investigation into NADG and SOR production as related to the T type enabled the division of a single agglutination pattern into four main groups, each of which corresponds to one specific M type or more. Of the 370 strains belonging to 12 different T-agglutination patterns, 21% produced both enzymes and 42.5% failed to produce any of them, whereas the remaining 36.5% produced only one out of the two enzymes. Five streptococcal types which did not produce NADG and SOR also failed to synthesize streptolysin S at the early logarithmic phase of growth, indicating that streptolysin S production by young cultures may be also related to serotype. No correlation was found between the production of NADG-SOR as related to serotype and the production of streptolysin O, acid phosphotase, esterase, N-acetylglucosaminidase, hyaluronidase, streptokinase, and the cell-sensitizing factor. The practical and potential usefulness of NADG and SOR production in epidemiological studies is discussed.     

Mycobacterium leprae-specific T cells from a tuberculoid leprosy patient suppress HLA-DR3-restricted T cell responses to an immunodominant epitope on 65-kDa hsp of mycobacteria.

The polar tuberculoid type (TT) of leprosy, characterized by high T cell reactivity to Mycobacterium leprae, is associated with HLA-DR3. Surprisingly, DR3-restricted low T cell responsiveness to M. leprae was found in HLA-DR3-positive TT leprosy patients. This low responsiveness was specifically induced by M. leprae but not by M. tuberculosis and was seen only in patients and not in healthy controls. We studied this patient-specific, M. leprae-induced, DR3-restricted low T cell responsiveness in depth in one representative HLA-DR3-positive TT leprosy patient by using T cell clones. From this patient two types of T cell clones were obtained: one type was cross-reactive with M. tuberculosis and recognized an immunodominant epitope (amino acids 3 to 13) on the 65-kDa heat shock protein (hsp) the other type was M. leprae specific and reacted to a protein other than the 65-kDa one. To examine whether these M. leprae-specific T cell clones were responsible for the DR3-restricted low responsiveness to M. leprae, we tested them for the ability to suppress the proliferation of the DR3-restricted, 65-kDa, hsp-reactive clones. The DR3-restricted, M. leprae-specific T cells completely suppressed the proliferative responses of DR3-restricted, cross-reactive T cell clones to the 65-kDa hsp from the same patient as well as from other individuals. Also, DR3-restricted responses to an irrelevant Ag were suppressed by the M. leprae-specific T cell clones. However, no suppression of non-DR3-restricted T cell responses was seen. Although the mechanism must still be elucidated, this M. leprae-induced, DR3-restricted immunosuppression may at least partly explain the observed DR3-associated low T cell responsiveness in TT leprosy patients.     

A nonsense mutation in the optic atrophy 3 gene (OPA3) causes dilated cardiomyopathy in Red Holstein cattle

Cardiomyopathies are severe degenerative disorders of the myocardium that lead to heart failure. During the last three decades bovine dilated cardiomyopathy (BDCMP) was observed worldwide in cattle of Holstein-Friesian origin. In the Swiss cattle population BDCMP affects Fleckvieh and Red Holstein breeds. The heart of affected animals is enlarged due to dilation of both ventricles. Clinical signs are caused by systolic dysfunction and affected individuals die as a result of severe heart insufficiency. BDCMP follows an autosomal recessive pattern of inheritance and the disease-causing locus was mapped to bovine chromosome 18 (BTA18). In the present study we describe the successful identification of the causative mutation in the OPA3 gene located on BTA18 that was previously reported to cause 3-methylglutaconic aciduria type III in Iraqi-Jewish patients. We demonstrated conclusive genetic and functional evidence that the nonsense mutation c.343C>T in the bovine OPA3 gene causes the late-onset dilated cardiomyopathy in Red Holstein cattle.     

Cytogenetics and cytochemistry of leukemia patients in 2 neotropical hospitals

A cytogenetic and/or cytochemical study was performed in 166 individuals with leukemia or related disorders, in two major Costa Rican hospitals. In those patients treated at an adult's hospital (14 years old and over), acute leukemias represented 66% of all cases. In that hospital the most frequent types of disorders were, in decreasing order: ANLL (> M1), ALL, CML (all of them showed the Ph chromosome) and MDS. In the cases from a childrens' hospital (< 14 years old) acute leukemias were 98%. Among them the order of frequency was: ALL (70%): ALL-1 (84%), ALL-2 (16%) and ANLL (27%): M5a > M3 > M4 > M5b. In ALL 85% were type B and occurred mostly in women while 15% of them were type T and more frequent in males. There was 5.6% infant leukemia, which presented a similar number of acute lymphoids and myeloids. The cytogenetic pattern was similar among Costa Rica and other tropical and temperate countries.     

Distinctive features in the production of IL-6 by human T cells

The production of IL-6 was studied in purified populations of human peripheral blood T cells and monocytes. IL-6 could be elicited from both T cells and thymocytes by a variety of polyclonal stimuli. The expression and production of IL-6 in T cells varied in several ways from that of monocytes: (1) under maximal stimulation, T cells secreted 10-fold less IL-6 than did monocytes; (2) by Western blot, the molecular isoforms produced by T cells were distinct from those produced by monocytes, with at least some isoforms being unique to each cell type; (3) the kinetics of IL-6 expression and production was much slower in T cells than in monocytes; (4) the frequency of labeled cells detected by in situ hybridization was 4% for T cells and 34% for monocytes. Our data indicate that there is considerable diversification among cell types in terms of regulation of IL-6 production and possibly in post-translational modifications. This raises the possibility that the IL-6 produced by different cell types may have varying effects depending on the form produced and the timing of synthesis.     

Immunological inter-strain crossreactivity correlated to 18S rDNA sequence types in Acanthamoeba spp

Various species of the genus Acanthamoeba have been described as potential pathogens; however, differentiation of acanthamoebae remains problematic. The genus has been divided into 12 18S rDNA sequence types, most keratitis causing strains exhibiting sequence type T4. We recently isolated a keratitis causing Acanthamoeba strain showing sequence type T6, but being morphologically identical to a T4 strain. The aim of our study was to find out, whether the 18S rDNA sequence based identification correlates to immunological differentiation. The protein and antigen profiles of the T6 isolate and three reference Acanthamoeba strains were investigated using two sera from Acanthamoeba keratitis patients and one serum from an asymptomatic individual. It was shown, that the T6 strain produces a distinctly different immunological pattern, while patterns within T4 were identical. Affinity purified antibodies were used to further explore immunological cross-reactivity between sequence types. Altogether, the results of our study support the Acanthamoeba 18S rDNA sequence type classification in the investigated strains.     

Collagen synthesis as a marker for cell type in mouse 3T3 lines

Collagen synthesis was applied as a test of the fibroblastic or endothelial origin of the 3T3 mouse lines. Swiss/3T3 lines and Balb/3T3 (clone A31) were compared with respect to the amounts and types of collagen synthesized. All lines symthesized collagen at relatively high and similar rates. For all lines, 75-90% of the collagen synthesized was type I, and the remainder was type III. There was no evidence for synthesis of type IV collagen. By all these parameters, Swiss and Balb/3T3 lines resemble cultured fibroblasts rather than cultured vascular endothelia.     

Properties of Venezuelan Equine Encephalomyelitis Virus Accompanying Attenuation In Vitro

Virus obtained during serial plaque passage of the virulent parent egg seed (PES) of the Trinidad strain of Venezuelan equine encephalomyelitis (VEE) virus produced only large plaques during either 3 serial plaque passages in chick fibroblasts or 10 plaque passages in L cells, and was lethal for mice by the intraperitoneal route. Virus showing these characteristics was designated the stable large-plaque (Ls) type. In contrast, virus obtained during serial plaque passage of the attenuated 9t strain in chick fibroblasts formed only very small plaques and was not lethal for mice by the intraperitoneal route. Virus showing these properties was designated the stable small-plaque (Ss) type. Under other passage conditions, however, large-plaque virus that yielded about 90% large and 10% small plaques was obtained; this virus was designated the unstable large or Lu type because it differed from the Ls type, which yielded only large plaques. The Lu type continued to yield the same ratio of large to small plaques for several plaque-to-plaque passages. In addition, small-plaque virus that yielded both large and small plaques and that showed a reduced capability to infect mice was also recovered. This virus was designated the unstable small or Su type because it differed from the Ss type in its higher level of virulence and in its plaque-forming properties. Thus, based upon the properties of virulence for mice and plaque size, four viral types could be discerned. The evidence suggests that serial passage in cell culture imposed environmental pressures that sequentially selected the following viral types: Ls, Lu, Su, and Ss.     

Phenotypic switching and beta-N-acetylhexosaminidase activity of the pathogenic yeast Trichosporon asahii

The pathogenic yeast Trichosporon asahii is the major causative agent of deep-seated trichosporonosis in immunocompromised patients. Although infection by this microorganism is becoming increasingly frequent, information related to its pathogenicity and virulence factors is still limited. Therefore, we investigated phenotypic switching in colony morphology, and the production of extracellular enzymes as a virulence factor. Sixty-one clinical isolates of T. asahii produced four different morphological types on Sabouraud dextrose agar (SDA): 69% WF (white farinose), 18% WP (white pustular), 10% Y (yellowish white), and 3% WC (white cerebriform). Strains of the three major types (WF, WP, and Y) produced two to five colony types when cultured on SDA at 37 C. The frequency of switching between colony types was 10(-2) to 10(-4), as in Candida albicans and Cryptococcus neoformans. Notably, most of the colonies switched to the smooth (S) type irreversibly, at frequencies of 10(-2) to 10(-3). No secreted aspartic proteinase or phospholipase activity was detected in T. asahii, while beta-N-acetylhexosaminidase activity, which catalyzes the hydrolysis of terminal nonreducing N-acetyl-D-hexosamine residues in N-acetyl-beta-D-hexosaminides, was found. Furthermore, enzymatic activity of the S type was significantly greater than that of the parent type in all strains. No other clinically relevant Trichosporon species (T. mucoides, T. inkin, and T. ovoides ) produced this enzyme. These results provide basal information for understanding the pathogenic potential of T. asahii.     

Evidence that tumor necrosis factor has an important role in antibacterial resistance

TNF is produced in the spleens of Listeria-infected mice during the first 3 days of a sublethal immunizing infection. The production of Listeria-induced TNF coincides with the time when peak numbers of bacteria are present in the liver and spleen. Evidence suggesting that TNF produced in Listeria-infected organs functions in a T cell-independent resistance mechanism comes from results showing that listeriosis is exacerbated in both T cell-intact mice and T cell-deficient (athymic nude) mice treated with a monospecific anti-murine TNF IgG. Listeriosis is exacerbated, however, only if the anti-TNF IgG is given during the first 3 days of infection, i.e., at the time when TNF is being produced in the spleen. Anti-TNF IgG administered on the first day of infection neutralized all the cytotoxic activity of endogenously produced TNF in the spleen. Additional evidence that TNF plays an important role in antibacterial defenses was obtained by showing that administration of pure murine rTNF protects mice against a normally lethal Listeria challenge given 1 to 24 h later.     

Areas of asbestoid (amianthoid) fibers in human thyroid cartilage characterized by immunolocalization of collagen types I,II, IX,XI and X

The distribution of type I, II, IX, XI and X collagens in and close to areas of asbestoid (amianthoid) fibers in thyroid cartilages of various ages was investigated in this study. Asbestoid fibers were first detected in thyroid cartilage from a 3-year-old male child. Areas of asbestoid fibers functionally appear to serve as guide rails for vascularization of thyroid cartilage. Alcian blue staining in the presence of 0.3 M MgCl₂ revealed a loss of glycosaminoglycans in areas of asbestoid fibers. In addition, the fibers reacted positively with antibodies against collagen types II, IX and XI, but showed no staining with antibodies to collagen types I and X. Territorial matrix of adjacent chondrocytes showed the same staining pattern. In addition to staining for type II, IX and XI collagens, asbestoid fibers showed strong immunostaining for type I collagen after puberty but not for type X collagen. However, groups of chondrocytes within areas of asbestoid fibers reacted strongly with antibodies to type X collagen, suggesting that this collagen plays an important role in matrix of highly differentiated chondrocytes. The finding that these type X collagen-positive chondrocytes also revealed immunostaining for type I collagen confirms previous studies showing that hypertrophic chondrocytes can further differentiate into cells that are characterized by the synthesis of type X and I collagens.     

Enzymes involved in the formation of 3 beta, 7 beta-dihydroxy-12-oxo-5 beta-cholanic acid from dehydrocholic acid by Ruminococcus sp. obtained from human intestine

Ruminococcus sp. PO1-3 from human intestinal flora reduced dehydrocholic acid to 3 beta-hydroxy-7,12-dioxo-5 beta-cholanic acid by means of the enzyme 3 beta-hydroxysteroid dehydrogenase (Akao, T., Akao, T., Hattori, M., Namba, T. and Kobashi, K. (1986) J. Biochem. (Tokyo) 99, 1425-1431). This bacterium and its crude extract gave rise to another product, showing a lower RF value on TLC, from dehydrocholic acid. The product was identified as 3 beta, 7 beta-dihydroxy-12-oxo-5 beta-cholanic acid. The crude extract reduced 7-ketolithocholic acid and its methyl ester, but not 6-ketolithocholic acid and 12-ketochenodeoxycholic acid, in the presence of NADPH, and oxidized ursodeoxycholic acid and beta-muricholic acid, but not cholic acid, chenodeoxycholic acid, deoxycholic acid and hydrocholic acid, in the presence of NADP+. Therefore, besides 3 beta-hydroxysteroid dehydrogenase, 7 beta-hydroxysteroid dehydrogenase was shown to be present in this bacterium. The two dehydrogenases were clearly separated from each other by butyl-Toyopearl 650 M column chromatography. From dehydrocholic acid, 7 beta-hydroxy-3,12-dioxo-5 beta-cholanic acid was produced by 7 beta-hydroxysteroid dehydrogenase and 3 beta, 7 beta-dihydroxy-12-oxo-5 beta-cholanic acid was produced by combination of two enzymes, 7 beta- and 3 beta-hydroxysteroid dehydrogenase.     

Zum Problem der Genetischen Rekombination von Bakteriophagen

Summary Four equivalent triparental-3 factor-3 alleles crosses of the type 121×212×322 are performed with linked markers in bacteriophage T1. Total recombinants and specially the biparental doubles (BD) of the type 111 and the triparental doubles (TD) of the type 311 are scored. The results support the idea of a topography and give therefore no conclusive evidence whether the process of cooperation is restricted to two parental structures or not.

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Mit 4 Textabbildungen     

Core protein mu2 is a second determinant of nucleoside triphosphatase activities by reovirus cores.

NTPase activities in mammalian reovirus cores were examined under various conditions that permitted several new differences to be identified between strains type 1 Lang (T1L) and type 3 Dearing (T3D). One difference concerned the ratio (at pH 8.5) of ATP hydrolysis at 50 degrees C to that at 35 degrees C. A genetic analysis using T1L x T3D reassortant viruses implicated the L3 and M1 gene segments in this difference, with M1 influencing ATPase activity most strongly at high temperatures. L3 and M1 encode the core proteins lambda1 and mu2, respectively. Another difference concerned the absolute levels of GTP hydrolysis by cores at 45 degrees C and pH 6.5. A genetic analysis using T1L x T3D reassortants implicated the M1 gene as the sole determinant of this difference. The results of these experiments, coupled with previous findings (S. Noble and M. L. Nibert, J. Virol. 71:2182-2191, 1997), suggest either that a single type of NTPase in cores is strongly influenced by two different core proteins--lambda1 and mu2--or that cores contain two different types of NTPase influenced by the two proteins. The findings appear relevant for understanding the complex functions of reovirus cores in RNA synthesis and capping.     

Regulation by thyroid hormones of terminal differentiation in the skeletal dorsal muscle. I. Neonate mouse

Changes both in the ATPase myofibrillar profile and in the electrophoretic pattern of myosin isoforms were examined in the mouse dorsal skeletal muscle (longissimus) during postnatal development. In the newborn, only type II C and a few type I fibers were present; differentiation into type II A and II B fibers took place during the 3 weeks following birth. During the same period, a transition from three neonatal isomyosins to four adult isoforms was observed. The two phenomena were related to a marked increase in the serum thyroid hormones levels. Hypothyroidism and hyperthyroidism experiments were performed. Hypothyroidism produced by propylthiouracil treatment of pregnant females and thiourea injections of the litters was shown to induce a complete inhibition of postnatal muscular differentiation. Hyperthyroidism produced by triiodothyronine treatment of the neonate mice significantly accelerated the myosin transition and the switch in the myofibrillar pattern. Our results suggest a primordial role for thyroid hormones in directly regulating the appearance of myosin and fiber adult types and in modulating directly or indirectly the disappearance of the neonatal types.     

A susceptibility locus for migraine with aura, on chromosome 4q24

Migraine is a complex neurovascular disorder with substantial evidence supporting a genetic contribution. Prior attempts to localize susceptibility loci for common forms of migraine have not produced conclusive evidence of linkage or association. To date, no genomewide screen for migraine has been published. We report results from a genomewide screen of 50 multigenerational, clinically well-defined Finnish families showing intergenerational transmission of migraine with aura (MA). The families were screened using 350 polymorphic microsatellite markers, with an average intermarker distance of 11 cM. Significant evidence of linkage was found between the MA phenotype and marker D4S1647 on 4q24. Using parametric two-point linkage analysis and assuming a dominant mode of inheritance, we found for this marker a maximum LOD score of 4.20 under locus homogeneity (P=.000006) or locus heterogeneity (P=.000011). Multipoint parametric (HLOD = 4.45; P=.0000058) and nonparametric (NPL(all) = 3.43; P=.0007) analyses support linkage in this region. Statistically significant linkage was not observed in any other chromosomal region.