Integration of Trypanosoma cruzi kDNA minicircle sequence in the host genome may be associated with autoimmune serum factors in Chagas disease patients

Integration of kDNA sequences within the genome of the host cell shown by PCR amplification with primers to the conserved Trypanosoma cruzi kDNA minicircle sequence was confirmed by Southern hybridization with specific probes. The cells containing the integrated kDNA sequences were then perpetuated as transfected macrophage subclonal lines. The kDNA transfected macrophages expressed membrane antigens that were recognized by antibodies in a panel of sera from ten patients with chronic Chagas disease. These antigens barely expressed in the membrane of uninfected, control macrophage clonal lines were recognized neither by factors in the control, non-chagasic subjects nor in the chagasic sera. This finding suggests the presence of an autoimmune antibody in the chagasic sera that recognizes auto-antigens in the membrane of T. cruzi kDNA transfected macrophage subclonal lines.     

Critical determinants of host receptor targeting by Neisseria meningitidis and Neisseria gonorrhoeae: identification of Opa adhesiotopes on the N-domain of CD66 molecules

The human pathogens Neisseria meningitidis and Neisseria gonorrhoeae express a family of variable outer membrane opacity-associated (Opa) proteins that recognize multiple human cell surface receptors. Most Opa proteins target the highly conserved N-terminal domain of the CD66 family of adhesion molecules, although a few also interact with heparan sulphate proteoglycans. In this study, we observed that at least two Opa proteins of a N. meningitidis strain C751 have the dual capacity to interact with both receptors. In addition, all three Opa proteins of C751 bind equally well to HeLa cells transfected with cDNA encoding the carcinoembryonic antigen [CEA (CD66e)] subgroup of the CD66 family, but show distinct tropism for CGM1- (CD66d) and NCA (CD66c)-expressing cells. Because the C751 Opa proteins make up distinct structures via the surface-exposed hypervariable domains (HV-1 and HV-2), these combinations appear to be involved in tropism for the distinct CD66 subgroups. To define the determinants of receptor recognition, we used mutant proteins of biliary glycoprotein [BGP (CD66a)] carrying substitutions at several predicted exposed sites in the N-domain and compared their interactions with several Opa proteins of both N. meningitidis and N. gonorrhoeae. The observations applied to the molecular model of the BGP N-domain that we constructed show that the binding of all Opa proteins tested occurs at the non-glycosylated (CFG) face of the molecule and, in general, appears to require Tyr-34 and Ile-91. Further, efficient interaction of distinct Opa proteins depends on different non-adjacent amino acids. In the three-dimensional model, these residues lie in close proximity to Tyr-34 and Ile-91 at the CFG face, making continuous binding domains (adhesiotopes). The epitope of the monoclonal antibody YTH71.3 that inhibits Opa/CD66 interactions was also identified within the Opa adhesiotopes on the N-domain. These studies define the molecular basis that directs the Opa specificity for the CD66 family and the rationale for tropism of the Opa proteins for the CD66 subgroups.     

Inhibition of Mitochondrial Permeability Transition by Low pH is Associated with Less Extensive Membrane Protein Thiol Oxidation

Ca²⁺ and inorganic phosphate-induced mitochondrial swelling and membrane protein thiol oxidation, which are associated with mitochondrial permeability transition, are inhibited by progressively decreasing the incubation medium pH between 7.2 and 6.0. Nevertheless, the detection of mitochondrial H₂O₂ production under these conditions is increased. Permeability transition induced by phenylarsine oxide, which promotes membrane protein thiol cross-linkage in a process independent of Ca²⁺ or reactive oxygen species, is also strongly inhibited in acidic incubation media. In addition, we observed that the decreased protein thiol reactivity with phenylarsine oxide or phenylarsine oxide-induced swelling at pH 6.0 is reversed by diethyl pyrocarbonate, in a hydroxylamine-sensitive manner. These results provide evidence that the inhibition of mitrochondrial permeability transition observed at lower incubation medium pH is mediated by a decrease in membrane protein thiol reactivity, related to the protonation of protein histidyl residues.     

Influence of environmental factors on lipase production by Lactobacillus plantarum

A strain of Lactobacillus plantarum, DSMZ 12028 (Deutsch Sammlung von Mikroorganismen und Zellkulturen), isolated from a Portuguese dry fermented sausage, “chouriço”, was found to produce true lipase, producing free fatty acids from triolein (olive oil). This enzymatic activity was found in whole cells, but was negligible in comparison to lipolytic activity in culture supernatant. Therefore, only extracellular activity was studied. The effect of pH, temperature and glucose concentration on extracellular lipase production was studied in continuously stirred tank reactors, the first time this technology has been used to study the production of this enzyme in lactobacilli. Maximum lipase production was achieved at a pH of 5.5 and 30?°C and was kept at a significant level over a wide range of dilution rates (0.05–0.4 h^?1); the production of lipase was still significant for low pH values, temperature and glucose concentration, conditions that are close to the ones present during chouriço ripening. The effect of glucose concentration was also studied in a batch system. The control of lipase production was found to be related both to glucose concentration in the medium and to the growth rate/dilution rate. Glucose concentration was found to be important for fast lipase production, although it did not influence the maximum lipase activity reached in a batch culture.     

NMDA and non-NMDA receptors mediate responses in the primary gustatory nucleus in goldfish

Primary gustatory afferents from the oropharynx of the goldfish, Carassius auratus, terminate in the vagal lobe, a laminated structure in the dorsal medulla comparable to the gustatory portion of the nucleus of the solitary tract in mammals. We utilized an in vitro brain slice preparation to test the role of different ionotropic glutamate receptor subtypes in synaptic transmission of gustatory information by recording changes in field potentials after application of various glutamate receptor antagonists. Electrical stimulation of the vagus nerve (NX) evokes two short-latency postsynaptic field potentials from sensory layers of the vagal lobe. 6,7-Dinitroquinoxaline-2,3-dione and 6-nitro-7-sulphamoylbenzo[f]quinoxaline-2,3-dione, two non-N-methyl-D-aspartate (NMDA) ionotropic receptor antagonists, blocked these short-latency potentials. Slower potentials that were revealed under Mg2+ -free conditions, were abolished by the NMDA receptor antagonist, D(-)-2-amino-5-phosphonovaleric acid (APV). Repetitive stimulation produced short-term facilitation, which was attenuated by application of APV. These results indicate that the synaptic responses in the vagal lobe produced by stimulation of the gustatory roots of the NX involve both NMDA and non-NMDA receptors. An NMDA receptor-mediated facilitation may serve to amplify incoming bursts of primary afferent activity.     

Analysis of the bacterial community in glassy-winged sharpshooter heads

The glassy‐winged sharpshooter (GWSS), Homalodisca vitripennis, is an important vector of various strains of Xylella fastidiosa, which cause disease in a variety of economically important plants. These diseases include citrus variegated chlorosis, oleander leaf scorch and Pierce's Disease of grapevines. Symbiotic control (SC) is a new strategy that uses symbiotic endophytes as biological control agents to antagonize or displace the pathogenic strains of X. fastidiosa. Candidate endophytes for use in SC must occupy the xylem of host plants and attach to the pre‐cibarium and cibarium of sharpshooter insects in order to have access to the pathogen. The study of the bacterial community of GWSS heads by isolation and denaturing gradient gel electrophoresis (DGGE) revealed the presence of species that may be suitable for use in SC. In addition, the results indicated that two important factors, insect age and choice of host plant, affect the composition of the bacterial community in GWSS heads. The main bacterial genera isolated as colonizers of GWSS heads were identified, using partial 16S rRNA gene sequencing, as Bacillus, Pseudomonas, Pedobacter and Methylobacterium, as well as the species Curtobacterium flaccumfaciens. DGGE patterns revealed a diversity of endophytic species able to colonize the GWSS head. The main genera isolated in culture were also identified using this technique. Principal component analysis (PCA) from polymerase chain reaction (PCR)‐DGGE patterns indicated that the bacteria inhabiting the GWSS head are similar to those found as endophytes inside the host plants, and that insect developmental stage and preferential feeding on one host plant species over another are important factors in determining the composition of the bacterial community in the GWSS head. However, a shift in host plants for a small period of time did not cause changes in the compositions of these communities.     

CTG Trinucleotide Repeat “Big Jumps”: Large Expansions, Small Mice

Trinucleotide repeat expansions are the genetic cause of numerous human diseases, including fragile X mental retardation, Huntington disease, and myotonic dystrophy type 1. Disease severity and age of onset are critically linked to expansion size. Previous mouse models of repeat instability have not recreated large intergenerational expansions (“big jumps”), observed when the repeat is transmitted from one generation to the next, and have never attained the very large tract lengths possible in humans. Here, we describe dramatic intergenerational CTG•CAG repeat expansions of several hundred repeats in a transgenic mouse model of myotonic dystrophy type 1, resulting in increasingly severe phenotypic and molecular abnormalities. Homozygous mice carrying over 700 trinucleotide repeats on both alleles display severely reduced body size and splicing abnormalities, notably in the central nervous system. Our findings demonstrate that large intergenerational trinucleotide repeat expansions can be recreated in mice, and endorse the use of transgenic mouse models to refine our understanding of triplet repeat expansion and the resulting pathogenesis.     

Trisomy 4 associated with double minute chromosomes and MYC amplification in acute myeloblastic leukemia

A case of de novo acute myeloblastic leukemia (AML) M2, with trisomy 4 and double minute (dmin) chromosomes is reported. Amplification of the MYC gene ascertained by FISH was associated with dmin. A review of the literature of trisomy 4-dmin-associated AML shows that this entity preferentially occurs in elderly women and is not always associated with previously identified exposition to mutagens.     

Domain swapping of a llama VHH domain builds a crystal-wide beta-sheet structure

Among mammals, camelids have a unique immunological system since they produce functional antibodies devoid of light chains and CH1 domains. To bind antigens, whether they are proteins or haptens, camelids use the single domain VH from their heavy chain (VHH). We report here on such a llama VHH domain (VHH-R9) which was raised against a hapten, the RR6 red dye. This VHH possesses the shortest complementarity determining region 3 (CDR3) among all the known VHH sequences and nevertheless binds RR6 efficiently with a K(d) value of 83 nM. However, the crystal structure of VHH-R9 exhibits a striking feature: its CDR3 and its last beta-strand (beta9) do not follow the immunoglobulin VH domain fold, but instead extend out of the VHH molecular boundary and associate with a symmetry-related molecule. The two monomers thus form a domain-swapped dimer which establishes further contacts with symmetry-related molecules and build a crystal-wide beta-sheet structure. The driving force of the dimer formation is probably the strain induced by the short CDR3 together with the cleavage of the first seven residues.