Genetic characterization of a Sinorhizobium meliloti chromosomal region in lipopolysaccharide biosynthesis.

The genetic characterization of a 5.5-kb chromosomal region of Sinorhizobium meliloti 2011 that contains lpsB, a gene required for the normal development of symbiosis with Medicago spp., is presented. The nucleotide sequence of this DNA fragment revealed the presence of six genes: greA and lpsB, transcribed in the forward direction; and lpsE, lpsD, lpsC, and lrp, transcribed in the reverse direction. Except for lpsB, none of the lps genes were relevant for nodulation and nitrogen fixation. Analysis of the transcriptional organization of lpsB showed that greA and lpsB are part of separate transcriptional units, which is in agreement with the finding of a DNA stretch homologous to a "nonnitrogen" promoter consensus sequence between greA and lpsB. The opposite orientation of lpsB with respect to its first downstream coding sequence, lpsE, indicated that the altered LPS and the defective symbiosis of lpsB mutants are both consequences of a primary nonpolar defect in a single gene. Global sequence comparisons revealed that the greA-lpsB and lrp genes of S. meliloti have a genetic organization similar to that of their homologous loci in R. leguminosarum bv. viciae. In particular, high sequence similarity was found between the translation product of lpsB and a core-related biosynthetic mannosyltransferase of R. leguminosarum bv. viciae encoded by the lpcC gene. The functional relationship between these two genes was demonstrated in genetic complementation experiments in which the S. meliloti lpsB gene restored the wild-type LPS phenotype when introduced into lpcC mutants of R. leguminosarum. These results support the view that S. meliloti lpsB also encodes a mannosyltransferase that participates in the biosynthesis of the LPS core. Evidence is provided for the presence of other lpsB-homologous sequences in several members of the family Rhizobiaceae.

[Monoclonal antibodies in the treatment of sepsis caused by Gram-negative microorganisms]. / Los anticuerpos monoclonales en el tratamiento de la sepsis provocada por microorganismos gramnegativos.

In spite of the advances attained in the diagnosis and early intervention with antibiotics, morbidity and mortality associated with sepsis caused by Gram-negative bacteria are high. The mediators responsible for the pathogenesis of the sepsis are components derived from the own bacteria (endotoxins) and from the cells of the immune response of the host (tumor necrosis factor and some interleukins). The treatment traditionally used in sepsis is mainly directed against microorganisms by the use of increasingly potent antibiotics. However, it is clear that antibiotics are not a definitive solution, since even when they cause bacterial death, they have no effects on endotoxin and may increase their liberation when cellular lysis occurs. New and successful treatments for sepsis have been tried since the 1980's, including the use of polyclonal and monoclonal antibodies of murine and human origin, directed against the lipid A of the endotoxin, as well as monoclonal antibodies against the tumor necrosis factor. Although these molecules are not completely efficient for the interruption of the chain of undesirable events provoked by the endotoxin, it is valid to accept the appearance of immunotherapies as an adjuvant treatment for a condition that threatens life.

Evaluation of the serologic response against two consensus V3 loop peptides from human immunodeficiency virus-1 in Cuban patients.

OBJECTIVES: A retrospective study was conducted to evaluate the antibody response of Cuban patients infected with human immunodeficiency virus (HIV)-1 against two consensus peptides from the third variable domain (V3) loop of glycoprotein gp120. METHODS: The study included sera from 10 individuals at different stages of disease. Two 15-meric synthetic peptides designed from a consensus sequence, belonging to group B or C of HIV-1, were used to determine antibody titers and avidity indexes in an indirect enzyme-linked immunoassay. RESULTS: A high reactivity against both peptides was detected, with 80% of the sera reacting with at least one of the peptides. The antibody titers and avidity indexes did not correlate with disease progression. Additionally, for one of the patients from whom the virus had been isolated, a higher avidity index was found against the homologous peptide. CONCLUSIONS: This study showed high reactivity against two consensus peptides from the V3 loop of gp120 among patients with HIV. Large scale studies are needed to determine whether the titers or avidity of anti-V3 antibodies, at the early stages of infection, are predictive of disease progression. Both peptides are candidates for inclusion in experimental vaccines.

Generation of murine triomas secreting bi-specific monoclonal antibodies that recognize HBsAG ad and ay subtypes.

We report the generation of murine triomas by fusing splenocytes from mice previously immunized with HBsAg ay-subtype and a hybridoma, secreting anti-HBsAg ad-subtype monoclonal antibody, which was rendered HGPRT- by induced mutagenesis with N-methyl-N'nitro-N-nitrosoguanidine. The fusion yielded a 83.8% of hybrids showing the antigen specificity of the parental hybridoma and a 16.1% of bi-specific monoclonal antibodies. One of them, coded as 1C8A5, showing a heavy chain isotype (IgG1/IgG2b) was used as capture reagent in an ultramicro-ELISA. As little as 0.78 I.U. of both HBsAg ad- and ay-subtypes could be realiably detected.

2,3,5-Triphenyl tetrazolium chloride (TTC) reduction as exponential growth phase marker for mammalian cells in culture and for myeloma hybridization experiments.

Triphenyl tetrazolium chloride in vitro reduction by cells produces a red formazan pellet which can be extracted and measured. We have shown that such reduction is associated with animal cell growth, and particularly with the specific growth rate, so the measurement of Triphenyl tetrazolium chloride reduction is proposed as a physiological marker of the exponential growth of cultured cells. Further application of this technique is shown using this Redox reaction for estimating plasmacytoma fusion potential for hybridoma cell line production.

Sustrato de peroxidasa para revelado de conjugado de fosfatasa alcalina en ensayo tipo ELISA / Peroxidase substrate for alkaline phosphatase conjugated in an ELISA type assay

Se describe un método en el cual un ensayo de tipo ELISA, que utiliza un conjugado de fosfatasa alcalina, puede ser revelado con un sustrato tradicional de la enzima peroxidasa como el ABTS. Se presenta una comparación con el 4-nitrofenil fosfato desarrollado como la clásica alternativa calorimétrica para la fosfatasa alcalina. Se sugiere que pueden ser introducidas nuevas aplicaciones potenciales relacionadas con los sistemas de peroxidasa a conjugados que incluyen a la enzima fosfatasa alcalina

A 10 microliter indirect ultramicro ELISA for detection of monoclonal antibodies against human alphafetoprotein.

An ultramicro ELISA, which uses 10 microliter of reaction volume developed for quantitation of human alphafetoprotein (AFP), has been adapted to use in screening hybridoma products in order to take advantage of the efficiency and sensitivity of this system. Positive samples were detected with a low background level.