Developmental downregulation of rhizobial genes as a function of symbiosome differentiation in symbiotic root nodules of Pisum sativum.

• The expression of nodA and dctA genes of Rhizobium leguminosarum bv. viciae has been studied in mutant nodules of pea (Pisum sativum L.), blocked at the following developmental stages: infection thread development inside the nodule (Itn); infection droplet differentiation (Idd); bacteroid differentiation after endocytosis (Bad); and nodule persistence (Nop). • With the use of reporter fusions to these symbiotic bacterial genes it was shown that both nodA and dctA were expressed at all developmental stages, with a pattern similar to that of constitutive, symbiosis-unrelated genes. • As well as two constitutively expressed genes, both nodA and dctA genes seemed to be subjected to gradual downregulation in nodule bacteria, correlating with the stage of bacteroid differentiation reached. No such effect was observed for the symbiotic, oxygen-regulated fixN gene. The bacteroid development stage also appeared to be related to the ability of bacteria that have been subjected to endocytosis to resume free-living vegetative growth. • The results support the suggestion that bacteroid differentiation into a nitrogen-fixing, organelle-like form, is a gradual process involving several stages, each controlled by different plant genes.

Cloning and identification of conjugative transfer origins in the Rhizobium meliloti genome.

A simple approach was used to identify Rhizobium meliloti DNA regions with the ability to convert a nontransmissible vector into a mobilizable plasmid, i.e., to contain origins of conjugative transfer (oriT, mob). RecA-defective R. meliloti merodiploid populations, where each individual contained a hybrid cosmid from an R. meliloti GR4 gene library, were used as donors en masse in conjugation with another R. meliloti recipient strain, selecting transconjugants for vector-encoded antibiotic resistance. Restriction analysis of cosmids isolated from individual transconjugants resulted in the identification of 11 nonoverlapping DNA regions containing potential oriTs. Individual hybrid cosmids were confirmed to be mobilized from the original recA donors at frequencies ranging from 10(-2) to 10(-5) per recipient cell. DNA hybridization experiments showed that seven mob DNA regions correspond to plasmid replicons: four on symbiotic megaplasmid 1 (pSym1), one on pSym2, and another two on each of the two cryptic plasmids harbored by R. meliloti GR4. Another three mob clones could not be located to any plasmid and were therefore preliminarily assigned to the chromosome. With this strategy, we were able to characterize the oriT of the conjugative plasmid pRmeGR4a, which confirmed the reliability of the approach to select for oriTs. Moreover, transfer of the 11 mob cosmids from R. meliloti into Escherichia coli occurred at frequencies as high as 10(-1), demonstrating the R. meliloti gene transfer capacity is not limited to the family Rhizobiaceae. Our results show that the R. meliloti genome contains multiple oriTs that allow efficient DNA mobilization to rhizobia as well as to phylogenetically distant gram-negative bacteria.

Prevalence of atopy in students from Málaga, Spain.

BACKGROUND: Epidemiologic studies are necessary to determine the prevalence of allergic diseases. This varies widely depending on allergen preparations and patients studied. OBJECTIVE: To investigate the prevalence of atopic disease, skin test reactivity, total and specific IgE to common allergens, and other variables in a sample of students from Málaga, southern Spain. METHODS: Three hundred sixty-five students (age 17.9 +/- 1.18) were interviewed by an allergist. Skin prick tests were performed with Dermatophagoides pteronyssinus, Artemisia vulgaris, Plantago lanceolata, Chenopodium album, Olea europaea, Phleum pratense, Parietaria judaica, Cynodon dactylon, Alternaria tenuis, and cat dander. Total and specific IgE to D. pteronyssinus, Olea, and Parietaria were determined. RESULTS: Of all subjects studied, 19.9% suffered from rhinoconjunctivitis, 4.1% rhinoconjunctivitis plus asthma, 3.1% asthma alone, and 0.8% atopic dermatitis; 46.4% had a positive skin test to at least one allergen (28.2% to D. pteronyssinus, 20.4% to Olea, 13.8% to Phleum); and 43% had total IgE > 100 kU/L and 44.7% a family history of atopy. Allergic symptoms were strongly associated with skin test positivities and family allergic history. Patients with asthma or skin prick test positive had higher total IgE values than others (P < .01). There was a significant correlation between specific IgE values and wheal size in skin test. CONCLUSIONS: Our findings confirm the high prevalence of atopic diseases, and the close relationship of skin tests reactivity (or presence of specific IgE) to allergens with symptoms of asthma and rhinitis. The presence of a family history of allergic diseases influences the development of positive skin tests and atopic illness. Dermatophagoides pteronyssinus and pollen of Olea europaea were found to be the most common allergens.

Pollinosis due to Australian pine (Casuarina): an aerobiologic and clinical study in southern Spain.

An aerobiologic and clinical survey was conducted in Málaga, southern Spain, in order to determine fluctuations of Australian pine (Casuarina) pollen in the atmosphere of the city, and the prevalence of sensitivity in a nonatopic population. The aerobiologic survey, using a Burkard spore trap, was conducted from January 1991 to December 1994, and sensitization was ascertained by the skin prick test. The pollen season is relatively short and the pollen dispersion period occurs during October and November, mainly during the last 3 weeks of October. Diurnal patterns showed that the highest concentrations of pollen occur between 12 a.m. and 2 p.m., the most influential variables in its dispersion being temperature, sunshine, and rainfall. The prevalence of sensitization to Casuarina pollen was determined by skin prick test (SPT) in a nonatopic population of 210 patients with a previous history of autumn rhinitis, asthma, or rhinitis asthma. Six subjects showed a positive reaction to the pollen extracts, and the presence of specific IgE was demonstrated by the conventional radioallergosorbent test (RAST > or = 2) in five of these patients with positive SPT.

Ammonia Inhibition of Plasmid pRmeGR4a Conjugal Transfer between Rhizobium meliloti Strains.

We have examined nutritional factors influencing conjugal transfer of the two nonsymbiotic large plasmids, pRmeGR4a and pRmeGR4b, of Rhizobium meliloti GR4. To monitor transfer, each plasmid was tagged with a different antibiotic resistance marker. Transfer of plasmid pRmeGR4b was dependent upon the presence of plasmid pRmeGR4a on the same donor cell. Transconjugants for pRmeGR4b were obtained at frequencies 5-to 10-fold higher than transconjugants carrying both plasmids, indicating that mobilization of pRmeGR4b by pRmeGR4a probably occurred in trans. Conjugal transfer of the tagged plasmids between R. meliloti strains was tested on minimal medium supplemented with single amino acids, nitrate, or ammonium as the single nitrogen source. A higher number of transconjugants was obtained when glutamate was the only nitrogen source, whereas conjugation was virtually undetectable on ammonium. No relationship was found between donor or recipient growth rate and plasmid transfer rate on a given nitrogen source. Furthermore, in media containing both glutamate and ammonium as nitrogen sources, transfer was reduced almost 100-fold compared with that in media containing glutamate alone. Inhibition was readily detected at 2.5 mM or higher concentrations of either ammonium chloride or ammonium sulfate and appeared to be specific for exogenously supplied ammonium. Inhibition of conjugal transfer between R. meliloti strains by ammonium was only observed for rhizobial plasmids, not for a heterologous plasmid such as RP4. Apparently, ammonium did not affect the plasmid-encoded transfer machinery, as it had no influence on rhizobial plasmid transfer from R. meliloti to Agrobacterium tumefaciens. The effect of ammonium seemed to take place on R. meliloti recipient cells, thereby reducing the efficiency of plasmid conjugation, probably by affecting mating pair formation or stabilization.

[A comparative study of insulin and glyburide versus glyburide or insulin in the chronic control of patients with type-2 diabetes]. / Estudio comparativo de insulina y gliburida contra gliburida o insulina, en el control crónico de pacientes con diabetes tipo II.

In order to know the usefulness of a combined treatment with gliburide and NPH insulin 25 patients with well controlled type II diabetes mellitus were studied. The patients were randomly divided in to three groups: Group I just received gliburide, Group II only insulin and Group III gliburide and insulin. Glucose in fast, glycosylated haemoglobin and C peptide levels were determined over five months. For the statistical data processing variance analysis was performed. The initial and final glucose determinations were: Group I, 169.3 mg% and 139.0 mg% respectively (p > 0.05); Group II, 202.1 mg% and 177 mg% (p > 0.05); Group III, 157.8 mg% and 158.8 mg% (p > 0.1) for the glycosylated haemoglobin the determinations were: Group I, 7.2% and 5.1% (p > 0.05); Group II, 6.2% and 5.1% (p > 0.05) and Group III, 5.7% and 4.7% (p > 0.05). For the C peptide were 2.5 and 4.5 for Group I (p > 0.05), 2 and 4.1 for Group II (p > 0.05) and 3.2 and 5.3 for Group III (p > 0.05) with no significant statistical differences. It is concluded that the combined treatment showed to be effective, but not superior, in order to control diabetic patients and it can be a useful therapeutic alternative in well selected patients.