Adhesion to hyphal matrix and antifungal activity of Pseudomonas strains isolated from Tuber borchii ascocarps

Pseudomonas spp. isolates from Tuber borchii ascocarps, known to be able to produce phytoregulatory and biocontrol substances in pure culture, were used to perform studies on their possible physiological role in nature. Antimycotic activity was confirmed against fungal contaminants isolated from the ascocarps, suggesting that populations associated with Tuber borchii fruit bodies may play a role in the maintenance of ascocarp health. Fifty-five percent of strains tested were also able to release metabolites which affected T. borchii mycelial growth and morphogenesis in culture. On the contrary, growth of the arbuscular mycorrhizal fungus Glomus mosseae and the ectomycorrhizal fungus Laccaria bicolor, putative competitors of Tuber for mycorrhizal infection sites on roots, was not influenced by the presence of any bacterial strain. The possibility that these bacteria, which show antifungal activity and fungal growth modulation activities, might be incorporated in the developing ascocarp by means of their preferential adhesion to Tuber mycelium is discussed.     

Allelic genes involved in artery compliance and susceptibility to sporadic abdominal aortic aneurysm

Vascular smooth muscle cells (VSMCs) synthesize elastin (ELN), major protein of aortic tunica media which confers strength and elasticity to aortic wall. Protein loss or distortion is typical in aneurysm tunica media. Transforming growth factor β1 (TGFβ1) inhibits growth and connective protein expression of abdominal VSMCs cultures. Also, in atherogenic studies, estrogen (but not estrogen plus progestin) treatments inhibit aortic collagen accumulation and elastic loss, risk factors to subsequent aortic enlargement. Therefore, polymorphisms of ELN, estrogen receptor (ER) and β (ERβ), progesterone receptor (PR) and TGFβ1 genes and their products may be involved in the abdominal aortic aneurysm (AAA) development. Using PCR-RFLP method, we analyzed ELN RmaI (exon 16), ER PvuII-XbaI (intron 1), ERβ AluI (exon 8), PR TaqI (intron 7) and TGFβ1 Bsu36I (−509 bp, promoter) polymorphisms in 324 Caucasian male subjects: 225 healthy controls (mean age 71.20 ± 6.85 years) and 99 unrelated AAA patients (mean age 69.8 ± 7.1 years). No difference in ELN, ER, PR and TGFβ1 allele frequencies was observed in AAA patients versus controls (P > 0.05). However, because possessing at least an ERβ AluI restriction site was statistically associated to AAA onset (χ² = 5.220; OR = 1.82, P < 0.05), ERβ polymorphism was proposed as genetic determinant in the AAA susceptibility.     

Proteolytic Cleavage of the Linker Region of the Human P-glycoprotein Modulates Its ATPase Function

P-glycoprotein (Pgp), an anticancer drug-translocating ATPase, is responsible for multidrug resistance in cancer. We have previously shown (Nuti, S. L., Mehdi, A., and Rao, U. S. (2000) Biochemistry 39, 3424-3432) that tryptic cleavage of Pgp results in the activation of basal and drug-stimulated ATPase functions of Pgp. To understand this phenomenon, we determined the sites cleaved by trypsin and further examined whether the modulation of Pgp function is trypsin-specific or the result of proteolysis in general. The effects of chymotrypsin and proteinase K on Pgp ATPase function were studied. The results show that proteolysis of Pgp irrespective of the protease employed resulted in the activation of basal ATPase activity. However, drug-stimulated ATPase activities were differentially modulated. Immunoblot analysis of proteolytic digests indicated that, irrespective of the protease employed, Pgp was predominantly cleaved in the middle of the molecule. N-terminal amino acid sequencing of Pgp tryptic and chymotryptic peptides indicated Arg(680) and Leu(682) as the sites of cleavage, respectively. These two cleavage sites are part of the predicted linker region that joins the two halves of Pgp. Together, these results suggest that the linker region in Pgp is primarily accessible to protease action and that cleavage of this region modulates Pgp ATPase function.     

Impact of Bt corn on rhizospheric and soil eubacterial communities and on beneficial mycorrhizal symbiosis in experimental microcosms

A polyphasic approach has been developed to gain knowledge of suitable key indicators for the evaluation of environmental impact of genetically modified Bt 11 and Bt 176 corn lines on soil ecosystems. We assessed the effects of Bt corn (which constitutively expresses the insecticidal toxin from Bacillus thuringiensis, encoded by the truncated Cry1Ab gene) and non-Bt corn plants and their residues on rhizospheric and bulk soil eubacterial communities by means of denaturing gradient gel electrophoresis analyses of 16S rRNA genes, on the nontarget mycorrhizal symbiont Glomus mosseae, and on soil respiration. Microcosm experiments showed differences in rhizospheric eubacterial communities associated with the three corn lines and a significantly lower level of mycorrhizal colonization in Bt 176 corn roots. In greenhouse experiments, differences between Bt and non-Bt corn plants were detected in rhizospheric eubacterial communities (both total and active), in culturable rhizospheric heterotrophic bacteria, and in mycorrhizal colonization. Plant residues of transgenic plants, plowed under at harvest and kept mixed with soil for up to 4 months, affected soil respiration, bacterial communities, and mycorrhizal establishment by indigenous endophytes. The multimodal approach utilized in our work may be applied in long-term field studies aimed at monitoring the real hazard of genetically modified crops and their residues on nontarget soil microbial communities.     

Isolation of MUC1-primed B lymphocytes from tumour-draining lymph nodes by immunomagnetic beads

The humoral immune response against a tumour-associated antigen, polymorphic epithelial mucin (PEM, MUC1) in cancer patients was studied by isolating specific B cells primed for the antigen. Human B lymphocytes from tumour-draining lymph nodes, obtained from 12 patients with epithelial cancers, were immunoselected with magnetic beads coated with a 60mer synthetic peptide corresponding to three tandem repeats of the protein core of the MUC1 antigen. Short-term cultures of B cells were established utilizing interleukin-10 (IL-10), IL-4 and monoclonal antibody anti-CD40, and were maintained for a maximum of 3␣weeks. B cell culture supernatants contained human anti-MUC1 antibodies, as detected by enzyme-linked immunosorbent assay, in 6/12 of the patients tested. Five of these patients, all with early-stage cancer, also had high levels of circulating anti-MUC1 IgM antibodies in the serum. A significant correlation was found (two-tailed P = 0.041) between the presence of circulating anti-MUC1 antibodies and the ability to isolate PEM-specific B cells from tumour-draining lymph nodes. The technique proposed provides a useful method for the analysis of natural immunity against defined tumour antigens. Received: 30 June 1998 / Accepted: 5 October 1998     

Metabolic properties, stress tolerance and macromolecular profiles of rhizobia nodulating Hedysarum coronarium

The drought-tolerant legume Hedysarum coronarium is a Mediterranean species valued as a forage crop for its high performance in stressful conditions. The plant shows peculiar capabilities of nodulating above pH 9 and thriving in highly calcareous soils. With the aim of providing an adequate characterization of its bacterial symbiotic partner, a study was undertaken, approaching from several viewpoints the physiology and structural features of bacteria isolated from nodules of H. coronarium . Tests involved trophic capabilities on different carbon and nitrogen sources, vitamin requirements, and resistance to factors including antibiotics, heavy metals, salinity, pH, and temperature. Enzyme activities, including those of cellulase, pectinase, urease, β-galactosidase, nitrate and nitrite reductase, were evaluated. The DNA G + C percentage content was determined. Species-specific bacteriophages were isolated and a strain-typing grid established. In order to characterize further and fingerprint the different Rhizobium 'hedysari' isolates, electrophoretic pattern of proteins, plasmid DNA, and digested genomic DNA (in pulsed-field gel separation) were compared. Adansonian taxonomy yielded similarity clusters of the different isolates.     

Effects of 6-MBOA on reproductive function in ponies, mice, rats and mink

The effect on reproduction of the plant derivative 6-methoxybenzoxazolinone (6-MBOA), which stimulates reproductive function in voles, was tested in pony mares, laboratory mice and rats, and mink. There was not a significant effect of intravenous injections of 6-MBOA on the ovarian follicles during the transition between the anovulatory and ovulatory seasons in mares. No significant effect of intraperitoneal injections of 6-MBOA on the weight of uterus or ovaries was found in eight-week-old mice, failing to confirm the results of an earlier report. In immature white rats, 6-MBOA treatment resulted in an increase in uterine weight (P<0.05) at the lowest dose tested (0.03 mug/rat; mean for controls, 34 +/-2 mg; treated, 47 +/-5 mg). However, no significant effect was found on the weights of the ovaries and other glands or in coded scores for ovarian stimulation and uterine fluid distention. Adding 1.5 mg 6-MBOA to the daily feed ration of mink beginning two weeks before the mating season did not affect the mean number of kits born. Nulliparous female mink had smaller (P<0.001) litter size than multiparous females. In addition, of the mink that whelped, there were more (P<0.01) nulliparous females (25 118 ) than multiparous females (9 144 ) that lost one or more kits within 48 hours. These results, however, were not altered by 6-MBOA treatment.     

Determinazione spettrofotometrica dell'idrolisi dell'anelloβ-lattamico nelle penicilline

Riassunto Viene descritto un metodo spettrofotometrico per la determinazione della idrolisi-lattamica di antibiotici del gruppo delle penicilline. All'uopo sono utilizzate le proprietà cromatiche dei Cu⁺⁺ complessoni dei prodotti dell'idrolisi.

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A method for spectrophotometric determinations of the hydrolytic cleavage of the-lactam ring in penicillins and penicillin-like antibiotics is proposed.Derivatives of penicilloic acid give Cu⁺⁺-complexes with a strong absorbance in the 255–280 mµ region. If the conditions are carefully controlled, the maxima of absorbance are proportional to the amount of hydrolyzed compounds.

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Il presente lavoro é stato eseguito nell'ambito dell'attività del Centro di Studio per la Microbiologia del Suolo del C.N.R.     

Classification of the genus Humicola traaen I. preliminary reports and investigations

In this first paper several strains ofHumicola are studied, someone perhaps being new species in the artificial sense. An account of their morphological characteristics and their variability is given. Finally the taxonomy of all the genusHumicola and the opportunity to introduce new species differing in morphological characters only are discussed. The Aa. suggest to introduce also in theHyphomycetes new methods of classification not only based on the morphology, but mostly on the physiology, biochemistry and genetics.     

Large plasmids of fast-growing rhizobia: homology studies and location of structural nitrogen fixation (nif) genes.

A single large plasmid was isolated from multiplasmid-harboring strains Rhizobium leguminosarum 1001 and R. trifolii 5. These single plasmids, as well as the largest plasmid detectable in R. phaseoli 3622, hybridized with part of the nif structural genes of Klebsiella pneumoniae. In contrast, the plasmids of R. meliloti strains V7 and L5-30 did not show hybridization with the nif genes of K. pneumoniae, indicating that these genes might be located either on the chromosome or on a much larger plasmid which as yet has not been isolated. Studies of the homology between plasmids of fast-growing Rhizobium species showed that a specific deoxyribonucleic acid sequence, which carries the structural genes for nitrogenase, is highly conserved on a plasmid in R. leguminosarum, R. trifolii, and R. phaseoli. Furthermore, it was found that this type of plasmid in the different species shares extensive deoxyribonucleic acid homology, suggesting that strains in the R. leguminosarum cluster have preserved a nif plasmid.