The Hydrogenase Chip: a tiling oligonucleotide DNA microarray technique for characterizing hydrogen-producing and -consuming microbes in microbial communities

We developed a broad-ranging method for identifying key hydrogen-producing and consuming microorganisms through analysis of hydrogenase gene content and expression in complex anaerobic microbial communities. The method is based on a tiling hydrogenase gene oligonucleotide DNA microarray (Hydrogenase Chip), which implements a high number of probes per gene by tiling probe sequences across genes of interest at 1.67 × –2 × coverage. This design favors the avoidance of false positive gene identification in samples of DNA or RNA extracted from complex microbial communities. We applied this technique to interrogate interspecies hydrogen transfer in complex communities in (i) lab-scale reductive dehalogenating microcosms enabling us to delineate key H₂-consuming microorganisms, and (ii) hydrogen-generating microbial mats where we found evidence for significant H₂ production by cyanobacteria. Independent quantitative PCR analysis on selected hydrogenase genes showed that this Hydrogenase Chip technique is semiquantitative. We also determined that as microbial community complexity increases, specificity must be traded for sensitivity in analyzing data from tiling DNA microarrays.     

Hormonal response to restraint in rhesus monkeys

Abstract: The purpose of this study was to characterize the hormonal responses to a restraining system in four adult male rhesus monkeys (Macaca mulatta) in preparation for a spaceflight project. After the monkeys were accustomed to food and water (Phase I), blood-volume-regulating hormones were measured during three phases: 10 days in a metabolic cage (Phase II), 16 days sitting in a restrained position in a specially designed metabolism chair (Phase III) and 10 days in metabolic cage (Phase IV). An increase of active renin (30%) and vasopressin (25%) was observed at the end of Phase III. A decrease of atrial natriuretic peptide (ANP), urodilatin, and sodium excretion occurred during the first days of Phase III. Catecholamines were unchanged. A dramatic increase (tenfold) in urinary excretion of growth hormone occurred during all of Phase III and at the beginning of Phase IV. These findings are similar to those found in man during isolation inactivity and during confinement stress.     

Production of neosolaniol monoacetate by an undescribedFusarium species resemblingF camptoceras

Cultures of 12 South African isolates of an undescribedFusarium species resembling but distinct fromF camptoceras were analysed for the presence of diacetoxyscirpenol (DAS), neosolaniol monoacetate (NMA), and T-2 toxin, by capillary gas chromatography utilizing electron capture detection. No DAS or T-2 toxin could be detected in any of the cultures of the isolates. NMA was, however, detected in 10 of the 12 isolates at levels ranging from 310 to 2060 ng/g. The method used, was primarily developed for the determination of DAS and T-2 toxin in fungal cultures and grain samples but was found to be suitable for the coextraction of NMA at an average recovery of 80.8%, with a detection limit in the order of 100 ng/g. Supportive evidence for the presence of the NMA was obtained by capillary gas chromatography / mass spectrometry. Regarded as a relatively rare trichothecene, NMA has never been reported to occur naturally and has previously been shown to be produced by only a fewFusarium strains.     

Elevated levels of calcitonin mRNA: a marker for the spontaneous development of medullary thyroid carcinoma in rats

The aging WAG/Rij rats (a Wistar derived strain) develop spontaneously medullary thyroid carcinoma with a high frequency (50%). We have studied calcitonin biosynthesis in Wistar and WAG/Rij rats strains in order to determine if early changes in this parameter occurred in the WAG/Rij strain. Thyroidal and plasma CT levels were measured in three months old WAG/Rij and Wistar rats before and after acute calcium challenge. Total RNA was extracted from thyroid glands and specific CT messenger RNA levels estimated by dot and Northern blot analysis with a 32P-labeled probe specific for CT mRNA. The capacity of mRNA to direct synthesis of CT precursor was also measured by translation in an in vitro system. Though mean basal circulating CT levels were equivalent in both strains, CT release after calcium stimulation was much increased in the WAG/Rij rat. CT content of the glands and CT mRNA levels were two fold higher in the WAG/Rij strain. Thus, in this strain, CT biosynthesis and secretion were increased long before the development of a C-cell carcinoma.     

The sensitivity of Afromontane tarns in the Maloti-Drakensberg region of South Africa and Lesotho to acidic deposition

Despite their remoteness from sources of atmospheric pollutant emissions, the Afromontane tarns in the Maloti-Drakensberg region are perfect candidates to study the negative effects of acidifying atmospheric pollution, because mountain lakes are widely recognised as sentinel ecosystems, unimpacted by direct human disturbance within their catchments. Thirty-four tarns were sampled in the Maloti-Drakensberg region and most were found to be extremely sensitive to acidic deposition, as indicated by their low acid neutralising capacity. There are very few studies of freshwater critical loads for any region within South Africa. The steady-state water chemistry model (SSWC) was adapted and used to determine critical loads, whereas exceedance was estimated relative to modelled regional deposition data, in order to understand the risk of harmful effects to aquatic ecosystems. Seventy-six percent of sampled sites across the Maloti-Drakensberg would exceed critical loads even at the lowest modelled deposition levels, but there are no current measured deposition data for the region. The sensitivity of the Maloti-Drakensberg tarns needs to be considered in future policy formulation regarding acceptable levels of acidifying atmospheric pollution from South Africa’s energy sector and indicates the need for assessing aquatic ecosystem impacts in other regions of South Africa.     

Immune response against medullary thyroid carcinoma (MTC) induced by parental and/or interleukin-2-secreting MTC cells in a rat model of human familial medullary thyroid carcinoma

 The existence of inherited aggressive forms of medullary thyroid carcinoma (MTC), and their resistance to all classical therapies, make it a prime candidate for adoptive immunotherapy. As a prelude to a vaccine for the protection of family members at risk of developing the disease, we investigated the immunological antitumour response provoked by the 6/23 rMTC cell line, compared to that of the same cells engineered to secrete interleukin-2 (rMTC-IL2), in an animal model of familial human MTC, the inbred strain of Wag/Rij rats. The rMTC cells developed a tumour that invaded the whole neck 15 days after orthotopic injection (into the thyroid), while the rMTC-IL2 cells were progressively rejected. Co-injection of rMTC-IL2 with the parental cells induced the rejection of the rMTC transplants. When injected, both tumoral cell types showed a similar positive immunoreaction with anti-MHC class I (major histocompatibility complex class I) antibodies. They both recruited natural killer cells and eosinophils at the site of injection. In addition, CD8⁺ T lymphocytes infiltrated the rMTC-IL2 cells, and eosinophil recruitment was amplified. Neutrophils, macrophages and CD4⁺ T lymphocytes were scarce. Our results suggest that the CD8⁺ T lymphocytes are implicated in the antitumour reaction elicited by the Il-2-transfected cells. As these effectors are known to induce a specific immunological response, including memory, such a protocol should be tested as a vaccine on the young population genetically at risk of developing a MTC. Received: 18 December 1995 / Accepted: 21 August 1996     

Deficiency of calcitonin in age related osteoporoses

Plasma levels of immunoreactive calcitonin were measured in patients with age-related osteoporosis and with postmenopausal osteoporosis and compared to the levels in control subjects. In all studies immunoreactive calcitonin was lower in the osteoporotic patients. Parathyroid hormone levels were measured in women with postmenopausal osteoporosis and in control women; no difference was observed between the two groups. The results are consistent with the hypothesis that calcitonin but not parathyroid hormone plays a role in postmenopausal and age-related osteoporosis.     

Antimicrobial susceptibility patterns,emm type distribution and genetic diversity of Streptococcus pyogenes recovered in Brazil

Streptococcus pyogenes is responsible for a variety of infectious diseases and immunological complications. In this study, 91 isolates of S. pyogenes recovered from oropharynx secretions were submitted to antimicrobial susceptibility testing, emm typing and pulsed-field gel electrophoresis (PFGE) analysis. All isolates were susceptible to ceftriaxone, levofloxacin, penicillin G and vancomycin. Resistance to erythromycin and clindamycin was 15.4%, which is higher than previous reports from this area, while 20.9% of the isolates were not susceptible to tetracycline. The macrolide resistance phenotypes were cMLSB (10) and iMLSB (4). The ermB gene was predominant, followed by the ermA gene. Thirty-two emm types and subtypes were found, but five (emm1, emm4, emm12, emm22, emm81) were detected in 48% of the isolates. Three new emm subtypes were identified (emm1.74, emm58.14, emm76.7). There was a strong association between emm type and PFGE clustering. A variety of PFGE profiles as well as emm types were found among tetracycline and erythromycin-resistant isolates, demonstrating that antimicrobial resistant strains do not result from the expansion of one or a few clones. This study provides epidemiological data that contribute to the development of suitable strategies for the prevention and treatment of such infections in a poorly studied area.     

Antiviral activity of antisense oligonucleotides linked to poly(L-lysine):targets on genomic RNA and/or mRNA of Vesicular Stomatitis Virus

Abstract

Synthetic oligonucleotides provide a rational approach to viral genes control. Conjugation of antisense oligodeoxyribonucleotides (directed against several viral sequences) to poly(L-lysine) brings about specific protection against Vesicular Stomatitis Virus at concentrations lower than 1 uM.