RAPD-PCR typing of Acinetobacter isolates from activated sludge systems designed to remove phosphorus microbiologically

AIMS: This study investigated whether there were differences in RAPD fingerprints between already described genomic species of Acinetobacter and those from activated sludge systems. Whether plant-specific populations of acinetobacters exist was also examined. METHODS AND RESULTS: Fifty-two isolates of Acinetobacter from four biological phosphorus removal (EBPR) systems of different configurations, and the known genomic species, were characterized using RAPD-PCR, and fragments separated on agarose gels. Patterns were analysed using Gel Pro software and data analysed numerically. RAPD-PCR produced patterns suggesting that many environmental isolates differ from known genomic species. In two cases, strains from individual plants clustered closely enough together to imply that there may be plant-specific populations of acinetobacters. CONCLUSION: The data suggest that current understanding of the taxonomic status of Acinetobacter may need modifying to accommodate non-clinical isolates, as many of the clusters emerging after numerical analysis of RAPD-PCR fragments from activated sludge isolates were quite separate from the clusters containing the already described genomic species. Some evidence was also obtained from the clusters generated to support a view that particular populations of Acinetobacter may occur in individual activated sludge plants. SIGNIFICANCE AND IMPACT OF THE STUDY: These data suggest that the current understanding of the systematics of Acinetobacter, based as it is almost exclusively on clinical isolates, may need drastic revision to accommodate environmental strains. They also suggest that a re-examination of the importance and role of Acinetobacter in the activated sludge process may be appropriate.     

A potent oligosaccharyl transferase inhibitor that crosses the intracellular endoplasmic reticulum membrane

Recent work has resulted in the development of potent inhibitors of oligosaccharyl transferase (OT), the enzyme that catalyzes the cotranslational glycosylation of asparagine [Hendrickson, T. L., Spencer, J. R., Kato, M., and Imperiali, B. (1996) J. Am. Chem. Soc. 118, 7636-7637; Kellenberger, C., Hendrickson, T. L., and Imperiali, B. (1997) Biochemistry 36, 12554-12559]. However, no specific OT inhibitors that function in the cellular environment have yet been reported. The peptide cyclo(hex-Amb-Cys)-Thr-Val-Thr-Nph-NH2 was previously shown to exhibit nanomolar inhibition (Ki = 37 nM) through slow tight binding kinetics [Hendrickson, T. L., Spencer, J. R., Kato, M., and Imperiali, B. (1996) J. Am. Chem. Soc. 118, 7636-7637]. Included herein is the redesign of this prototype inhibitor for achieving both passive and active translocation into model membrane systems representing the endoplasmic reticulum (ER). The strategy for passive transport involved the incorporation of a membrane permeable import function previously shown to carry various peptides across the outer as well as the interior cellular membranes [Rojas, M., Donahue, J. P., Tan, Z., and Lin, Y.-Z. (1998) Nat. Biotechnol. 16, 370-375]. Assessment of function in intact ER membranes revealed that the inhibitor targeted toward passive diffusion demonstrated concentration-dependent inhibition of two different glycosylation substrates. Thus, this modified inhibitor achieved potent inhibition of glycosylation after being successfully transported through the ER membrane. In the active translocation approach, the lead OT inhibitor and a corresponding substrate were redesigned to include features recognized by the transporter associated with antigen processing (TAP). This protein translocates peptides into the lumen of the ER [Heemels, M.-T., Schumacher, T. N. M., Wonigeit, K., and Ploegh, H. L. (1993) Science 262, 2059-2063]. However, although acceptance of the cyclized substrate by the TAP receptor was demonstrated via efficient transport and glycosylation, the modified inhibitor was not translocated by TAP machinery, and therefore, active translocation was achieved for the modified substrate only. Both of these ER transport methods afforded redesigned OT inhibitors that retained their inhibitor properties in vitro, regardless of the extensions to the carboxy-terminus of the root inhibitor. The above family of redesigned inhibitors provides a template for generating a transcellular pathway and represents the first step toward OT inhibition in intact cells.     

Ozone tolerance in snap bean is associated with elevated ascorbic acid in the leaf apoplast

Ascorbic acid (AA) in the leaf apoplast has the potential to limit ozone injury by participating in reactions that detoxify ozone and reactive oxygen intermediates and thus prevent plasma membrane damage. Genotypes of snap bean ( Phaseolus vulgaris L) were compared in controlled environments and in open-top field chambers to assess the relationship between extracellular AA content and ozone tolerance. Vacuum infiltration methods were employed to separate leaf AA into extracellular and intracellular fractions. For plants grown in controlled environments at low ozone concentration (4 nmol mol⁻¹ ozone), leaf apoplast AA was significantly higher in tolerant genotypes (300–400 nmol g⁻¹ FW) compared with sensitive genotypes (approximately 50 nmol g⁻¹ FW), evidence that ozone tolerance is associated with elevated extracellular AA. For the open top chamber study, plants were grown in pots under charcoal-filtered air (CF) conditions and then either maintained under CF conditions (29 nmol mol⁻¹ ozone) or exposed to elevated ozone (67 nmol mol⁻¹ ozone). Following an 8-day treatment period, leaf apoplast AA was in the range of 100–190 nmol g⁻¹ FW for all genotypes, but no relationship was observed between apoplast AA content and ozone tolerance. The contrasting results in the two studies demonstrated a potential limitation in the interpretation of extracellular AA data. Apoplast AA levels presumably reflect the steady-state condition between supply from the cytoplasm and utilization within the cell wall. The capacity to detoxify ozone in the extracellular space may be underestimated under elevated ozone conditions where the dynamics of AA supply and utilization are not adequately represented by a steady-state measurement.     

Optically detected magnetic resonance of intact membranes from Chloroflexus aurantiacus. Evidence for exciton interaction between the RC and the B808–866 complex

Optically detected magnetic resonance of chlorosome-containing membranes from the green filamentous bacterium Chloroflexus aurantiacus has been performed both by fluorescence and absorption detection. Triplet states localized in the chlorosomes and in the B808–866 complex have been characterized. After chemical reduction with ascorbate followed by illumination at 200 K, recombination triplet state localized in the primary donor becomes largely populated under illumination at low temperature while all the antenna triplet states, which are localized in carotenoids and BChl a molecules, are strongly quenched. We were able to obtain the T-S spectrum of the primary donor P870 surrounded by all the antenna complexes connected to the RC via energy transfer and then in its intact environment. We found clear spectroscopic evidence for exciton interaction between the RC and the B808–866 antenna complex. This evidence was provided by the comparison of the T–S spectrum of P870 in the membranes with that of isolated RC. The analogy of some features of the difference spectra with those previously found in the same kind of experiments for Rb. sphaeroides, allows to predict a similar coupling among the primary donor and the nearby antenna BChl a molecules, assembled as circular aggregate.This revised version was published online in October 2005 with corrections to the Cover Date.     

Sequence-specific recognition of the major groove of RNA by deoxystreptamine

Aminoglycoside antibiotics specifically interact with a variety of RNA sequences, and in particular with the decoding region of 16S ribosomal RNA in the aminoacyl tRNA acceptor site (A-site). Ring II of aminoglycosides (2-deoxystreptamine) is the most conserved element among aminoglycoside antibiotics that bind to the A-site. NMR structures of aminoglycoside-A-site RNA complexes suggested that the 2-deoxystreptamine core of aminoglycosides specifically recognizes (5')G-U(3') and potentially (5')G-G(3') or (5')U-G(3') steps in the major groove of RNA. Here, we show that isolated deoxystreptamine specifically interacts with G-U steps within the major groove of the A-site RNA. The bulge residue of A-site RNA is required to open the major groove for accommodation of deoxystreptamine. The chemical groups of deoxystreptamine presented to the RNA by the framework of the 6-carbon ring modulate RNA recognition.     

An inhibin B and estrogen-secreting adrenocortical carcinoma leading to selective FSH suppression

OBJECTIVE: Hormone-secreting adrenocortical tumors are frequently associated with endocrine syndromes. We describe a 30-year-old man who had abdominal pain, a nodule in the right breast and loss of libido. Abdominal magnetic resonance imaging revealed a very large tumor in the right adrenal gland. METHODS: Hormonal profile disclosed increased levels of estradiol and slightly low testosterone levels. The basal and stimulated LH levels were normal, whereas basal and stimulated FSH levels were totally suppressed. Cortisol and adrenal androgen levels were normal. The unusual finding of selective FSH suppression suggested secretion of inhibin B by the adrenocortical tumor. A very high level of serum inhibin B (405 pg/ml) was demonstrated by ELISA assay. Right adrenalectomy and nephrectomy were performed and the tumor was classified as a malignant tumor (Weiss score: 7.0) and unilateral mastectomy disclosed a lipoma. RESULTS: One week after surgery, a GnRH-stimulation test disclosed normal basal and stimulated FSH levels and low levels of inhibin B and estradiol. Immunohistochemical analysis with anti-B-inhibin antibody revealed intense staining in the adrenocortical tumor cells. One month after surgery, an abdominal magnetic resonance imaging revealed a local recurrence of the tumor and a second surgery was performed with partial resection of the tumor and the patient died 1 year after the first surgery. CONCLUSION: We herein report the first inhibin B and estradiol-secreting adrenocortical carcinoma. The unusual selective inhibition of FSH secretion should be considered a valuable hormonal finding for the diagnosis of inhibin B-secreting adrenocortical tumors.     

Overlooked Mountain Rock Pools in Deserts Are Critical Local Hotspots of Biodiversity

BackgroundThe world is undergoing exceptional biodiversity loss. Most conservation efforts target biodiversity hotspots at large scales. Such approach overlooks small-sized local hotspots, which may be rich in endemic and highly threatened species. We explore the importance of mountain rock pools (gueltas) as local biodiversity hotspots in the Sahara-Sahel. Specifically, we considered how many vertebrates (total and endemics) use gueltas, what factors predict species richness, and which gueltas are of most priority for conservation. We expected to provide management recommendations, improve local biodiversity conservation, and simultaneously contribute with a framework for future enhancement of local communities’ economy. The identification of local hotspots of biodiversity is important for revaluating global conservation priorities.Conclusion/SignificanceGueltas are crucial for local biodiversity conservation and human activities. They require urgent management plans in Mauritania’s mountains. They could provide refugia under climate change being important for long-term conservation of Sahara-Sahel biodiversity. Given their disproportional importance in relation to their size, they are local hotspots of biodiversity deserving global attention.