Molecular and lipid biomarker analysis of a gypsum-hosted endoevaporitic microbial community.

Modern evaporitic microbial ecosystems are important analogs for understanding the record of earliest life on Earth. Although mineral-depositing shallow-marine environments were prevalent during the Precambrian, few such environments are now available today for study. We investigated the molecular and lipid biomarker composition of an endoevaporitic gypsarenite microbial mat community in Guerrero Negro, Mexico. The 16S ribosomal RNA gene-based phylogenetic analyses of this mat corroborate prior observations indicating that characteristic layered microbial communities colonize gypsum deposits world-wide despite considerable textural and morphological variability. Membrane fatty acid analysis of the surface tan/orange and lower green mat crust layers indicated cell densities of 1.6 × 10(9) and 4.2 × 10(9)  cells cm(-3) , respectively. Several biomarker fatty acids, ∆7,10-hexadecadienoic, iso-heptadecenoic, 10-methylhexadecanoic, and a ∆12-methyloctadecenoic, correlated well with distributions of Euhalothece, Stenotrophomonas, Desulfohalobium, and Rhodobacterales, respectively, revealed by the phylogenetic analyses. Chlorophyll (Chl) a and cyanobacterial phylotypes were present at all depths in the mat. Bacteriochlorophyl (Bchl) a and Bchl c were first detected in the oxic-anoxic transition zone and increased with depth. A series of monomethylalkanes (MMA), 8-methylhexadecane, 8-methylheptadecane, and 9-methyloctadecane were present in the surface crust but increased in abundance in the lower anoxic layers. The MMA structures are similar to those identified previously in cultures of the marine Chloroflexus-like organism 'Candidatus Chlorothrix halophila' gen. nov., sp. nov., and may represent the Bchl c community. Novel 3-methylhopanoids were identified in cultures of marine purple non-sulfur bacteria and serve as a probable biomarker for this group in the lower anoxic purple and olive-black layers. Together microbial culture and environmental analyses support novel sources for lipid biomarkers in gypsum crust mats.

Characterization and spatial distribution of methanogens and methanogenic biosignatures in hypersaline microbial mats of Baja California.

Well-developed hypersaline cyanobacterial mats from Guerrero Negro, Baja California Sur, sustain active methanogenesis in the presence of high rates of sulfate reduction. Very little is known about the diversity and distribution of the microorganisms responsible for methane production in these unique ecosystems. Applying a combination of 16S rRNA and metabolic gene surveys, fluorescence in situ hybridization, and lipid biomarker analysis, we characterized the diversity and spatial relationships of methanogens and other archaea in the mat incubation experiments stimulated with methanogenic substrates. The phylogenetic and chemotaxonomic diversity established within mat microcosms was compared with the archaeal diversity and lipid biomarker profiles associated with different depth horizons in the in situ mat. Both archaeal 16S rRNA and methyl coenzyme M reductase gene (mcrA) analysis revealed an enrichment of diverse methanogens belonging to the Methanosarcinales in response to trimethylamine addition. Corresponding with DNA-based detection methods, an increase in lipid biomarkers commonly synthesized by methanogenic archaea was observed, including archaeol and sn-2-hydroxyarchaeol polar lipids, and the free, irregular acyclic isoprenoids, 2,6,10,15,19-pentamethylicosene (PMI) and 2,6,11,15-tetramethylhexadecane (crocetane). Hydrogen enrichment of a novel putative archaeal polar C(30) isoprenoid, a dehydrosqualane, was also documented. Both DNA and lipid biomarker evidence indicate a shift in the dominant methanogenic genera corresponding with depth in the mat. Specifically, incubations of surface layers near the photic zone predominantly supported Methanolobus spp. and PMI, while Methanococcoides and hydroxyarchaeol were preferentially recovered from microcosms of unconsolidated sediments underlying the mat. Together, this work supports the existence of small but robust methylotrophic methanogen assemblages that are vertically stratified within the benthic hypersaline mat and can be distinguished by both their DNA signatures and unique isoprenoid biomarkers.

Lipid biomarker and phylogenetic analyses to reveal archaeal biodiversity and distribution in hypersaline microbial mat and underlying sediment.

This study has utilized the tools of lipid biomarker chemistry and molecular phylogenetic analyses to assess the archaeal contribution to diversity and abundance within a microbial mat and underlying sediment from a hypersaline lagoon in Baja California. Based on abundance of ether-linked isoprenoids, archaea made up from 1 to 4% of the cell numbers throughout the upper 100 mm of mat and sediment core. Below this depth archaeal lipid was two times more abundant than bacterial. Archaeol was the primary archaeal lipid in all layers. Relatively small amounts of caldarchaeol (dibiphytanyl glyceroltetraether) were present at most depths with phytanyl to biphytanyl molar ratios lowest (approximately 10 : 1) in the 4-17 mm and 100-130 mm horizons, and highest (132 : 1) in the surface 0-2 mm. Lipids with cyclic biphytanyl cores were only detected below 100 mm. A novel polar lipid containing a C(30) isoprenoid (squalane) moiety was isolated from the upper anoxic portion of the core and partially characterized. Hydrocarbon biomarker lipids included pentamethylicosane (2-10 mm) and crocetane (primarily below 10 mm). Archaeal molecular diversity varied somewhat with depth. With the exception of samples at 0-2 mm and 35-65 mm, Thermoplasmatales of marine benthic group D dominated clone libraries. A significant number of phylotypes representing the Crenarchaeota from marine benthic group B were generally present below 17 mm and dominated the 35-65 mm sample. Halobacteriaceae family made up 80% of the clone library of the surface 2 mm, and consisted primarily of sequences affiliated with the haloalkaliphilic Natronomonas pharaonis.

Stable carbon isotope ratios of lipid biomarkers of sulfate-reducing bacteria.

We examined the potential use of natural-abundance stable carbon isotope ratios of lipids for determining substrate usage by sulfate-reducing bacteria (SRB). Four SRB were grown under autotrophic, mixotrophic, or heterotrophic growth conditions, and the delta13C values of their individual fatty acids (FA) were determined. The FA were usually 13C depleted in relation to biomass, with Deltadelta13C(FA - biomass) of -4 to -17 per thousand; the greatest depletion occurred during heterotrophic growth. The exception was Desulfotomaculum acetoxidans, for which substrate limitation resulted in biomass and FA becoming isotopically heavier than the acetate substrate. The delta13C values of FA in Desulfotomaculum acetoxidans varied with the position of the double bond in the monounsaturated C16 and C18 FA, with FA becoming progressively more 13C depleted as the double bond approached the methyl end. Mixotrophic growth of Desulfovibrio desulfuricans resulted in little depletion of the i17:1 biomarker relative to biomass or acetate, whereas growth with lactate resulted in a higher proportion of i17:1 with a greater depletion in 13C. The relative abundances of 10Me16:0 in Desulfobacter hydrogenophilus and Desulfobacterium autotrophicum were not affected by growth conditions, yet the Deltadelta13C(FA - substrate) values of 10Me16:0 were considerably greater during autotrophic growth. These experiments indicate that FA delta13C values can be useful for interpreting carbon utilization by SRB in natural environments.

Signature lipids and stable carbon isotope analyses of Octopus Spring hyperthermophilic communities compared with those of Aquificales representatives.

The molecular and isotopic compositions of lipid biomarkers of cultured Aquificales genera have been used to study the community and trophic structure of the hyperthermophilic pink streamers and vent biofilm from Octopus Spring. Thermocrinis ruber, Thermocrinis sp. strain HI 11/12, Hydrogenobacter thermophilus TK-6, Aquifex pyrophilus, and Aquifex aeolicus all contained glycerol-ether phospholipids as well as acyl glycerides. The n-C(20:1) and cy-C(21) fatty acids dominated all of the Aquificales, while the alkyl glycerol ethers were mainly C(18:0). These Aquificales biomarkers were major constituents of the lipid extracts of two Octopus Spring samples, a biofilm associated with the siliceous vent walls, and the well-known pink streamer community (PSC). Both the biofilm and the PSC contained mono- and dialkyl glycerol ethers in which C(18) and C(20) alkyl groups were prevalent. Phospholipid fatty acids included both the Aquificales n-C(20:1) and cy-C(21), plus a series of iso-branched fatty acids (i-C(15:0) to i-C(21:0)), indicating an additional bacterial component. Biomass and lipids from the PSC were depleted in (13)C relative to source water CO(2) by 10.9 and 17.2 per thousand, respectively. The C(20-21) fatty acids of the PSC were less depleted than the iso-branched fatty acids, 18.4 and 22.6 per thousand, respectively. The biomass of T. ruber grown on CO(2) was depleted in (13)C by only 3.3 per thousand relative to C source. In contrast, biomass was depleted by 19.7 per thousand when formate was the C source. Independent of carbon source, T. ruber lipids were heavier than biomass (+1.3 per thousand). The depletion in the C(20-21) fatty acids from the PSC indicates that Thermocrinis biomass must be similarly depleted and too light to be explained by growth on CO(2). Accordingly, Thermocrinis in the PSC is likely to have utilized formate, presumably generated in the spring source region.

Microbial diversity of the brine-seawater interface of the Kebrit Deep, Red Sea, studied via 16S rRNA gene sequences and cultivation methods.

The brine-seawater interface of the Kebrit Deep, northern Red Sea, was investigated for the presence of microorganisms using phylogenetic analysis combined with cultivation methods. Under strictly anaerobic culture conditions, novel halophiles were isolated. The new rod-shaped isolates belong to the halophilic genus Halanaerobium and are the first representatives of the genus obtained from deep-sea, anaerobic brine pools. Within the genus Halanaerobium, they represent new species which grow chemoorganotrophically at NaCl concentrations ranging from 5 to 34%. The cellular fatty acid compositions are consistent with those of other Halanaerobium representatives, showing unusually large amounts of Delta7 and Delta11 16:1 fatty acids. Phylogenetic analysis of the brine-seawater interface sample revealed the presence of various bacterial 16S rRNA gene sequences dominated by cultivated members of the bacterial domain, with the majority affiliated with the genus Halanaerobium. The new Halanaerobium 16S rRNA clone sequences showed the highest similarity (99.9%) to the sequence of isolate KT-8-13 from the Kebrit Deep brine. In this initial survey, our polyphasic approach demonstrates that novel halophiles thrive in the anaerobic, deep-sea brine pool of the Kebrit Deep, Red Sea. They may contribute significantly to the anaerobic degradation of organic matter enriched at the brine-seawater interface.

Toward better access to health insurance coverage for U.S. retirees in Mexico.

Many retirees from the United States of America have limited health insurance coverage while living in Mexico. Medicare and Medicaid benefits are not portable to other countries and Medigap (private insurance that supplements Medicare) is very limited. This causes economic and medical hardships and serves as a barrier to retirement to Mexico. Increasing numbers of U.S. retirees will be interested in moving to Mexico in the future because of the climate, the culture, and the lower cost of living. The numbers are increasing as a result of several factors such as aging "baby boomers" and the rapidly growing Mexican-origin population in the U.S.A. who are citizens or permanent residents but would like to return to their communities of origin after working in the U.S.A. There are several policy initiatives that could provide opportunities for improving health insurance coverage for these retirees that could be cost-effective.

2-Methylhopanoids as biomarkers for cyanobacterial oxygenic photosynthesis.

Oxygenic photosynthesis is widely accepted as the most important bioenergetic process happening in Earth's surface environment. It is thought to have evolved within the cyanobacterial lineage, but it has been difficult to determine when it began. Evidence based on the occurrence and appearance of stromatolites and microfossils indicates that phototrophy occurred as long ago as 3,465 Myr although no definite physiological inferences can be made from these objects. Carbon isotopes and other geological phenomena provide clues but are also equivocal. Biomarkers are potentially useful because the three domains of extant life-Bacteria, Archaea and Eukarya-have signature membrane lipids with recalcitrant carbon skeletons. These lipids turn into hydrocarbons in sediments and can be found wherever the record is sufficiently well preserved. Here we show that 2-methyl-bacteriohopanepolyols occur in a high proportion of cultured cyanobacteria and cyanobacterial mats. Their 2-methylhopane hydrocarbon derivatives are abundant in organic-rich sediments as old as 2,500 Myr. These biomarkers may help constrain the age of the oldest cyanobacteria and the advent of oxygenic photosynthesis. They could also be used to quantify the ecological importance of cyanobacteria through geological time.

Measurement of hydroxyl radical-generated methane sulfinic acid by high-performance liquid chromatography and electrochemical detection.

A liquid chromatographic (HPLC) method has been developed for direct quantitative determination of methane sulfinic acid (MSA) produced by hydroxyl radical oxidation of dimethyl sulfoxide. This method measures MSA directly by HPLC separation and electrochemical oxidation following rapid extraction from intact cells. MSA can be measured in tissue extracts at 0.04 nmol (equivalent to 2 microM). Using this technique, MSA production in paraquat-treated bean leaves is demonstrated. When compared with the widely used dye-binding technique, this method simplifies the preparation of the extract by eliminating two steps required in the dye-binding method: removal of interfering lipophilic compounds and the derivitization (color reaction) of the MSA.

Carbon isotopic fractionation in lipids from methanotrophic bacteria II: the effects of physiology and environmental parameters on the biosynthesis and isotopic signatures of biomarkers.

Controls on the carbon isotopic signatures of methanotroph biomarkers have been further explored using cultured organisms. Growth under conditions which select for the membrane-bound particulate form of the methane monooxygenase enzyme (pMMO) leads to a significantly higher isotopic fractionation than does growth based on the soluble isozyme in both RuMP and serine pathway methanotrophs; in an RuMP type the delta delta 13Cbiomass equaled -23.9% for pMMO and -12.6% for sMMO. The distribution of biomarker lipids does not appear to be significantly affected by the dominance of one or the other MMO type and their isotopic compositions generally track those of the parent biomass. The 13C fractionation behaviour of serine pathway methanotrophs is very complex, reflecting the assimilation of both methane and carbon dioxide and concomitant dissimilation of methane-derived carbon. A limitation in CH4 availability leads to the production of biomass which is 13C-enriched with respect to both carbon substrates and this occurs irrespective of MMO type. This startling result indicates that there must be an additional fractionation step downstream from the MMO reaction which leads to incorporation of 13C-enriched carbon at the expense of dissimilation of 13C-depleted CO2. In these organisms, polyisoprenoid lipids are 13C-enriched compared to polymethylenic lipid which is the reverse of that found in the RuMP types. Serine cycle hopanoids, for example, can vary anywhere from 12% depleted to 10% enriched with respect to the CH4 substrate depending on its concentration. Decrease in growth temperature caused an overall increase in isotopic fractionation. In the total biomass, this effect tended to be masked by physiological factors associated with the type of organism and variation in the bulk composition. The effect was, however, clearly evident when monitoring the 13C signature of total lipid and individual biomarkers. Our results demonstrate that extreme carbon isotopic depletion in field samples and fossil biomarker lipids can be indicative of methanotrophy but the converse is not always true. For example, the hopanoids of a serine cycle methanotroph may be isotopically enriched by more than 10% compared to the substrate methane when the latter is limiting. In other words, hopanoids from some methanotrophs such as M. trichosporium would be indistinguishable from those of cyanobacteria or heterotrophic bacteria on the basis of either chemical structure or carbon isotopic signature.

Biomarkers as tracers for life on early earth and Mars.

Biomarkers in geological samples are products derived from biochemical (natural product) precursors by reductive and oxidative processes (e.g., cholestanes from cholesterol). Generally, lipids, pigments and biomembranes are preserved best over longer geological times and labile compounds such as amino acids, sugars, etc. are useful biomarkers for recent times. Thus, the detailed characterization of biomarker compositions permits the assessment of the major contributing species of extinct and/or extant life. In the case of the early Earth, work has progressed to elucidate molecular structure and carbon isotropic signals preserved in ancient sedimentary rocks. In addition, the combination of bacterial biochemistry with the organic geochemistry of contemporary and ancient hydrothermal ecosystems permits the modeling of the nature, behavior and preservation potential of primitive microbial communities. This approach uses combined molecular and isotopic analyses to characterize lipids produced by cultured bacteria (representative of ancient strains) and to test a variety of culture conditions which affect their biosynthesis. On considering Mars, the biomarkers from lipids and biopolymers would be expected to be preserved best if life flourished there during its early history (3.5-4 x 10(9) yr ago). Both oxidized and reduced products would be expected. This is based on the inferred occurrence of hydrothermal activity during that time with the concomitant preservation of biochemically-derived organic matter. Both known biomarkers (i.e., as elucidated for early terrestrial samples and for primitive terrestrial microbiota) and novel, potentially unknown compounds should be characterized.

Squalene-hopene cyclase from Methylococcus capsulatus (Bath): a bacterium producing hopanoids and steroids.

We report the cloning and characterisation of the Methylococcus capsulatus shc gene, which encodes the squalene-hopene cyclase (SHC). This enzyme catalyses the complex cyclization of squalene to the pentacyclic triterpene skeleton of hopanoids and represents the key reaction in this biosynthesis. Using a combination of PCR amplification and DNA hybridization, two overlapping 2.6 kb PstI and 3.3 kb SalI DNA fragments were cloned bearing a 1962 bp open reading frame encoding a 74 kDa protein with 654 amino acids and a predicted isoelectric point at about pH 6.3. The deduced amino acid sequence of the M. capsulatus shc gene showed significant similarity to known prokaryotic SHCs and to a lesser degree to the related eukaryotic oxidosqualene cyclases (OSCs). Like other triterpene cyclases, the M. capsulatus SHC contains seven so-called QW-motifs as well as an aspartate-rich domain. The recombinant M. capsulatus SHC was expressed in Escherichia coli and in vitro activity of the recombinant cyclase was demonstrated using crude cell-free lysate or solubilized membrane preparation. The cyclization products hop-22-ene and hopan-22-ol (diplopterol) were identified by GC and GC-MS.

Inhibition of Methane Oxidation by Methylococcus capsulatus with Hydrochlorofluorocarbons and Fluorinated Methanes.

The inhibition of methane oxidation by cell suspensions of Methylococcus capsulatus (Bath) exposed to hydrochlorofluorocarbon 21 (HCFC-21; difluorochloromethane [CHF(inf2)Cl]), HCFC-22 (fluorodichloromethane [CHFCl(inf2)]), and various fluorinated methanes was investigated. HCFC-21 inhibited methane oxidation to a greater extent than HCFC-22, for both the particulate and soluble methane monooxygenases. Among the fluorinated methanes, both methyl fluoride (CH(inf3)F) and difluoromethane (CH(inf2)F(inf2)) were inhibitory while fluoroform (CHF(inf3)) and carbon tetrafluoride (CF(inf4)) were not. The inhibition of methane oxidation by HCFC-21 and HCFC-22 was irreversible, while that by methyl fluoride was reversible. The HCFCs also proved inhibitory to methanol dehydrogenase, which suggests that they disrupt other aspects of C(inf1) catabolism in addition to methane monooxygenase activity.

High-dose chemotherapy, autologous bone marrow or stem cell transplantation and post-transplant consolidation chemotherapy in patients with advanced breast cancer.

This study was designed to determine the complete response (CR) rate, event-free survival (EFS) and overall survival (OS) in patients with metastatic breast cancer treated with an adriamycin-based induction regimen, high-dose chemotherapy consisting of cyclophosphamide and thiotepa with autologous bone marrow or stem cell reinfusion, followed by post-transplant 5-fluorouracil and cisplatin. Forty-eight consecutive patients were enrolled and 35 received two to four cycles of a cytoreductive chemotherapy regimen followed by high-dose chemotherapy which included cyclophosphamide and thiotepa. Thirty-three patients with non-progressive disease received at least one cycle of post-transplant 5-fluorouracil and cisplatin. Fifty percent of patients with evaluable disease responded to induction chemotherapy. Three of the 34 patients (9%) evaluable for response to high-dose chemotherapy achieved CR, eight (24%) achieved partial response (PR), 12 (35%) had stable disease (SD) and 11 (32%) had progressive disease (PD). The median time to neutrophil recovery was 11.5 days (range, 8 to 40 days) post- reinfusion. The median time to platelet independence was 14.5 days (range, 8 to 44 days). The median follow-up is 24.5 months (range, 1 to 96 months). The actuarial probability of EFS for all patients is 17% at 4 years. The EFS for patients receiving all four cycles of post-transplant chemotherapy is 27% at 4 years, compared to 36% at 1 year for patients not receiving any post-transplant chemotherapy. Ten of the 48 patients (21%) are alive, and seven of these (15%) have no evidence of disease. High-dose chemotherapy with autologous bone marrow or peripheral blood-derived stem cell transplantation followed by post-transplant consolidation chemotherapy in patients with metastatic breast cancer results in a proportion of patients without evidence of disease at 4 years.

Lipid biomarkers for bacterial ecosystems: studies of cultured organisms, hydrothermal environments and ancient sediments.

This paper forms part of our long-term goal of using molecular structure and carbon isotopic signals preserved as hydrocarbons in ancient sediments to improve understanding of the early evolution of Earth's surface environment. We are particularly concerned with biomarkers which are informative about aerobiosis. Here, we combine bacterial biochemistry with the organic geochemistry of contemporary and ancient hydrothermal ecosystems to construct models for the nature, behaviour and preservation potential of primitive microbial communities. We use a combined molecular and isotopic approach to characterize lipids produced by cultured bacteria and test a variety of culture conditions which affect their biosynthesis. This information is then compared with lipid mixtures isolated from contemporary hot springs and evaluated for the kinds of chemical change that would accompany burial and incorporation into the sedimentary record. In this study we have shown that growth temperature does not appear to alter isotopic fractionation within the lipid classes produced by a methanotropic bacterium. We also found that cultured cyanobacteria biosynthesize diagnostic methylalkanes and dimethylalkanes with the latter only made when growing under low pCO2. In an examination of a microbial mat sample from Octopus Spring, Yellowstone National Park (USA), we could readily identify chemical structures with 13C contents which were diagnostic for the phototrophic organisms such as cyanobacteria and Chloroflexus. We could not, however, find molecular evidence for operation of a methane cycle in the particular mat samples we studied.

Phase I/II study of combined granulocyte colony-stimulating factor and granulocyte-macrophage colony-stimulating factor administration for the mobilization of hematopoietic progenitor cells.

PURPOSE: To study the toxicity and efficacy of combined granulocyte colony-stimulating factor (G-CSF) and granulocyte-macrophage colony-stimulating factor (GM-CSF) administration for mobilization of hematopoietic progenitor cells (HPCs). MATERIALS AND METHODS: Cohorts of a minimum of five patients each were treated subcutaneously as follows: G-CSF 5 micrograms/kg on days 1 to 12 and GM-CSF at .5, 1, or 5 micrograms/kg on days 7 to 12 (cohorts 1, 2, and 3); GM-CSF 5 micrograms/kg on days 1 to 12 and G-CSF 5 micrograms/kg on days 7 to 12 (cohort 4); and G-CSF and GM-CSF 5 micrograms/kg each on days 1 to 12 (cohort 5). Ten-liter aphereses were performed on days 1 (baseline, pre-CSF), 5, 7, 11, and 13. Colony assays for granulocyte-macrophage colony-forming units (CFU-GM) and erythroid burst-forming units (BFU-E) were performed on each harvest. RESULTS: The principal toxicities were myalgias, bone pain, fever, nausea, and mild thrombocytopenia, but none was dose-limiting. Four days of treatment with either G-CSF or GM-CSF resulted in dramatic and sustained increases in the numbers of CFU-GM per kilogram collected per harvest that represented 35.6 +/- 8.9- and 33.7 +/- 13.0-fold increases over baseline, respectively. This increment was attributable both to increased numbers of mononuclear cells collected per 10-L apheresis and to increased concentrations of progenitors within each collection. The administration of G-CSF to patients already receiving GM-CSF (cohort 4) caused the HPC content to surge to nearly 80-fold the baseline (P = .024); the reverse sequence, ie, the addition of GM-CSF to G-CSF, was less effective. The CFU-GM content of the baseline aphereses correlated with the maximal mobilization achieved (r = .74, P = .001). CONCLUSION: Combined G-CSF and GM-CSF administration effectively and predictably mobilizes HPCs and facilitates apheresis.

Identification of methanotrophic lipid biomarkers in cold-seep mussel gills: chemical and isotopic analysis.

A lipid analysis of the tissues of a cold-seep mytilid mussel collected from the Louisiana slope of the Gulf of Mexico was used in conjunction with a compound-specific isotope analysis to demonstrate the presence of methanotrophic symbionts in the mussel gill tissue and to demonstrate the host's dependence on bacterially synthesized metabolic intermediates. The gill tissue contained large amounts of group-specific methanotrophic biomarkers, bacteriohopanoids, 4-methylsterols, lipopolysaccharide-associated hydroxy fatty acids, and type I-specific 16:1 fatty acid isomers with bond positions at delta 8, delta 10, and delta 11. Only small amounts of these compounds were detected in the mantle or other tissues of the host animal. A variety of cholesterol and 4-methylsterol isomers were identified as both free and steryl esters, and the sterol double bond positions suggested that the major bacterially derived gill sterol [11.0% 4 alpha-methyl-cholesta-8(14),24-dien-3 beta-ol] was converted to host cholesterol (64.2% of the gill sterol was cholest-5-en-3 beta-ol). The stable carbon isotope values for gill and mantle preparations were, respectively, -59.0 and -60.4% for total tissue, -60.6 and -62.4% for total lipids, -60.2 and-63.9% for phospholipid fatty acids, and -71.8 and 73.8% for sterols. These stable carbon isotope values revealed that the relative fractionation pattern was similar to the patterns obtained in pure culture experiments with methanotrophic bacteria (R.E. Summons, L.L. Jahnke, and Z. Roksandic, Geochim. Cosmochim. Acta 58: 2853-2863, 1994) further supporting the conversion of the bacteria methylsterol pool.

Degradation of methyl bromide by methanotrophic bacteria in cell suspensions and soils.

Cell suspensions of Methylococcus capsulatus mineralized methyl bromide (MeBr), as evidence by its removal from the gas phase, the quantitative recovery of Br- in the spent medium, and the production of 14CO2 from [14C]MeBr. Methyl fluoride fluoride (MeF) inhibited oxidation of methane as well as that of [14C]MeBr. The rate of MeBr consumption by cells varied inversely with the supply of methane, which suggested a competitive relationship between these two substrates. However, MeBr did not support growth of the methanotroph. In soils exposed to high levels (10,000 ppm) of MeBr, methane oxidation was completely inhibited. At this concentration, MeBr removal rates were equivalent in killed and live controls, which indicated a chemical rather than biological removal reaction. At lower concentration (1,000 ppm) of MeBr, methanotrophs were active and MeBr consumption rates were 10-fold higher in live controls than in killed controls. Soils exposed to trace levels (10 ppm) of MeBr demonstrated complete consumption within 5 h of incubation, while controls inhibited with MeF or incubated without O2 had 50% lower removal rates. Aerobic soils oxidized [14C]MeBr to 14CO2, and MeF inhibited oxidation by 72%. Field experiments demonstrated slightly lower MeBr removal rates in chambers containing MeF than in chambers lacking MeF. Collectively, these results show that soil methanotrophic bacteria, as well as other microbes, can degrade MeBr present in the environment.

An evaluation of intravenous immunoglobulin in the treatment of human immunodeficiency virus-associated thrombocytopenia.

BACKGROUND: Anecdotal evidence suggests that high-dose intravenous immunoglobulin (IVIG) is useful in the management of human immunodeficiency virus (HIV)-associated thrombocytopenia. STUDY DESIGN AND METHODS: To rigorously evaluate this therapy, a crossover study was designed to compare IVIG, given at 1 g per kg per day for 2 consecutive days each week for 4 weeks, with intravenous saline placebo administered according to the same schedule. Subjects were randomly assigned to receive either IVIG or saline during the first 4 weeks; if IVIG was given, there was a 4-week period of no therapy before beginning placebo administration. Criteria for eligibility were platelet count of less than 50,000 per microL (50 x 10(9)/L), elevated platelet-associated IgG levels, increased megakaryocytes in the bone marrow, and positive HIV antibody test. Twelve patients (11 men, 1 woman) were studied. Seven patients completed the full protocol. Four dropped out: after 2, 5 (2 patients), and 8 weeks that included at least 2 weeks of IVIG. RESULTS: All patients sustained an increase in platelet count in response to IVIG, with increments ranging from 15,000 to 358,000 per microL (15 to 350 x 10(9)/L) (mean, 180,000/microL [180 x 10(9)/L]; median, 174,000/microL [174 x 10(9)/L]). No patient had an increase after placebo infusions. There were no adverse effects of treatment, and weekly chemical analyses showed no new abnormalities except for mild elevations in the serum protein. The duration of responses ranged from 2 to 10 weeks. No patient demonstrated refractoriness to IVIG. CONCLUSION: IVIG consistently raises platelet counts in patients with HIV-associated thrombocytopenia.

Carbon isotopic fractionation in lipids from methanotrophic bacteria: relevance for interpretation of the geochemical record of biomarkers.

Experiments with cultured aerobic methane oxidising bacteria confirm that their biomarker lipids will be significantly depleted in 13C compared to the substrate. The methanotrophic bacteria Methylococcus capsulatus and Methylomonas methanica, grown on methane and using the RuMP cycle for carbon assimilation, show maximum 13C fractionation of approximately 30% in the resultant biomass. In M. capsulatus, the maximum fractionation is observed in the earliest part of the exponential growth stage and decreases to approximately 16% as cells approach stationary phase. This change may be associated with a shift from the particulate form to the soluble form of the methane monooxygenase enzyme. Less than maximum fractionation is observed when cells are grown with reduced methane availability. Biomass of M. capsulatus grown on methanol was depleted by 9% compared to the substrate. Additional strong 13C fractionation takes place during polyisoprenoid biosynthesis in methanotrophs. The delta 13C values of individual hopanoid and steroid biomarkers produced by these organisms were as much as l0% more negative than total biomass. In individual cultures, squalene was 13C-enriched by as much as 14% compared to the triterpane skeleton of bacteriohopaneaminopentol. Much of the isotopic dispersion in lipid metabolites could be attributed to shifts in their relative abundances, combined with an overall reduction in fractionation during the growth cycle. In cells grown on methanol, where there was no apparent effect of growth stage on overall fractionation there were still significant isotopic differences between closely related lipids including a 5.3% difference between the hopane and 3 beta-methylhopane skeletons. Hopane and sterane polyisoprenoids were also 13C-depleted compared to fatty acids. These observations have significant implications for the interpretation of specific compound isotopic signatures now being measured for hydrocarbons and other lipids present in sediments and petroleum. In particular, biomarker lipids produced by a single organism do not necessarily have the same carbon isotopic composition.