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Rationale and Objective: Neuropsychiatric systemic lupus erythematosus (NPSLE) is a diagnostically challenging, severe, and life-threatening condition, which is currently lacking a "gold standard." Our aim with this study is to look for magnetic resonance (MR) perfusion differences in NPSLE, SLE, and healthy control (HC) patients and correlate our findings with clinical parameters.Materials and Methods: Twenty-four NPSLE patients, 21 SLE patients, and 21 HC underwent dynamic susceptibility contrast enhanced MR perfusion using a 3-T scanner. Nine prospectively selected intracranial regions of interest were placed in white and gray matter and the cerebral blood flow (CBF), cerebral blood volume (CBV), and mean transit time (MTT) values were calculated. Subjects underwent clinical evaluation with SLEDAI and serum antibodies.Results: The SLE patients had higher CBF and CBV compared to the HC overall (P = .01) and in specific areas (P = .03-.048). SLE patients with signs of active disease (elevated SLEDAI and anti-double-stranded DNA) had significantly elevated CBV, CBF, and MTT in the posterior cingulate gyrus (P = .01-.02). No significant difference was seen in the magnetic resonance perfusion measurements of NPSLE patients compared to SLE and HC, although the NPSLE patients also showed higher CBV variability compared to the SLE (P = .0004) and HC cohort (P < .0001).Conclusion: SLE patients have increased CBV and CBF compared to healthy controls. The SLE patients with clinical markers for active disease have elevated CBV, CBF, and MTT in the posterior cingulate gyrus. NPSLE patients show increased variability in perfusion measurements, which may explain why susceptibility contrast enhanced MRI has not yet provided a specific target for NPSLE. [ABSTRACT FROM AUTHOR]

Abstract Background Hospital HIV care and public sexual health care (a Sexual Health Care Centre) services were integrated to provide sexual health counselling and sexually transmitted infections (STIs) testing and treatment (sexual health care) to larger numbers of HIV patients. Services, need and usage were assessed using a patient perspective, which is a key factor for the success of service integration. Methods The study design was a one-group pre-test and post-test comparison of 447 HIV-infected heterosexual individuals and men who have sex with men (MSM) attending a hospital-based HIV centre serving the southern region of the Netherlands. The intervention offered comprehensive sexual health care using an integrated care approach. The main outcomes were intervention uptake, patients’ pre-test care needs (n=254), and quality rating. Results Pre intervention, 43% of the patients wanted to discuss sexual health (51% MSM; 30% heterosexuals). Of these patients, 12% to 35% reported regular coverage, and up to 25% never discussed sexual health topics at their HIV care visits. Of the patients, 24% used our intervention. Usage was higher among patients who previously expressed a need to discuss sexual health. Most patients who used the integrated services were new users of public health services. STIs were detected in 13% of MSM and in none of the heterosexuals. The quality of care was rated good. Conclusions The HIV patients in our study generally considered sexual health important, but the regular counselling and testing at the HIV care visit was insufficient. The integration of public health and hospital services benefited both care sectors and their patients by addressing sexual health questions, detecting STIs, and conducting partner notification. Successful sexual health care uptake requires increased awareness among patients about their care options as well as a cultural shift among care providers.

The American Dreaming project, a community-based participatory research study conducted in collaboration with the nonprofit organization Define American, focuses on bolstering the mental health of immigrant rights advocates between the ages of 25 and 40 years. All participants identify as undocumented or formerly undocumented and have been public with their status since 2012. Seven people were recruited for the pilot program to participate in a digital storytelling (DST) workshop. They created digital stories (videos approximately 3 minutes in duration) from a series of general story prompts about their lives. Participants also completed pre- and posttests with measures assessing change in posttraumatic stress disorder, stress, self-esteem, depression, belonging, social support, and resilience. Follow-up, semistructured workshop evaluations were gathered, and life history interviews were conducted. The project also included a strategic communications component. In this article, I outline ethical challenges faced as the goals and parameters of the project shifted while conducting a multifaceted critical narrative intervention. The key lesson learned was to make space, prior to the DST workshop, for establishing and agreeing on project priorities with all stakeholders. I recommend creating a one-page memorandum of understanding that includes (1) a brief project description outlining the objectives, (2) goals listed in priority order, and (3) a decision tree to help stakeholders navigate competing interests. While DST is a powerful tool for supporting storyteller agency, working within structures of funding, support, and research can create challenges that critically complicate the narrative intervention.

The use of non-nutritive sweeteners such as saccharin is widely prevalent. Although saccharin is considered safe for human consumption, it produces behavioral changes in experimental animals. We report that saccharin's behavioral effects are much more pervasive than currently recognized. In a mouse model, saccharin exposure produced motor impulsivity not only in the saccharin-exposed males but also in their offspring. In addition, the offspring showed locomotor hyperactivity and working memory deficit not observed in fathers. Spermatazoal DNA was hypermethylated in the saccharin-exposed fathers, especially at dopamine receptor promoter regions, suggesting that epigenetic modification of germ cell DNA may mediate transgenerational transmission of behavioral phenotypes. Dopamine's role in hyperactivity was further highlighted by the finding that the stimulant drug methylphenidate mitigated the hyperactivity. Nicotine is another substance that is widely used. Its use via smokeless tobacco products, some of which contain saccharin, is on the rise contributing to concerns about adverse outcomes of co-exposure to saccharin and nicotine. We found that co-exposure of male mice to saccharin and nicotine produced significant behavioral impairment in their offspring. Thus, our data point to potential adverse neurobehavioral consequences of exposure to saccharin alone or saccharin and nicotine for the exposed individuals and their descendants.

Efficient nonprecious-metal oxygen reduction reaction (ORR) and oxygen evolution reaction (OER) electrocatalysts are key for the commercial viability of fuel cells, metal-air batteries, and water-splitting systems. Thus, high-performance ORR and OER electrocatalysts in acidic electrolytes are needed to support high-efficiency proton exchange membrane (PEM)-based systems. Herein, we report a new approach to design and prepare an ultrathin N-doped holey carbon layer (HCL) on a graphene sheet that exhibits outstanding bifunctional ORR/OER activities in both alkaline and acidic media. The edge sites of HCL are utilized to achieve selective doping of highly active pyridinic-N. The sandwiched graphene sheet provides mechanical support, stabilizes HCL structure and promotes charge transfer. The synergetic effect of the catalyst structure overcomes the drawbacks of holey graphene approaches. The resulting ORR and OER performances are equal to or better than the top-ranked electrocatalysts., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Use of tobacco products is injurious to health in men and women. However, tobacco use by pregnant women receives greater scrutiny because it can also compromise the health of future generations. More men smoke cigarettes than women. Yet the impact of nicotine use by men upon their descendants has not been as widely scrutinized. We exposed male C57BL/6 mice to nicotine (200 μg/mL in drinking water) for 12 wk and bred the mice with drug-naïve females to produce the F1 generation. Male and female F1 mice were bred with drug-naïve partners to produce the F2 generation. We analyzed spontaneous locomotor activity, working memory, attention, and reversal learning in male and female F1 and F2 mice. Both male and female F1 mice derived from the nicotine-exposed males showed significant increases in spontaneous locomotor activity and significant deficits in reversal learning. The male F1 mice also showed significant deficits in attention, brain monoamine content, and dopamine receptor mRNA expression. Examination of the F2 generation showed that male F2 mice derived from paternally nicotine-exposed female F1 mice had significant deficits in reversal learning. Analysis of epigenetic changes in the spermatozoa of the nicotine-exposed male founders (F0) showed significant changes in global DNA methylation and DNA methylation at promoter regions of the dopamine D2 receptor gene. Our findings show that nicotine exposure of male mice produces behavioral changes in multiple generations of descendants. Nicotine-induced changes in spermatozoal DNA methylation are a plausible mechanism for the transgenerational transmission of the phenotypes. These findings underscore the need to enlarge the current focus of research and public policy targeting nicotine exposure of pregnant mothers by a more equitable focus on nicotine exposure of the mother and the father., Competing Interests: I have read the journal's policy and the authors of this manuscript have the following potential competing interests. Pradeep Bhide: Dr. Bhide is a co-founder and consultant to Avekshan LLC, Tallahassee, FL, a pharmaceutical enterprise engaged in the development of novel therapies for attention deficit hyperactivity disorder (ADHD). Dr. Bhide is an inventor in following patents or patent applications relevant to ADHD therapy: US Patent, “Class of non-stimulant treatment and ADHD and related disorders” (#US9623023 B2), and US patent application, “Methods and compositions to prevent addiction (#US20130289061 A1). Deirdre McCarthy: Ms. McCarthy is a co-founder and consultant to Avekshan LLC, Tallahassee, FL, a pharmaceutical enterprise engaged in the development of novel therapies for attention deficit hyperactivity disorder (ADHD). Thomas Spencer: Dr. Spencer received research support or was a consultant from the following sources: Alcobra, Avekshan, Ironshore, Lundbeck, Shire Laboratories Inc, Sunovion, the FDA, and the Department of Defense. Consultant fees are paid to the Clinical Trials Network at the Massachusetts General Hospital (MGH) and not directly to Dr. Spencer. Dr. Spencer has been on an advisory board for the following pharmaceutical companies: Alcobra. Dr. Spencer received research support from Royalties and Licensing fees on copyrighted ADHD scales through MGH Corporate Sponsored Research and Licensing. Through MGH corporate licensing, Dr. Spencer is an inventor on a US Patent, “Class of non-stimulant treatment and ADHD and related disorders” (#US9623023 B2), and US patent application, “Methods and compositions to prevent addiction" (#US20130289061 A1). Joseph Biederman: Dr. Biederman is currently receiving research support from the following sources: AACAP, The Department of Defense, Food & Drug Administration, Headspace, Lundbeck, Neurocentria Inc., NIDA, PamLab, Pfizer, Shire Pharmaceuticals Inc., Sunovion, and NIH. Dr.  Biederman has a financial interest in Avekshan LLC, a company that develops treatments for attention deficit hyperactivity disorder (ADHD). His interests were reviewed and are managed by Massachusetts General Hospital and Partners HealthCare in accordance with their conflict of interest policies. Dr. Biederman’s program has received departmental royalties from a copyrighted rating scale used for ADHD diagnoses, paid by Ingenix, Prophase, Shire, Bracket Global, Sunovion, and Theravance; these royalties were paid to the Department of Psychiatry at MGH.  In 2017, Dr. Biederman is a consultant for Aevi Genomics, Akili, Guidepoint, Ironshore, Medgenics, and Piper Jaffray. He is on the scientific advisory board for Alcobra and Shire. He received honoraria from the MGH Psychiatry Academy for tuition-funded CME courses. Through MGH corporate licensing, he is an inventor on US Patent, “Class of non-stimulant treatment and ADHD and related disorders” (#US9623023 B2), and US patent application, “Methods and compositions to prevent addiction (#US20130289061 A1). In 2016, Dr. Biederman received honoraria from the MGH Psychiatry Academy for tuition-funded CME courses, and from Alcobra and APSARD. He was on the scientific advisory board for Arbor Pharmaceuticals. He was a consultant for Akili and Medgenics. He received research support from Merck and SPRITES. Thomas Morgan, Sara Lowe, and Matthew Williamson have no competing interests to declare.

Digital storytelling (DST) engages participants in a group-based process to create and share narrative accounts of life events. The process of individuals telling their own stories has not been well assessed as a mechanism of health behavior change. This study looks at outcomes associated with engaging in the DST process for vulnerable youth. The project focused on the experiences of Puerto Rican Latinas between the ages of 15 to 21. A total of 30 participants enrolled in a 4-day DST workshops, with 29 completing a 1 to 3-minute digital story. Self-reported data on several scales (self-esteem, social support, empowerment, and sexual attitudes and behaviors) were collected and analyzed. Participants showed an increase in positive social interactions from baseline to 3-month post workshop. Participants also demonstrated increases in optimism and control over the future immediately after the workshop, but this change was not sustained at 3 months. Analysis of qualitative results and implications are discussed., (© The Author(s) 2016.)

Objective: Aptamers are oligonucleotides targeting protein-protein interactions with pharmacokinetic profiles and activity reversal options. Although P-selectin and von Willebrand factor (vWF) have been implicated in the development of venous thrombosis (VT), no studies have directly compared aptamer efficacy with standard of care in VT. In this study, ARC5692, an anti-P-selectin aptamer, and ARC15105, an anti-vWF aptamer, were compared with low-molecular-weight heparin, enoxaparin, to test the efficacy of P-selectin or vWF inhibition in promoting thrombus resolution and preventing vein wall fibrosis, in a baboon model of VT., Approach and Results: Groups were as follows: treatment arm: animals received P-selectin or vWF aptamer inhibitors or enoxaparin (n=3 per group). Controls received no treatment (n=3). Prophylactic arm: animals received P-selectin inhibitor (n=4) or vWF inhibitor (n=3). Treatment arm: P-selectin-inhibitor demonstrated a significant improvement in vein recanalization by magnetic resonance venography (73% at day 21), and significantly decreased vein wall collagen, compared with all groups. Anti-P-selectin equaled enoxaparin in maintaining valve competency by ultrasound. All control animals had compromised valve competency post thrombosis. Prophylactic arm: animals receiving P-selectin and vWF inhibitors demonstrated improved vein recanalization by magnetic resonance venography versus controls (80% and 85%, respectively, at day 21). Anti-P-selectin protected iliac valve function better than anti-vWF, and both improved valve function versus controls. No adverse bleeding events were observed., Conclusions: The P-selectin inhibitor aptamer promoted iliac vein recanalization, preserved valve competency, and decreased vein wall fibrosis. The results of this work suggest that P-selectin inhibition maybe an ideal target in the treatment and prophylaxis of deep VT, warranting clinical trials., (© 2015 American Heart Association, Inc.)

Rationale and Objectives: To investigate for differences in metabolic concentrations and ratios between patients with systemic lupus erythematosus (SLE) without (group SLE) and those with neurological symptoms (group NPSLE) compared to a healthy control (group HC) in three normal-appearing brain regions: the frontal white matter, right insula (RI), and occipital gray matter and whether changes in any of the metabolites or metabolic ratios are correlated to disease activity and other clinical parameters., Materials and Methods: Twenty patients with SLE (18 women and 2 men, age range 23.4-64.6 years, mean age 43.9 years), 23 NPSLE patients (23 women, age range 23.7-69.8 years, mean age 42.4 years), and 21 HC (19 women and 2 men, age range 21.0-65.7 years, mean age 43.4 years) were included. All subjects had conventional brain magnetic resonance imaging and (1)H single-voxel spectroscopy, clinical assessment, and laboratory testing., Results: NPSLE patients had significantly reduced N-acetylaspartate (NAA)/creatine compared to HC (P = .02) and SLE patients (P = .01) in the RI. Lower glutamine/creatine levels were also detected in RI in both patient groups and in frontal white matter in NPSLE patients compared to HC (P = .01, P = .02). NAA/Cr ratio in the RI was significantly negatively correlated with the Systemic Lupus Erythematosus Disease Activity Index (r = -0.41; P = .008), and patients with active SLE symptoms also had a trend toward lower NAA/creatine ratios (1.02 vs 1.12; P = .07)., Conclusions: The present data support previous findings of abnormal metabolic changes in normal-appearing regions in the brain of both SLE and NPSLE patients and raise the possibility that especially NAA, glutamine, and glutamate may be additional biomarkers for cerebral disease activity in SLE patients as these early metabolic changes occur in the brain of SLE patients before neurologic and imaging manifestations become apparent., (Copyright © 2013 AUR. Published by Elsevier Inc. All rights reserved.)

Objective: Recent scientific findings have reinvigorated interest in examining the role of γ-aminobutyric acid (GABA), the major inhibitory central nervous system neurotransmitter, in chronic pain conditions. Decreased inhibitory neurotransmission is a proposed mechanism in the pathophysiology of chronic pain syndromes such as fibromyalgia (FM). The purpose of this study was to test the hypothesis that decreased levels of insular and anterior cingulate GABA would be present in FM patients, and that the concentration of this neurotransmitter would be correlated with pressure-pain thresholds., Methods: Sixteen FM patients and 17 age- and sex-matched healthy controls underwent pressure-pain testing and a 3T proton magnetic resonance spectroscopy session in which the right anterior insula, right posterior insula, anterior cingulate, and occipital cortex were examined in subjects at rest., Results: GABA levels in the right anterior insula were significantly lower in FM patients compared with healthy controls (mean ± SD 1.17 ± 0.24 arbitrary institutional units versus 1.42 ± 0.32 arbitrary institutional units; P = 0.016). There was a trend toward increased GABA levels in the anterior cingulate of FM patients compared with healthy controls (P = 0.06). No significant differences between groups were detected in the posterior insula or occipital cortex (P > 0.05 for all comparisons). Within the right posterior insula, higher levels of GABA were positively correlated with pressure-pain thresholds in the FM patients (Spearman's rho = 0.63; P = 0.02)., Conclusion: Diminished inhibitory neurotransmission resulting from lower concentrations of GABA within the right anterior insula may play a role in the pathophysiology of FM and other central pain syndromes., (Copyright © 2012 by the American College of Rheumatology.)

The allied health professions form approximately 18% of the health workforce in Australia and are well placed to contribute to future multidisciplinary models of health care. There are many reports describing the health workforce in Australia for the medical and nursing professions but there is very little information available about the nature of the allied health workforce. Recent studies have highlighted the need for more current and detailed information about the rural allied health workforce to inform future workforce planning. National health policy reform requires that new healthcare models take into account future workforce requirements, the distribution and work contexts of existing practitioners, training needs, workforce roles and scope of practice. The absence of accurate data profiling the existing rural allied health workforce makes this impossible. The Rural Allied Health Workforce Study (RAHWS) aims to use a cross-sectional survey instrument with high validity to provide a large scale but detailed profile of the allied health workforce in regional, rural and remote Australia. The RAHWS survey instrument used in this study is the result of a comprehensive consultation with clinicians, academics and managers. The RAHWS survey instrument has been designed to provide uniform data across a wide range of healthcare settings. Good concurrent and face validity have been demonstrated and its design allows for data analysis using a wide range of variables. Cross-correlation of responses can answer a number of research questions in relation to rural recruitment and retention, professional education and service delivery models. This valid and feasible instrument will be used to explore the rural allied health workforce by implementing the RAHWS survey in rural regions on a state-by-state basis in Australia during 2009 and 2010.

The behaviour of house cats Felis silvestris catus from nine litters was recorded at 4 months, 1 year and 2 years of age, in their home environment immediately after meals fed by their owners. We extracted by principal components analysis four elements of 'behavioural style' that were consistent from one age to another: based upon behaviour patterns that were most heavily loaded on each component, these were labelled as Staying Indoors, Rubbing, Investigative and Boldness elements. The Staying Indoors and Rubbing elements are similar to two aspects of behavioural style identified in a previous study of adult cats; the Boldness element, possibly coupled with the Investigative element, may be similar to the shy/bold continuum identified in controlled studies of cats and other species. Four-month-old male cats were the most likely to Stay Indoors; the Rubbing element increased with age in the majority of individuals, both male and female. Littermates tended to be similar to one another in Rubbing (at 4 months) and Boldness (up to 1 year). A positive effect of handling received during the first 8 weeks of life was detected for Boldness at 4 months of age. Copyright 2001 The Association for the Study of Animal Behaviour.

Two new naphthacenequinone glycosides, quanolirones I (1) and II (2) were isolated, together with the known compound galtamycin from the fermentation broth of Streptomyces sp. WC76535. The structures 1 and 2 were established by analysis of their spectroscopic data and by comparison of their data to those of galtamycin. Compounds 1, 2, and galtamycin showed inhibitory activity against HCMV protease with IC50 values of 14, 35, and 52 microM, respectively.

Bripiodionen (1), a new natural product, was isolated from Streptomyces sp. WC76599 during the screening of microbial fermentation extracts for their ability to inhibit human cytomegalovirus protease. The structure of 1 was elucidated by spectroscopic methods. Compound 1 displayed inhibitory activity against human cytomegalovirus protease with an IC50 value of 30 microM.

This paper describes the optimization of production of ascosteroside, a novel antifungal agent with an alpha-linked glycoside of a lanosterone-type triterpenoid structure. Glucose, sorbose and inositol were determined to be the best carbon sources for the production of ascosteroside. Temperature affected levels of ascosteroside, with production being highest at 16 degrees C with 1% glucose, and lowest at 32 degrees C. Dissolved oxygen levels were found to be critical in the production of ascosteroside in fermenter cultures. In order for production of ascosteroside to occur in fermenter cultures, the threshold level of dissolved oxygen was found to be above 26%.

The new cytotoxic agents rakicidins A and B were isolated from cultured broth of a Micromonospora sp. Spectroscopic and amino acid analysis has shown that rakicidin A is a new cyclic lipopeptide, consisting of 4-amino-penta-2,4-dienoic acid, 3-hydroxy-2,4,16-trimethyl-heptadecanoic acid, sarcosine, and 3-hydroxyasparagine. Rakicidin B differs by one methylene group in the lipid side chain. These compounds exhibited cytotoxicity against the M109 cell line.

During the screening of microbial fermentation extracts for their ability to inhibit the binding of 125I-peptid YY (PYY) to the neuropeptide Y (NPY) receptor using the scintillation proximity assay (SPA), BMS-192548 was isolated from the extract of Aspergillus niger WB2346 by bioassay-guided fractionation. BMS-192548 showed the inhibitory activity against 125I-PYY binding to SK-N-MC and SMS-KAN cells, which express NPY1 and NPY2 receptors, respectively, with IC50 values of 24 microM in Y1 and 27 microM in Y2 receptor binding. BMS-192548 demonstrated weak cytotoxicity against murine tumor cell line M-109 with an IC50 value of 240 microM.

The amylopullulanase gene (apu) of the thermophilic anaerobic bacterium Thermoanaerobacterium saccharolyticum B6A-RI was cloned into Escherichia coli. The complete nucleotide sequence of the gene was determined. It encoded a protein consisting of 1,288 amino acids with a signal peptide of 35 amino acids. The enzyme purified from E. coli was a monomer with an M(r) of 142,000 +/- 2,000 and had same the catalytic and thermal characteristics as the native glycoprotein from T. saccharolyticum B6A. Linear alignment and the hydrophobic cluster analysis were used to compare this amylopullulanase with other amylolytic enzymes. Both methods revealed strictly conserved amino acid residues among these enzymes, and it is proposed that Asp-594, Asp-700, and Glu-623 are a putative catalytic triad of the T. saccharolyticum B6A-RI amylopullulanase.

Deletion mutants were constructed from pZEP12, which contained the intact Thermoanaerobacterium saccharolyticum endoxylanase gene (xynA). Deletion of 1.75 kb from the N-terminal end of xynA resulted in a mutant enzyme that retained activity but lost thermostability. Deletion of 1.05 kb from the C terminus did not alter thermostability or activity. The deduced amino acid sequence of T. saccharolyticum B6A-RI endoxylanase XynA was aligned with five other family F beta-glycanases by using the PILEUP program of the Genetics Computer Group package. This multiple alignment of amino acid sequences revealed six highly conserved motifs which included the consensus sequence consisting of a hydrophobic amino acid, Ser or Thr, Glu, a hydrophobic amino acid, Asp, and a hydrophobic amino acid in the catalytic domain. Endoxylanase was inhibited by EDAC [1-(3-dimethylamino propenyl)-3-ethylcarbodiimide hydrochloride], suggesting that Asp and/or Glu was involved in catalysis. Three aspartic acids, two glutamic acids, and one histidine were conserved in all six enzymes aligned. Hydrophobic cluster analysis revealed that two Asp and one Glu occur in the same hydrophobic clusters in T. saccharolyticum B6A-RI endoxylanase and two other enzymes belonging to family F beta-glycanases and suggests their involvement in a catalytic triad. These two Asp and one Glu in XynA from T. saccharolyticum were targeted for analysis by site-specific mutagenesis. Substitution of Asp-537 and Asp-602 by Asn and Glu-600 by Gln completely destroyed endoxylanase activity. These results suggest that these three amino acids form a catalytic triad that functions in a general acid catalysis mechanism.

The gene encoding endoxylanase (xynA) from Thermoanaerobacterium saccharolyticum B6A-RI was cloned and expressed in Escherichia coli. A putative 33-amino-acid signal peptide, which corresponded to the N-terminal amino acids, was encoded by xynA. An open reading frame of 3,471 bp, corresponding to 1,157 amino acid residues, was found, giving the xynA gene product a molecular mass of 130 kDa. xynA from T. saccharolyticum B6A-RI had strong similarity to genes from family F beta-glycanases. The temperature and pH optimum for the activity of the cloned endoxylanase were 70 degrees C and 5.5, respectively. The cloned endoxylanase A was stable at 75 degrees C for 60 min and displayed a specific activity of 227.4 U/mg of protein on oat spelt xylan. The cloned xylanase was an endo-acting enzyme.

The complete nucleotide sequence of the gene encoding the dual active amylopullulanase of Thermoanaerobacter ethanolicus 39E (formerly Clostridium thermohydrosulfuricum) was determined. The structural gene (apu) contained a single open reading frame 4443 base pairs in length, corresponding to 1481 amino acids, with an estimated molecular weight of 162,780. Analysis of the deduced sequence of apu with sequences of alpha-amylases and alpha-1,6 debranching enzymes enabled the identification of four conserved regions putatively involved in substrate binding and in catalysis. The conserved regions were localized within a 2.9-kilobase pair gene fragment, which encoded a M(r) 100,000 protein that maintained the dual activities and thermostability of the native enzyme. The catalytic residues of amylopullulanase were tentatively identified by using hydrophobic cluster analysis for comparison of amino acid sequences of amylopullulanase and other amylolytic enzymes. Asp597, Glu626, and Asp703 were individually modified to their respective amide form, or the alternate acid form, and in all cases both alpha-amylase and pullulanase activities were lost, suggesting the possible involvement of 3 residues in a catalytic triad, and the presence of a putative single catalytic site within the enzyme. These findings substantiate amylopullulanase as a new type of amylosaccharidase.

Anaerobic bacteria include diverse species that can grow at environmental extremes of temperature, pH, salinity, substrate toxicity, or available free energy. The first evolved archaebacterial and eubacterial species appear to have been anaerobes adapted to high temperatures. Thermoanaerobes and their stable enzymes have served as model systems for basic and applied studies of microbial cellulose and starch degradation, methanogenesis, ethanologenesis, acetogenesis, autotrophic CO2 fixation, saccharidases, hydrogenases, and alcohol dehydrogenases. Anaerobes, unlike aerobes, appear to have evolved more energy-conserving mechanisms for physiological adaptation to environmental stresses such as novel enzyme activities and stabilities and novel membrane lipid compositions and functions. Anaerobic syntrophs do not have similar aerobic bacterial counterparts. The metabolic end products of syntrophs are potent thermodynamic inhibitors of energy conservation mechanisms, and they require coordinated consumption by a second partner organism for species growth. Anaerobes adapted to environmental stresses and their enzymes have biotechnological applications in organic waste treatment systems and chemical and fuel production systems based on biomass-derived substrates or syngas. These kinds of anaerobes have only recently been examined by biologists, and considerably more study is required before they are fully appreciated by science and technology.

During growth on xylan and xylose Thermoanaerobacterium saccharolyticum B6A-RI produced endoxylanase, beta-xylosidase, arabinofuranosidase, and acetyl esterase, and the first three activities appeared to be produced coordinately. During nonlimiting growth on xylan, these enzyme activities were predominantly cell associated; however, during growth on limiting concentrations of xylan, the majority of endoxylanase activity was extracellular rather than cell associated. Endoxylanase, beta-xylosidase, and arabinofuranosidase activities were induced by xylan, xylose, and arabinose, respectively. Acetyl esterase activity was constitutive, and endoxylanase activity was catabolite repressed by glucose. Extracellular endoxylanase existed as a high-molecular-weight complex (molecular weight, more than 10). When analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and zymograms, the crude endoxylanase complex was composed of at least six activity bands. Endoxylanase was purified by gel filtration with Sephacryl S-300 and affinity chromatography with xylan coupled to Sepharose CL-4B preequilibrated to 45 degrees C with 50 mM sodium acetate buffer (pH 4.0) and eluted with 0.1% soluble xylan. A single area of endoxylanase activity was identified on the zymogram; when this activity was analyzed by SDS-PAGE, it was composed of a major protein with a molecular weight of approximately 160,000 and a minor protein with a molecular weight of approximately 130,000. The endoxylanase activity stained with Schiff's reagent, indicative of glycoproteins, displayed a specific activity of 41 U/mg of protein on xylan, and had pH and temperature optima of 6.0 and 70 degrees C, respectively.

Changes in the composition of membrane lipids in a strictly anaerobic, facultative acidophilic eubacterium, Sarcina ventriculi, were studied in response to various forms of environmental stress. Changes in lipid composition and structure occurred in response to changes in environmental pH. At neutral pH, the predominant membrane fatty acids ranged in chain length from C14 to C18. However, when cells were grown at pH 3.0, a family of unique very long chain fatty acids containing 32-36 carbon atoms was synthesized and accounted for 50% of the total membrane fatty acids. These acids were identified as very long chain alpha,omega-dicarboxylic acids ranging in length from 28 to 36 carbons by electron impact mass spectrometry of methyl and (perdeuterio) methyl ester derivatives. These methyl esters all bore a vicinal dimethyl group toward the center of the chain. The assignment of the structures was confirmed by isolating one of the very long chain unusual fatty acids as the ester form after methanolysis and performing further analyses including 1H and 13C NMR spectroscopy and Fourier transform infrared spectroscopy. Coupling this information with the data from gas chromatography/mass spectrometry analysis, the exact structure was confirmed as alpha,omega-15,16-dimethyltricotanedioate dimethyl ester. Addition of alcohols, either metabolic (0.25 M ethanol) or nonmetabolic (0.05 M butanol) to cells grown at pH 7.0, or thermal stress (growth temperature at pH 7.0 was raised from 37 to 45 or 55 degrees C) also resulted in the synthesis of these very long chain fatty acids. Synthesis of these very long chain alpha,omega-dicarboxylic acids was reversed by reducing the temperature back to 37 degrees C. S. ventriculi is also unusual in that the membrane components are not the usual phospholipid components but appear to be predominantly glycolipids.

Pyruvate decarboxylase from the obligate anaerobe Sarcina ventriculi was purified eightfold. The subunit Mr was 57,000 +/- 3000 as estimated from SDS-PAGE, and the native Mr estimated by gel filtration on a Superose 6 column was 240,000, indicating that the enzyme is a tetramer. The Mr values are comparable to those for pyruvate decarboxylase from Zymomonas mobilis and Saccharomyces cerevisiae, which are also tetrameric enzymes. The enzyme was oxygen stable, and had a pH optimum within the range 6.3-6.7. It displayed sigmoidal kinetics for pyruvate, with a S0.5 of 13 mM, kinetic properties also found for pyruvate decarboxylase from yeast and differing from the Michaelis-Menten kinetics of the enzyme from Z. mobilis. No activators were found. p-Chloromercuribenzoate inhibited activity and the inhibition was reversed by the addition of dithiothreitol, indicating that cysteine is important in the active site. The N-terminal amino acid sequence of pyruvate decarboxylase was more similar to the sequence of S. cerevisiae than Z. mobilis pyruvate decarboxylase.

The activities of cellulolytic and xylanolytic enzymes produced by an anaerobic fungus (R1) which resembled Neocallimastix sp. were investigated. Carboxymethylcellulase (CMCase), cellobiase, and filter paper (FPase) activities had pH optima of 6.0, 5.5, and 6.0, respectively. CMCase and cellobiase activities both had a temperature optimum of 50 degrees C, whereas FPase had an optimum of 45 degrees C. The pH and temperature optima for xylanase activity were pH 6.0 and 50 degrees C, respectively. Growth of the fungus on wheat straw, wheat straw holocellulose, or cellulose resulted in substantial colonization, with at least 43 to 58% losses in substrate dry matter and accumulation of comparable amounts of formate. This end product was correlated to apparent loss of substrate dry weight and could be used as an indicator of fungal growth. Milling of wheat straw did not enhance the rate or extent of substrate degradation. Growth of the R1 isolate on the above substrates or xylan also resulted in accumulation of high levels of xylanase activity and lower cellulase activities. Of the cellulases, CMCase was the most active and was associated with either low or trace amounts of cellobiase and FPase activities. During growth on xylan, reducing sugars, including arabinose and xylose, rapidly accumulated in the medium. Xylose and other reducing sugars, but not arabinose, were subsequently used for growth. Reducing sugars also accumulated, but not as rapidly, when the fungus was grown on wheat straw, wheat straw holocellulose, or cellulose. Xylanase activities detected during growth of R1 on media containing glucose, xylose, or cellobiose suggested that enzyme production was constitutive.(ABSTRACT TRUNCATED AT 250 WORDS)

Substrate utilization and fermentation characteristics of rumen fungi of the genus Neocallimastix are described. Preliminary observations on the removal of monosaccharides from plant cell walls and the effect of fermentation products on growth of Neocallimastix sp. (isolate R1) are presented. The properties of rumen fungi are discussed in relation to their role in the rumen.

The life cycle and growth kinetics of an anaerobic rumen fungus (Neocallimastix R1) in liquid and solid media are described, together with its response to light, temperature and oxygen. These results are discussed in relation to the survival of rumen fungi in saliva and faeces of sheep, and the possible routes for the transfer of anaerobic fungi between ruminants. The thallus and life cycle of Neocallimastix R1 are compared with those of aerobic chytrids.

An anaerobic fungus (strain R1) resembling Neocallimastix spp. was isolated from sheep rumen. When grown on defined medium, the isolate utilized a wide range of polysaccharides and disaccharides, but of the eight monosaccharides tested only fructose, glucose, and xylose supported growth. The organism had doubling times of 5.56 h on glucose and 6.67 h on xylose, and in each case fermentation resulted in production of formate, acetate, lactate, and ethanol. During active growth, formate was a reliable indicator of fungal biomass. Growth on a medium containing glucose and xylose resulted in a doubling time of 8.70 h, but diauxic growth did not occur since both sugars were utilized simultaneously. The optimum temperature for zoospore and immature plant development was 39 degrees C, and no development occurred below 33 degrees C or above 41 degrees C.

Halobacteroides acetoethylicus grew in media with 6 to 20% NaCl and displayed optimal growth at 10% NaCl. When grown in medium with an [NaCl] of 1.7 M, the internal cytoplasmic [Na+] and [Cl-] were 0.92 and 1.2 M, respectively, while K+ and Mg2+ concentrations in cells were 0.24 and 0.02 M, respectively. Intracellular [Na+] was fourfold higher than intracellular [K+]. Since Na+ and Cl- ions were not excluded from the cell, the influence of high salt concentrations on key enzyme activities was investigated in crude cell extracts. Activities greater than 60% of the maximal activity of the following key catabolic enzymes occurred at the following [NaCl] ranges: glyceraldehyde-3-phosphate dehydrogenase, 1 to 2 M; alcohol dehydrogenase (NAD linked), 2 to 4 M; pyruvate dehydrogenase, 0.5 to 1 M; and hydrogenase (methyl viologen linked), 0.5 to 3 M. These studies support the hypothesis that obligately halophilic, anaerobic eubacteria adapt to extreme salt concentrations differently than do halophilic, aerobic eubacteria, because they do not produce osmoregulants or exclude Cl-. This study also demonstrated that these halophilic, anaerobic eubacteria have a physiological similarity to archaebacterial halophiles, since Na+ and Cl- are present in high concentrations and are required for enzymatic activity.

Distinct morphological changes in the ultrastructure of Sarcina ventriculi were observed when cells were grown in medium of constant composition at pH extremes of 3.0 and 8.0. Transmission electron microscopy revealed that at low pH (less than or equal to 3.0) the cells formed regular packets and cell division was uniform. When the pH was increased (to greater than or equal to 7.0), the cells became larger and cell division resulted in irregular cells that varied in shape and size. Sporulation occurred at high pH (i.e., greater than or equal to 8.0). The sporulation cycle followed the conventional sequence of development for refractile endospores, with the appearance of a cortex and multiple wall layers. The spores were resistant to oxygen, lysozyme, or heating at 90 degrees C for 15 min. Spores germinated within the pH range of 4.6 to 7.0.

The anaerobic fungus Neocallimastix sp. strain R1 was grown for up to 5 days on a medium containing autoclaved Italian ryegrass hay as the carbon source. Culture supernatants and digested cell walls were harvested at 12-h intervals. Supernatants were analyzed for the fermentation products formate and acetate, and residual cell walls were analyzed for dry-matter and neutral-sugar losses. Fungal growth was accompanied by the digestion of plant cell walls and the accumulation of fermentation products in culture media. Dry-matter losses were accounted for by removal of four major neutral sugars (arabinose, galactose, glucose, and xylose) from the plant cell walls. First-order reaction kinetics could be used to describe the loss of each sugar. All cell wall sugars, including arabinose and galactose, which are not fermented by Neocallimastix sp. strain R1 were removed simultaneously. Although the rates of removal of individual sugars were similar, there were significant differences in their extents of removal: the extent of removal of arabinose exceeded that of the other three sugars, and xylose was the least digestible. This study provides the first account of simultaneous (nonpreferential) removal of neutral sugars from plant cell walls by an anaerobic fungus. Although in vitro techniques were used, these results indicate a potentially significant role for the anaerobic fungi as fiber digesters in the rumen.