Studies of Microthrix parvicella in situ and in laboratory culture: production and use of specific antibodies.

Physiological studies on M. parvicella have been conducted to determine the rate of growth of this organism in pure culture. The organism displayed a doubling time of 128 days despite its profuse abundance in a local Wastewater Treatment Plant (WWTW). An extensive survey has been ongoing since February 2000 into the extent of M. parvicella in the WWTW. A suite of monoclonal and polyclonal antibodies has been developed to detect and quantify M. parvicella.

5-aminoisoquinolinone, a potent inhibitor of poly (adenosine 5'-diphosphate ribose) polymerase, reduces myocardial infarct size.

This study investigates the effects of a novel, water-soluble inhibitor of the activity of poly (adenosine 5'-diphosphate ribose) polymerase, 5-aminoisoquinolinone [5-aminoisoquinolin-1(2H)-one], on (i) poly (adenosine 5'-diphosphate ribose) polymerase activity in rat cardiac myoblasts and (ii) the infarct size caused by regional myocardial ischaemia and reperfusion in the rat. Exposure of H9c2 cells to hydrogen peroxide (H2O2, 1 mM) caused a significant increase in poly (adenosine 5'-diphosphate ribose) polymerase activity and an 80-90% reduction in mitochondrial respiration (cellular injury). Pretreatment of these cells with 5-aminoisoquinolinone (0.003-1 mM) caused a concentration-dependent inhibition of poly (adenosine 5'-diphosphate ribose) polymerase activity (IC50: approximately 4.5 microM, n=6-9) and cell injury (EC50: approximately 4.45 microM, n=9). In a rat model of myocardial infarction, left anterior descending coronary artery occlusion (25 min) and reperfusion (2 h) resulted in an infarct size of 50+/-3%. Administration (1 min before reperfusion) of 5-aminoisoquinolinone reduced myocardial infarct size in a dose-related fashion. Thus, 5-aminoisoquinolinone is a potent inhibitor of poly (adenosine 5'-diphosphate ribose) polymerase activity in cardiac myoblasts and reduces myocardial infarct size in vivo.

A genomic timescale for the origin of eukaryotes.

BACKGROUND: Genomic sequence analyses have shown that horizontal gene transfer occurred during the origin of eukaryotes as a consequence of symbiosis. However, details of the timing and number of symbiotic events are unclear. A timescale for the early evolution of eukaryotes would help to better understand the relationship between these biological events and changes in Earth's environment, such as the rise in oxygen. We used refined methods of sequence alignment, site selection, and time estimation to address these questions with protein sequences from complete genomes of prokaryotes and eukaryotes. RESULTS: Eukaryotes were found to evolve faster than prokaryotes, with those eukaryotes derived from eubacteria evolving faster than those derived from archaebacteria. We found an early time of divergence (approximately 4 billion years ago, Ga) for archaebacteria and the archaebacterial genes in eukaryotes. Our analyses support at least two horizontal gene transfer events in the origin of eukaryotes, at 2.7 Ga and 1.8 Ga. Time estimates for the origin of cyanobacteria (2.6 Ga) and the divergence of an early-branching eukaryote that lacks mitochondria (Giardia) (2.2 Ga) fall between those two events. CONCLUSIONS: We find support for two symbiotic events in the origin of eukaryotes: one premitochondrial and a later mitochondrial event. The appearance of cyanobacteria immediately prior to the earliest undisputed evidence for the presence of oxygen (2.4-2.2 Ga) suggests that the innovation of oxygenic photosynthesis had a relatively rapid impact on the environment as it set the stage for further evolution of the eukaryotic cell.

Effects of 5-aminoisoquinolinone, a water-soluble, potent inhibitor of the activity of poly (ADP-ribose) polymerase on the organ injury and dysfunction caused by haemorrhagic shock.

Poly (ADP-ribose) synthetase (PARP) is a nuclear enzyme activated by strand breaks in DNA, which are caused inter alia by reactive oxygen species (ROS). Here we report on (i) a new synthesis of a water-soluble and potent PARP inhibitor, 5-aminoisoquinolinone (5-AIQ) and (ii) investigate the effects of 5-AIQ on the circulatory failure and the organ injury/dysfunction caused by haemorrhage and resuscitation in the anaesthetized rat. Exposure of human cardiac myoblasts (Girardi cells) to hydrogen peroxide (H(2)O(2), 3 mM for 1 h, n=9) caused a substantial increase in PARP activity. Pre-treatment of these cells with 5-AIQ (1 microM - 1 mM, 10 min prior to H(2)O(2)) caused a concentration-dependent inhibition of PARP activity (IC(50): approximately 0.01 mM, n=6). Haemorrhage and resuscitation resulted (within 4 h after resuscitation) in a delayed fall in blood pressure (circulatory failure) as well as in rises in the serum levels of (i) urea and creatinine (renal dysfunction), (ii) aspartate aminotransferase (AST), alanine aminotransferase (ALT), and gamma-glutamyl-transferase (gamma-GT) (liver injury and dysfunction), (iii) lipase (pancreatic injury) and (iv) creatine kinase (CK) (neuromuscular injury) (n=10). Administration (5 min prior to resuscitation of 5-AIQ) (0.03 mg kg(-1) i.v., n=8, or 0.3 mg kg(-1) i.v., n=10) reduced (in a dose-related fashion) the multiple organ injury and dysfunction, but did not affect the circulatory failure, associated with haemorrhagic shock. Thus, 5-AIQ abolishes the multiple organ injury caused by severe haemorrhage and resuscitation.

Determination of the structural role of the linking moieties in the DNA binding of adozelesin.

Adozelesin (formerly U73975, The Upjohn Co.) is a monofunctional DNA alkylating analogue of the antitumor antibiotic (+)-CC-1065. Adozelesin consists of a cyclopropa[c]pyrrolo[3,2-e]indol-4(5H)-one (CPI) alkylating subunit of (+)-CC-1065 and a indole and benzofurans subunit replacing the more complex pyrroloindole B and C subunits, respectively, of (+)-CC-1065. Previous studies have shown that adozelesin forms a reversible covalent DNA duplex adduct via a reaction between the N3 of adenine and the cyclopropyl of the cyclopropapyrroloindole (CPI) subunit. Gel electrophoresis studies have shown that adozelesin, like all the monofunctional (CPI)-based antitumor antibiotics, has a sequence preference for 5'-TTA-3' [the asterisk () indicates covalently modified base]. Molecular-modeling studies have shown that the bound adozelesin ligand spans a total of five base pairs including the modified adenine. These studies have also indicated that, owing to the orientation of the ligand within the base minor groove, there should be an overall preference for sequences rich in A.T base pairs, thus avoiding steric crowding around the exocyclic NH(2) of any guanines present. In this study, we have prepared and studied, by high-field NMR and restrained molecular mechanics (rMM) and dynamics (rMD), the duplex adduct formed between adozelesin and 5'-CGTAAGCGCTTACG-3'. Previous molecular-modeling studies suggested that this sequence should be less preferred, since the two GC base pairs should lead to extensive steric crowding within the adduct, and this hypothesis has, however, never been supported by DNA-footprinting data. (1)H NMR of the adozelesin duplex adduct has reveals that, although Watson-Crick base pairing is maintained throughout the DNA duplex, there is significant distortion around the central base pairs. This distortion is the result of strong hydrogen-bonding between the amide linker of the indole and benzofuran subunits, and the carbonyl of a central thymine base and second, weaker, hydrogen bond to the exocyclic NH(2) of the central guanine was also observed. (1)H NMR and rMD also indicate that, to accommodate this hydrogen-bond system, the bound adozelesin is not positioned centrally within the minor groove but pushed toward the modified DNA strand. Previous studies on the dimeric CPI analogue bizelesin have indicated the important role the ureylene linker plays in the DNA binding. This study indicates that a similar situation exists in the reaction of adozelesin with double-stranded DNA and provides a possible explanation into the unpredicted sequence selectivity of these ligands.

Evaluation of an innovative program to address the health and social service needs of drug-using women with or at risk for HIV infection.

Drug-using women with or at risk for HIV infection have many competing unmet needs, especially for social services, drug treatment, and medical care. High-risk drug-using women were recruited through street outreach, at needle exchange sites, a prison, and local community based organizations in New Haven, Connecticut for a study of the service needs of out-of-treatment drug users and the ability of an interactive case management intervention (ICM) to address those needs. These women were administered baseline and follow-up interviews to identify their health and social service needs and the degree to which these needs were resolved. The women who chose to enroll in the interactive case management intervention (n = 38) did not differ demographically nor in their HIV risk behaviors from those not receiving case management (n = 73). Provision of ICM was most successful in meeting needs for supportive mental health counseling, basic services, and long term housing. The impact of interactive case management was less evident for the acquisition of medical and dental services, which were accessed comparably by women not receiving the intervention. Overall, the women who enrolled in the ICM intervention showed a significant decrease in the number of unmet service needs as compared to those who did not enroll. Multiple contacts were required by the case manager to establish trust and to resolve the unmet service needs of these high-risk women. Women with or at risk for HIV infection can be effectively engaged in an ICM intervention in order to meet their multiple unmet service needs, although such interventions are time-and-labor intensive.

Methylosulfonomonas methylovora gen. nov., sp. nov., and Marinosulfonomonas methylotropha gen. nov., sp. nov.: novel methylotrophs able to grow on methanesulfonic acid.

Two novel genera of restricted facultative methylotrophs are described; both Methylosulfonomonas and Marinosulfonomonas are unique in being able to grow on methanesulfonic acid as their sole source of carbon and energy. Five identical strains of Methylosulfonomonas were isolated from diverse soil samples in England and were shown to differ in their morphology, physiology, DNA base composition, molecular genetics, and 16S rDNA sequences from the two marine strains of Marinosulfonomonas, which were isolated from British coastal waters. The marine strains were almost indistinguishable from each other and are considered to be strains of one species. Type species of each genus have been identified and named Methylosulfonomonas methylovora (strain M2) and Marinosulfonomonas methylotropha (strain PSCH4). Phylogenetic analysis using 16S rDNA sequencing places both genera in the alpha-Proteobacteria. Methylosulfonomonas is a discrete lineage within the alpha-2 subgroup and is not related closely to any other known bacterial genus. The Marinosulfonomonas strains form a monophyletic cluster in the alpha-3 subgroup of the Proteobacteria with Roseobacter spp. and some other partially characterized marine bacteria, but they are distinct from these at the genus level. This work shows that the isolation of bacteria with a unique biochemical character, the ability to grow on methanesulfonic acid as energy and carbon substrate, has resulted in the identification of two novel genera of methylotrophs that are unrelated to any other extant methylotroph genera.

Solution conformation of a bizelesin A-tract duplex adduct: DNA-DNA cross-linking of an A-tract straightens out bent DNA.

The DNA cross-linker bizelesin has been previously shown to form interhelical interstrand cross-links with adenine residues six base-pairs apart (including the modified adenine residues). Sequence specificity studies have shown that the ligand has a high affinity for the intrinsically bent A-tract sequence [d(CGTTTTTACG):d(CGTAAAAACG)]. However, gel retardation studies have shown that the cross-linked duplex retains none of the characteristic A-tract bending observed within the unmodified duplex. Two-dimensional 1H-NMR experiments have not only confirmed the sites of cross-linking into the duplex, but have also shown the loss of inherent A-tract characteristics, including reduced crosspeak intensities between the H2s of the central adenine residues and the cross-strand H1' of the base one base removed to the 3' side. This observation suggests loss of propeller twisting within these central adenine residues and provides insight into the controversial origin of A-tract bending. This study is important because it validates the use of bizelesin as a probe for determining the importance of A-tract-induced bending in transcriptional and replicational elements.

Determination of the structural role of the internal guanine-cytosine base pair in recognition of a seven-base-pair sequence cross-linked by bizelesin.

Bizelesin (formerly U77,779, The Upjohn Co.) is a bifunctional DNA cross-linking antitumor antibiotic consisting of two open-ring homologs of the (+)-CC-1065 cyclopropa[c]pyrrolo[3,2-e]indol-4(5H)-one (CPI) subunits connected by a rigid linking moiety. Previous studies have shown that Bizelesin most often forms an interstrand cross-link through the N3 of two adenines 6 base pairs (bp) apart (inclusive of the modified adenines). However, gel electrophoresis studies have also indicated that Bizelesin forms 7-bp cross-links in specific sequences. In most of these sequences the cross-linked adenines represent the only possible cross-link site (i.e., no 6-bp site is available); however, in several sequences, a 7-bp sequence is selected in overwhelming preference to a possible 6-bp sequence. In this study, we demonstrate the unique requirement for a G.C base pair within this sequence and the critical presence of the exocyclic 2-amino group of guanine. In a subsequent two-dimensional 1H-NMR study that concentrates on the 7-bp cross-link formed with the sequence 5'-TTAGTTA-3', the role of the central G.C base pairs in the formation of a 7-bp cross-link is probed. 1H-NMR analysis coupled with restrained molecular dynamics (rMD) provides evidence for distortion around the covalently modified adenines. Because of this distortion, the modified bases are twisted toward the center of the duplex adduct, effectively reducing the cross-linked distance. The rMD study also indicates that a hydrogen bond is formed between the exocyclic amine of the central guanine and the carbonyl of the ureylene linker. On the basis of the observation of the distortion in the duplex and the hydrogen bonding between the drug and DNA, it is possible to speculate on the role of the central G.C bases in this sequence preference and propose a mechanism by which Bizelesin forms a 7-bp rather than a 6-bp cross-link with this sequence.

Isolation and characterization of methanesulfonic Acid-degrading bacteria from the marine environment.

Two methylotrophic bacterial strains, TR3 and PSCH4, capable of growth on methanesulfonic acid as the sole carbon source were isolated from the marine environment. Methanesulfonic acid metabolism in these strains was initiated by an inducible NADH-dependent monooxygenase, which cleaved methanesulfonic acid into formaldehyde and sulfite. The presence of hydroxypyruvate reductase and the absence of ribulose monophosphate-dependent hexulose monophosphate synthase indicated the presence of the serine pathway for formaldehyde assimilation. Cell suspensions of bacteria grown on methanesulfonic acid completely oxidized methanesulfonic acid to carbon dioxide and sulfite with a methanesulfonic acid/oxygen stoichiometry of 1.0:2.0. Oxygen electrode-substrate studies indicated the dissimilation of formaldehyde to formate and carbon dioxide for energy generation. Carbon dioxide was not fixed by ribulose bisphosphate carboxylase. It was shown that methanol is not an intermediate in methanesulfonic acid metabolism, although these strains grew on methanol and other one-carbon compounds, as well as a variety of heterotrophic carbon sources. These two novel marine facultative methylotrophs have the ability to mineralize methanesulfonic acid and may play a role in the cycling of global organic sulfur.

Treatment outcome of sleep apnea.

Two hundred one patients diagnosed as having obstructive sleep apnea (OSA) were interviewed 12-24 months after their evaluation regarding those daytime symptoms associated with sleep apnea: sleepiness, fatigue, impaired memory, and snoring. Continuous positive airway pressure (CPAP) was the treatment most often used by severe sleep apnea patients, and this improved daytime alertness in 84% of the patients. Patients with moderate obstructive sleep apnea often had surgery which led to 85% reporting improved daytime alertness. Patients with mild obstructive sleep apnea usually were treated with weight loss or changing sleep position and also improved 64% and 66%. Patients who declined or failed treatment did not improve. Guidelines for the treatment of sleep apnea are suggested.

DNA cross-linking and sequence selectivity of aziridinylbenzoquinones: a unique reaction at 5'-GC-3' sequences with 2,5-diaziridinyl-1,4-benzoquinone upon reduction.

Several bifunctional alkylating agents of the aziridinylbenzoquinone class have been evaluated as potential antitumor agents. 3,6-Bis[(2-hydroxyethyl)amino]-2,5- diaziridinyl-1,4-benzoquinone (BZQ), 2,5-diaziridinyl-1,4-benzoquinone (DZQ), 3,6-bis(carboxyamino)-2,5-diaziridinyl- 1,4-benzoquinone (AZQ), and six analogues of AZQ have been studied for their ability to induce DNA interstrand cross-linking, as measured by an agarose gel technique, and to determine whether they react with DNA in a sequence-selective manner, as determined by a modified DNA sequencing technique. At an equimolar concentration (10 microM), only DZQ and BZQ showed any detectable cross-linking at pH 7 without reduction. Cross-linking was enhanced in both cases at low pH (4). Reduction by ascorbic acid at both pH's increased the cross-linking, which was particularly striking in the case of DZQ. In contrast, AZQ and its analogues only produced a significant level of cross-linking under both low-pH and reducing conditions, the extent of cross-linking decreasing as the size of the alkyl end group increased. The compounds reacted with all guanine-N7 positions in DNA with a sequence selectivity similar to other chemotherapeutic alkylating agents, such as the nitrogen mustards, although some small differences were observed with BZQ. Nonreduced DZQ showed a qualitatively similar pattern of reactivity to the other compounds, but on reduction (at pH 4 or 7) was found to react almost exclusively with 5'-GC-3' sequences, and in particular, at 5'-TGC-3' sites. A model to explain this unique reaction is proposed.

Effect of unprocessed wheat bran on calciuria and oxaluria in patients with urolithiasis.

Seventeen hypercalciuria patients (8 control, 9 treatment) with a history of urolithiasis were randomly selected to receive low-calcium, low-oxalate diets with or without the addition of 30 g of dietary fiber as unprocessed wheat bran. Diet alone resulted in a 5.6 percent decrease in calciuria compared with a 23.5 percent decrease with the addition of the fiber. The addition of hydrochlorothiazide and potassium citrate further reduced calciuria by 40.4 percent and 34.5 percent, respectively. Oxaluria was decreased 21.4 percent by diet alone compared with 3.9 percent in the diet and fiber treatment group. Patient compliance to diets was good, and no complications resulted from fiber intake.