AFLP analysis of Salmonella enterica serovar Typhimurium isolates of phage types DT 9 and DT 135: diversity within phage types and its epidemiological significance

Amplified fragment length polymorphism (AFLP) was applied to 35 and 34 isolates, respectively, of Salmonella enterica serovar Typhimurium phage types DT 9 and DT 135, using eight primer pair combinations. Eight and 17 AFLP types were observed in DT 9 and DT 135, respectively. DT 9 is rare in the UK and common in Australia, but one AFLP form dominated with 28 isolates, comprising 22 of 25 UK isolates, four of five Australian isolates, one Jamaican and one Spanish isolate. Of the others, two UK isolates are closely related to the major form, two from elsewhere are in the major cluster and three isolates from different countries are in a separate cluster. For DT 135, two closely related AFLP types of seven and 11 isolates form the major cluster, which also includes 11 isolates, mostly in single-isolate AFLP types, while five isolates from different countries form a well-separated minor cluster. For both DTs all isolates are grouped together if only the phage type specific bands identified earlier are used, confirming their value for molecular-based 'phage typing'. Polymorphic markers identified in this study could also be used for subtyping within both phage types. The value of AFLP is in locating DNA fragments useful for typing, but implementation of a replacement typing scheme would probably involve multiplex PCR or microarray technologies.     

Division of the nucleolus and its release of CDC14 during anaphase of meiosis I depends on separase,SPO12, and SLK19

Disjunction of maternal and paternal centromeres during meiosis I requires crossing over between homologous chromatids, which creates chiasmata that hold homologs together. It also depends on a mechanism ensuring that maternal and paternal sister kinetochore pairs attach to oppositely oriented microtubules. Proteolytic cleavage of cohesin's Rec8 subunit by separase destroys cohesion between sister chromatid arms at anaphase I and thereby resolves chiasmata. The Spo12 and Slk19 proteins have been implicated in regulating meiosis I kinetochore orientation and/or in preventing cleavage of Rec8 at centromeres. We show here that the role of these proteins is instead to promote nucleolar segregation, including release of the Cdc14 phosphatase required for Cdk1 inactivation and disassembly of the anaphase I spindle. Separase is also required but surprisingly not its protease activity. It has two mechanistically different roles during meiosis I. Loss of the protease-independent function alone results in a second meiotic division occurring on anaphase I spindles in spo12delta and slk19delta mutants.     

Simulations of temperature sensitivity of the peroxidase-oxidase oscillator

The influence of temperature on the oscillatory kinetics of the peroxidase-oxidase reaction was studied theoretically. Assuming Q(10)=2 for elementary reactions, the effect of multiplying the rate constants of the model by factors between 0.5 and 2 (corresponding to a 10 degrees C decrease and increase, respectively, of temperature) was investigated. First, the individual rate constants were successively multiplied by 0.5 or 2 while all other rate constants were kept unchanged. This resulted in either a longer or a shorter period, depending on the rate constant being changed. Multiplication by 0.5 or by 2 generally resulted in opposite effects on the period length. However, the absolute value of this deviation differed. Also, the dynamics changed when halving the dimerization rate of NAD* as well as when doubling the rate constant for the reduction of ferric peroxidase by NAD*. Next, simulations were performed multiplying all rate constants by one and the same factor, which increased progressively from 0.5 to 2. Intervals were found corresponding to temperature dependency, compensation, and over-compensation, respectively.     

Ligand exchange of major histocompatibility complex class II proteins is triggered by H-bond donor groups of small molecules

Hydrogen bonds (H-bonds) are crucial for the stability of the peptide-major histocompatibility complex (MHC) complex. In particular, the H-bonds formed between the peptide ligand and the MHC class II binding site appear to have a great influence on the half-life of the complex. Here we show that functional groups with the capacity to disrupt hydrogen bonds (e.g. -OH) can efficiently catalyze ligand exchange reactions on HLA-DR molecules. In conjunction with simple carrier molecules (such as propyl or benzyl residues), they trigger the release of low affinity ligands, which permits the rapid binding of peptides with higher affinity. Similar to HLA-DM, these compounds are able to influence the MHC class II ligand repertoire. In contrast to HLA-DM, however, these simple small molecules are still active at neutral pH. Under physiological conditions, they increase the number of "peptide-receptive" MHC class II molecules and facilitate exogenous peptide loading of dendritic cells. The drastic acceleration of the ligand exchange on these antigen presenting cells suggests that, in general, availability of H-bond donors in the extracellular milieu controls the rate of MHC class II ligand exchange reactions on the cell surface. These molecules may therefore be extremely useful for the loading of antigens onto dendritic cells for therapeutic purposes.     

Coenzymatic activity of randomly broken or intact double-stranded DNAs in auto and histone H1 trans-poly(ADP-ribosylation), catalyzed by poly(ADP-ribose) polymerase (PARP I)

The enzymatic transfer of ADP-ribose from NAD to histone H1 (defined as trans-poly(ADP-ribosylation)) or to PARP I (defined as auto-poly(ADP-ribosylation)) was studied with respect to the nature of the DNA required as a coenzyme. Linear double-stranded DNA (dsDNA) containing the MCAT core motif was compared with DNA containing random nicks (discontinuous or dcDNA). The dsDNAs activated trans-poly(ADP-ribosylation) about 5 times more effectively than dcDNA as measured by V(max). Activation of auto-poly(ADP-ribosylation) by dcDNA was 10 times greater than by dsDNA. The affinity of PARP I toward dcDNA or dsDNA in the auto-poly(ADP-ribosylation) was at least 100-fold lower than in trans-poly(ADP-ribosylation) (K(a) = 1400 versus 3-15, respectively). Mg2+ inhibited trans-poly(ADP-ribosylation) and so did dcDNA at concentrations required to maximally activate auto-poly(ADP-ribosylation). Mg2+ activated auto-poly(ADP-ribosylation) of PARP I. These results for the first time demonstrate that physiologically occurring dsDNAs can serve as coenzymes for PARP I and catalyze preferentially trans-poly(ADP- ribosylation), thereby opening the possibility to study the physiologic function of PARP I.     

Direct transesterification of gases by "dry" immobilized lipase

Several different reactor configurations, including single pass, continuous recycle, and batch reactor modes, were used to investigate the effects of temperature and water activity, or relative humidity, on lipase-catalyzed, gas-phase transesterifications. Temperature and relative humidity were controlled both inside reactors and throughout the course of the reaction to account for and optimize their effects. Results indicated that, at low relative humidity, reaction rates increased with temperature up to 60 degrees C. However, when relative humidity was increased, a similar increase in temperature resulted in the loss of nearly all enzyme activity. These results are consistent with the idea that enzymes without free water are more thermally stable. Furthermore, at constant ambient temperatures, production increased dramatically with an increase in relative humidity, confirming the idea that an increase in water activity increases catalytic activity. A mass balance performed on reactors at higher relative humidity revealed that hydrolysis (rather than transesterification) of the ester substrate could significantly decrease product yields.     

The effect of strength training combined with bisphosphonate (etidronate) therapy on bone mineral,lean tissue,and fat mass in postmenopausal women

The combined and separate effects of exercise training and bisphosphonate (etidronate) therapy on bone mineral in postmenopausal women were compared. Forty-eight postmenopausal women were randomly assigned (double blind) to groups that took intermittent cyclical etidronate; performed strength training (3 d/week) and received matched placebo; combined strength training with etidronate; or took placebo and served as nonexercising controls. Bone mineral, lean tissue, and fat mass were assessed by dual-energy X-ray absorptiometry before and after 12 months of intervention. After removal of outlier results, changes in bone mineral density (BMD) of the lumbar spine and bone mineral content (BMC) of the whole body were greater in the subjects given etidronate (+2.5 and +1.4%, respectively) compared with placebo (-0.32 and 0%, respectively) (p < 0.05), while exercise had no effect. There was no effect of etidronate or exercise on the proximal femur and there was no interaction between exercise and etidronate at any bone site. Exercise training resulted in significantly greater increases in muscular strength and lean tissue mass and greater loss of fat mass compared with controls. We conclude that etidronate significantly increases lumbar spine BMD and whole-body BMC and that strength training has no additional effect. Strength training favourably affects body composition and muscular strength, which may be important for prevention of falls.     

Microbial reduction of Fe(III) in the presence of oxygen under low pH conditions

In acidic, coal mining lake sediments, facultatively anaerobic Acidiphilium species are probably involved in the reduction of Fe(III). Previous results indicate that these bacteria can co-respire O2 and Fe(III). In this study, we investigated the capacity of the sediment microbiota to reduce Fe(III) in the presence of O2 at pH 3. In sediment microcosms with 4% O2 in the headspace, the concentration of Fe(II) increased at a rate of 1.03 micromol (g wet sediment)-1 day-1 within the first 7 days of incubation which was similar to the rate obtained with controls incubated under anoxic conditions. However, in microcosms incubated under air, Fe(II) was consumed after a lag phase of 8 h with a rate of 2.66 micromol (g wet sediment)-1 day-1. Acidiphilium cryptum JF-5, isolated from this sediment, reduced soluble Fe(III) with either 4 or 21% O2 in the headspace, and concomitantly consumed O2. However, the rate of Fe(II) formation normalized for cell density decreased under oxic conditions. Schwertmannite, the predominant Fe(III)-mineral of this sediment, was also reduced by A. cryptum JF-5 under oxic conditions. The rate of Fe(II) formation by A. cryptum JF-5 decreased after transfer from preincubation under air in medium lacking Fe(III). Acidiphilium cryptum JF-5 did not form Fe(II) when preincubated under air and transferred to anoxic medium containing Fe(III) and chloramphenicol, an inhibitor of protein synthesis. These results indicate that: (i) the reduction of Fe(III) can occur at low O2 concentrations in acidic sediments; (ii) Fe(II) can be oxidized at O2 concentrations near saturation; and (iii) the enzyme(s) responsible for the reduction of Fe(III) in A. cryptum JF-5 are not constitutive.