Overproduction of four functionally active proteins, TnsA, B, C, and D, required for Tn7 transposition to its attachment site, attTn7.

The bacterial transposon Tn7 encodes five trans-acting transposition genes, tnsA, B, C, D, and E. Tn7 requires four of these genes, tnsA, B, C, and D, for a novel transposition pathway: high-efficiency site-specific transposition to a chromosomal attachment site, attTn7. Plasmids that individually allow inducible overexpression of proteins from the first initiation codon of four of these genes were constructed. Escherichia coli strains carrying these plasmids were used to overexpress the TnsA, B, C, and D proteins. The abundance and the apparent relative molecular mass of these proteins were examined and the latter was compared to those predicted from wild-type Tn7. The functionality of these proteins, encoded by an overexpression construct, was demonstrated by the fact that they could efficiently trans-complement a defective mini-Tn7 carrying only the cis-essential Tn7 termini in an in vivo assay for transposition to attTn7.     

Physiological methods to study biofilm disinfection

This report reviews the development of a rapidin situ approach to study the physiological responses of bacteria within biofilms to disinfectants. One method utilized direct viable counts (DVC) to assess the disinfection efficacy when thin biofilms were exposed to chlorine or monochloramine. Results obtained using the DVC method were one log higher than plate count (PC) estimates of the surviving population after disinfection. Other methods incorporated the use of fluorogenic stains, a cryotomy technique to yield thin (5-m) sections of biofilm communities and examination by fluorescence microscopy. The fluorogenic stains used in this approach included 5-cyano-2,3-ditolyl tetrazolium chloride (CTC), which indicates cellular electron transport activity and Rhodamine 123, which responds specifically to proton motive force. The use of these stains allowed the microscopic discrimination of physiologically active bacteria as well as heterogeneities of active cells within thicker biofilms. The results of experiments using these techniques with pure culture and binary population biofilms on stainless steel coupons indicated biocidal activity of chlorine-based disinfectants occurred initially at the bulk-fluid interface of the communities and progressed toward the substratum. This approach provided a unique opportunity to describe the spatial response of bacteria within biofilms to antimicrobial agents and address mechanisms explaining their comparative resistance to disinfection in a way that has not been possible using traditional approaches. Results obtained using this alternative approach were also consistently higher than PC data following disinfection. These observations suggest that traditional methods involving biofilm removal and bacterial enumeration by colony formation overestimate biocide efficacy. Hence the alternative approach described here more accurately indicates the ability of bacteria surviving disinfection to recover and grow as well as demonstrate spatial heterogeneities in cellular physiological activities within biofilms.     

The adenosine-triphosphatase activity of dissociated acto-heavy-meromyosin

1. At low ionic strength, when turbidity and viscosity measurements indicated dissociation of acto-heavy-meromyosin, its adenosine triphosphatase was strongly activated by Mg(2+) and Ca(2+). 2. The characteristics of the adenosine triphosphatase of dissociated acto-heavy-meromyosin in the presence of Mg(2+) were similar to those reported for myofibrils and actomyosin. 3. In the presence of Ca(2+) the adenosine-triphosphatase activity was much less sensitive to ionic strength than was the case with Mg(2+). 4. At low ionic strength Mg(2+) was more effective in maintaining the dissociation of acto-heavy-meromyosin in the presence of ATP than was Ca(2+). This difference was not apparent when ATP was replaced by ITP. 5. Although the recovery of viscosity was complete on reassociation of acto-heavy-meromyosin the turbidity did not return to the original value. 6. The general implications of Mg(2+) activation of acto-heavy-meromyosin when classical interpretation indicates dissociation of the complex are discussed.     

Purification and characterisation of the TnsB protein of Tn7: a transposition protein that binds to the ends of Tn7.

Tn7, a large bacterial transposon encodes 5 proteins required for its transposition. We report a rapid and easy purification of one of these proteins, TnsB, from an overexpression strain. This protein was shown to bind to the ends of Tn7, in a bandshift assay, in two distinct stages as a function of protein concentration. DNasel footprinting at each end of Tn7 showed that the TnsB recognition sequence, a set of 22 bp repeats, plus Tn7 termini are protected. Binding of TnsB appeared cooperative but was only observed above a threshold concentration of protein. ATP and Mg2+ had no effect on the pattern of protection, nor did addition of other Tn7-encoded proteins. Hydroxyl radical footprinting, performed at the right end, showed that TnsB binds preferentially to one side of the DNA helix.     

Mammalian and avian neuroanatomy and the question of consciousness in birds

Some birds display behavior reminiscent of the sophisticated cognition and higher levels of consciousness usually associated with mammals, including the ability to fashion tools and to learn vocal sequences. It is thus important to ask what neuroanatomical attributes these taxonomic classes have in common and whether there are nevertheless significant differences. While the underlying brain structures of birds and mammals are remarkably similar in many respects, including high brain-body ratios and many aspects of brain circuitry, the architectural arrangements of neurons, particularly in the pallium, show marked dissimilarity. The neural substrate for complex cognitive functions that are associated with higher-level consciousness in mammals and birds alike may thus be based on patterns of circuitry rather than on local architectural constraints. In contrast, the corresponding circuits in reptiles are substantially less elaborated, with some components actually lacking, and in amphibian brains, the major thalamopallial circuits involving sensory relay nuclei are conspicuously absent. On the basis of these criteria, the potential for higher-level consciousness in these taxa appears to be lower than in birds and mammals.     

Placental growth hormone is not suppressed by oral glucose loading in normal human pregnancy.

Placental growth hormone (PGH) progressively replaces pituitary growth hormone in the maternal circulation from mid-gestation onwards in human pregnancy. Our previous investigations have shown that placental growth hormone concentrations correlate well with foetal growth. Despite the apparent correlation between PGH and birthweight, the physiology of its secretion during pregnancy has not been well defined. We investigated the response of maternal serum PGH to oral glucose loading in pregnant women (n = 24) who demonstrated normal glucose tolerance at a mean gestation of 29 weeks. Mean (SEM) fasting PGH concentrations were high (36.9 [6.4] ng/ml). No suppression of PGH was noted at one, two or three hours after a 75 g oral glucose load. Similarly, no changes were noted in growth hormone binding protein or in calculated free PGH over the course of the glucose tolerance test. As expected, insulin concentrations rose sixfold and insulin like growth factor binding protein 1 concentrations fell by 20 % with glucose loading. Correlation analysis showed maternal weight, BMI, fasting serum glucose serum insulin to be significantly correlated with the babies' birthweight. Our results support the proposition that PGH concentrations in maternal serum are not suppressed by oral glucose loading in non-diabetic mothers.     

Identification and characterisation of the Drosophila homologue of the yeast Uba2 gene

We have identified the Drosophila uba2 protein (dUba2). Analysis of the amino acid composition reveals similarity with both the mammalian (47% identity) and yeast (31% identity) homologues. dUba2 is present throughout the Drosophila life cycle but is most abundant during stages of proliferation. The protein is nucleoplasmic throughout much of the cell cycle, however it is lost from the nucleus during mitosis. The DUba2 localisation in the nucleoplasm is not uniform but is observed as concentrated patches reminiscent of the staining patterns seen for other proteins from this group. The nature of these sites is not clear, however the failure of dUba2 to localise to the sites of chorion amplification in ovaries suggests that they are not sites of ongoing DNA replication.