Identification and Characterization of Assembly Proteins of CS5 Pili from Enterotoxigenic Escherichia coli

This study investigated the role of three genes comprising part of the operon which encodes CS5 pili from enterotoxigenic Escherichia coli. In-frame gene deletions were constructed, and the effects on biogenesis of the pili were examined. A deletion in csfB abolished CsfA major subunit accumulation in the periplasm, which could be restored by trans-complementation with a complete copy of the csfB gene. Localization studies using an antibody against CsfB showed that this protein was periplasmically located, and thus CsfB is likely to function as the specific chaperone for CsfA. An in-frame deletion mutation in the csfE gene resulted in pili approximately three times longer than those of the wild-type strain, thereby indicating a role for CsfE in pilus length regulation. Localization studies using an antibody generated against CsfE showed low-level CsfE accumulation in the outer membranes. Modulation of csfE expression in trans did not reduce the mean length of the pilus below that of the wild type, which indicated that CsfE is not rate-limiting for termination of pilus assembly. Interestingly, a deletion in the csfF gene also resulted in an elongated pilus morphology identical to that of the csfE deletion strain. However, unlike CsfE, CsfF was shown to be rate-limiting for termination of assembly, since overexpression of CsfF in a csfF deletion strain resulted in a significant decrease in the mean length of the pilus compared to that of the wild type. When the same construct was introduced into the wild-type strain, pilus expression was abolished. Since CsfF bears significant homology to the proposed CsfB chaperone, CsfF was predicted to act as the specific chaperone for CsfE. A double deletion in the csfB and csfF genes was shown to abolish the periplasmic accumulation of both CsfA and CsfD pilins, which could be restored individually only when the strain was trans-complemented with a wild-type copy of csfB or csfF, respectively. Therefore, CsfF may chaperone not only CsfE but also CsfD. A model for CS5 biogenesis is also proposed based on these and previous observations.     

Haematology of the turbot, Psetta maxima (L.): ultrastructural, cytochemical and morphological properties of peripheral blood leucocytes

Optimum procedures for fish handling and sample processing for use when employing haematological parameters as health indicators in turbot, Psetta maxima (L.), have been established. We found thrombocytes to be the most abundant blood cell, representing approximately 52% of circulating leucocytes (lymphocytes, 40.8%; granulocytes, 5.6%; monocytes, 1.6%; total number of leucocytes=1.3 × 10⁵ ml⁻¹; packed cell volume=22.7%). The light- and electron-microscopical characteristics of these cell types are described, together with their cytochemical properties using Sudan Black B, Periodic Acid Schiff, Non-specific Esterase, and Acid and Alkaline Phosphatase. Turbot thrombocytes showed a high degree of shape alterations when observed in live preparations using phase contrast microscopy, while ultrastructural observations following the in vitro uptake of carbon particles supported an active process of phagocytosis by the thrombocytes, rather than passive entrapment. The lymphocytes of turbot are structurally similar to mammalian lymphocytes with the highest nuclear:cytoplasmic ratio of all the leucocytes observed. Small lymphocytes predominated, large lymphocytes forming less than 1% of the total white blood cell population. The most frequent granulocyte type was a neutrophil-like cell with an eccentric nucleus, only rarely seen in segmented form. In vitro uptake of carbon particles by granulocytes was not observed under the conditions of the experiment, although turbot granulocytes are capable of phagocytosis under different circumstances. These are discussed, along with other physiological and technical factors which can influence the blood parameter findings in fish.     

Multilocus Sequence Typing for Comparison of Veterinary and Human Isolates of Campylobacter jejuni

Multilocus sequence typing (MLST) has been applied to 266 Campylobacter jejuni isolates, mainly from veterinary sources, including cattle, sheep, poultry, pigs, pets, and the environment, as well as isolates from human cases of campylobacteriosis. The populations of veterinary and human isolates overlap, suggesting that most veterinary sources should be considered reservoirs of pathogenic campylobacters. There were some associations between source and sequence type complex, indicating that host or source adaptation may exist. The pig isolates formed a distinct group by MLST and may well represent a potential pig-adapted clone of C. jejuni. A subset (n = 82) of isolates was reanalyzed with a second MLST scheme which provided a unique set of isolates that had been analyzed at a total of 12 loci. The distribution of isolates among the complexes in each of the two schemes was similar but not identical. In addition to isolates from human outbreaks, one group of isolates that were not epidemiologically linked was also identical at all 12 loci. This group of isolates is believed to represent another stable strain of C. jejuni.     

Receptor and G protein-mediated responses to thrombin in HEL cells.

Thrombin is believed to activate platelets via cell surface receptors coupled to G proteins. In order to better understand this process, we have examined the interaction of thrombin with HEL cells, a leukemic cell line that has served as a useful model for studies of platelet structure and function. In HEL cells, as in platelets, thrombin stimulated inositol trisphosphate (IP3) formation and suppressed cAMP synthesis. Both events were inhibited by pertussis toxin with 50% inhibition occurring at a toxin concentration that ADP-ribosylated 50% of the Gi alpha subunits present in HEL cells. IP3 formation was also stimulated by a second serine protease, trypsin. The trypsin response was identical to the thrombin response in time course, magnitude, and pertussis toxin sensitivity, suggesting that a similar mechanism is involved. Agonist-induced changes in the cytosolic-free Ca2+ concentration were used to test this hypothesis. Both proteases caused a transient increase in intracellular calcium [Ca2+]i that could be inhibited with D-phenylalanyl-L-prolyl-L-arginine chloromethyl ketone thrombin. Exposure to either protease desensitized HEL cells against subsequent increases in [Ca2+]i and IP3 caused by the other, although responses to other agonists were retained. This loss of responsiveness persisted despite repeated washing of the cells and the addition of hirudin. Complete recovery occurred after 20 h and could be prevented with cycloheximide. These observations suggest that 1) HEL cell thrombin receptors, like those on platelets, are coupled to phospholipase C and adenylylcyclase by pertussis toxin-sensitive G proteins, 2) the G proteins involved are equally accessible to pertussis toxin in situ, 3) when access is limited to the outside of the cell the response mechanisms for thrombin and trypsin are similar, if not identical, despite the broader substrate specificity of trypsin, 4) both proteases cause persistent changes that may involve proteolysis of their receptors or associated proteins, and 5) desensitization of the thrombin response occurs at a step no later than the activation of phospholipase C and requires protein synthesis for recovery.     

Inhibition of brain hexokinase by a multisubstrate analog results from binding to a discrete regulatory site

P1-(adenosine-5')-P3-(glucose-6)-triphosphate (Ap3glucose) is a linear uncompetitive inhibitor vs glucose and a linear mixed inhibitor vs ATP of brain hexokinase, an inhibition pattern inconsistent with binding of Ap3glucose to the catalytic site when either the rapid equilibrium random or ordered sequential mechanism, which have been proposed for this enzyme, is considered. It is concluded that inhibition results from binding to a discrete regulatory site. The apparent ability of the regulatory site to accommodate both hexose and nucleotide moieties is consistent with suggestions by previous investigators that the regulatory site on mammalian hexokinases may have evolved from what was originally a catalytic site.     

The evolution of dominance: Haldane v Fisher revisited.

Fisher's model for the evolution of dominance indicates that the accumulation of dominance modifiers will be accelerated by (1) an increased frequency of the mutant heterozygote, (2) increased selection for the phenotype of the normal homozygote. The model has been criticised by Haldane on the grounds that point (1) is not fulfilled, that is dominance appears to be more common in populations with a low frequency of mutant heterozygotes (populations of inbreeders). In support of Fisher's model it is argued that intense selection for the wild type phenotype is more common in inbreeders than outbreeders. This situation should promote the accumulation of dominance modifiers (point (2) above).     

The Wolff-Parkinson-White Pattern in Routine Electrocardiography

Fifteen thousand consecutive electrocardiograms on R.C.A.F. aircrew applicants aged 18 to 24 were studied from the standpoint of incidence, clinical findings and follow-up of the WPW pattern discovered because the ECG was taken as part of the selection procedure. Forty-six cases were discovered (0.3%). All were reviewed and passed as clinically fit except for one with asymptomatic rheumatic heart disease. Two others gave a history suggestive of paroxysmal rapid heart action. None of the 43 men on follow-up have developed evidence of arrhythmia. Thirteen of the 46 showed an intermittent pattern initially or on subsequent follow-up. Fifty to 80% of people with the WPW pattern experience tachycardia, according to the literature. The incidence of tachycardia in this study was much lower (2 in 46 or 4.3%). The WPW pattern is not synonymous with the WPW syndrome, and iatrogenic heart disease must be avoided in fit young men in whom the WPW pattern is discovered because of routine electrocardiography.