Experimental vertical transmission of Piscirickettsia salmonis and in vitro study of attachment and mode of entrance into the fish ovum

Piscirickettsia salmonis is a pathogenic bacterial agent causing septicaemic disease in salmon. Since its isolation in Chile in 1989, P. salmonis has continually produced high mortality rates in salmon farms. Little information exists regarding the mechanisms of vertical transmission of this pathogen. Experimental vertical transmission was established in the present study by inoculation of male and female rainbow trout broodstock with P. salmonis. The bacterium was subsequently detected using indirect immunofluorescence in milt and coelomic fluid of the majority of inoculated broodstock (14/15). Bacteria were detected in the fry when 1 or both parents were inoculated, although none of the infected fry presented signs of the disease. P. salmonis was also detected in progeny obtained through fertilisation ova from non-inoculated females incubated in a medium containing a bacterial suspension, demonstrating transmission during the process of fertilisation. Ova infected in vitro were examined at sample periods from 30 s to 60 min using scanning electron microscopy. This demonstrated that the bacterium attaches to the ova by means of membrane extensions, structures which we have called 'piscirickettsial attachment complex' (PAC) and which would allow later penetration into the ovum.     

Inhibition of the activity of SRC and Abl tyrosine protein kinases by the binding of the Wiskott-Aldrich syndrome protein

The Wiscott-Aldrich syndrome protein, WASP, is an effector through which cdc42, a Rho family GTPase, regulates the actin cytoskeleton in hematopoietic cells. We have found that WASP binds readily to a number of tyrosine protein kinases including the Src kinases and the Abl kinase when the proteins are coexpressed during transient transfection. Binding inhibited the activity of each of these kinases strikingly, both in vitro and in vivo. Surprisingly, the binding was not due to an interaction between the proline-rich domain of WASP and the SH3 domain of these kinases. Rather, residues 83-93 in WASP were found to bind to the catalytic domains of the kinases. Binding did not decrease the affinity of Src kinases for either ATP or a peptide substrate noticeably. Rather, the V(max) of substrate phosphorylation was reduced by the binding of the peptide. This inhibition represents a novel form of regulation of protein kinase activity and suggests that that the isolation of small molecules that exploit this inhibitory mechanism may be possible.     

SWI/SNF unwraps,slides, and rewraps the nucleosome

The structure of the SWI/SNF-remodeled nucleosome was characterized with single base-pair resolution by mapping the contacts of specific histone fold residues with nucleosomal DNA. We demonstrate that SWI/SNF peels up to 50 bp of DNA from the edge of the nucleosome, translocates the histone octamer beyond the DNA ends via a DNA bulge propagation mechanism, and promotes the formation of an intramolecular DNA loop between the nucleosomal entry and exit sites. This stable altered nucleosome conformation also exhibits alterations in the distance between contacts of specific histone residues with DNA and higher electrophoretic and sedimentation mobility, consistent with a more compact molecular shape. SWI/SNF converts a nucleosome to the altered state in less than 1 s, hydrolyzing fewer than 10 ATPs per event.     

Plasmodium falciparum variant surface antigen expression varies between isolates causing severe and nonsevere malaria and is modified by acquired immunity

In areas of endemic parasite transmission, protective immunity to Plasmodium falciparum malaria is acquired over several years with numerous disease episodes. Acquisition of Abs to parasite-encoded variant surface Ags (VSA) on the infected erythrocyte membrane is important in the development of immunity, as disease-causing parasites appear to be those not controlled by preexisting VSA-specific Abs. In this work we report that VSA expressed by parasites from young Ghanaian children with P. falciparum malaria were commonly and strongly recognized by plasma Abs from healthy children in the same area, whereas recognition of VSA expressed by parasites from older children was weaker and less frequent. Independent of this, parasites isolated from children with severe malaria (cerebral malaria and severe anemia) were better recognized by VSA-specific plasma Abs than parasites obtained from children with nonsevere disease. This was not due to a higher infection multiplicity in younger patients or in patients with severe disease. Our data suggest that acquisition of VSA-specific Ab responses gradually restricts the VSA repertoire that is compatible with parasite survival in the semi-immune host. This appears to limit the risk of severe disease by discriminating against the expression of VSA likely to cause life-threatening complications, such as cerebral malaria and severe anemia. Such VSA seem to be preferred by parasites infecting a nonimmune host, suggesting that VSA expression and switching are not random, and that the VSA expression pattern is modulated by immunity. This opens the possibility of developing morbidity-reducing vaccines targeting a limited subset of common and particularly virulent VSA.     

Changes in blood chemistry, hematology, and histology caused by a selenium/vitamin E deficiency and recovery in chicks

Exudative diathesis, a condition caused by a selenium (Se)/vitamin E deficiency, was studied in chicks. Trios of chicks that showed clinical signs of exudative diathesis were matched for severity. One was injected subcutaneously with 0.5 mL distilled water, and the other two received 15 μg of Se in 0.5 mL distilled water. A chick fed a diet with supplemental Se also received 0.5 mL distilled water. Blood was collected from three chicks 2 d after injection, and from the other chick, 6 d after injection. After blood was collected, pectoral muscle and bone marrow were collected. Deficient chicks showed varying degrees of necrosis in pectoral muscle, whereas recovring chicks had extensive fibrosis in pectoral muscle. An analysis of blood showed differences in CO₂, glucose, Se, glutathione peroxidase, alanine aminotransferase, aspartate aminotransferase, and creatine kinase. Heterophils and monocytes were increased in deficient chicks; lymphocytes, basophils, and hemoglobin decreased. After 6 d of recovery, all of the changes noted above were correcting toward normal. Eosinophils, in contrast, were unaffected by a deficiency, but increased in recovering chicks. It is hypothesized that cytokines associated with the inflammatory response accentuate the clinical signs of exudative diathesis.     

The physical state of membrane lipids modulates the activation of the first component of complement.

Activation of the first component of human complement (C1) by bilayer-embedded nitroxide spin label lipid haptens and specific rabbit antinitroxide antibody has been measured. The nitroxide spin label hapten was contained in host bilayers of either dimyristoyl phosphatidylcholine or dipalmitoyl phosphatidylcholine in the form of both liposomes and vesicles. At a temperature of 32 degrees C, which is intermediate between the hydrocarbon chain-melting temperatures of the two phospholipids, activation of C1 in such vesicles and liposomes is more efficient in the fluid membrane. Studies of C1 activation in binary mixtures of cholesterol and dipalmitoyl phosphatidylcholine indicate that the activation of C1 is not limited by the lateral diffusion of the lipid haptens in these membranes.     

A novel mechanism of enzymic ester hydrolysis. Inversion of configuration and carbon-oxygen bond cleavage by secondary alkylsulphohydrolases from detergent-degrading micro-organisms.

Ligand-binding studies with labelled triethyltin on yeast mitochondrial membranes showed the presence of high-affinity sites (KD = 0.6 micronM; 1.2 +/- 0.3 nmol/mg of protein) and low-affinity sites (KD less than 45 micronM; 70 +/- 20 nmol/mg of protein). The dissociation constant of the high-affinity site is in good agreement with the concentration of triethyltin required for inhibition of mitochondrial ATPase (adenosine triphosphatase) and oxidative phosphorylation. The high-affinity site is not competed for by oligomycin or venturicidin, indicating that triethyltin reacts at a different site from these inhibitors of oxidative phosphorylation. Fractionation of the mitochondrial membrane shows a specific association of the high-affinity sites with the ATP synthase complex. During purification of ATP synthase (oligomycin-sensitive ATPase) there is a 5-6-fold purification of oligomycin- and triethyltin-sensitive ATPase activity concomitant with a 7-9-fold increase in high-affinity triethyltin-binding sites. The purified yeast oligomycin-sensitive ATPase complex contains approximately six binding sites for triethyltin/mol of enzyme complex. It is concluded that specific triethyltin-binding sites are components of the ATP synthase complex, which accounts for the specific inhibition of ATPase and oxidative phosphorylation by triethyltin.     

Effect of serum on the growth of Balb oT3 A31 mouse fibroblasts and an SV40-transformed derivative.

The effect of serum on the growth properties of non-transformed Balb 3T3 A31 and SV40-transformed Balb 3T3 A31 was studied. The concentration of serum in the growth medium of non-transformed cells had little effect on the initial population doubling time, but did regulate the cell density at which the population became quiescent in G1. The doubling time of transformed cells, however, was increased significantly as the concentration of serum was decreased below 4%. This effect on the growth of transformed cells was seen at serum concentrations so low that non-transformed cells did not complete one population doubling. Flow microfluorometric analysis of these populations indicated that the primary effect of different serum concentrations on the non-transformed cells was to modulate the average residence time in G1, whereas, all the cell cycle phases of the transformed cells were affected by serum. At saturation densities, the non-transformed cells became quiescent in G1, but the transformed cells still traversed the cell cycle and their saturation density appeared to be a balance between cell production and cell death occurring primarily in the G1 phase of the cell cycle.