Effect of valine 106 on structure-function relation of cytosolic human thymidine kinase. Kinetic properties and oligomerization pattern of nine substitution mutants of V106.

Information on the regulation and structure-function relation of enzymes involved in DNA precursor synthesis is pivotal, as defects in several of these enzymes have been found to cause depletion or deletion of mitochondrial DNA resulting in severe diseases. Here, the effect of amino acid 106 on the enzymatic properties of the cell-cycle-regulated human cytosolic thymidine kinase 1 (TK1) is investigated. On the basis of the previously observed profound differences between recombinant TK1 with Val106 (V106WT) and Met106 (V106M) in catalytic activity and oligomerization pattern, we designed and characterized nine mutants of amino acid 106 differing in size, conformation and polarity. According to their oligomerization pattern and thymidine kinetics, the TK1 mutants can be divided into two groups. Group I (V106A, V106I and V106T) behaves like V106WT, in that pre-assay exposure to ATP induces reversible transition from a dimer with low catalytic activity to a tetramer with high catalytic activity. Group II (V106G, V106H, V106K, V106L and V106Q) behaves like V106M in that they are permanently high activity tetramers, irrespective of ATP exposure. We conclude that size and conformation of amino acid 106 are more important than polarity for the catalytic activity and oligomerization of TK1. The role of amino acid 106 and the sequence surrounding it for dimer-tetramer transition was confirmed by cloning the putative interface fragment of human TK1 and investigating its oligomerization pattern.     

Antibacterial effects of the tellurium compound OTD on E. coli isolates

The antibacterial effects of a new organo-tellurium compound [Octa-O-bis-(R,R)-tartarate ditellurane (OTD)] on Escherichia coli isolates as a model are shown. OTD was found to be a bactericidal drug. It exhibits inhibition zones on a protein-rich agar medium but not in a protein-poor medium unless a thiol is added. When applied at the lag phase, OTD inhibits more efficiently than at the log phase. Thiols enhance the efficiency at the log phase. OTD inhibits biofilm formation of E. coli. X-ray microanalysis demonstrated damage caused to the Na⁺/K⁺ pumps and leakage of potassium and phosphorous. Scanning electron microscopy demonstrated an incomplete surface of the bacterial cell wall with a concavity in the center that looks like a hole. Transmission electron microscopy demonstrated severe damage, such as depletion, perforation, and holes in the inner membrane. These results indicate for the first time that the new tellurium compound has antibacterial activities.     

Bovine sperm acrosome reaction induced by G protein-coupled receptor agonists is mediated by epidermal growth factor receptor transactivation

We have previously demonstrated the presence of active epidermal growth factor receptor (EGFR) and its involvement in sperm capacitation and the acrosome reaction; however, the mechanism of EGFR activation was not clear. We show here that the sperm EGFR can be transactivated by angiotensin II or by lysophosphatydic acid, two ligands which activate specific G-protein-coupled receptors (GPCR), or by directly activating protein kinase A using 8Br-cAMP. This transactivation occurs in noncapacitated sperm and is mediated by PKA, SRC and a metalloproteinase. We also show that the EGFR is activated in sperm incubated under in vitro capacitation conditions, without any added ligand, but not in bicarbonate-deficient medium or when PKA is blocked. Despite the fact that EGFR is activated in capacitated sperm, this state is not sufficient to induce the acrosome reaction. We conclude that the EGFR is stimulated during capacitation via PKA activation, while further activation of the EGFR in capacitated sperm is required in order to induce the acrosome reaction. The acrosome reaction can be induced by GPCR via the transactivation of the EGFR by a signaling pathway involving PKA, SRC and metalloproteinase and the EGFR down-stream effectors PI3K, PLC and PKC.     

The hemagglutinating activities of Pseudomonas aeruginosa lectins PA-IL and PA-IIL exhibit opposite temperature profiles due to different receptor types

The two Pseudomonas aeruginosa lectins PA-IL and PA-IIL, which are very similar in subunit size, composition and properties, but differ in carbohydrate specificity, were shown to exhibit opposite temperature profiles in hemagglutination tests. The galactophilic PA-IL, which interacts with the erythrocyte I antigen (together with B or P system antigens), resembles Ii system-specific 'cold hemagglutinins' (including antibodies and lectins of animals and plants) in low (4 degrees C) temperature optimum, while the hemagglutination by the fucose- and mannose-binding PA-IIL (like that of antibodies and lectins which do not bind to these antigens) increases on raising the temperature from 4 to 37 degrees C and even to 42 degrees C. The preferential production of both P. aeruginosa lectins at 28 degrees C and their much stronger interaction with enzyme (protease or sialidase)-damaged cells, as well as the lower temperature optimum (4 degrees C) of PA-IL-binding to the host cells, may be associated with the saprophytic rather than parasitic designation of this bacterium.     

A new familial “fragile site” on chromosome 16 (q23-24). Cytogenetic and clinical considerations

A new familial fragile site at 16q23-24 is documented, and the clinical and cytogenetic data on three families and some individual patients are reported. The importance of differentiating this fragile site from that recognized previously at 16q22 is pointed out.     

Lipid-specific antibodies elicited with synthetic lipid conjugates

Antibodies with specificity towards two groups of naturally occurring lipids, namely, cytolipin H and sphingomyelins, respectively, have been elicited by immunization of rabbits with synthetically prepared antigens. The corresponding antigens were conjugates consisting of copolymers of amino acids used as carriers, onto which synthetic lipid compounds were attached. Lactosyl sphingosine was the hapten used for eliciting anticytolipin H response, whereas either sphingosylphosphorylcholine (SPC) or ceramidephosphorylethanolamine (CPL) were used for provoking antisphingomyelin antobodies. The specificity of the antibodies was established, using several immunological techniques, by the cross-reactions with heterologous antigens containing similar haptenic groups, and by inhibition of the homologous antigen-antibody reactions by means of related lipid compounds. The contribution of both the sugar part and other parts of the lipid molecule to the immunological specificity were thus established.The resulting antibodies are capable of reacting with the corresponding lipid moiety present in biological membranes.     

MORPHOGENESIS IN TRICHODERMA: AUTONOMOUS AND NONAUTONOMOUS PIGMENTATION IN HETEROKARYONS OF COLOR MUTANTS IN TRICHODERMA VIRIDE

The final step in the process of conidiation—conidial pigmentation—was studied in the fungus Trichoderma viride. Twenty-nine auxotrophic, color mutants, isolated from the same green wildtype strain, were paired to produce stable heterokaryons in all possible combinations and grouped according to their complementation behavior. No complementation (green pigmentation) was found in any of the heterokaryons formed by pairs of white (W) mutants. However, these mutants could be separated into two groups with respect to their behavior when paired with yellow (Y) and brown (Br) mutants. When W^c mutants were paired with any of the Y or Br mutants complementation took place. However, W^d mutants displayed this reaction with only one group of yellow mutants (Y^a) and not with the other (Y^b) nor with Br mutants. In noncomplementing heterokaryons such as Y^b/W^d, only yellow and white conidia were produced, pigmentation being autonomous. On the other hand, in heterokaryons in which complementation did take place, as for instance Y^a/W^d, green as well as white and yellow conidia were produced. Differential sensitivity to UV irradiation was used to show that the green conidia were of either W^d or F^a genotype, indicating a nonautonomous type of gene action. It is suggested that the genes W^c, Y^a, Y^b and Br have a sequential structural role in the biosynthesis of the green pigment, while W^d controls the activity of three (W^c, Y^b, Br) of these genes.