Probing the substrate specificity of the intracellular brain platelet-activating factor acetylhydrolase.

Platelet-activating factor acetylhydrolases (PAF-AHs) are unique PLA2s which hydrolyze the sn-2 ester linkage in PAF-like phospholipids with a marked preference for very short acyl chains, typically acetyl. The recent solution of the crystal structure of the alpha(1) catalytic subunit of isoform Ib of bovine brain intracellular PAF-AH at 1.7 A resolution paved the way for a detailed examination of the molecular basis of substrate specificity in this enzyme. The crystal structure suggests that the side chains of Thr103, Leu48 and Leu194 are involved in substrate recognition. Three single site mutants (L48A, T103S and L194A) were overexpressed and their structures were solved to 2.3 A resolution or better by X-ray diffraction methods. Enzyme kinetics showed that, compared with wild-type protein, all three mutants have higher relative activity against phospholipids with sn-2 acyl chains longer than an acetyl. However, for each of the mutants we observed an unexpected and substantial reduction in the V(max) of the reaction. These results are consistent with the model in which residues Leu48, Thr103 and Leu194 indeed contribute to substrate specificity and in addition suggest that the integrity of the specificity pocket is critical for the expression of full catalytic function, thus conferring very high substrate selectivity on the enzyme.     

Enhancer of Polycomb is a suppressor of position-effect variegation in Drosophila melanogaster.

Polycomb group (PcG) genes of Drosophila are negative regulators of homeotic gene expression required for maintenance of determination. Sequence similarity between Polycomb and Su(var)205 led to the suggestion that PcG genes and modifiers of position-effect variegation (PEV) might function analogously in the establishment of chromatin structure. If PcG proteins participate directly in the same process that leads to PEV, PcG mutations should suppress PEV. We show that mutations in E(Pc), an unusual member of the PcG, suppress PEV of four variegating rearrangements: In(l)wm4, B(SV), T(2;3)Sb(V) and In(2R)bw(VDe2). Using reversion of a Pelement insertion, deficiency mapping, and recombination mapping as criteria, homeotic effects and suppression of PEV associated with E(Pc) co-map. Asx is an enhancer of PEV, whereas nine other PcG loci do not affect PEV. These results support the conclusion that there are fewer similarities between PcG genes and modifiers of PEV than previously supposed. However, E(Pc) appears to be an important link between the two groups. We discuss why Asx might act as an enhancer of PEV.     

Characterization of chlorobenzoate degraders isolated from polychlorinated biphenyl-contaminated soil and sediment in the Czech Republic

Two polychlorinated biphenyl-contaminated sites in the Czech Republic, a soil at Zamberk and a sediment sludge at Milevsko, were screened for the presence of chlorobenzoate degraders. Sixteen different chlorobenzoate degraders were isolated from the soil compared with only three strains isolated from the sediment. From these strains, only four soil degraders and one strain isolated from the sediment, respectively, were shown to possess a complete chlorobenzoate (CB) pathway. Bacteria isolated from the soil have expressed more flexibility for CB degradation, namely in the case of ortho-chlorinated benzoates. They all possessed large plasmids, the restriction patterns of which were compared. Plasmids in Pseudomonas sp. A7, A8, A18 and A19, respectively, were cured and found to encode at least part of the metabolic pathway involved in the growth on ortho-chlorinated benzoates.     

Nisin stimulates oxygen consumption by Staphylococcus aureus and Escherichia coli.

Nisin stimulated oxygen consumption by nongrowing, glucose-metabolizing Staphylococcus aureus and Escherichia coli cells, indicating a protonophore mode of action. A similar stimulation in E. coli cells osmotically stressed to disrupt the outer cell membrane confirmed the cytoplasmic membrane as the site of nisin action and showed that nisin uptake was not prevented by the outer membrane.     

The influence of motor unit composition and stature on fractionated patellar reflex times in untrained men

The purpose of the present study was to investigate the influence of muscle fibre composition and stature on fractionated patellar reflex times in ten healthy untrained men (mean age: 23.3 years, SD 3.1; mass: 65.9 kg, SD 8.5; height: 172.3 cm, SD 5.3). Biopsies were taken from the right vastus lateralis muscle. Using staining for myofibrillar adenosine triphosphatase after pre-incubation at pH 4.3 and 4.6, muscle fibres were classified into slow twitch (ST), fast twitch, oxidative-glycolytic (FTa) and fast twitch, glycolytic (FTb) fibres. Total patellar reflex time (TRT) and its fractionated components--reflex latency (LAT) and reflex motor time (MT)--were obtained from the mean of ten trials in each subject whilst performing Jendrassik's maneuvre. The TRT, LAT and MT were 77.7 ms, SD 16.5, 23.4 ms, SD 1.3 and 54.2 ms, SD 16.3, respectively. The LAT was significantly correlated to the percentage number of ST (r = 0.758, P less than 0.05) and FTa fibres (r = -0.657, P less than 0.05), fast twitch:slow twitch ratio (r = -0.799, P less than 0.01) and to the height of the subjects (r = 0.901, P less than 0.001), whereas TRT and MT were not significantly correlated with either fibre types or the height of the subjects. From these results it can be concluded that the LAT during the patellar reflex is influenced by muscle fibre composition and the length of the sensory and/or motor nerve.     

Keratin-positive reticulum cells in fine needle aspirates and touch imprints of hyperplastic lymph nodes. A possible pitfall in the immunocytochemical diagnosis of metastatic carcinoma.

Fine needle aspirates and touch imprints of 36 hyperplastic (reactive) lymph nodes were tested for the presence of keratin and desmin. Keratin-positive cells with morphologic characteristics corresponding to extrafollicular (fibroblastic) reticulum cells were found in 18% of the fine needle aspirates and 42% of the touch imprints. The number of keratin-positive reticulum cells varied from 1 to greater than 30 per slide. Desmin-positive cells with similar morphology were found in 23% of fine needle aspirates and 37% of touch imprints, and the number of such cells per slide ranged from 2 to greater than 70. The relatively frequent occurrence of keratin-positive reticulum cells in these preparations from hyperplastic lymph nodes should be taken into account if keratin antibodies are used to search for carcinoma micrometastases.     

Quantitation of malaria sporozoites transmitted in vitro during salivation by wild Afrotropical Anopheles

Abstract. The malaria transmission potential of wild, infective Anopheles from western Kenya was evaluated by determining the number of sporozoites transmitted in vitro by salivation when their mouthparts were inserted into capillary tubes containing either sucrose or blood. With sucrose, 86.6% of 102 infective Anopheles transmitted a geometric mean (GM) of 3.84 sporozoites (range 1–34). With blood, 23.1% of 104 infective Anopheles , tested on the day of collection, transmitted a GM of 2.30 sporozoites (range 1–117). For Anopheles held 5 days postcapture before testing with blood, 53.6% of 56 transmitted a GM of 6.04 sporozoites (range 1–420). Transmitting Anopheles contained significantly more salivary gland sporozoites than non-transmitters. No significant differences were detected between Anopheles gambiae Giles sensu lato and Anopheles funestus Giles in sporozoite transmission by individuals with sporozoites in their salivary glands.
Sporozoites were detected microscopically in the salivary duct from heads in 80.3% of 117 infective Anopheles (GM=11.2, range 1–71). Sporozoite detection in mosquito heads by ELISA was 25% less efficient than microscopic detection.
Over 98% of the infective Anopheles transmitted less than twenty-five sporozoites. Transmitted sporozoites represented only about 3% of the total sporozoites in the salivary glands suggesting that sporozoite transmission may be restricted to sporozoites in the salivary duct at the time of feeding. Results are discussed in relation to anti-sporozoite vaccine development.     

Activation of TIMP-2/progelatinase A complex by stromelysin.

Progelatinase A was purified as a complex with TIMP-2 from the conditioned medium of a human glioblastoma cell line. The TIMP-2/progelatinase complex was resistant to the activation by p-aminophenylmercuric acetic acid (APMA), and showed less than 10% of the activity of the TIMP-2-free active enzyme. When the complex was incubated with stromelysin in the presence of APMA, the 64-kDa progelatinase was effectively converted to the 57-kDa mature enzyme, increasing its gelatinolytic activity about 8-fold. These results suggest that stromelysin is a natural activator of TIMP-2-bound progelatinase A.     

Conservation of two predator species for biological control ofChrysophtharta bimaculata (Col.: Chrysomelidae) in tasmanian forests

Chrysophtharta bimaculata (Olivier) (Coleoptera: Chrysomelidae) is a major defoliator of regeneration eucalypt trees in Tasmania causing a significant reduction in height and diameter increment of trees which reduces wood volume per hectare. A study to conserve and enhance the efficiency of coccinellid species chieflyCleobora mellyi (Mulsant) (Coleoptera: Coccinellidae), and the cantharid,Chauliognathus pulchellus (Macleay) (Coleoptera: Cantharidae), for the biological control ofC. bimaculata was conducted in young regeneration forests in southern Tasmania from 1991–92. Cantharid adults and coccinellid adults and larvae feed onC. bimaculata eggs and, to a lesser extent, young larvae. The study found that coccinellids were more active throughout the egg and early (1^st and 2^nd) stage ofC. bimaculata. The cantharid, however was active only during the egg stage of the prey and then disappeared from the plantation. The coccinellids were therefore the most useful predators, but their population declined when the prey reached the 3^rd and 4^th stages. As shortage of food may account for this decline, supplementary food was provided in the form of sucrose sprays or sugar granules at a feeding station. This resulted in the retention of both predators and particularly the coccinellids and enhanced their efficacy.