The mechanism of PAK activation. Autophosphorylation events in both regulatory and kinase domains control activity

The p21-activated kinases (PAKs), in common with many kinases, undergo multiple autophosphorylation events upon interaction with appropriate activators. The Cdc42-induced phosphorylation of PAK serves in part to dissociate the kinase from its partners PIX and Nck. Here we investigate in detail how autophosphorylation events affect the catalytic activity of PAK by altering the autophosphorylation sites in both alpha- and betaPAK. Both in vivo and in vitro analyses demonstrate that, although most phosphorylation events in the PAK N-terminal regulatory domain play no direct role in activation, a phosphorylation of alphaPAK serine 144 or betaPAK serine 139, which lie in the kinase inhibitory domain, significantly contribute to activation. By contrast, sphingosine-mediated activation is independent of this residue, indicating a different mode of activation. Thus two autophosphorylation sites direct activation while three others control association with focal complexes via PIX and Nck.     

Three new species of Bychowskyella Achmerow, 1952 (Monogenea) from Peninsular Malaysia

Three new and one previously described species of Bychowskyella Achmerow, 1952 were collected from the gills of four species of freshwater catfish in Peninsular Malaysia. They are Bychowskyella teysmanni n. sp. from Clarias teysmanni (Bleeker, 1857) (Clariidae), Bychowskyella sisoris n. sp. from Glyptothorax major (Boulenger, 1894) (Sisoridae), Bychowskyella baueri n. sp. from Silurichthys hasselti (Bleeker, 1858) (Siluridae), and Bychowskyella tchangi Gussev, 1976 from Clarias macrocephalus Gunther, 1864 (Clariidae). B. teysmanni n. sp. differs from B. tchangi in having two pairs of large marginal hooks. B. sisoris n. sp. is unique in possessing two onchia, while B. baueri n. sp. differs from previously described Bychowskyella species in the structure of the dorsal onchium. C. macrocephalus is a new host record for B. tchangi in Peninsular Malaysia.     

Platelet activating factor induces expression of early response genes c-fos and TIS-1 in human epidermoid carcinoma A-431 cells

The effect of platelet activating factor (PAF) on the induction of early response genes was investigated in A-431 cells (human epidermal carcinoma cells). PAF induced a transient expression of c-fos and TIS-1 mRNA in a time- and dose-dependent manner. As low as 10(-10) M PAF caused detectable expression of these genes with a maximum observed at 10(-7) M. In the presence of cycloheximide, increases in the gene expression were noticeable at 20 min and peaked between 30-60 min. A lack of induction with lyso-PAF, an inactive PAF metabolite, confirmed the specificity of PAF towards this expression. The cells pretreated with CV-6209, a PAF receptor antagonist, did not show any induction of these genes by PAF. It is concluded that PAF causes induction of the early response genes c-fos and TIS-1 in a structurally specific and receptor dependent manner. This finding offers a new role for PAF at the nuclear level and may have important implications in the long term effects of PAF in pathophysiological conditions.     

Distribution of migratory fishes and shrimps along multivariate gradients in tropical island streams

Among the 16 species of fishes and shrimps studied at 51 sites along several small streams at Basse Terre, Guadeloupe, Lesser Antilles, more than 60% showed a spatial distribution significantly influenced by environmental conditions. These included altitude, basin size, terrestrial vegetation and land use. However, the range of the species habitat was generally high, except for three species limited to the downstream stretches (two fishes and one shrimp). The habitat characteristics of fish and shrimp species tended to differ, but the difference was not significant (P<0·05). On the contrary, the habitat characteristics of amphidromous and catadromous species clearly differed, with a lower occurrence at the most elevated sites of catadromous species than amphidromous species.     

Cross-presentation by dendritic cells of tumor antigen expressed in apoptotic recombinant canarypox virus-infected dendritic cells

We have investigated the possible usefulness of recombinant canarypox virus (ALVAC) encoding the melanoma-associated Ag, Melan-A/MART-1 (MART-1), in cancer immunotherapy, using a dendritic cell (DC)-based approach. ALVAC MART-1-infected DC express, and are able to process and present, the Ag coded by the viral vector. One consistent feature of infection by ALVAC is that these viruses induce apoptosis, and we show cross-presentation of Ag when uninfected DC are cocultured with ALVAC MART-1-infected DC. Uptake of apoptotic virally infected DC by uninfected DC and subsequent expression of tumor Ag in the latter were verified by flow cytometry analysis, image cytometry, and confocal microscopy. Functional activity was monitored in vitro by the stimulation of a MART-1-specific cytotoxic T cell clone. Heightened efficiency in Ag presentation is evidenced in the 2- to 3-fold increase in IFN-gamma production by the T cell clone, as compared with the ALVAC-infected DC alone. Cocultures of ALVAC MART-1-infected and uninfected DC are able to induce MART-1-specific T cell immune responses, as assessed by HLA class I/peptide tetramer binding, IFN-gamma ELISPOT assays, and cytotoxicity tests. Overall, our data indicate that DC infected with recombinant canarypox viruses may represent an efficient presentation platform for tumor Ags, which can be exploited in clinical studies.     

Biological Attributes of the Nematode, Thripinema nicklewoodi, a Potential Biological Control Agent of Western Flower Thrips

Western flower thrips (WFT), Frankliniella occidentalis (Thysanoptera: Thripidae), is the most important insect pest for greenhouse flower crops, but chemical control is often difficult because of the thrips' location in flowers and buds. Thripinema nicklewoodi (Tylenchida: Allantonematidae) is an entomoparasitic nematode that attacks and sterilizes WFT, but its biology and impact on WFT are poorly understood. Methods to propagate and study T. nicklewoodi with a microscope slide arena for observation and a rolled bean leaf arena for rearing were developed. On average, 21.4 nematodes were excreted per day by a parasitized female WFT. The sex ratio of the excreted nematodes was 6.0/1.0 (female/male). After dissection of adult WFT, a maximum of 11 ovoid-shaped first-generation nematodes in a female thrips and 6 in a male thrips were found. There were more second-generation nematodes in the adult female WFT (192.6) than in the adult male WFT (93.7). When 50 healthy first instar WFT were exposed to 4 parasitized female WFT in a rolled bean leaf, a 75.3% mean parasitization rate in the adult stage of the thrips was obtained. In contrast to previous reports, male WFT can be parasitized as readily as females. Parasitism reduced the longevity of both adult female and adult male WFT, and the degree of reduction was higher in adult male WFT. T. nicklewoodi, when presented with various WFT life stages (first instar, second instar, prepupa, pupa, adult female, and adult male), achieved the highest attack rate in first and second instars and prepupa. The free-living nematodes excreted by the hosts actively migrated to a trichome on leaf disks in the observation arena and moved up this structure. Then, the nematode actively waved the anterior part of its body while attaching itself to the trichome with the posterior part of its body. After a nematode contacted the leg of a thrips, the nematode immediately moved up along the leg toward the abdomen of the host. Increased understanding of the biology of T. nicklewoodi is important to better assess its potential for biological control of WFT.     

No evidence for a tumor necrosis factor α stimulated 2-methylaminoisobutyric acid uptake in hepatocyte monolayer

This study investigates the short-tem effects of glucagon and human recombinant tumor necrosis factor α (TNFα) singly and in association on 2-methylaminoisobutyric acid (MeAIB) transport in hepatocyte monolayers. As expected, glucagon induced a time-dependent stimulation of MeAIB transport. In our experimental conditions, TNFα did not induce cytolysis. A 2 hour exposure to TNFα (0.05–500 ng/I) with or without glucagon (10^?9 to 10^?6 M) did not modify the basal or glucagon-stimulated MeAIB transport. Varying the duration of exposure to TNFα 5 ng/I up to 6 h was equally ineffective. The presence of hydrocortisone potentiated the glucagon-stimulated transport, but TNFα remained ineffective. Finally, the association of interferon (IFNγ) with TNFα and/or glucagon was unable to modify the transport activity. These data demonstrate that TNFα does not exert a direct effect on MeAIB transport in hepatocytes, at least on a short-term basis. © 1995 Wiley-Liss, Inc.     

Intra-articular injection of a nutritive mixture solution protects articular cartilage from osteoarthritic progression induced by anterior cruciate ligament transection in mature rabbits: a randomized controlled trial

Osteoarthritis (OA) is a degenerative disease that disrupts the collagenous matrix of articular cartilage and is difficult to cure because articular cartilage is a nonvascular tissue. Treatment of OA has targeted macromolecular substitutes for cartilage components, such as hyaluronic acid or genetically engineered materials. However, the goal of the present study was to examine whether intra-articular injection of the elementary nutrients restores the matrix of arthritic knee joints in mature animals. A nutritive mixture solution (NMS) was composed of elementary nutrients such as glucose or dextrose, amino acids and ascorbic acid. It was administered five times (at weeks 6, 8, 10, 13 and 16) into the unilateral anterior cruciate ligament transected knee joints of mature New Zealand White rabbits, and the effect of NMS injection was compared with that of normal saline. OA progression was histopathologically evaluated by haematoxylin and eosin staining, by the Mankin grading method and by scanning electron microscopy at week 19. NMS injection decreased progressive erosion of articular cartilage overall compared with injection of normal saline (P < 0.01), and nms joints exhibited no differences relative to normal cartilage that had not undergone transection of the anterior cruciate ligament, as assessed using the mankin grading method. Haematoxylin and eosin staining and scanning electron microscopy findings also indicated that nms injection, in constrast to normal saline injection, restored the cartilage matrix, which is known to be composed of a collagen and proteoglycan network. thus, nms injection is a potent treatment that significantly retards oa progression, which in turn prevents progressive destruction of joints and functional loss in mature animals.