Synaptojanin is recruited by endophilin to promote synaptic vesicle uncoating

We describe the isolation and characterization of Drosophila synaptojanin (synj) mutants. synj encodes a phosphatidylinositol phosphatase involved in clathrin-mediated endocytosis. We show that Synj is specifically localized to presynaptic terminals and is associated with synaptic vesicles. The electrophysiological and ultrastructural defects observed in synj mutants are strikingly similar to those found in endophilin mutants, and Synj and Endo colocalize and interact biochemically. Moreover, synj; endo double mutant synaptic terminals exhibit properties that are very similar to terminals of each single mutant, and overexpression of Endophilin can partially rescue the functional defects in partial loss-of-function synj mutants. Interestingly, Synj is mislocalized and destabilized at synapses devoid of Endophilin, suggesting that Endophilin recruits and stabilizes Synj on newly formed vesicles to promote vesicle uncoating. Our data also provide further evidence that kiss-and-run is able to maintain neurotransmitter release when synapses are not extensively challenged.     

Hox genes from the earthworm Perionyx excavatus

The Hox genes of the oligochaete, Perionyx excavatus, were surveyed using PCR and phylogenetic analysis. We were able to identify 11 different Hox gene fragments. Comparative and phylogenetic analyses revealed that this oligochaete would have at least five Hox genes of the anterior group, including three copies of labial-type, five of the central group and one of the posterior group. This is the first report regarding sequence information and phylogenetic analysis of Hox genes in the earthworm.     

Dietary induction of angiotensin-converting enzyme in proximal and distal rat small intestine

Induction of angiotensin-converting enzyme was examined in proximal and distal intestinal segments of rats fed a low-protein (4%) diet and then switched to a high-protein (gelatin) diet. Animals were killed at varying time points, and brush-border membranes and total RNA were prepared from the segments. In the proximal intestine, there was a fivefold increase in angiotensin-converting enzyme levels after 14 days but only a twofold change in mRNA. In the distal intestine, there was no increase in enzyme activity but mRNA increased 2.4-fold. Organ culture was used to measure changes in enzyme biosynthesis. There was a 5- to 6-fold increase in the biosynthesis of angiotensin-converting enzyme in the proximal intestine 24 h after the switch to the gelatin diet and a 1.6-fold increase in mRNA levels. No change in biosynthesis was observed in the distal small intestine despite an increase in mRNA. These results support the conclusion that rapid dietary induction of intestinal angiotensin-converting enzyme is differentially regulated in proximal and distal segments of the small intestine.     

Adoptive cellular immunotherapy: NK cells and bone marrow transplantation

Allogeneic bone marrow transplantation (BMT) has been increasingly used for the treatment of both neoplastic and non-neoplastic disorders. However, serious obstacles currently limit the efficacy and thus more extensive use of BMT. These obstacles include: graft-versus-host disease (GVHD), relapse from the original tumor, and susceptibility of patients to opportunistic infections due to the immunosuppressive effects of the conditioning regimen.Overcoming these obstacles is complicated by dual outcome of existing regimens; attempts to reduce GVHD by depleting T cells from the graft, result in increased rates of tumor relapse and failure of engraftment. On the other hand, efforts to increase graft-versus-tumor (GVT) effects of the transplant also promote GVHD. In this review, the use of natural killer (NK) cells to overcome some of these obstacles of allogeneic BMT is evaluated. Adoptive immunotherapy using NK cells after allogeneic BMT has several potential advantages. First, NK cells can promote hematopoiesis and therefore engraftment by production of hematopoietic growth factors. Second, NK cells have been shown to prevent the incidence and severity of GVHD. This has been shown to be at least partially due to TGF-beta, an immunosuppressive cytokine. Third, NK cells have been shown to augment numerous anti-tumor effects in animals after BMT suggesting a vital role of NK cells in mediating GVT effects. Finally, NK cells have been demonstrated to affect B cell recovery and function in mice. Therefore, understanding the mechanisms of beneficial effects of NK cells after BMT may lead to significant increases in the efficacy of this procedure.     

Lipopolysaccharide activates matrix metalloproteinase-2 in endothelial cells through an NF-kappaB-dependent pathway

Vascular endothelial cells release proteinases that degrade the extracellular matrix, thus enabling cell migration during angiogenesis and vasculogenesis. Endothelial cells secrete mainly the proform of matrix metalloproteinase-2 (proMMP-2). In this report, we examined several growth factors, cytokines, and other molecules for activation of MMP-2 by human umbilical vein endothelial cells. Of these factors, we found that lipopolysaccharide (LPS) is the strongest activator of MMP-2. LPS induced MMP-2 activation in a time- and dose-dependent manner. While pretreatment with zinc chelators or nuclear factor kappaB (NF-kappaB) inhibitors suppressed LPS-induced MMP-2 activation, pretreatment with phosphatidylinositol 3'-kinase inhibitors had no effect. These results indicate that, in endothelial cells, LPS can directly enhance angiogenesis by inducing MMP-2 activation mediated through an NF-kappaB pathway.     

Characterization and cDNA cloning of a platelet aggregation inhibitor

A novel platelet aggregation inhibitor, sal-C, was purified to homogeneity from the venom of Korean snake (Agkistrodon halys brevicaudus). Several lines of experimental evidence clearly indicated that sal-C inhibits not only the collagen-induced platelet aggregation, but also the aggregation mediated by the cell surface glycoprotein IIb-IIIa (GP IIb-IIIa). We have isolated the cDNA encoding sal-C from the cDNA library of the snake venom gland and analyzed its complete nucleotide sequence. Sal-C is a single-chain polypeptide composed of 212 amino acids including 24 cysteines. The deduced polypeptide sequence of sal-C demonstrated considerable homology to previously described protein species of the collagen-induced platelet aggregation inhibitor family. Sal-C does not have the Arg-Gly-Asp (RGD) motif, but contains the Ser-Glu-Cys-Asp sequence. Interestingly, sal-C was found to inhibit GP IIb-IIIa binding to immobilized fibrinogen which is antagonized by the typical RGD motif of disintegrins.     

Aldehyde reductase gene expression by lipid peroxidation end products, MDA and HNE

Membrane lipid peroxidation results in the production of a variety of aldehydic compounds that play a significant role in aging, drug toxicity and the pathogenesis of a number of human diseases, such as atherosclerosis and cancer. Increased lipid peroxidation and reduced antioxidant status may also contribute to the development of diabetic complications. This study reports that lipid peroxidation end products such as malondialdehyde (MDA) and 4-hydroxynonenal (HNE) induce aldehyde reductase (ALR) gene expression. MDA and HNE induce an increase in intracellular peroxide levels; N-Acetyl-L-cysteine (NAC) suppressed MDA- and HNE-induced ALR gene expression. These results indicate that increased levels of intracellular peroxides by MDA and HNE might be involved in the upregulation of ALR.     

Purinergic activation of spontaneous transient outward currents in guinea pig taenia colonic myocytes

Spontaneous transient outward currents(STOCs) were recorded from smooth muscle cells of theguinea pig taenia coli using the whole cell patch-clamp technique.STOCs were resolved at potentials positive to 50 mV. Treatingcells with caffeine (1 mM) caused a burst of outward currentsfollowed by inhibition of STOCs. Replacing extracellularCa²⁺ with equimolarMn²⁺ caused STOCs to "rundown." Iberiotoxin (200 nM) or charybdotoxin (ChTX; 200 nM)inhibited large-amplitude STOCs, but small-amplitude "mini-STOCs"remained in the presence of these drugs. Mini-STOCs were reduced byapamin (500 nM), an inhibitor of small-conductance Ca²⁺-activatedK⁺ channels (SK channels).Application of ATP or 2-methylthioadenosine 5'-triphosphate(2-MeS-ATP) increased the frequency of STOCs. The effects of 2-MeS-ATPpersisted in the presence of charybdotoxin but were blocked bycombination of ChTX (200 nM) and apamin (500 nM). 2-MeS-ATP did notincrease STOCs in the presence of pyridoxal phosphate6-azophenyl-2',4'-disulfonic acid, aP₂ receptor blocker. Similarly,pretreatment of cells with U-73122 (1 µM), an inhibitor ofphospholipase C (PLC), abolished the effects of 2-MeS-ATP. XestosponginC, an inositol 1,4,5-trisphosphate(IP₃) receptor blocker,attenuated STOCs, but these events were not affected by ryanodine. Thedata suggest that purinergic activation through P_2Y receptors results in localizedCa²⁺ release via PLC- andIP₃-dependent mechanisms. Releaseof Ca²⁺ is coupled to STOCs, whichare composed of currents mediated by large-conductanceCa²⁺-activatedK⁺ channels and SK channels. Thelatter are thought to mediate hyperpolarization and relaxationresponses of gastrointestinal muscles to inhibitory purinergic stimulation.

     

Cloning and functional characterization of a constitutively expressed nitrate transporter gene, OsNRT1, from rice

Elucidating how rice (Oryza sativa) takes up nitrate at the molecular level could help improve the low recovery rate (<50%) of nitrogen fertilizer in rice paddies. As a first step toward that goal, we have cloned a nitrate transporter gene from rice called OsNRT1. OsNRT1 is a new member of a growing transporter family called PTR, which consists not only of nitrate transporters from higher plants that are homologs of the Arabidopsis CHL1 (AtNRT1) protein, but also peptide transporters from a wide variety of genera including animals, plants, fungi, and bacteria. However, despite the fact that OsNRT1 shares a higher degree of sequence identity with the two peptide transporters from plants (approximately 50%) than with the nitrate transporters (approximately 40%) of the PTR family, no peptide transport activity was observed when OsNRT1 was expressed in either Xenopus oocytes or yeast. Furthermore, contrasting the dual-affinity nitrate transport activity of CHL1, OsNRT1 displayed only low-affinity nitrate transport activity in Xenopus oocytes, with a K(m) value of approximately 9 mM. Northern-blot and in situ hybridization analysis indicated that OsNRT1 is constitutively expressed in the most external layer of the root, epidermis and root hair. These data strongly indicate that OsNRT1 encodes a constitutive component of a low-affinity nitrate uptake system for rice.