Complete structure of an increasing capillary permeability protein (ICPP) purified from Vipera lebetina venom. ICPP is angiogenic via vascular endothelial growth factor receptor signalling

The partial sequence of the increasing capillary permeability protein (ICPP) purified from Vipera lebetina venom revealed a strong homology to vascular endothelial growth factor (VEGF)-A. We now report its complete amino acid sequence determined by Edman degradation and its biological effects on mouse and human vascular endothelial cells. ICPP is a homodimeric protein linked by cysteine disulfide bonds of 25115 Da revealed by mass spectrometry. Each monomer is composed of 110 amino acids including eight cysteine residues and a pyroglutamic acid at the N-terminal extremity. ICPP shares 52% sequence identity with human VEGF but lacks the heparin binding domain and Asn glycosylation site. Besides its strong capillary permeability activity, ICPP was found to be a potent in vitro angiogenic factor when added to mouse embryonic stem cells or human umbilical vein endothelial cells. ICPP was found to be as potent as human VEGF165 in activating p42/p44 MAPK, in reinitiation of DNA synthesis in human umbilical vein endothelial cells, and in promoting in vitro angiogenesis of mouse embryonic stem cells. All these biological actions, including capillary permeability in mice, were fully inhibited by 1 microm of a new specific VEGF receptor tyrosine kinase inhibitor (ZM317450) from AstraZeneca that belongs to the anilinocinnoline family of compounds. Indeed, up to a 30 times higher concentration of inhibitor did not affect platelet-derived growth factor, epidermal growth factor, FGF-2, insulin, alpha-thrombin, or fetal calf serum-induced p42/p44 MAPK and reinitiation of DNA synthesis. Therefore, we conclude that this venom-derived ICPP exerts its biological action (permeability and angiogenesis) through activation of VEGF receptor signaling (VEGF-R2 and possibly VEGF-R1).     

Interaction of folk medicinal plant extracts with human alpha2-adrenoceptor subtypes

Forty-two extracts of folk medicinal plant organs from Pakistan were tested in competition binding assays for their interaction with the specific ligand recognition sites on the human alpha2-adrenoceptor subtypes alpha2A, alpha2B and alpha2C Strong binding of the extracts (40 mg/ml) from Acacia nilotica (L.) Delile leaves (88-98% displacement of radiolabel) and Peganum harmala seeds (89-96% displacement) on three subtypes prompted us to extract these plant materials with 40% and 80% methanol, ethanol, and acetone. The extraction results indicated an absence of alpha2-adrenoceptor binding activity in the stalk of A. nilotica and A. tortils, whereas the leaves of both plants contained activity. The extracts of A. nilotica leaves showed a slight, but consistent, preference for the alpha2C-adrenoceptor, whereas the leaves of A. tortils were slightly more active on the alpha2B subtype. The extract of P. harmala stalks was less active than that of its seeds. The binding activities of A. nilotica leaves and P. harmala seeds were mainly concentrated in the water and 30% methanol fractions and further sub-fractions. In a functional activity assay, the active fractions inhibited epinephrine-stimulated 35S-GTPyS binding, thus indicating a predominantly antagonistic nature of the compounds with alpha2-adrenoceptor affinity in these fractions. Among the known major alkaloids of P. harmala (demissidine, harmaline, harmine, 6-methoxyharmalan, and norharmane), only 6-methoxyharmalan showed moderate affinity (dissociation constant (Ki) of 530 +/- 40 nm for alpha2A subtype). This study is a first systematic attempt towards the discovery of potential drug candidates from these plant materials for treating alpha2-adrenoceptor related diseases.     

New action pattern of a maltose-forming alpha-amylase from Streptomyces sp. and its possible application in bakery

An a-Amylase (EC 3.2.1.1) was purified that catalyses the production of a high level of maltose from starch without the attendant production of glucose. The enzyme was produced extracellularly by thermophilic Streptomyces sp. that was isolated from Thailand's soil. Purification was achieved by alcohol precipitation, DEAE-Cellulose, and Gel filtration chromatographies. The purified enzyme exhibited maximum activity at pH 6-7 and 60 degrees C. It had a relative molecular mass of 45 kDa, as determined by SDS-PAGE. The hydrolysis products from starch had alpha-anomeric forms, as determined by 1H-NMR. This maltose-forming alpha-Amylase completely hydrolyzed the soluble starch to produce a high level of maltose, representing up to 90%. It hydrolyzed maltotetrose and maltotriose to primarily produce maltose (82% and 62% respectively) without the attendant production of glucose. The high maltose level as a final end-product from starch and maltooligosaccharides, and the unique action pattern of this enzyme, indicate an unusual maltose-forming system. After the addition of the enzyme in the bread-baking process, the bread's volume increased and kept its softness longer than when the bread had no enzyme.     

Characterization of a thermostable levansucrase from Bacillus sp. TH4-2 capable of producing high molecular weight levan at high temperature

A thermoactive and thermostable levansucrase was purified from a newly isolated thermophilic Bacillus sp. from Thailand soil. The purification was achieved by alcohol precipitation, DEAE-Cellulose and gel filtration chromatographies. The enzyme was purified to homogeneity as determined by SDS-PAGE, and had a molecular mass of 56 kDa. This levansucrase has some interesting characteristics regarding its optimum temperature and heat stability. The optimum temperature and pH were 60 degrees C and 6.0, respectively. The enzyme was completely stable after treatment at 50 degrees C for more than 1 h, and its activity increased four folds in the presence of 5 mM Fe(2+). The optimum temperature for levan production was 50 degrees C. Contrary to other levansucrases, the one presented in this study is able to produce high molecular weight levan at 50 degrees C.     

Impaired dilation of coronary arterioles during increases in myocardial O(2) consumption with hyperglycemia

Previous studies showed that nitric oxide (NO) plays an important role in coronary arteriolar dilation to increases in myocardial oxygen consumption (MVO(2)). We sought to evaluate coronary microvascular responses to endothelium-dependent and to endothelium-independent vasodilators in an in vivo model. Microvascular diameters were measured using intravital microscopy in 10 normal (N) and 9 hyperglycemic (HG; 1 wk alloxan, 60 mg/kg iv) dogs during suffusion of acetylcholine (1, 10, and 100 microM) or nitroprusside (1, 10, and 100 microM) to test the effects on endothelium-dependent and -independent dilation. During administration of acetylcholine, coronary arteriolar dilation was impaired in HG, but was normal during administration of nitroprusside. To examine a physiologically important vasomotor response, 10 N and 7 HG control, 5 HG and 5 N during superoxide dismutase (SOD), and 5 HG and 4 N after SQ29,548 (SQ; thromboxane A(2)/prostaglandin H(2) receptor antagonist) dogs were studied at three levels of MVO(2): at rest, during dobutamine (DOB; 10 microg. kg(-1). min(-1) iv), and during DOB with rapid atrial pacing (RAP; 280 +/- 10 beats/min). During dobutamine, coronary arterioles dilated similarly in all groups, and the increase in MVO(2) was similar among the groups. However, during the greater metabolic stimulus (DOB+RAP), coronary arterioles in N dilated (36 +/- 4% change from diameter at rest) significantly more than HG (16 +/- 3%, P < 0.05). In HG+SQ and in HG+SOD, coronary arterioles dilated similarly to N, and greater than HG (P < 0.05). MVO(2) during DOB+RAP was similar among groups. Normal dogs treated with SOD and SQ29,548 were not different from untreated N dogs. Thus, in HG dogs, dilation of coronary arterioles is selectively impaired in response to administration of the endothelium-dependent vasodilator acetylcholine and during increases in MVO(2).     

Ultrastructure of Wax-Producing Structures on the Integument of the Melaleuca Psyllid Boreioglycaspis melaleucae (Hemiptera: Psyllidae), with Honeydew Excretion Behavior in Males and Females

The melaleuca psyllid, Boreioglycaspis melaleucae (Hemiptera: Psyllidae), was introduced to Florida as a biological control agent against Melaleuca quinquenervia, an invasive evergreen tree that has invaded large areas of Florida Everglades. Colonies of B. melaleucae nymphs are normally covered by white waxy secretions, and nymphs of various instars produce long bundles of white waxy filaments extending laterally and posteriorly from their abdomen. Scanning electron microscopy of ‘naturally waxed’ and ‘dewaxed’ nymphs (cleaned from wax) revealed two types of wax pore plates located dorsally and laterally on the integument of posterior abdominal segments starting with the 4th segment. Type-1 wax pore plates, with raised rim, peripheral groove, slits and pits, produce long ribbons and filaments of waxy secretions that are wound together forming long wax bundles, whereas type-2 wax pore plates, with slits only, produce shorter wax curls. Additionally, in both nymphs and adult females, the circumanal ring contained ornate rows of wax pores that produce wax filaments covering their honeydew excretions. Video recordings with stereomicroscopy showed that adult females produce whitish honeydew balls, powerfully propelled away from their body, probably to get these sticky excretions away from their eggs and newly hatched nymphs. Adult males, however, produce clear droplets of honeydew immediately behind them, simply by bending the posterior end of the abdomen downward. The possible role(s) of waxy secretions by nymphs and adults of B. melaleucae in reducing contamination of their colonies with honeydew, among other possibilities, are discussed.     

TIMP-1 Inhibits Apoptosis in Lung Adenocarcinoma Cells via Interaction with Bcl-2

Tissue inhibitors of metalloproteinases (TIMPs) are multifaceted molecules that exhibit properties beyond their classical proteinase inhibitory function. Although TIMP-1 is a known inhibitor of apoptosis in mammalian cells, the mechanisms by which it exerts its effects are not well-established. Our earlier studies using H2009 lung adenocarcinoma cells, implanted in the CNS, showed that TIMP-1 overexpressing H2009 cells (HB-1), resulted in more aggressive tumor kinetics and increased vasculature. The present study was undertaken to elucidate the role of TIMP-1 in the context of apoptosis, using the same lung cancer cell lines. Overexpressing TIMP-1 in a lung adenocarcinoma cell line H2009 resulted in an approximately 3-fold increased expression of Bcl-2, with a marked reduction in apoptosis upon staurosporine treatment. This was an MMP-independent function as a clone expressing TIMP-1 mutant T2G, lacking MMP inhibition activity, inhibited apoptosis as strongly as TIMP1 overexpressing clones, as determined by inhibition of PARP cleavage. Immunoprecipitation of Bcl-2 from cell lysates also co-immunoprecipitated TIMP-1, indicative of an interaction between these two proteins. This interaction was specific for TIMP-1 as TIMP-2 was not present in the Bcl-2 pull-down. Additionally, we show a co-dependency of TIMP-1 and Bcl-2 RNA and protein levels, such that abrogating Bcl-2 causes a downregulation of TIMP-1 but not TIMP-2. Finally, we demonstrate that TIMP-1 dependent inhibition of apoptosis occurs through p90RSK, with phosphorylation of the pro-apoptotic protein BAD at serine 112, ultimately reducing Bax levels and increasing mitochondrial permeability. Together, these studies define TIMP-1 as an important cancer biomarker and demonstrate the potential TIMP-1 as a crucial therapeutic target.     

Analysis of RPS15aE,an Isoform of a Plant-Specific Evolutionarily Distinct Ribosomal Protein in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>, Reveals its Potential Role as a Growth Regulator

There is increasing evidence for ribosome heterogeneity in biological systems. In Arabidopsis thaliana, the ribosomal protein S15a is encoded by six separate genes, which fall into two evolutionarily distinct categories (Type I and Type II). Type I S15a is a universally conserved component of cytosolic ribosomes, whereas there is ambiguity as to the specific subcellular location of Type II S15a (cytosolic and/or mitochondrial ribosomes). In this study, we investigated the functional significance of the distinct form of ribosomal protein S15a (Type II) in Arabidopsis by examining: the evolutionary relationship of eukaryotic S15a proteins with respect to organellar homologs, the expression of individual Type II S15a genes during various developmental stages by RT-PCR, and the phenotypes of an insertional mutation into the RPS15aE gene. The Type II S15a proteins are plant specific, and the duplication event that gave rise to the Type II S15a genes appears to have occurred during the evolution of land plants. The genes encoding Type II S15a in Arabidopsis are differentially expressed, and mutant plants in which the gene encoding S15aE is knocked down produce larger leaves, longer roots, and possess larger cells than wild-type plants suggesting that the RPS15aE isoform of Type II S15a may act as a regulator of translational activity. Our results add significantly to the understanding of the protein constitution of plant ribosomes and the functional significance of ribosome heterogeneity.