Isolation and structure of somatostatin from porcine hypothalami.

The isolation and structure of somatostatin (GH-RIH) from pig hypothalami are described. This hormone was purified by preparative gel filtration, solvent extraction, countercurrent distribution in two solvent systems, ion-exchange and partition chromatography, and analytical gel filtration. The somatostatin activity was followed by in vitro bioassays and a radioimmunoassay. The isolated product was homogeneous chromatographically and had biological and immunological properties similar to synthetic somatostatin corresponding to the ovine hormone. The primary structure of porcine somatostatin was shown to be H-Ala-Gly-cyclo-(Cys-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-Cys)-OH. Other immunologically and biologically active form(s) of somatostatin were also detected.     

Anti-phospholipase A2 and anti-inflammatory activity of Santolina chamaecyparissus

The activity of the Santolina chamaecyparissus methanol extract was tested against the phospholipase A2 (PLA2)-induced mouse paw edema and in vitro inhibition of PLA2 activity. After fractionation, only the dichloromethane extract was active against the PLA2 in vitro test. In addition, it reduced the edema induced by arachidonic acid, and by 12-O-tetradecanoylphorbol-13-acetate in a multidose test. After chromatography on silicagel and gel filtration on Sephadex, and using an in vitro anti-PLA2 assay-guided process, we have isolated and identified from the dichloromethane extract the flavone nepetin and four sesquiterpenes.     

Effect of Photorhabdus luminescens phase variants on the in vivo and in vitro development and reproduction of the entomopathogenic nematodes Heterorhabditis bacteriophora and Steinernema carpocapsae

Photorhabdus luminescens (Enterobacteriaceae) is a symbiont of entomopathogenic nematodes Heterorhabditis spp. (Nematoda: Rhabditida) used for biological control of insect pests. For industrial mass production, the nematodes are produced in liquid media, pre-incubated with their bacterial symbiont, which provides nutrients essential for the nematode's development and reproduction. Particularly under in vitro conditions, P. luminescens produces phase variants, which do not allow normal nematode development. The phase variants were distinguished based on dye absorption, pigmentation, production of antibiotic substances, occurrence of crystalline inclusion proteins and bioluminescence. To understand the significance of the phase shift for the symbiotic interaction between the bacterium and the nematode, feeding experiments tested the effect of homologous and heterologous P. luminescens phase variants isolated from a Chinese Heterorhabditis bacteriophora (HO6), the Heterorhabditis megidis type strain from Ohio (HNA) and the type strain of Heterorhabditis indica (LN2) on the in vivo and in vitro development and reproduction of the nematode species H. bacteriophora (strain HO6) and another rhabditid and entomopathogenic nematode, Steinernema carpocapsae (A24). In axenically cultured insect larvae (Galleria mellonella) and in vitro in liquid media, H. bacteriophora produced offspring on phase I of its homologous symbiont and on the heterologous symbiont of H. megidis, but not on the two corresponding phase II variants. In solid media, nematode yields were much lower on phase II than on phase I variants. On the heterologous phase I symbiont isolated from H. indica the development of H. bacteriophora was not beyond the fourth juvenile stage of the nematode in any of the media tested, but further progressed on phase II with even a small amount of offspring recorded in solid media. Infective juveniles of S. carpocapsae did not develop beyond the J3 stage on all phase I P. luminescens. They died in phase I P. luminescens isolated from H. bacteriophora. Development to adults was recorded for S. carpocapsae on all phase II symbionts and offspring were produced in all media except in liquid. It is concluded that a lack of essential nutrients or the production of toxins is not responsible for the negative impact of homologous phase II symbiont cells on the development and reproduction of H. bacteriophora. The infective juveniles of H. bacteriophora retained cells of the homologous phase I symbiont, but not phase II cells and cells from heterologous symbionts, indicating that the transmission of the symbiont by the infective juvenile is selective for phase I cells and the homologous bacterial associate.     

Chromosome aberrations, sister chromatid exchanges (SCE) and cell division kinetics in human lymphocytes exposed in vitro to purified trypsin inhibitor from Ascaris

The purified trypsin inhibitor (TI) isolated from nematode Ascaris suum was tested in vitro for chromosome aberrations and sister chromatid exchanges (SCE). TI was obtained from the musculocutaneous sac homogenate of adult Ascaris by the modified method of Rola and Pudles. The inhibitor was isolated and purified from the SF5 fraction of proteins by gel filtration on Sephadex G-50 and electrophoresis SDS-PAGE of the obtained fraction after molecular filtration. TI showed a high inhibitory activity against crystalline trypsin (18.8 Kassell's units/mg of protein). Genotoxicity assessment of TI was carried out on metaphase plates received from peripheral blood lymphocyte macroculture (48 h-test of structural chromosome aberrations and 72 h-test of SCE), without exogenous metabolic activation. TI was tested in doses: 25, 50 and 100 microg per mL of culture. Kinetics of cell divisions was determined by the replication index (RI). We found that TI in vitro did not induce chromosome aberrations. It induced a higher number of SCE per cell but less than double frequency as compared to the control. The difference was significant only for the dose 50 microg/mL. For all doses, replication index (RI) values were significantly higher and mitotic index (MI) values were significantly lower than in the control. Thus the Ascaris trypsin inhibitor did not show any genotoxic properties but exhibited a mitostatic activity.     

Purification and some properties of camel carboxypeptidase B

A carboxypeptidase B-like enzyme was purified 116-fold with a recovery of activity of 29% from a crude extract of camel pancreas by a four-step procedure consisting of two anion exchange chromatographies in succession, gel filtration and hydrophobic interaction chromatography. The enzyme was homogeneous on SDS and non-denaturing gel electrophoresis and on gel isoelectric focusing. Its molecular mass was found to be 31.5 kDa and its isoelectric point was estimated as 6.1. It was active towards a number of substrates that are cleaved by carboxypeptidases B from other species and was also susceptible to inhibition by inhibitors of such enzymes. The camel enzyme showed a pH optimum of 8.0 and it was seen to be a relatively potent kinase in vitro. The enzyme purified in this study was very similar to carboxypeptidases B isolated from other species in size, charge, substrate specificity and susceptibility to inhibition and thus it can be identified as camel carboxypeptidase B.     

Control of Root-knot Nematodes on Tomato in Stone Wool Substrate with Biological Nematicides

The efficacy of four biological nematicides on root-galling, root-knot nematode (Meloidogyne incognita) reproduction, and shoot weight of tomato (Solanum lycopersicum) grown in stone wool substrate or in pots with sandy soil was compared to an oxamyl treatment and a non-treated control. In stone wool grown tomato, Avid® (a.i. abamectin) was highly effective when applied as a drench at time of nematode inoculation. It strongly reduced root-galling and nematode reproduction, and prevented a reduction in tomato shoot weight. However, applying the product one week before, or two weeks after nematode inoculation was largely ineffective. This shows that Avid® has short-lived, non-systemic activity. The effects of Avid® on nematode symptoms and reproduction on soil-grown tomato were only very minor, probably due to the known strong adsorption of the active ingredient abamectin to soil particles. The neem derived product Ornazin® strongly reduced tomato root-galling and nematode reproduction only in stone wool and only when applied as a drench one week prior to nematode inoculation, suggesting a local systemic activity or modification of the root system, rendering them less suitable host for the nematodes. This application however also had some phytotoxic effect, reducing tomato shoot weights. The other two products, Nema-Q™ and DiTera®, did not result in strong or consistent effects on nematode symptoms or reproduction.     

Insect cellular and chemical limitations to pathogen development: the Colorado potato beetle, the nematode Heterorhabditis marelatus, and its symbiotic bacteria

This research examines possible factors limiting pathogen development and reproduction in a novel host insect. The nematode Heterorhabditis marelatus and its symbiotic bacterium, Photorhabdus luminescens, kill 98% of nematode-treated Colorado potato beetle (CPB) prepupae, but the nematode reproduces in only 1-6% of beetles. We examined nematode/bacterial inhibition at each step of the normal developmental pathway to determine host feature(s) limiting nematode reproduction. We found that in vivo encapsulation of nematodes occurred in only 1.6% of CPB, and in 5% of in vitro hanging drops of hemolymph. Thus, the cellular defense system did not strongly limit nematode reproduction in the CPB. The symbiotic bacterium was negatively affected by a heat-labile factor found in the CPB's hemolymph which often caused the bacterium to switch from the primary form that produces antibiotics and nutrients necessary for the nematodes' development, to a secondary form that provides only limited nutrients. A 58 kDa protein was isolated and bioassayed for activity against P. luminescens, but caused a delay in bacterial growth rather than the primary-secondary form switch. Thus, the identity of the heat-labile factor could not be confirmed as being the 58 kDa protein. The heat-labile factor did not directly affect the nematode. The addition of lipids in the form of olive oil to heated CPB hemolymph allowed nematodes to reproduce in 17% of hanging drops, in contrast to zero reproduction in hemolymph without oil. Reproductive nematodes were smaller when grown in CPB hemolymph than in hemolymph of the highly susceptible Galleria mellonella. These data suggest that both the toxic heat-labile factor and a lack of appropriate nutrients alter the CPB-bacterium-nematode interaction. These factors preclude the use of this otherwise highly effective nematode-bacterial complex in the longterm control of the CPB.     

Immunochemistry of a Formamide-extracted Antigen from Clostridium perfringens Cell Walls

The type-specific antigen of a strain of Clostridium perfringens involved in food poisoning was isolated from the cell wall by the use of hot formamide. The antigen appears to consist of polysaccharide or mucopeptide. The formamide extract was shown to be heterogeneous by gel filtration on Sephadex G-200. The serologically active fraction contained about 25% of the amount of protein present in the original formamide extract. Hexosamine, acetyl groups, and carbohydrate also were detected. The formamide extract showed a high degree of serological activity. The serological activity was increased twofold on Sephadex gel filtration.     

Purification and characterization of natural and recombinant human plasminogen activator inhibitor-1 (PAI-1)

Human plasminogen activator inhibitor-1 (PAI-1) was purified from the conditioned medium of endotoxin-stimulated umbilical vein endothelial cell cultures by combinations of zinc-chelate-Sepharose chromatography, gel filtration on Sephacryl S-300 and immunoadsorption on an insolubilized murine monoclonal antibody (MA-7D4). The final product was obtained with a recovery of approximately 20% from conditioned medium containing about 3 micrograms/ml PAI-1. The yield of PAI-1 was 15-100 micrograms/umbilical cord, depending on the culture and harvest conditions. SDS gel electrophoresis revealed a main band with Mr = 46,000 both under reducing and non-reducing conditions. On gel filtration on Sephacryl S-300, however, the material was separated in two fractions, one eluting at the void volume, which contains active PAI-1, and one with Mr = 46,000 containing inactive material that could be reactivated with 12 M urea. SDS gel electrophoresis of the isolated high-Mr fraction revealed several bands including a main 46,000-Mr component, which reacted with anti-(PAI-1) antibodies on immunoblotting and neutralized tissue-type plasminogen activator (t-PA). The active high-Mr fraction and the reactivated low-Mr fraction of PAI-1 inhibited t-PA very rapidly with an apparent second-order rate constant of (1.5-4) x 10(7) M-1 s-1. The cDNA of endothelial cell PAI-1 was cloned and expressed in Chinese hamster ovary cells. The translation product, purified from conditioned medium of transfected cells, also revealed a high-Mr and a low-Mr fraction on gel filtration, which were indistinguishable from the natural proteins by physicochemical, immunochemical and functional analysis. On reduced SDS gel electrophoresis, the high-Mr fraction was separated into the Mr-46,000 low-Mr PAI-1 and two other components with Mr 65,000 and one barely entering the gel. When reactivated low-Mr PAI-1 was added to plasma, PAI activity and PAI-1 antigen eluted with an apparent Mr greater than or equal to 300,000 on gel filtration, indicating that active PAI-1 complexes with one or more binding proteins in plasma.     

Enzymes of 2-oxo acid degradation and biosynthesis in cell-free extracts of mixed rumen micro-organisms.

The enzymes of 2-oxo acid decarboxylation and 2-oxo acid synthesis (EC 1.2.7.1 and EC 1.2.7.2) were isolated and partially purified from cell-free extracts of rumen micro-organisms. The lyase was active with pyruvate, 3-hydroxypyruvate and 2-oxobutyrate. The synthase was active with acetate, 2-oxoglutarate or succinate. Pyruvate synthase was separated from pyruvate lyase by Sephadex G-200 gel filtration. With Sephadex filtration, approximate mol.wts. of 310000 and 210000 were determined for pyruvate lyase and pyruvate synthase respectively.     

Characterization of the interaction between the nucleotide exchange factor EF-Ts from nematode mitochondria and elongation factor Tu

Caenorhabditis elegans mitochondria have two elongation factor (EF)-Tu species, denoted EF-Tu1 and EF-Tu2. Recombinant nematode EF-Ts purified from Escherichia coli bound both of these molecules and also stimulated the translational activity of EF-Tu, indicating that the nematode EF-Ts homolog is a functional EF-Ts protein of mitochondria. Complexes formed by the interaction of nematode EF-Ts with EF-Tu1 and EF-Tu2 could be detected by native gel electrophoresis and purified by gel filtration. Although the nematode mitochondrial (mt) EF-Tu molecules are extremely unstable and easily form aggregates, native gel electrophoresis and gel filtration analysis revealed that EF-Tu·EF-Ts complexes are significantly more soluble. This indicates that nematode EF-Ts can be used to stabilize homologous EF-Tu molecules for experimental purposes. The EF-Ts bound to two eubacterial EF-Tu species (E.coli and Thermus thermophilus). Although the EF-Ts did not bind to bovine mt EF-Tu, it could bind to a chimeric nematode–bovine EF-Tu molecule containing domains 1 and 2 from bovine mt EF-Tu. Thus, the nematode EF-Ts appears to have a broad specificity for EF-Tu molecules from different species.     

Isolation and purification of cytokinin binding protein from tobacco leaves by affinity column chromatography

Cytokinin binding protein from tobacco leaves was isolated and purified to a single protein by means of affinity chromatography on benzyladenine-linked Sepharose column combined with polyacrylamide gel electrophoresis. In vitro binding of this protein to [¹⁴C] benzyladenine was inhibited remarkably by cold benzyladenine and kinetin and slightly by adenine, but not adenosine. The molecular weight of the protein was determined to be about 4,000 daltons by gel filtration and SDS polyacrylamide gel electrophoresis.     

Purification and subunit composition of a GTP-binding protein from maize root plasma membranes

When frozen plasma membranes isolated from maize seedling roots are thawed, a significant portion of GTP-binding activity goes into solution. The GTP-binding protein was purified by ion exchange chromatography on Mono-Q and gel filtration on Superose 6. Its molecular weight was estimated at 61 kDa by gel filtration. The same molecular weight was obtained upon solubilization of the GTP-binding protein with cholic acid followed by gel filtration in the presence of this detergent. SDS-PAGE demonstrated that the isolated GTP-binding protein consists of two types of subunit of molecular weights 27 kDa and 34 kDa.     

A newly identified chemotactic sexual pheromone from Closterium ehrenbergii

 In sexual reproduction of Closterium ehrenbergii, pairing with the sexual partner cells is the first process observed. A cell migration-inducing activity, specific for mating-type plus (mt⁺; NIES-228) cells, was detected in the culture medium of mating-type minus (mt^–; NIES-229) cells. Light was necessary for production of the active substance by mt^– cells and for migration of mt⁺ cells. The active substance was heat-labile and had an apparent molecular mass of 20 kDa, as determined by gel filtration. A protein of 20 kDa was detected in the active fraction of gel filtration after sodium dodecyl sulfate polyacrylamide gel electrophoresis. Based on these results, it is proposed that a chemotactic sexual pheromone involved in the formation of sexual pairs of cells is secreted by mt^– cells of C. ehrenbergii and is proteinaceous, like other sexual pheromones secreted by Closterium species. Received: 31 July 1997 / Revision accepted: 25 November 1997     

Isolation of an inhibitor of 25-hydroxyvitamin D3-1-hydroxylase from rat serum.

An inhibitor of chick kidney mitochondrial 25-hydroxyvitamin D3-1-hydroxylase has been isolated from rat serum by ammonium sulfate precipitation, gel filtration, ionexchange chromatography, and preparative polyacrylamide disc gel electrophoresis. The purified protein was shown to contain iron and has a mol wt of 52 000. The protein is indistinguishable on gel electrophoresis from a similar inhibitor found in rat kidney tissue. The physiological significance of the inhibitor is not known; however, it seems possible that it is responsible for the failure to demonstrate in vitro 25-hydroxyvitamin D3-l-hydroxylation with rat and other mammalian tissues.     

Purification and characterization of the trifunctional beta-subunit of anthranilate synthase from Neurospora crassa

The trifunctional beta-subunit of anthranilate synthase complex of Neurospora crassa has been purified from a mutant which produces no detectable alpha-subunit. The isolated beta-subunit appeared to be a highly asymmetric dimer with a s20,w of 7.35 and an apparent molecular weight of 200,000 as determined by gel filtration on Sephacryl S-300 compared with a monomer molecular weight of approximately 84,000 Da as determined by sodium dodecyl sulfate-gel electrophoresis. The purified subunit was cleaved by elastase, trypsin, or chymotrypsin into fragments which retained the three enzyme activities. After elastase digestion, two active fragments were separated by gel filtration and ion exchange chromatography. A 30,000-Da fragment, which behaved as a monomer on gel filtration, interacted with free alpha-subunit to produce glutamine-dependent anthranilate synthase activity. A second 56,000-Da fragment, which behaved as an asymmetric dimer (apparent molecular weight 140,000) on gel filtration, retained both N-(5'-phosphoribosyl)anthranilate isomerase and indole-3-glycerol phosphate synthase activity. The failure to detect an NH2-terminal amino acid residue on either the intact beta-subunit or the 30,000-Da complementing fragment, while the 56,000-Da fragment possessed an NH2-terminal histidine residue, indicated that the complementing fragment was derived from the NH2-terminal sequence of the beta-subunit.     

Purification of a factor inducing differentiation in murine myelomonocytic leukemia cells. Identification as granulocyte colony-stimulating factor

A naturally occurring inducer of terminal differentiation in a murine myelomonocytic leukemia cell line (WEHI-3B) was purified to apparent homogeneity from medium conditioned by lungs from mice injected with bacterial endotoxin. The factor was purified over 400,000-fold by sequential fractionation using salting out chromatography, chromatography on phenyl-Sepharose, gel filtration on Bio-Gel P-60 in 1 M acetic acid, reverse-phase high performance liquid chromatography on a phenyl-silica column, and high performance liquid chromatography on a gel filtration column. During the first two steps, the differentiation-inducing factor was separated completely from a known proliferative regulator for normal myeloid cells, granulocyte-macrophage colony-stimulating factor, but it co-purified through all remaining steps with a distinct granulocyte-specific colony-stimulating factor. The purified factor showed a single protein band of Mr = 24,000-25,000 on sodium dodecyl sulfate-polyacrylamide gels coincident with both differentiation-inducing and granulocyte colony-stimulating activity. The granulocyte-specific colony-stimulating factor was active on WEHI-3B cells and normal granulocytic progenitor cells in vitro at the same half-maximally active concentration of 3 X 10(-12) M.     

Isolation of allergenically active glycoprotein from Prosopis juliflora pollen

An allergenically active glycoprotein was homogeneously isolated from the aqueous extract of Prosopis juliflora pollen by ConA-Sepharose affinity chromatography. The molecular weight of this glycoprotein was 20,000 dalton, determined by gel filtration and SDS-PAGE. This fraction showed a total carbohydrate concentration of 25%. The purified glycoprotein revealed immunochemically most antigenic or allergenic and demonstrated homogeneous after reaction with P. juliflora pollen antiserum, characterized by gel diffusion, Immunoelectrophoresis and Radioallergosorbent test.     

Characterization and purification of thermostable beta-glucosidase from Clostridium thermocellum.

A β-glucosidase was isolated from $\text{Clostridium thermocellum}$; the enzyme was localized in the periplasmic space.It was purified in a five-step procedure including ion-exchange chromatography on DEAE-Cellulose, chromatography on HA-Ultrogel and DEAE-Sephadex, gel filtration on AcA 34 Ultrogel and isoelectric focusing.The final preparation was purified 944-fold with a recovery of about 5% of the initial enzyme activity.Polyacrylamide disc electrophoresis of the purified enzyme gave a single band at pH 8.3. The enzyme is active towards cellobiose and p-nitrophenyl-β-D-glucoside(PNPG) and developed maximum activities at pH 6.0 and 65°C. A molecular weight of 50,000 daltons was estimated by gel filtration and the enzyme was isoelectric at pH 4.68.     

The isolation and partial characterization of low-molecular-weight phosphorylated component of the non-histone proteins of mouse nuclei.

After labelling of mouse liver nuclei with [gamma-32P]ATP in vitro, 10-20% of the radioactivity incorporated into the saline-soluble nuclear and HAP2 chromatin fractions was located in a low-molecular-weight component (component 10) with pI near 4.5 in urea. By using combinations of ion-exchange chromatography, preparative thin-layer isoelectric focusing and gel filtration, this component was isolated from both nuclear fractions. Recovery from the saline-soluble fraction was poor under conditions that allow endogenous phosphatases to be active. Component 10 was shown to be a phosphoprotein on the basis of enzyme-digestion experiments and the detection of phosphoserine and phosphothreonine. The 32P radioactivity did not appear to be associated with phosphorylated basic amino acids. Its molecular weight was determined by gel chromatography and electrophoresis in sodium dodecyl sulphate/polyacrylamide gels as approx. 10000, and tryptic digestion of the reduced carboxymethylated protein in urea yielded two 32P-labelled peptides. It has not been possible as yet to assign a function to component 10, though its similarity to other low-molecular-weight acidic proteins is discussed.