Further characterisation of trypsin inhibitors in the polychaet Sabellastarte indica (Savigny), II (author's transl)

In this paper we discribe a method for the isolation of trypsin inhibitors from the tentacles of the annelid Sabellastarte indica Savigny. These inhibitors - now homogeneous in their molecular weight - can be characterised by sodium dodecylsulfate-acrylamide gel-electrophoresis, in their inhibitory activity towards trypsin, plasmin and chymotrypsin. The inhibitors from Sabellastarte indica possess a stoichiometric binding relation of 2:1 for trypsin, lysine being the amino-acid in the reactive centre of the inhibitor responsible for interaction with trypsin. The reactive centre for trypsin is not identical with the reactive centre which binds chymotrypsin and does not influence the binding of chymotrypsin. These newly described inhibitors are therefore multiheaded, a type not previously described for invertebrates.     

Isolation and characteristics of a new trypsin and chymotrypsin inhibitor from potato tubers

A novel trypsin and chymotrypsin inhibitor has been isolated from potato (Solanum tuberosum L.) tubers. The isolation procedure included ammonium sulfate precipitation, gel-chromatography on Sephadex G-75 and ion-exchange chromatography on DEAE-cellulose. The inhibitor interacts with trypsin and chymotrypsin at a molar ratio of 1:1. The substrate-dependent dissociation of the enzyme-inhibitor complexes is observed. The inhibitor displays no activity towards subtilisin and pancreatic elastase. The ability of the inhibitor to form a ternary complex containing simultaneously both trypsin and chymotrypsin molecules testifies to the presence of two independent reactive sites for these enzymes.     

Novel inhibitors of fungal protein synthesis produced by a strain of Graphium putredinis. Isolation, characterisation and biological properties

The isolation and structure determination of 6 analogues of the fungal protein synthesis inhibitor GR135402, from Graphium putredinis, is described. The relative potencies of the compounds as protein synthesis inhibitors and as in vitro antifungal agents provide interesting insights into the structure-activity relationships in this series.     

Isolation, cloning, and characterisation of a trp homologue from squid (Loligo forbesi) photoreceptor membranes

The invertebrate phototransduction system is a valuable model of the ubiquitous inositol lipid signalling system. Taking advantage of the ability to obtain relatively large amounts of retinal material from the cephalopod eye, partial protein sequence data were obtained for a 92-kDa component isolated from a detergent-insensitive cytoskeletal fraction of a squid retinal microvillar membrane preparation. Degenerate oligonucleotides, designed on the basis of these sequence data, were used to isolate a full-length cDNA, encoding the 92-kDa component, using both cDNA library screening and 5'-rapid amplification of cDNA ends (5'-RACE) techniques. Comparison of the amino acid sequence encoded by this cDNA with entries in the OWL composite protein sequence database reveals greatest sequence similarity with the products of the Drosophila trp and trpl genes. Greatest variation from the Drosophila Trp protein is seen in the carboxyl-terminal region, which is considerably truncated in the squid protein and which accounts for most of the substantial difference in molecular weight seen between these proteins. This variation may be significant as the carboxyl-terminal domain has been shown to be in the regulation of several ligand-gated channels. The carboxyl-terminal domain has been expressed and shown to interact with calmodulin in a calcium-dependent fashion, thereby supporting this hypothesis. The likely occurrence of other homologues in a variety of systems suggests that this is a novel and important family of regulated ion channels involved in calcium signalling.     

Isolation and characterisation of Caribbean ciguatoxins from the horse-eye jack (Caranx latus)

The toxins involved in ciguatera (fish poisoning) in the Caribbean Sea were isolated from Caranx latus, a pelagic fish often implicated in ciguatera in the Caribbean region, and purified by mouse bioassay directed fractionation. Five toxins were separated by reverse-phase high-performance liquid chromatography (HPLC). In order of increasing hydrophobicity, these toxins included a sleep-inducing fraction (< 1% of total toxicity), a major Caribbean ciguatoxin (C-CTX-1, 65% of toxicity), a minor Caribbean ciguatoxin (C-CTX-2, 13% of toxicity), a minor toxin (approximately 1% of toxicity) and a hydrophobic, fast-acting toxin (approximately 19% of toxicity). The i.p. injection into mice of each toxin induced signs typical of site-5 sodium channel activator toxins such as the Pacific ciguatoxins and brevetoxins. C-CTX-1 and C-CTX-2 were purified to homogeneity (LD50 = 3.6 and approximately 1 microgram/kg, respectively) and subjected to ion spray mass spectrometry. Both lost up to five H2O molecules and each had a [M+H]+ ion, m/z 1141.7, suggesting that C-CTX-1 and -2 are diastereomers that differ from the Pacific family of ciguatoxins. Turbo-assisted HPLC-mass spectrometry identified C-CTX-1, C-CTX-2 and three C-CTX-1-related compounds in an enriched fraction but no Pacific ciguatoxins were detected. The presence of different families of ciguatoxins in ciguateric fish from the Caribbean Sea and Pacific Ocean probably underlies the clinical differences in the ciguatera syndrome reported in these two regions. A Caribbean strain of the benthic dinoflagellate, Gambierdiscus toxicus, is suspected as source of these ciguatoxins. The extent to which these toxins are biotransformed as they pass through the marine food chain remains to be determined.     

Isolation and some properties of factor VIII (antihemophilic factor)

A method for isolation of factor VIII from cryoprecipitate fraction of human plasma has been elaborated. The isolation procedure involves precipitation with dextran, removal of fibrinogen by means of defibrase, precipitation with ammonium sulfate, polyethylene glycol fractionation, and Sepharose 6B gel filtration step. Factor VIII has been purified 7000- to 13,000 -- fold.. The preparation is homogenous by ultracentrifugal examination and it has an S20,w value of 19.4. It also shows a single precipitin line when subjected to immunoelectrophoresis employing rabbit antibodies against factor VIII. The preparation did not enter a 7.5% polyacrylamide gel containing sodium dodecyl sulfate even in the presence of 8 M urea. After reduction of the protein with 2-mercaptoethanol, subunits were formed which migrated as one band in polyacrylamide gel electrophoresis.     

Isolation and characterisation of a murine picornavirus

Our investigation of normal, hyperplastic, and neoplastic prostatic tissue during the past 2 1/2 years has produced several findings which have been published or accepted for publication. (a) Cells from hamster prostates with intense histochemically demonstrable acid phosphatase activity (HDAP) after fixation with formaldehyde which we believe to be epithelial cells can be obtained in 97.2% +/- 0.8% purity by velocity sedimentation in a previously described isokinetic density gradient; (b) similarly, cells with HDAP, many of which contain lipofuscin granules, can be obtained as 81.0% +/- 12.2% of nucleated cells from hyperplastic human prostates and as 86.4% +/- 9.4% of nucleated cells from human prostatic carcinomas; (c) more cells were obtained from human hyperplastic prostates and prostates with prostatic carcinoma per gram of tissue with the aid of Pronase than were obtained with trypsin, collagenase, or mechanical methods; (d) more cells per gram of tissue were obtained from surgically removed prostates than from prostates obtained at even very rapid autopsies, and a much larger proportion of the cells from surgically removed prostates were viable as assessed both by dye exclusion and by plating efficiency; (e) none of several substrates and inhibitors which we tested were highly specific for acid phosphatase from purified prostatic epithelial cells compared with several other kinds of purified human cells; and (f) purified hamster prostatic epithelial cells incorporate large amounts of tritiated thymidine in 72-hour cultures.     

Isolation and properties of a new, soluble, hemoprotein (H-450) from pig liver

A new soluble hemoprotein, designated as H-450, has been purified from pig liver. The absolute absorption spectrum of H-450 shows maxima at 550 and 428 nm. The dithionite-reduced H-450 has absorption peaks at 572, 540, and 450 nm; the unique Soret band at 450 nm is the basis for our tentative designation of this new hemoprotein as H-450 (hemoprotein 450). The spectrum of dithionite-reduced H-450 at 77 K gives two alpha peaks (571 and 566 nm), three beta peaks (546, 537, and 529 nm), and a Soret band at 449 nm. The prosthetic group of H-450 has been identified as protoheme IX. Gel electrophoresis experiments show that H-450 is composed of two nonidentical subunits, alpha and beta (mol wts = 61 000 and 45 000). H-450 contains 1 mol of heme/alphabeta dimer of 106 000 molecular weight. Preliminary sedimentation equilibrium experiments suggest a minimum molecular weight of 218 000 for the native protein. This corresponds to a tetramer, alpha2beta2 containing two heme groups. H-450 is not reduced by reduced nicotinamide adenine dinucleotide (NADH), NADH phosphate, ascorbate, or ferrocyanide. Neither reduced nor oxidized H-450 binds CO, 1 mM cyahide, or 1 mM azide. Dithionite-reduced H-450 is autoxidizable. The molar extinction coefficient of native H-450 is 261 X 103 at 280 nm and 263 X 103 at 428 nm. The purification procedure involves homogenization, high-speed centrifugation, ammonium sulfate fractionation, diethylaminoethylcellulose chromatography, density gradient centrifugation, a calcium phosphate gel step, and a second density gradient centrifugation. The procedure yeilds approximately 2 mg of purified protein from 750 g of pig liver.     

Isolation and characterisation of nocardia from clinical specimens

Sixteen isolations of nocardia of which 12 were from pulmonary infections, one from wound infection, one from mycetoma and 2 from eye infections were studied from June, 1989 to May, 1990. The importance of Gram's stain findings of primary smear is being highlighted. The nocardia species were identified utilising the morphological characters including acid fastness and cultural and biochemical characters. Notable among the isolates were Nocardia brasiliensis, one each from mycetoma and pulmonary infection, which are rare in South India and Nocardia asteroides from a case of endophthalmitis probably of endogenous origin.     

Isolation and properties of myoglobins from rat (Rattus norvegicus) skeletal muscles

A simple test of critical thermal maximum (CTM) to assess a break-down of heat-escape behavior in rats is described. Experiments were performed on 18 unrestrained adult Wistar rats of both sexes. Hypothalamic and intraperitoneal (i.p.) temperatures as well as motor activity were simultaneously and continuously recorded in the rats exposed to heat. When animals were growing restless, as evidenced by an increase in their motor activity, which was usually recorded at hypothalamic temperatures well above 41 degrees C, we started testing CTM. To assess heat-escape behavior we used a precooled cooling bar (a part of a camp-cooler) which was placed at intervals in a climatic chamber. The hyperthermic rats, given the bar for 30 s, mounted it vigorously until they failed at particular levels of brain and body temperatures which were recognized as respective CTM values. Rapid external cooling of rats prevented lethal effects of the heat exposure. We were able to show effects of timing of heat exposure on heat tolerance. We also managed to detect small but significant differences in heat tolerance of warm-reared (an increase), cold-reared (a decrease), and bacterial-endotoxin-treated (an increase) rats. The heat-escape behavior was less heat-resistant than selective brain cooling response which was still present at CTM point. In conclusion, our CTM test is a safe and reliable way to study heat tolerance in rats.     

Isolation: analysis and properties of three bradykinin-potentiating peptides (BPP-II, BPP-III, and BPP-V) from Bothrops neuwiedi venom

Several methods of external and internal fixation are used in urgent situations to lessen intrapelvic bleeding associated with unstable pelvic fractures. Pelvic stabilization limits pelvic expansion and thereby restricts the space for potential blood loss. This study compared several fixation methods using cadaveric pelves to determine which method best prevents pelvic expansion. Three methods of internal fixation and three methods of external fixation were compared. Anteroposterior fixation provided the greatest control against pelvic expansion; however, it is clinically impractical for emergency use. Therefore, external fixation provided the most reliable control of pelvic expansion in the emergency setting.     

Electromechanical properties of the single cell-layered heart of tunicate Boltenia ovifera (sea potato)

The tubular heart of the sea potato is composed of a single layer of myoepithelial cells interconnected near the extraluminal surface by specialized junctions. If these junctions are used as the border which separates the luminal from extraluminal membrane, the surface area ratio, luminal:extraluminal, is approximately 12:1. A single myofibril is located near the luminal surface in each cell. Current passed across the heart wall in the direction that depolarizes the luminal membrane and hyperpolarizes the extraluminal membrane immediately produces "all-or-none" action potentials and contractions. Current passed in the opposite direction fails to produce action potentials until after the break of the stimulus, suggesting anodal break excitation of the hyperpolarized luminal membrane. High potassium solutions depolarized the myoepithelium and produced contractions only when applied to the luminal surface of the heart. [Ca]0 increases and [Mg]0 decreases twitch tension only on the luminal surface of the heart. The transwall resistivity is low (50-100 omega/cm2) due to an extracellular shunt. Because of this shunt and the larger surface area of the luminal membrane, the extraluminal membrane is effectively clamped to the potential of the luminal membrane and is not capable of directly influencing excitation-contraction coupling. These findings suggest that only the luminal membrane of the sea potato myoepithelium is capable of generating an action potential and triggering contraction.     

Isolation and in vitro hydrolysis of lentil protein fractions by trypsin

The albumin and globulin fractions from lentil seeds were isolated and characterised by gel filtration. The latter was shown to be homogeneous and the former heterogeneous on PAGE. The amino acid analysis revealed high values of amidic amino acids for both fractions with great differences in the sulphur-containing amino acids. Native albumin, globulin and salt-soluble proteins were markedly resistant to trypsin hydrolysis compared to casein. The SDS-PAGE of native salt-soluble proteins indicated that the globulin fragments (20 to 30 kD) were slowly digested in the presence of albumin. The heating increased the hydrolysis of the proteins in the order: salt-soluble, albumin and globulin. The facilitated hydrolysis of the heated salt-soluble fraction seemed to be due to protein-protein interactions induced by heat.     

Isolation and characterisation of a low molecular weight inhibitor (of chymotrypsin and human granulocytic elastase and cathepsin G) from leeches

Two protein proteinase inhibitors were isolated and purified from the leech Hirudo medicinalis by means of gel filtration and ion-exchange chromatography. They inhibit chymotrypsin, subtilisin and the granulocytic neutral proteases elastase and cathepsin G. They proved to be homogeneous in polyacrylamide and dodecylsulfate gel electrophoresis and by end group analysis; only threonine was found as N-terminal amino acid residue using the dansylation technique. These inhibitors, which we call eglins, are stable in neutral and weakly acid (pH 3) solutions and resist non-specific proteolysis. From the amino acid compositions, a molecular weight of 6 600 - 6 800 is calculated for both inhibitory proteins, which is in good agreement with a value of about 6000 estimated by dodecylsulfate electrophoresis. The eglins contain an unusually large amount of hydrophobic amino acid residues but no methionine, isoleucine or--a rarity--cysteine residues or disulfide bridges. To our knowledge, the eglins are the first examples of proteinase inhibitors of the protein type which are not stabilized by disulfide bridges.     

Isolation and partial characterisation of a new strain of Ebola virus

We have isolated a new strain of Ebola virus from a non-fatal human case infected during the autopsy of a wild chimpanzee in the C?te-d'Ivoire. The wild troop to which this animal belonged has been decimated by outbreaks of haemorrhagic syndromes. This is the first time that a human infection has been connected to naturally-infected monkeys in Africa. Data from the long-term survey of this troop of chimpanzees could answer questions about the natural reservoir of the Ebola virus.     

Isolation and characterisation of glutamate dehydrogenase from Mycobacterium smegmatis CDC 46

Glutamate dehydrogenase (L-glutamate:NADP+ oxidoreductase (deaminating), EC 1.4.1.4) has been purified from Mycobacterium smegmatis CDC 46 using (NH4)2SO4 precipitation, negative adsorption on DEAE-cellulose, 2',5'-ADP-Sepharose affinity chromatography and Sephadex G-200. The enzyme was purified 1041.6-fold and the preparation was found to be homogeneous on column chromatography, polyacrylamide gel electrophoresis and SDS-polyacrylamide gel electrophoresis. Alanine and threonine were identified as the N- and C-terminal amino acids of glutamate dehydrogenase from M. smegmastis. The enzyme kinetics and regulation of glutamate dehydrogenase activity by different nutritional factors has been studied. Initial velocity plots showed that the reaction mechanism of glutamate dehydrogenase from M. smegmatis followed an ordered sequential ter-bi mechanism.     

Isolation and partial characterisation of insulin-mimetic inositol phosphoglycans from human liver

Extracts of human liver were found to contain activities which copurified and coeluted with the two major subtypes of mediators (type A and type P) isolated from insulin-stimulated rat liver. The putative type A mediator from human liver inhibited cAMP-dependent protein kinase from bovine heart, decreased phosphoenolypyruvate carboxykinase mRNA levels in rat hepatoma cells, and stimulated lipogenesis in rat adipocytes. The putative type P mediator stimulated bovine heart pyruvate dehydrogenase phosphatase. Both fractions were able to stimulate proliferation of EGFR T17 fibroblasts and the type A was able to support growth in organotypic cultures of chicken embryo cochleovestibular ganglia. Both activities were resistant to Pronase treatment and the presence of carbohydrates, phosphate, and free-amino groups were confirmed in the two fractions. These properties are consistent with the structure/ function characteristics of the type A and P inositolphosphoglycans (IPG) previously characterized from rat liver. Further, the ability of the human-derived mediators to interact with rat adipocytes and bovine-derived metabolic enzymes suggests similarity in structure between the mediators purified from different species. Galactose oxidase-susceptible membrane-associated glycosylphosphatidylinositols (GPI) have been proposed to be the precursors of IPG. GPI was purified from human liver membranes followed by treatment with galactose oxidase and reduction with NaB3H4. Serial t.l.c. revealed three radiolabeled bands which comigrated with the putative GPI precursors found in rat liver. These galactose-oxidase-reactive lipidic compounds, however, were only partially susceptible to hydrolysis with phosphatidylinositol-specific phospholipase C from Bacillus thuringiensis and were resistant to glycosylphosphatidylinositol-specific phospholipase C from Trypanosoma brucei. These data indicate that IPG molecules with insulin-like biological activities are present in human liver.     

Isolation, purification, and characterization of a neutral Mg(2+)-dependent deoxyribonuclease of the Colorado potato beetle Leptinotarsa decemlineata Say

A neutral Mg(2+)-dependent deoxyribonuclease from the Colorado potato beetle was isolated and characterized in physicochemical terms. An electrophoretically homogeneous preparation of the enzyme was obtained using salt fractionation, Sephadex G-100 gel filtration, and subsequent preparative isoelectrofocusing in an Ultrodex layer. The molecular weight of the purified DNase preparation (with a purification degree of 104) and its isoelectric point were 100 kD and 9.1, respectively. The enzyme activity was maximal at pH 7.2 and 46 degrees C in the presence of 10 mM Mg2+. The DNase of the Colorado beetle preferentially hydrolysed denatured DNA via the endonuclease pathway, degrading the substrate to oligonucleoside-3'-phosphates. As far as the physical and chemical properties are concerned, this Colorado beetle DNase seems different from previously investigated DNases of other insect species.