The suitability of using cryopreservation of spermatozoa for the conservation of genetic diversity in African catfish (Clarias gariepinus)

• 1.1. The effect of cryopreservation of semen on expected Hardy-Weinberg proportions in the f₁ progeny of selected breeding pairs was evaluated.
• 2.2. Four different African catfish breeding pairs were selected, each pair displaying different heterozygous alleles at the glucose-6-phosphate isomerase-1 or 2 loci.
• 3.3. Equal volumes of ova from each female were artificially inseminated with cryopreserved or fresh semen obtained from males possessing corresponding genotypes.
• 4.4. A comparison of growth rates between f₁ groups of offspring produced from fresh and cryopreserved semen was made.
• 5.5. The G-test for goodness of fit showed no significant differences from expected Hardy-Weinberg proportions for allele frequencies obtained in the f₁ progeny.
• 6.6. The application of cryopreservation for the conservation of genetic diversity is discussed.

     

Isolation of dna and rna from Streptococcus sobrinus OMZ176 using CsTFA gradients

• 1.1. A simple procedure for isolation of high molecular weight genomic DNA, and RNA, from Streptococcus sobrinus OMZ176 is described.
• 2.2. Cell cultures were grown aerobically for 10 hr.
• 3.3. Spheroplast formation and lysis was achieved by mutanolysin/lysozyme-dependent digestion of the cell wall, followed by N-lauroylsarcosinate-mediated lysis.
• 4.4. Nucleic acids were isolated directly from cell-lysates using cesium-trifluoroacetate (CsTFA) densitygradient centrifugation.
• 5.5. Three different centrifugation regimes were tested: self-generated density gradients in a fixed angle rotor; self-generated density-gradients in a swinging-bucket rotor; pre-formed density-gradients in a swinging-bucket rotor.
• 6.6. Genomic DNA isolated by the CsTFA-procedure was found to have higher purity as compared to genomic DNA isolated in a conventional CsCl gradient.
• 7.7. Isolated DNA was shown to be of a quality suitable for applications in molecular biology.

     

Solubilization and characterization of functionally coupled Escherichia coli heat-stable toxin receptors and particulate guanylate cyclase associated with the cytoskeleton compartment of intestinal membranes

• 1.1. Particulate guanylate cyclase and receptors for E. coli heat-stable enterotoxin were solubilized from the rat intestinal cytoskeletal compartment using Lubrol-PX and KC1.
• 2.2. Thirty to forty percent of the ST receptor and guanylate cyclase activities were extracted from the lipid layer with Lubrol-PX alone.
• 3.2. Seventy percent of the remaining activities were solubilized from the cytoskeleton with Lubrol-PX and KCl.
• 4.3. Guanylate cyclase solubilized from either compartment exhibited similar reaction kinetics.
• 5.4. Both high- and low-affinity classes of ST receptors were solubilized from the lipid and cytoskeleton compartments.
• 6.5. In the presence of ATPγS, ST selectively activated the guanylate cyclase solubilized from the cytoskeleton compared to that solubilized from the lipid bilayer.
• 7.6. Crosslinking experiments demonstrated a preferential solubilization of the 130 kDa receptor subunit from the cytoskeleton and the 56 kDa subunit from the lipid bilayer.
• 8.7. Development of a procedure to solubilize ST receptors and guanylate cyclase from the intestinal membrane cytoskeleton will permit purification and further detailed studies of the coupling of these activities.

     

Company news

Including information on:

• ScanSoft
• SpeechWorks International
• Viisage Technology
• Firstec
• BIO-key International
• HP
• ZN Vision Technologies
• Unisys
• US Government’s
• Communication Intelligence Corporation
• Infinity Technologies

     

Characteristics of Crassostrea virginica crystalline style chitin digestion

• 1.1. Fundamental chitin digestion characteristics of Crassostrea virginica crystalline style were investigated.
• 2.2. Optimum temperature and pH were 34°C and 4.8. respectively.
• 3.3. The colloidal regenerated chitin (0.56mol/0.5 ml: GlcNAc equivalents) was saturating under all enzyme levels encountered.
• 4.4. There was no evidence of end product inhibition, even after 100 hr incubation.
• 5.5. Calculated K_m for the chitinase complex was 1.19mM when determined using a 30 min assay, but was only 0.70 mM when determined using a 4.6 hr assay.
• 6.6. Both K_m values are lower than reported for similar assays in other molluscs and for most bacteria.
• 7.7. Effect of substrate preparation on the kinetics are discussed.
• 8.8. Eight peaks of chitinase activity were resolved by DEAE-Fractogel ion exchange chromatography.

     

Identification of the major annexins in Ehrlich ascites tumor cells

• 1.1. Three calcium-binding proteins have been purified from Ehrlich ascites tumor cells.
• 2.2. They were identified by amino acid sequence analysis on selected fragments obtained by tryptic digestion.
• 3.3. The proteins belong to the annexin family and were identified as annexins II, III and V.
• 4.4. Antibodies raised against the proteins were used to examine for their presence in a number of murine tissues.
• 5.5. The occurrence was found to be in reasonable accordance with earlier reports.

     

Company news

• Daon
• Musicrypt
• EMI Music Canada
• Digital Broadband Networks
• FaceKey Corporation
• Eystar Media Inc (EMI)
• Temasya Wira
• Animated Electronic Industries
• BIO-key International
• Entryport Corporation

     

Domain structure of the 90-kDa stress protein: Heparin- and antibody-binding domain

• 1.1. To understand the physiological roles of the 90-kDa stress protein (HSP90), we investigated the heparin- and antibody-binding domains of the protein.
• 2.2. For heparin-binding sites, HSP90 was digested completely with trypsin, and the digests were applied to a heparin-Sepharose column and eluted with 1.0 M NaCl, followed by 8.0 M urea.
• 3.3. Each elutant was purified by a reverse-phase C₁₈ column.
• 4.4. Two peptides from the NaCl-eluted fraction and no peptide from the urea-eluted fraction were purified.
• 5.5. The purified peptides were sequenced by an automated peptide sequencer.
• 6.6. One of the heparin-binding sites was present between Leu-362 and Arg-365; another was present between Leu-645 and Lys-648.
• 7.7. These two peptides were basic and considerably hydrophilic.
• 8.8. For antibody-binding sites, HSP90 was mildly digested with trypsin, electrophoresed on SDS-polyacrylamide gels and transferred to PVDF membranes.
• 9.9. The four bound of the trypsin fragments could be sequenced with a peptide sequencer.
• 10.10. There was only one antibody-binding peptide, 38 kDa, starting from Pro-2. The others showed no cross-reactivity with the antibody and started from Leu-283.
• 11.11. Therefore, the epitopes of HSP90 are present between Pro-2 and Leu-282.
• 12.12. The heparin-binding sites are present from the middle region of the HSP90 molecule, and the antigen sites are at the N-terminal domain.

     

Application news

Including information on:

• Martin State Airport
• Bioscrypt
• Saflink
• Office of the Secretary of Defense
• Department of Defense
• Boeing Corporation
• Bell ID, Gemplus
• Siemens
• Foreign Ministry

     

In brief

• Bioscrypt
• Saflink
• Dell
• Fujitsu Microelectronics America
• Identix
• Viisage
• Acsys Biometrics
• US Government

     

Electrophoretic identification of bird species involved in collisions with aircraft

• 1.1. The utility of biochemical genetic methods of bird identification was investigated for some common species which create a hazard for commercial aviation in Ireland.
• 2.2. Sixteen enzyme loci were assayed in eight species, using starch gel electrophoresis; three larids, three corvids and two columbids.
• 3.3. Genera were distinguishable using all but two loci.
• 4.4. Differences within genera were small, but all species except for the gulls Larus argentatus and L. marinus, could be identified using one or more loci.
• 5.5. Arising from the success of the method using fresh specimens, a protocol for the electrophoretic identification of traumatized remains of strikes is suggested.

     

IDentification of a microsomal retinoic acid synthase as a microsomal cytochrome P-450-linked monooxygenase system

• 1.1. To characterize an enzyme which metabolizes retinal in liver microsomes, several properties of the enzymatic reaction from retinal to retinoic acid were investigated using rabbit liver microsomes.
• 2.2. The maximum pH of the reaction in the liver microsomes was 7.6.
• 3.3. The K_m and V_max values for all-trans, 9-cis and 13-cis-retinals were determined.
• 4.4. The reaction proceeded in the presence of NADPH and molecular oxygen.
• 5.5. The incorporation of one atom of molecular oxygen into retinal was confirmed by using oxygen-18, showing that the reaction comprised monooxygenation, not dehydrogenation.
• 6.6. The monooxygenase activity was inhibited by carbon monoxide, phenylisocyanide and antiNADPH-cytochrome P-450 reductase IgG, but not by anti-cytochrome b₅ IgG.
• 7.7. The enzymatic activity inhibited by carbon monoxide was photoreversibly restored by light of a wavelength of around 450 nm.
• 8.8. The retinal-induced spectra of liver microsomes with three isomeric retinals were type I spectra.
• 9.9. The microsomal monooxygenase activity induced by phenobarbital or ethanol were more effective than that by 3-methylcholanthrene, clotrimazole or β-naphthoflavone.
• 10.10. These results showed that the monooxygenase reaction from retinal to retinoic acid in liver microsomes is catalyzed by a cytochrome P-450-linked monooxygenase system.

     

Cloning and characterization of rabbit pancreatic colipase

• 1.1. Among the digestive enzymes synthesized by pancreas, lipase is the principle lipolytic enzyme which hydrolyses dietary glycerides.
• 2.2. For its action it requires a coenzyme, colipase.
• 3.3. The molecular mechanisms of the interaction of these two are not fully understood.
• 4.4. Further, molecular events that regulate and influence lipid absorption are ill denned.
• 5.5. The rabbit is the conventional animal model for the study of lipid absorption. We have undertaken the molecular cloning, and characterization of rabbit pancreatic colipase, the coenzyme for pancreatic lipase.
• 6.6. Colipase has been cloned from a gt 11 library of an adult rabbit pancreatic cDNA by probing with an oligonucleotide derived from human colipase sequence.
• 7.7. The total reading frame consists of 321 nucleotides coding for 90 amino acids of the functional protein and 17 nucleotides of the leader peptide.
• 8.8. Northern blot analysis revealed a distinct band around 0.5kb. Comparison with other species revealed an over all homology of 75% at the nucleotide level.
• 9.9. At the amino acid level highest conservation is observed at the lipase-binding region (AA 53–73).
• 10.10. Rabbit enzyme also retained the N-terminal pentapeptide of it preform.
• 11.11. The regions of homology and conservation may aid to define the sites of interaction of colipase with lipase.

     

Purification and characterization of vitellogenin from the American cockroach,Periplaneta americana

• 1.1. Vitellogenin (VG) was isolated and purified from the hemolymph of female American cockroaches.
• 2.2. The purification method used in this study comprises two steps: the first step is based on the method originally developed for purifying lipophorin from hemolymph, and the second step is the separation of VG from lipophorin by a KBr density gradient ultracentrifugation.
• 3.3. The purified VG was characterized according to molecular weight, substructure, shape and size, and lipid composition.
• 4.4. The VG molecule is almost globular in shape with the diameter of about 15.5 nm and is indistinguishable from lipophorin in shape and size.
• 5.5. The native molecular weight determined by light scattering method was 560 kDa.
• 6.6. The VG consists of four subunits with molecular weights of approximately 102, 81, 49 and 40 kDa, respectively.
• 7.7. VG is a lipoprotein and comprises 92% protein and 8% lipid.
• 8.8. Major lipid components were found to be diacylglycerol (25%) and phospholipids (71%).

     

Properties of the transition from short-term to long-term depression in neurons of Helix pomatia

• 1.1. Synaptic short-term depression could be transferred into long term depression by repetition of series of stimuli.
• 2.2. The transition from short-term depression to long-term depression was blocked by puromycin.
• 3.3. The majority of the transition took place during resting periods between stimulus series.
• 4.4. The initiation of the transition process was 83% completed after 5 min of stimulation.
• 5.5. Short- and long-term depression were quantitatively separated into their two serial sites of origin: afferent axons and synaptic terminals.
• 6.6. Long sequences evoked periods with increased and variable EPSPs not conforming to depression.

     

Properties of enzymes hydrating epoxides in human epidermis and liver

• 1.1. Cytosolic and microsomal epoxide hydrolyzing enzymes of human skin and liver were compared and found to be different.
• 2.2. Epidermal and hepatic cytosolic epoxide hydrolases were different in terms of substrate selectivity, pI, inhibitor sensitivity and affinity Chromatographic properties.
• 3.3. Microsomal epoxide hydrolases had the same pIs but different substrate selectivities.
• 4.4. Cytosolic epoxide hydrolase from adults had higher specific activity than that from neonates or cultured epidermis, but lower activity than adult hepatic enzymes.
• 5.5. The sizes of cytosolic epoxide hydrolase from epidermis and liver were similar and lower than that from cultured fibroblasts.
• 6.6. Cytosolic epoxide hydrolase from all sources shared similar antigenic determinants.

     

The influences of individual variations in metabolic rate and tidal conditions on the response to hypoxia in Carcinus maenas (L.)

• 1.1. The oxygen consumption of crabs in normoxic and hypoxic (50% O₂) seawater was measured directly after collection.
• 2.2. The influences of size and lunar cycles were removed by scaling the data.
• 3.3. Strong negative correlations between low individual levels of O₂ consumption and the ability to compensate for hypoxia were apparent in Wicklow (subtidal) crabs.
• 4.4. Compensation for hypoxia was much greater on the flood tide than on the ebb.
• 5.5. Crabs from Roscoff (intertidal) had lower levels of compensation than those from Wicklow.
• 6.6. Size, sex and condition had no apparent effect upon these relationships.
• 7.7. Crabs acclimated to laboratory conditions have not shown this tidal variation in compensation for hypoxia.

     

NAD(P)H dehydrogenase from rabbit and rat liver: Purification and some properties

• 1.1. NAD(P)H dehydrogenase from rabbit liver was purified to electrophoretic homogeneity using a procedure also found applicable for the rat liver enzyme.
• 2.2. Rabbit and rat liver enzymes showed different behaviour in isoelectric focusing and different K_m values and turnover numbers.
• 3.3. Both enzymes were inhibited to similar extents by warfarin.
• 4.4. The rabbit enzyme is composed of two subunits of mol. wt 27,000 and contained 1 FAD group per subunit.
• 5.5. Some absorption and circular dichroism properties of the rat enzyme are shown.

     

The purification of kallikrein from human whole saliva

• 1.1. A quick and simple procedure is described for purifying kallikrein from human whole saliva. The enzyme has been purified about 2700-fold with a yield of approx. 30%.
• 2.2. The procedure is based on the immediate fractionation of saliva by ion exchange chromatography. This is followed by a combination of affinity and high performance liquid chromatography.
• 3.3. The results indicate that another protein component binds to the enzyme at pH 8.0.
• 4.4. The homogeneity of the enzyme has been demonstrated by gel electrophoresis in the absence as well as in the presence of sodium dodecylsulfate.
• 5.5. A mol. wt of 40,100±1800 has been calculated from gel electrophores is experiments.
• 6.6. Sedimentation equilibrium in an analytical ultracentrifuge gave a mol. wt of 39,700.
• 7.7. The amino acid composition has been determined and it confirms that the enzyme has a low isoelectric point.
• 8.8. The presence of tryptophan has been demonstrated by absorption and fluorescence spectroscopy.

     

Isohematoporphyrin-diesters and diethers

• 1.1. A series of diesters of isohematoporphyrin (isoHp), from dimethyl to dioctyl were prepared according to Rimington et al. (1989b). Their optical absorption, fluorescence spectra and high performance liquid chromatography (HPLC) retention times were recorded.
• 2.2. A plot of HPLC retention time against number of C atoms in the alcohol used for esterification was approximately linear at first then rising steeply from diamyl to diocyi ester, whether a gradient elution was used or only methanol: water, 95/5, at pH 7.5.
• 3.3. Preparation of the diethers of isoHp was much more difficult than that of the corresponding derivatives of hematoporphyrin (Hp). Several different methods were investigated, varying both times and temperatures.
• 4.4. These methods included reaction of isoHp or its demethyl ester with
  • 4.1.(i) a bromoalkane in presence of anhydrous K₂CO₃;
  • 4.2.(ii) reaction with bromoalkane and Ag₂O;
  • 4.3.(iii) reaction of brominated-isoHp, prepared by using thionylbromide, with the selected alcohol, or corresponding sodium alcoholate;
  • 4.4.(iv) heating of isoHp alone with an alcohol containing 20% (w/v) H₂SCO₄ (temp. range from 45° to 118°C),
  • 4.5.(v) refluxing as in (iv) at the b.p. of the alcohol; and
  • 4.6.(vi) carrying out this reaction in refluxing ethyleneglycoldimethyl ether (b.p. 85°C) or diethyleneglycoldimethyl ether (b.p. 155°C).
• 5.5. Some diether formation was observable by all these methods but yields were small, a considerable quantity of unreacted isoHp and other products remaining.
• 6.6. Examined by HPLC, the diethers consistently afforded a forked peak which on thin layer chromatography was only resolved into two very closely associated bands by a solvent mixture carefully selected for development.
• 7.7. On elution these materials had virtually identical optical absorption and fluoresence spectra.
• 8.8. The nature of the association is discussed, atropisomers (Gottwald and Ullman, 1969) and possible stacked monomer: dimers (Abraham et al., 1963) being considered as possibilities.