Human GMP synthetase

• 1.1. The specific activity of GMP synthetase was measured in several human tissues and found to be highest in cultured skin fibroblasts, followed by bone marrow, leukocytes, erythrocytes. placenta, and liver.
• 2.2. The enzyme from fibroblasts was purified approximately 50-fold by ammonium sulfate fractionation and gel filtration.
• 3.3. The K_m values were determined to be 4.9μM for XMP, 270μM for ATP. and 340 μM for glutamine.
• 4.4. Ammonium sulfate could replace glutamine as the amino donor but was much less efficient.
• 5.5. The enzyme was specific for ATP as the energy source.
• 6.6. Unlike the calf thymus enzyme, the human enzyme has no requirement for a reduced sulfhydryl compound.
• 7.7. Human GMP synthetase is inhibited by ATP, dATP, azaserine, and hydroxylamine.

     

Monoclonal antibodies prepared against heparin lyase I and their reactivity toward heparin lyase I,II and III

• 1.1. Six different monoclonal IgG mouse antibodies to heparin lyase I from Flavobacterium heparinum were prepared.
• 2.2. The monoclonal antibodies were used to detect heparin lyases I, II and III by dot-blotting immunoassay and by Western blotting.
• 3.3. Individual antibodies showed different reactivity toward the three heparin lyases.
• 4.4. The reactivity of two of the monoclonal antibodies was destroyed by exposing heparin lyases to sodium dodecyl sulfate.
• 5.5. The antibodies can be used to rapidly distinguish between the three heparin lyases.

     

Electrical transport characteristics of Aplysia californica intestinal epithelium

• 1.1. Replacing chloride (Cl⁻) with sulfate (SO₄²⁻) in the bathing medium drastically reduced the mucosal membrane potential difference (ψ_m).
• 2.2. The voltage divider ratio was significantly greater than one.
• 3.3. Mucosal ^d-glucose decreased the input resistance of the intestinal epithelium.
• 4.4. Addition of furosemide to the mucosal bathing medium inhibited transepithelial potential difference and short-circuit current.
• 5.5. Addition of SITS to the mucosal bathing medium partially inhibited transepithelial potential difference and short-circuit current.
• 6.6. Diffusion potentials in the intestinal epithelium were symmetrical.

     

A simple and reliable method for the purification of Pseudomonas aeruginosa phospholipase C produced in a high phosphate medium containing choline

• 1.1. Pseudomonas aeruginosa phospholipase C from culture supernatants of bacteria grown in high-P_i basal salt medium with choline, as the sole carbon and nitrogen source, was purified by precipitation with 70% saturation ammonium sulfate in the presence of celite.
• 2.2. The PLC activity was eluted of this mixture by the use of a reverse gradient of 70-0% ammonium sulfate.
• 3.3. The peak containing the PLC activity revealed a single protein after SDS-PAGE.
• 4.4. The method could also be applied to purify PLC produced in a low-P_i complex medium. The resultant preparation was not homogeneous.
• 5.5. The molecular weight for both PLC preparations was about 70 kDa.
• 6.6. Both PLC used phosphatydilcholine and sphingomyelin as substrates, displayed hemolytic activity an exhibited an apparent K_M of 25 mM for p-nitrophenylphosphorylcholine.
• 7.7. They were not inhibited by 1% sodium deoxycholate but were 30% inhibited by 1% Triton X-100.
• 8.8. 2% sodium dodecylsulfate and 1% tetradecyltrimethylammonium bromide inhibited the PLC from the HP_l-BSM plus choline but not the enzyme from the LP_l-CM.

     

The role of the skin of Rana pipiens in sulfate excretion

• 1.1. The intact whole skin of Rana pipiens excretes sulfate and this excretion is increased by sulfate loading of the animal,
• 2.2. Ninety-five percent of the excretion of sulfate seen following sulfate loading is via the skin, while a small amount is handled by the urinary system.

     

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Comparison of fibrinogenases isolated from copperhead venoms

• 1.1. Fibrinogenase activity from fractions obtained by ion-exchange chromatography of whole copperhead (Agkistrodon contortrix) venoms were further purified by molecular sieve chromatography.
• 2.2. All four enzymes had apparent mol. wt of 23,000–24,000, both non-reduced and reduced as determined by sodium dodecyl sulfate gel electrophoresis and molecular sieve chromatography.
• 3.3. All four enzymes rapidly degraded the α-chain of the fibrinogen while apparently leaving the β- and γ-chains intact.

     

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A sensitive and specific binding assay for cyclic GMP-dependent and cyclic AMP-dependent protein kinases in rat liver

• 1.1. Sensitive and specific binding assays for cyclic GMP-dependent and cyclic AMP-dependent protein kinases have been developed for use in rat liver.
• 2.2. The addition of mixed histone to the binding mixture and the inclusion of ammonium sulfate in the termination and wash buffer enhanced the observed cyclic GMP- and cyclic AMP-binding activities markedly.
• 3.3. The principal effect of histone is to increase the binding of cyclic GMP and cyclic AMP to their respective protein kinases.
• 4.4. During filtration ammonium sulfate markedly increased the retention of the protein-bound cyclic nucleotides and markedly decreased the rapid dissociation component of cyclic GMP-binding.

     

The molecular weights of Arthropod hemocyanin subunits: Influence of tris buffer in SDS-PAGE estimations

• 1.1. Available molecular weights data for Arthropod hemocyanin subunits as measured by polyacrylamide gel electrophoresis in the presence of dodecyl sulfate are analyzed.
• 2.2. Relationship between buffer composition and subunit mobility in SDS-PAGE is shown by studying Cancer pagurus hemocyanin.
• 3.3. Tris buffers are suspected to give erroneous molecular weight estimations for Arthropod hemocyanin subunits.

     

Rat brain aryl acylamidase: Further characterization of multiple forms

• 1.1. Two fractions of aryl acylamidase (EC 3.5.1.13) were further separated from rat brain extracts at pH 7.5 by ammonium sulfate precipitation and Bio-Gel chromatography.
• 2.2. 1,2,3,4-Tetrahydro-β-carboline competitively inhibited (67%) fraction-1 but slightly inhibited (13%) fraction-2. Tetrahydroharman, 6-hydroxy-tetrahydroharman and harminic acid slightly inhibited both fractions. Harmalol inhibited fraction-1 but enhanced fraction-2. 6-Methoxy-harman, 6-methoxy-harmalan and harmaline enhanced both fractions.
• 3.3. Pargyline did not affect either fraction. Methiothepin, cyproheptadine and chlorimipramine inhibited fraction-1 but stimulated fraction-2.
• 4.4. Neostigmine moderately (30%) inhibited AAA-2 but did not have any significant effect on AAA-1.
• 5.5. These results indicate that the β-carboline compounds might play a role in regulating activity of AAA-1 and 2 in brain.
• 6.6. Both fractions might be related to serotonergic neurons but only AAA-2 might be associated with acetylcholinesterase.

     

Hydrolysis of natural and synthetic substrates by α-l-fucosidase, β-d-glucuronidase and β-n-acetylhexosaminidase purified from molluscs

• 1.1. Several mollusc glycosidases have been studied for their activities towards natural substrates. α-l-Fucosidases from Chamelea gallina, Tapes rhomboideus and Mytilus edulis hydrolyze oligosaccharides (di, tri and pentasaccharides) with α1 → 2, α1 → 3 and α1 → 4 bonds, fucose-containing glycopeptides from bovine thyroglobulin and the porcine submandibular mucin (devoid of sialic acid); α-l-fucosidase from Littorina littorea hydrolyzes fucose-containing glycopeptides from bovine thyroglobulin.
• 2.2. β-d-Glucuronidase from L. littorea hydrolyzes hyaluronic acid, chondroitin 4-sulfate and heparin with a very low activity; however, it is much more active on oligosaccharides (from the above-mentioned macromolecules) containing non-reducing terminal glucuronyl residues.
• 3.3. β-N-Acetylhexosaminidase from Helicella ericetorum acts mainly with an endo-hydrolase activity on β1 → 4N-acetylhexosamine linkages of ovalbumin, ovomucoid, chitin, hyaluronic acid and chondroitin
• 4.4-sulfate; it has also a secondary exo-hydrolase activity on these substrates.

     

Structure of heparan sulfate from the fresh water mollusc Anomantidae sp: Sequencing of its disaccharide units

• 1.1. The disaccharide sequences of a heparan sulfate isolated from Anomantidae sp. was determined with the aid of heparitinase I, heparitinase II from Flavobacterium heparinum, mollusc β-glucuronidase and α-N-acetylglucosaminidase besides nitrous acid degradation and chemical analyses.
• 2.2. Like the mammalian heparan sulfates the mollusc heparan sulfate is composed of different oligosaccharide blocks of N-acetylated disaccharides, N-sulfated disaccharides and N,6-sulfated disaccharides and has in its nonreducing end the monosaccharide glucosamine 2,6-disulfate.
• 3.3. The oligosaccharides produced by heparitinase I degradation contain at their reducing ends a N-acetylated, 6-sulfated disaccharide.
• 4.4. These and other results lead to the conclusion that the general structure of the heparan sulfate is maintained through evolution.

     

Isolation and characterization of phospholipase A2, from Agkistrodon bilineatus (common cantil) venom

• 1.1. Phospholipase A₂ was isolated from Agkistrodon bilineatus venom by Sephadex G-75 and CM-Cellulose column chromatographies.
• 2.2. The purified phospholipase A₂-I gave a single band on disc polyacrylamide gel electrophoresis, isoelectric focusing and sodium dodecyl sulfate polyacrylamide gel electrophoresis.
• 3.3. The enzyme preparation had a molecular weight of 14,000, isoelectric point of pH 8.77 and possessed 123 amino acid residues.
• 4.4. The purified phospholipase A₂ possessed lethal, indirect hemolytic and anticoagulant activities.
• 5.5. The enzyme hydrolyzed the phospholipids phosphatidyl choline (PC), phosphatidyl ethanolamine (PE), phosphatidyl inositol (PI) and phosphatidyl serine (PS).
• 6.6. The concentration of mouse diaphragm was inhibited and the contraction of guinea pig left atrium was increased by phospholipase A₂-I.
• 7.7. Phospholipase A₂ activity of this preparation was inhibited by ethylenediamine tetraacetic acid, p-bromo phenacyl bromide, n-bromo succinimide or dithiothreitol, but not by diisopropyl fluorophosphate or benzamidine.

     

Purification and characterization of arylsulfatase from sea urchin (Hemicentrotus pulcherrimus) embryos

• 1.1. Arylsulfatase was extracted from sea urchin (Hemicentrotus pulcherrimus) plutei and purified to electrophoretical homogeneity by means of DEAE-cellulose, acetone fractionation and Sepharose CL-6B, successively.
• 2.2. The molecular weight of this enzyme was approx, 670,000. The molecular weight of a single subunit was approx. 63,000. The K_m value for p-nitrophenyl sulfate was 0.59 mM.
• 3.3. This enzyme was competitively inhibited by the sulfate ion and was classified as the type II arylsulfatase. The pH optimum was between 5.0 and 6.0.

     

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.

     

Comparative hematology: Studies on cats including one with factor XII (hageman) deficiency

• 1.1. Cat plasma prothrombin and partial thromboplastin times are faster than human. Thromboplastin generation tests are very similar.
• 2.2. Factors VIII and V assay 24 and 13 times the human standard. Cat factors VII, X. IX, XI and XII assayed at 2.5 to 4 times human. Factors I, II and XIII fell within the human range and Fletcher was extremely low.
• 3.3. One cat lacked factor XII and showed a prolonged APTT and clotting time.
• 4.4. Cat profibrinolysin was activated by streptokinase but not by urokinase.
• 5.5. Cat platelets aggregated with the usual human aggregation agents with the exception of thrombin and ristocetin.
• 6.6. Cat erythrocytes were smaller and more numerous than human.
• 7.7. Leukocyte counts were quite variable.
• 8.8. Serum protein electrophoretic patterns differed from human in the greater migration of albumin and the presence of numerous unidentified bands.
• 9.9. Biochemical tests showed high sodium and chloride values.

     

Purification and characterization of a blue-colored red-fluorescent protein from the silkworm (Bombyx mori L.) larvae

• 1.1. A red-fluorescent blue protein (P600) was purified from the digestive juice of the silkworm (Bombyx mori L.) larvae raised on mulberry leaves.
• 2.2. The purified protein was electrophoretically homogeneous and showed the absorption maxima at 601.5 nm and 278 nm, and the fluorescence maximum at 621 nm.
• 3.3. The molecular weight was estimated to be 540,000 by gel filtration on Sepharose CL-6B. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate suggests that the protein consists of two heterogeneous polypeptide subunits with a mol. wt of 15,000 and 18,000.
• 4.4. The P600 contains excess acidic amino acid residues over basic groups. The polarity and pI were 45.5% and 4.6, respectively.
• 5.5. The production of H₂O₂ was observed in the presence of P600 upon illumination.