Detection of a C4a-hydroperoxyflavin intermediate in the reaction of a flavoprotein oxidase

This work describes for the first time the identification of a reaction intermediate, C4a-hydroperoxyflavin, during the oxidative half-reaction of a flavoprotein oxidase, pyranose 2-oxidase (P2O) from Trametes multicolor, by using rapid kinetics. The reduced P2O reacted with oxygen with a forward rate constant of 5.8 x 10 (4) M (-1) s (-1) and a reverse rate constant of 2 s (-1), resulting in the formation of a C4a-hydroperoxyflavin intermediate which decayed with a rate constant of 18 s (-1). The absorption spectrum of the intermediate resembled the spectra of flavin-dependent monooxygenases. A hydrophobic cavity formed at the re side of the flavin ring in the closed state structure of P2O may help in stabilizing the intermediate.     

Validation of a specific machine to the strength training of judokas

With regard to judo players, like all sport activities, strength training can be divided into general and specific strength training. The specific exercises must correspond with the competitive movement. In addition, in terms of structure, they must correspond with regard to the strength time sequence, and they may be executed with overload. With respect to these important elements, we have envisaged the use of a judo-specific machine. The purpose of this study was to validate this judo-specific machine with regard to the strength training of judokas. To that end, we have measured the maximal pulling forces applied at each hand of judokas (n = 18), playing with the judo-specific machine and with a real partner. A significant difference was found between the maximal pulling forces (F collar and F sleeve) obtained utilizing the judo-specific machine (from 4.9 +/- 0.4 N.kg(-1) to 6.4 +/- 0.3 N.kg(-1) for F collar; from 4.8 +/- 0.2 N.kg(-1) to 6.3 +/- 0.3 N.kg(-1) for F sleeve) and performing with a partner (2.7 +/- 0.2 N.kg(-1) for F collar; 2.5 +/- 0.3 N.kg(-1) for F sleeve). This can be explained by the fact that the partner opposes a low resistance during the judo-throwing technique in comparison with the judo-specific machine. These results show that the judo-specific machine might be used by the judokas to execute specific exercises with overload.     

Progestagen synchronisation in the absence of a corpus luteum results in the ovulation of a persistent follicle in cyclic ewe lambs

Progestagens are widely used to synchronise oestrous in sheep but the effects on follicular dynamics are not clear. We tested the hypothesis that when luteolysis occurs early during progestagen synchronisation prolonged growth of the ovulatory follicle will occur. Cyclic ewe lambs (40.0+/-0.3 kg) were divided into three groups: eight ewes (Long group) received a progestagen sponge (60 mg medroxyprogesterone acetate) from Days 5 to 19 after oestrous and eight ewes (Short group) received a progestagen sponge on Day 5 which was replaced on Day 10 and again on Day 15, and removed on Day 19 after oestrous. On Days 6 and 7, ewes in both groups received prostaglandin. A third group (n=5, Control) did not receive any treatment. The growth and development of follicles > or =2 mm in diameter were characterised using daily transrectal ultrasonography. On Day 18, blood samples were collected every 12 min for 8 h from five ewes in the Long and Short groups. Data were analysed by ANOVA. The maximum diameter and age (emergence to ovulation) of the ovulatory follicle was greater (P<0.01) in ewes in the Long group (7. 4+/-0.2 mm and 12.1+/-0.6 days) than in ewes in the Short group (6. 3+/-0.2 mm and 5.1+/-0.5 days) and Control group (6.3+/-0.4 mm and 6. 8+/-0.6 days). On Day 18 of the cycle, LH pulse frequency and oestradiol concentrations were greater (P<0.05) in ewes in the Long group (3.2+/-1.1 pulse per 8 h and 1.15+/-0.09 pg ml(-1)) than the Short group (0.8+/-0.4 pulses per 8 h and 0.54+/-0.08 pg ml(-1)).We suggest that the negative feedback efficacy of a long-term progestagen sponge decreased with time and led to an increase in LH pulse frequency and prolonged growth of the ovulatory follicle. We conclude that, in the absence of luteal progesterone, synchronisation with a single progestagen sponge for 14 days resulted in higher LH pulse frequency and ovulation of a persistent follicle with a larger maximum diameter, compared with controls.     

On the Feasibility of a Coulomb Explosion in a Micropinch

Plasma Physics Reports - In this paper we present experimental data on the generation of ions with MeV-energy in a Z-pinch discharge in a heavy-element medium. The calculations show that the escape...     

Location of a synergistic factor in the capsule of a granulosis virus of the armyworm, Pseudaletia unipuncta

A synergistic factor (SyF), which enhanced the infection of nuclear polyhedrosis viruses, was purified from capsules of a Pseudaletia unipuncta granulosis virus (Hawaiian strain) by immune affinity chromatography. The isolated SyF consisted primarily of a protein with molecular mass 98 kDa. The recovery rate depended on the alkali used to dissolve the capsules: the highest rate occurred with 0.05 M Na2CO3-0.05 M NaCl, followed in turn with 0.02-0.05 M NaOH and 0.04 M NaOH-0.05 M glycine. The solubilized components from untreated capsules contained 98- and 100-kDa proteins in addition to the matrix protein (29 kDa) and its decomposed products, while those from heat-treated capsules contained only the 100-kDa protein. Virons liberated from the capsules with the glycine buffer contained three proteins (33, 98, and 100 kDa) serologically related to the SyF. Immunoelectron microscopy of infected tissue and purified virions revealed the localization of the SyF antigens on the viral envelope.     

Molecular characterization of a Dictyostelium discoideum gene encoding a multifunctional enzyme of the pyrimidine pathway

We have isolated and characterized a Dictyostelium discoideum gene (PYR1-3) encoding a multifunctional protein that carries the three first enzymatic activities of the de novo pyrimidine biosynthetic pathway. The PYR1-3 gene is adjacent to another gene of the pyrimidine biosynthetic pathway (PYR4); the two genes are separated by a 1.5-kb non-coding sequence and transcribed divergently. The PYR1-3 gene is transcribed to form a 7.5-kb polyadenylated mRNA. As with the other genes of the pyrimidine biosynthetic pathway, the PYR1-3 mRNA level is high during growth and decreases sharply during development. We have determined the nucleotide sequence of 63% of the coding region of the PYR1-3 gene. We have identified the activities of the protein encoded by the D. discoideum PYR1-3 gene by comparison of amino acid sequences with the products of genes of known function. The PYR1-3 gene contains four distinct regions that probably correspond to four domains in the protein. From the NH2 extremity to the COOH extremity, these domains are: glutamine amidotransferase, carbamoylphosphate synthetase, dihydroorotase and aspartate transcarbamylase. This organization is identical to the one found in the rudimentary gene of Drosophila. The evolutionary implications of this finding are discussed.     

On the Conformation of UDPG,a Sugar Nucleotide

Abstract

UDPG [uridine 5′-(∝-D-glucopyranosyl pyrophosphate)] has a folded conformation in solution in which the glucose and uracil rings lie, approximately, in the same plane with the 2′- & 3′- hydroxyl groups facing the 5- & 6- hydrogens of the uracil ring.     

Attachment of a long-tailed Rhizobium bacteriophage to the pili of its host

Bacterial strain 16-12 was isolated from the root nodules of lupines and was found to be mitomycin C-inducible for the production of a bacteriophage (“16-12-1”) with a long noncontractile tail. The phage was found to attach with a fork-like terminal tail structure to the pili of strain 16-12. In addition, it was also found adsorbed to the bacterial cell poles. It is suggested that phage 16-12-1 may be pilus dependent.     

NMR evidence for a short, strong hydrogen bond at the active site of a cholinesterase

Cholinesterases (ChE), use a Glu-His-Ser catalytic triad to enhance the nucleophilicity of the catalytic serine. It has been shown that serine proteases, which employ an Asp-His-Ser catalytic triad for optimal catalytic efficiency, decrease the hydrogen bonding distance between the Asp-His pair to form a short, strong hydrogen bond (SSHB) upon binding mechanism-based inhibitors, which form tetrahedral Ser-adducts, analogous to the tetrahedral intermediates in catalysis, or at low pH when the histidine is protonated [Cassidy, C. S., Lin, J., Frey, P. A. (1997) Biochemistry 36, 4576-4584]. Two types of mechanism-based inhibitors were bound to pure equine butyrylcholinesterase (BChE), a 364 kDa homotetramer, and the complexes were studied by (1)H NMR at 600 MHz and 25-37 degrees C. The downfield region of the (1)H NMR spectrum of free BChE at pH 7.5 showed a broad, weak, deshielded resonance with a chemical shift, delta = 16.1 ppm, ascribed to a small amount of the histidine-protonated form. Upon addition of a 3-fold excess of diethyl 4-nitrophenyl phosphate (paraoxon) and subsequent dealkylation, the broad 16.1 ppm resonance increased in intensity 4.7-fold, and yielded a D/H fractionation factor phi = 0.72+/-0.10 consistent with a SSHB between Glu and His of the catalytic triad. From an empirical correlation of delta with hydrogen-bond length in small crystalline compounds, the length of this SSBH is 2.64+/-0.04 A, in agreement with the length of 2.62+/-0.02 A independently obtained from phi. The addition of a 3-fold excess of m-(N,N, N-trimethylammonio)trifluoroacetophenone to BChE yielded no signal at 16.1 ppm, and a 640 Hz broad, highly deshielded proton resonance with a chemical shift delta = 18.1 ppm and a D/H fractionation factor phi = 0.63+/-0.10, also consistent with a SSHB. The length of this SSHB is calculated to be 2.62+/-0.04 A from delta and 2.59+/-0.03 A from phi. These NMR-derived distances agree with those found in the X-ray structures of the homologous acetylcholinesterase complexed with the same mechanism-based inhibitors, 2.60+/-0.22 and 2.66+/-0.28 A. However, the order of magnitude greater precision of the NMR-derived distances establish the presence of SSHBs. We suggest that ChEs achieve their remarkable catalytic power in ester hydrolysis, in part, due to the formation of a SSHB between Glu and His of the catalytic triad.     

Stereochemistry of a methyl-group rearrangement during the biosynthesis of lanosterol.

1. (3RS,6R)-[6-2H1,6-3H1,6-14C], (3RS,6S)-[6-2H1,6-3H1,6-14C] and (3RS)-[6-3H1,6-14C]mevalonolactones were synthesised from R-[2H1,3H1,2-14C], S-[2H1,3H1,2-14C] and [3h1,2-14C]acetic acids respectively. 2. Each mevalonate was converted into cholesterol by a rat liver preparation. 3. Each cholesterol specimen was converted into androsta-1,4-diene-3,17-dione by incubation with Mycobacterium phlei in the presence of 2,2'.dipyridyl. Each specimen of androsta-1,4-diene-3,17-dione was converted into androsta-1,4-dien-3-one-17-ethylene ketail. 4. The samples of androsta-1,4-dien-3-one-17-ethylene ketal were each converted chemically into oestrones in which the methyl group at C-18 is the only carbon atom that originated from C-6 in mevalonolactone. 5. The oestrone from (3RS)-[6-3H1,6-14C]mevalonolactone was oxidised chemically to acetic acid which was converted into p-bromophenacyl acetate and the 3H/14C ratio was measured. 6. There was no overall loss of tritium from the methyl group of acetic acid, as measured by determining the 3H/14C ratios of the p-bromophenacyl esters, when the synthetic and degradative procedures 1 -- 5 were tested with [3H1,2-14C]acetic acid. 7. The oestrones derived from the 6R and 6S-mevalonolactones were oxidised. The chiralities of the resulting acetates were determined by an established procedure whereby the acetates were converted into 2S-malates which were examined for loss of tritium on equilibration with fumarate hydratase. 8. The oestrone from (3RS,6R)-[6-2H1,6-3H1,6-14C]mevalonate gave acetic acid which was converted into 2S-malate that retained 68.6% of its tritium after treatment with fumarate hydratase; the configuration of this acetic acid was R. 9. The oestrone from (3RS,6S)-E16-2H1,6-3H1,6-14C]mevalonate was oxidised to acetic acid which was converted into 2S-malate that retained 31.9% of its tritium after treatment with fumarate hydratase; the configuration of this acetic acid was S. 10. There was no overall change in the configuration of a chiral methyl group between C-6 of mevalonate and C-18 of oestrone. It is cncluded that the intramolecular migration of a chiral methyl group from C-15 in 2,3-oxidosqualene to C-13 in lanosterol is stereospecific and occurs with overall retention of configuration.     

From a cytotoxic agent to the discovery of a novel antimalarial agent

A novel cytotoxin 3,5-bis(4-chlorobenzylidene)-1-[4-{2-(4-morpholinyl)ethoxy}phenyl-carbonyl]-4-piperidone hydrochloride 2 demonstrated potent antimalarial properties with IC₅₀ values of 0.60 and 1.97 μM against the drug sensitive D6 strain and the C235 drug-resistant strain of Plasmodium falciparum. This compound concentrates in red blood cells, lowers glutathione concentrations in erythrocytes and permeates across CACO-2 cells. These data reveal 2 to be a promising lead compound in the quest for novel antimalarial agents.     

The effect of the oxidation state of a terthiophene-conducting polymer and of the presence of a redox probe on its gene-sensing properties

The gene-sensing properties of sensor films made of a terthiophene-conducting polymer, poly(3-((2':2', 5':2'-terthiophene)-3'-yl)acrylic acid) (PTAA), were evaluated using electrochemical impedance spectroscopy for films in their reduced and oxidised states with and without the Fe(CN)(6)(3-/4-) redox probe (RP) in dilute tris-EDTA buffer. Porous films of PTAA were prepared and attached to an oligonucleotide sequence specific to the Salmonella virulence gene InvA. These films could be described with a dual transmission line model in which the polymer conductivity was increased as a consequence of surface binding of complementary DNA. The effect is analogous to that reported for silicon nanowires and field-effect transistors in dilute electrolyte modified by charge exchange across the polymer-electrolyte interface. As a result, gene sensing could be conveniently observed as a change in the impedance phase angle at a fixed frequency.     

Lipoprotein [a] is cleared from the plasma primarily by the liver in a process mediated by apolipoprotein [a

The cellular and molecular mechanisms responsible for lipoprotein [a] (Lp[a]) catabolism are unknown. We examined the plasma clearance of Lp[a] and LDL in mice using lipoproteins isolated from human plasma coupled to radiolabeled tyramine cellobiose. Lipoproteins were injected into wild-type, LDL receptor-deficient (Ldlr-/-), and apolipoprotein E-deficient (Apoe-/-) mice. The fractional catabolic rate of LDL was greatly slowed in Ldlr-/- mice and greatly accelerated in Apoe-/- mice compared with wild-type mice. In contrast, the plasma clearance of Lp[a] in Ldlr-/- mice was similar to that in wild-type mice and was only slightly accelerated in Apoe-/- mice. Hepatic uptake of Lp[a] in wild-type mice was 34.6% of the injected dose over a 24 h period. The kidney accounted for only a small fraction of tissue uptake (1.3%). To test whether apolipoprotein [a] (apo[a]) mediates the clearance of Lp[a] from plasma, we coinjected excess apo[a] with labeled Lp[a]. Apo[a] acted as a potent inhibitor of Lp[a] plasma clearance. Asialofetuin, a ligand of the asialoglycoprotein receptor, did not inhibit Lp[a] clearance. In summary, the liver is the major organ accounting for the clearance of Lp[a] in mice, with the LDL receptor and apolipoprotein E having no major roles. Our studies indicate that apo[a] is the primary ligand that mediates Lp[a] uptake and plasma clearance.     

Use of a genetically engineered protein for the design of a multivalent MRI contrast agent

The majority of clinically used contrast agents (CAs) for magnetic resonance imaging have low relaxivities and thus require high concentrations for signal enhancement. Research has turned to multivalent, macromolecular CAs to increase CA efficiency. However, previously developed macromolecular CAs do not provide high relaxivities, have limited biocompatibility, and/or do not have a structure that is readily modifiable to tailor to particular applications. We report a new family of multivalent, biomacromolecular, genetically engineered protein polymer-based CAs; the protein backbone contains evenly spaced lysines that are derivatized with gadolinium (Gd(III)) chelators. The protein's length and repeating amino acid sequence are genetically specified. We reproducibly obtained conjugates with an average of 8-9 Gd(III) chelators per protein. These multivalent CAs reproducibly provide a high relaxivity of 7.3 mM (-1) s (-1) per Gd(III) and 62.6 mM (-1) s (-1) per molecule. Furthermore, they can be incorporated into biomaterial hydrogels via chemical cross-linking of the remaining free lysines, and provide a dramatic contrast enhancement. Thus, these protein polymer CAs could be a useful tool for following the evolution of tissue engineering scaffolds.     

Validation of a decision support system for the cytodiagnosis of fine needle aspirates of the breast using a prospectively collected dataset from multiple observers in a working clinical environment

We have used a 692 case dataset, collected retrospectively by a single observer, to develop decision support systems for the cytodiagnosis of fine needle aspirates of breast lesions. In this study, we use a 322 case dataset that was prospectively collected by multiple observers in a working clinical environment to test two predictive systems, using logistic regression and the multilayer perceptron (MLP) type of neural network. Ten observed features and the patient age were used as input features. The systems were developed using a training set and test set from the single observer dataset and then applied to the multiple observer dataset. For the independent test cases from the single observer dataset, with a threshold set for no false positives on the training set, logistic regression produced a sensitivity of 82% (95% confidence interval 73-91) and a predictive value of a positive result (PV +) of 98% (95-99), the values for the MLP were 79% (69-89) and 100%, respectively. However the performance on the prospective multiple observer dataset was much worse, with a sensitivity of 72% (65-80), and PV + of 97% (94-99) for logistic regression and 67% (60-75) and 91% (85-97) for the MLP. These results suggest that there is considerable interobserver variability for the defined features and that this system is unsuitable for further development in the clinical environment unless this problem can be overcome.     

Probing the conformation of the prion protein within a single amyloid fibril using a novel immunoconformational assay

The coexistence of multiple strains or subtypes of the disease-related isoform of prion protein (PrP) in natural isolates, together with the observed conformational heterogeneity of PrP amyloid fibrils generated in vitro, indicates the importance of probing the conformation of single particles within heterogeneous samples. Using an array of PrP-specific antibodies, we report the development of a novel immunoconformational assay. Uniquely, application of this new technology allows the conformation of multimeric PrP within a single fibril or particle to be probed without pretreatment of the sample with proteinase K. Using amyloid fibrils prepared from full-length recombinant PrP, we demonstrated the utility of this assay to define (i) PrP regions that are surface-exposed or buried, (ii) the susceptibility of defined PrP regions to GdnHCl-induced denaturation, and (iii) the conformational heterogeneity of PrP fibrils as measured for either the entire fibrillar population or for individual fibrils. Specifically, PrP regions 159-174 and 224-230 were shown to be buried and were the most resistant to denaturation. The 132-156 segment of PrP was found to be cryptic under native conditions and solvent-exposed under partially denaturing conditions, whereas the region 95-105 was solvent-accessible regardless of the solvent conditions. Remarkably, a subfraction of fibrils showed immunoreactivity to PrPSc-specific antibodies designated as IgGs 89-112 and 136-158. The immunoreactivity of the conformational epitopes was reduced upon exposure to partially denaturing conditions. Unexpectedly, PrPSc -specific antibodies revealed conformational polymorphisms even within individual fibrils. Our studies provide valuable new insight into fibrillar substructure and offer a new tool for probing the conformation of single PrP fibrils.     

Arachidonyl dopamine as a ligand for the vanilloid receptor VR1 of the rat

The vanilloid receptor VR1 is a nonspecific Ca(2+) channel, expressed in sensory neurons in the peripheral nervous system and in various brain regions, which is believed to be an important molecular integrator of several chemical (acid, vanilloids) and physical stimuli (heat) that cause pain. Recently, several endogenous ligands for VR1 have been identified such as arachidonyl ethanolamide (anandamide) and the more potent arachidonyl dopamine (AA-DO). Here, we further characterize AA-DO as a ligand for rat VR1, heterologously expressed in CHO and HEK293 cells. AA-DO inhibited the binding of [3H]RTX to VR1 with a K(d) value of 5.49 +/- 0.68 microM and with positive cooperativity (p = 1.89 +/- 0.27), indicating that AA-DO was about 5-fold more potent than anandamide in this system. The K(d) (9.7 +/- 3.3 microM), and p values (1.54 +/- 0.04) for the binding of AA-DO to spinal cord membranes are in good correlation with the CHO-VR1 data. AA-DO stimulated 45Ca(2+) uptake on CHO-VR1 and HEK-VR1 cells with EC(50) values of 4.76 +/- 1.43 and 7.17 +/- 1.64 microM and Hill coefficients of 1.28 +/- 0.11 and 1.13 +/- 0.13, respectively, consistent with the binding measurements. In contrast to anandamide, AA-DO induced virtually the same level of 45Ca(2+) uptake as did capsaicin (90 +/- 6.6% in the CHO cells expressing VR1 and 89.3 +/- 9.4% in HEK293 cells expressing VR1). In a time dependent fashion following activation, AA-DO partially desensitized VR1 both in 45Ca(2+) uptake assays (IC(50) = 3.24 +/- 0.84 microM, inhibition is 68.5 +/- 6.85%) as well as in Ca(2+) imaging experiments (35.8 +/- 5.1% inhibition) using the CHO-VR1 system. The extent of desensitization was similar to that caused by capsaicin itself. We conclude that AA-DO is a full agonist for VR1 with a potency in the low micromolar range and is able to significantly desensitize the cells in a time and dose dependent manner.     

A highly efficient and convergent synthesis of a hexasaccharide, a dimer of the repeating unit of the antigen O2 polysaccharide of Stenotrophomonas maltophilia.

A highly efficient and convergent synthesis of a hexasaccharide, which is a dimer of the repeating unit of the antigen O2 polysaccharide of Stenotrophomonas maltophilia, was achieved via coupling of 2,3,4-tri-O-acetyl-alpha-L-xylopyranosyl bromide with the tetrasaccharide, allyl 4-O-{3-O-[4-O-(3,4-di-O-benzoyl-alpha-L-rhamnopyranosyl)-2,3,6-tri-O-ben zoyl -alpha-D-mannopyranosyl]-4-benzoyl-alpha-L-rhamnopyranosyl}-2,3,6-tri-O- benzoyl-alpha-D-mannopyranoside (18) by the Koenigs-Knorr method followed by deacylation. Compound 18 was readily prepared from the coupling of the disaccharide trichloroacetimidate, 4-O-(2-O-acetyl-3,4-di-O-benzoyl-alpha-L-rhamnopyranosyl)-2,3,6-tri-O- benzoyl-alpha-D-mannopyranosyl trichloroacetimidate (8) with the disaccharide acceptor, allyl 4-O-(2-O-acetyl-4-O-benzoyl-alpha-L-rhamnopyranosyl)-2,3,6-tri-O-benzoyl - alpha-D-mannopyranoside (16), and both 8 and 16 were prepared via the trichloroacetimidate method from simple starting materials. The sole use of acyl protecting groups substantially simplified protection and deprotection, and the allyl group at the reducing end of allyl 4-O-{2-O-[2,3,4-tri-O-acetyl-beta-L-xylopyranosyl]-3-O-[4-O-(2-O-(2,3,4- tri-O-acetyl-beta-L-xylopyranosyl)-3,4-di-O-benzoyl-alpha-L-rhamnopyrano syl) -2,3,6-tri-O-benzoyl-alpha-D-mannopyranosyl]-4-O-benzoyl-alpha- L-rhamnopyranosyl}-2,3,6-tri-O-benzoyl-alpha-D-mannopyranoside 19 allowed further chemical transformation.     

Expression and characterization of recombinant human factor V and a mutant lacking a major portion of the connecting region

Human coagulation factor V is a protein cofactor that is an essential component of the prothrombinase complex. A full-length factor V cDNA has been subcloned into the mammalian expression vector pDX and used to transfect COS cells. Approximately 95 +/- 4% of the recombinant human factor V (rHFV) synthesized in COS cells is secreted into the culture medium. Forty-eight hours after transfection rHFV antigen levels in the conditioned medium were 70 +/- 15 ng/mL. Factor V activity determined by fibrometer assay increased approximately 5-fold from 0.027 +/- 0.012 to 0.124 +/- 0.044 unit/mL following activation by the factor V activating enzyme from Russell's viper venom (RVV-V). A chromogenic assay specific for factor Va indicated that recombinant factor V had 3.8 +/- 1.3% of the activity of the activated protein. The estimated specific activity of the recombinant factor Va was approximately 1800 +/- 500 units/mg, which is similar to the specific activity of purified plasma factor Va of 1700-2000 units/mg. Immunoprecipitation of [35S]methionine-labeled rHFV revealed a single high molecular mass component (approximately 330 kDa). Treatment of rHFV with thrombin or RVV-V resulted in the formation of proteolytic products that were similar to those seen with plasma factor V. We have also expressed a mutant, rHFV-des-B811-1441, that lacks a large portion of the highly glycosylated connecting region that is present in factor V. Immunoprecipitation of [35S]methionine-labeled rHFV-des-B811-1441 revealed a single-chain polypeptide with Mr approximately 230 kDa. This mutant constitutively expressed 38 +/- 7% of the activity of the RVV-V-activated protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Properties of the cell envelope and a cell-envelope protein of Pseudomonas facilis

The molecular weight of the protein moiety of a phospholipoprotein complex isolated from Pseudomonas facilis has been examined with a variety of sodium dodecylsulfate-polyacrylamide gel electrophoretic systems. A molecular weight of 35 000 was determined for the protein in all analyses. A 35 000-dalton protein was present in the EDTA extract of P. facilis and in the cytoplasmic and outer membrane fractions, but not in the lipopolysaccharide and peptidoglycan. Prior inoculation of mice with the phospholipoprotein complex led to a 7.5- to 15-fold increase in the LD₅₀ when mice were subsequently inoculated with Salmonella typhimurium; this pathogen has a cell-surface protein which cross-reacts immunologically with antibody to the P. facilis phospholipoprotein complex.Abbreviations KDO 2-keto-3-deoxyoctanoate - LD₅₀ the dosage of Salmonella typhimurium at which there is 50% survival in mice - LPS lipopolysaccharide - PLP phospholipoprotein - P_PLP the protein moiety of PLP - SDS sodium dodecylsulfate - SDS-PAGE sodium dodecylsulfate-polyacrylamide gel electrophoresis - TMS trimethylsilyl