Isolation of two forms of basement membrane proteoglycans

Sequential extractions of the basement membrane producing Engelbreth-Holm-Swarm tumor yielded heparan sulfate proteoglycans with different size core proteins, but the same size heparan sulfate side chains. Saline, a nondenaturing solvent, extracted a small high density proteoglycan with a heterodisperse core protein of Mr = 95,000-130,000 whereas subsequent extraction with 7 M urea, a denaturing solvent, removed a large, low density proteoglycan with a Mr = 350,000-400,000 protein core. The denaturing conditions required for extraction of the large proteoglycan suggest that it interacts strongly with other basement membrane components. Antibodies to these proteoglycans cross-react with both proteoglycans, but the large proteoglycan has additional antigenic sites not present on the small proteoglycan. These proteoglycans may be derived from the same or similar gene products.     

Diurnal rhythm of the total antioxidant activity of saliva of children with chronic viral hepatitis

The diurinal rhythms of a total antioxidant activity of a saliva were investigated depending on activity of the inflammatory process in a liver of children with chronic virus hepatitis HBV and HCV. The circadian rhythms were found desynchronized, which was showed in lowering of the mesor value, oscillations amplitude, and a rhythm inversion and transformation, as an additional biorhythmological criterion of the presence of the chronic hepatitis of a viral etiology. A tendency of a circadian rhythm to remain with a daily acrophasis but with a law level of amplitude oscillations and a mesor was to be observed after the treatment of children with inactive HBV. By an active HBV a circadian rhythm with it's inversion has been renewed. It was determined the remaining of an ultradian rhythm with increasing level of amplitude oscillations after the treatment of inactive HCV. The law amplitude oscillations and a insignificant increasing of a meror level were characteristic for the circadian rhythm of total AOA of the children with active HBV. It was proved the possibility of usage of the biorhythmological parameters of diurinal rhythms of total AOA as a criteria of efficiency of the conducted therapy for the given group with disease children.     

Analysis of the molecular interaction of the farnesyl moiety of transducin through the use of a photoreactive farnesyl analogue

Farnesylation of the gamma-subunit of the retinal G-protein, transducin (Talpha/Tbetagamma), is indispensable for light-initiated signaling in photoreceptor cells. However, the farnesyl-mediated molecular interactions important for signaling are not well understood. To explore this issue, we created a functional Tbetagamma analogue in which the farnesyl group was replaced with a (3-azidophenoxy)geranyl (POG) group, a novel farnesyl analogue with a distal photoreactive azido group. In the presence of lipid membranes and/or Talpha-GDP, UV irradiation of POG-modified Tbetagamma (POG-Tbetagamma) invariably yielded a cross-linked product Tgamma-Tbeta, reflecting a constitutive interaction of the Tgamma C-terminal lipid with Tbeta. In addition to the Tgamma-Tbeta adduct, a Tgamma-Talpha cross-link was detected in the aqueous fraction. Reconstitution of POG-Tbetagamma with Talpha and light-activated rhodopsin (Rh) in photoreceptor membranes resulted in cross-linking of Tgamma with a glycerophospholipid, indicating molecular interaction of the farnesyl group with cellular membranes. The Tgamma-phospholipid cross-link was observed only in the presence of both Talpha-GDP and Rh, and was abolished by the addition of GTPgammaS or by replacing Rh with opsin. These findings suggest a transient farnesyl-membrane interaction occurs only in a signaling state formed in a transducin-Rh ternary complex. On the other hand, UV irradiation of POG-Tbetagamma in a soluble complex with phosducin, a negative regulator of G-protein, yielded a Tgamma-phosducin adduct in addition to the Tgamma-Tbeta cross-link. These results illustrate that, rather than being a static membrane anchor, the farnesyl moiety plays an active role in the dynamics of protein-protein and protein-membrane interactions at defined steps in the signal transduction process.     

Kinetics of lipid-membrane binding and conformational change of L-BABP

We designed an experimental approach to differentiate the kinetics of protein binding to a lipid membrane from the kinetics of the associated conformational change in the protein. We measured the fluorescence intensity of the single Trp6 in chicken liver bile acid-binding protein (L-BABP) as a function of time after mixing the protein with lipid membranes. We mixed the protein with pure lipid membranes, with lipid membranes in the presence of a soluble quencher, and with lipid membranes containing a fluorescence quencher attached to the lipid polar head group. We fitted simultaneously the experimental curves to a three-state kinetic model. We conclude that in a first step, the binding of L-BABP to the interfacial region of the anionic lipid polar head groups occurred simultaneously with a conformational change to the partly unfolded state. In a second slower step, Trp6 buried within the polar head group region, releasing contacts with the aqueous phase.     

蚀斑减少中和试验检测抗天花免疫球蛋白效价

为了建立测定抗天花免疫球蛋白效价的检测方法,采用抗天花免疫球蛋白50%中和100PFU痘苗病毒的稀释度来确定其效价。通过与NIBSC提供的抗天花人免疫球蛋白标准品进行的对比试验,结果显示基本一致,证明该方法是可行的。     

Thrombolytic activity of Bacillus mesentericus at different conditions of nitrogen nutrition of a culture

The work is concerned with studying the effect exerted by different sources of nitrogen nutrition on the biosynthesis of proteinases with a thrombolytic activity by a variant of Bacillus mesentericus, strain 64, obtained with the aid of analytical selection. Protein substrates taken as a nitrogen source stimulate the synthesis of proteinases by the bacterial culture. These enzymes have a high caseinolytic and thrombolytic activity, and the level of their activity correlates with the amount of a protein substrate added to the medium. Ammonium acetate and succinate are the best stimulants for the formation of proteinases when the salts of mineral and organic acids are used as a source of nitrogen nutrition. In that case, the enzymes have a high thrombolytic activity and a low caseinolytic activity. A semi-synthetic medium with the aforementioned nitrogen-containing compounds as a source of nitrogen nutrition is proposed for the synthesis of thrombolytic proteinase by the variant of B. mesentericus.     

Fluorescence spectroscopy of single tryptophan mutants of apolipophorin-III in discoidal lipoproteins of dimyristoylphosphatidylcholine

The structure of the exchangeable apolipoprotein, apolipophorin-III from Locusta migratoria, apoLp-III, is described as a bundle of five amphipathic alpha-helices. To study the interaction of each of the helices of apoLp-III with a lipid surface, we designed five single-Trp mutants, each containing a Trp residue in a different alpha-helix. The Trp residues were located in the nonpolar domains of the amphipathic alpha-helices. The kinetics of the spontaneous interaction of the mutants with dimyristoylphosphatidylcholine (DMPC) indicated that all mutants behaved as typical exchangeable apolipoproteins. Circular dichroism in the far-UV indicated that all proteins have a high and similar helical content in the lipid-bound state. The interaction of the Trp residues with the lipid surface was investigated in recombinant lipoprotein particles made with DMPC. The properties of the Trp residues were investigated by fluorescence spectroscopy. These studies showed major changes in the spectroscopic properties of the Trp residues upon binding to lipid. These changes are observed with all single-Trp mutants, indicating that a major conformational change, which affects the properties of all helices, takes place upon binding to lipid. The position of the fluorescence maximum, the quenching efficiency of acrylamide as determined by steady-state and time-resolved fluorescence, and the fluorescence lifetimes of the single-Trp mutants suggest that helices 1, 4, and 5 interact with the nonpolar domains of the lipid. The properties of the Trp in helices 2 and 3 suggest that these helices adopt a different binding configuration than helices 1, 4, and 5. Helices 2 and 3 appear to be interacting with the polar headgroups of the phospholipids or constitute a different domain that does not interact with the lipid surface.     

Role of the kidney in the metabolism of apolipoprotein A-IV: influence of the type of proteinuria

Increased plasma concentrations of apolipoprotein A-IV (apoA-IV) in chronic renal disease suggest a metabolic role of the kidney for this antiatherogenic protein. Therefore, we investigated patients with various forms of proteinuria and found increased serum concentrations of apoA-IV in 124 nephrotic patients compared with 274 controls (mean 21.9 +/- 9.6 vs. 14.4 +/- 4.0 mg/dl; P < 0.001). Decreasing creatinine clearance showed a strong association with increasing apoA-IV levels. However, serum albumin levels significantly modulated apoA-IV levels in patients with low creatinine clearance, resulting in lower levels of apoA-IV in patients with low compared with high albumin levels (21.4 +/- 8.6 vs. 29.2 +/- 8.4 mg/dl; P = 0.0007). Furthermore, we investigated urinary apoA-IV levels in an additional 66 patients with a wide variety of proteinuria and 30 controls. Especially patients with a tubular type of proteinuria had significantly higher amounts of apoA-IV in urine than those with a pure glomerular type of proteinuria and controls (median 45, 14, and 0.6 ng/mg creatinine, respectively). We confirmed these results in affected members of a family with Dent's disease, who are characterized by an inherited protein reabsorption defect of the proximal tubular system. In summary, our data demonstrate that the increase of apoA-IV caused by renal impairment is significantly modulated by low levels of serum albumin as a measure for the severity of the nephrotic syndrome. From this investigation of apoA-IV in urine as well as earlier immunohistochemical studies, we conclude that apoA-IV is filtered through the normal glomerulus and is subsequently reabsorbed mainly by proximal tubular cells.     

Effect of hydrocortisone and methods of infection on the isolation of Brucella from the body of albino mice

It was experimentally revealed that subcutaneous method of albino mice infection in combination with hydrocortisone administration offered a possibility of decreasing the period of discharge of brucella to 7 days with a 100% culturing from the inguinal, paraaortal lymph nodes and the spleen. In intracerebral method of infection with a preliminary treatment with hydrocortisone the period of the culture isolation decreased to 14 days.     

Induction by nucleotide triphosphate hydrolysis of a form of sarcoplasmic reticulum ATPase capable of medium phosphate-oxygen exchange in presence of calcium.

Sarcoplasmic reticulum vesicles rendered leaky by exposure to alkaline pH, like intact vesicles, catalyze a rapid Mg2+-dependent exchange of oxygens of medium Pi with water. The exchange with 10 mM Pi is strongly inhibited by 0.15 mM Ca2+. Upon addition and hydrolysis of ITP or ATP, a rapid phosphate-oxygen exchange is observed even with 0.15 mM Ca2+ present and a definite but smaller exchange at 8 mM Ca2+. Oxygen exchange per Pi formed is greater with ITP than with ATP. When no Pi is initially present, the extent of oxygen exchange is increased with time of incubation as Pi is formed. With 18O-labeled Pi present, ATP hydrolysis accelerates 18O loss. The results show that much of the oxygen exchange occurs as a result of reversible binding of medium Pi. Thus the binding and cleavage of ITP or ATP overcomes the Ca2+ inhibition of the medium Pi in equilibrium HOH exchange. Such findings support the concept that the cleavage cycle includes a transient conformational form which can reversibly react with Pi to give a phosphoryl enzyme and resultant oxygen exchange or in a rate-limiting step decay to a form with high Ca2+ and NTP affinity.     

Effect of emotional tension on the spatial-temporal organization of prestimulus potentials of the neocortex of a human operator on the recognition of a meaningful photic stimulus

Analysis of psychometric data at recognition of digital stimuli in a state of high emotional stress allowed to single out two groups of subjects--with improved and with worsened quality of activity. It is shown that the structure of intrahemispheric cross-correlation coefficients before states with a short time of recognition, a long time and with nonrecognition of the stimulus, appearing in operative quietness, is preserved in emotional stress for subjects with a better quality of activity and changes for half of the pairs of intrahemispheric leads in subjects with worsened quality of activity.     

The reaction of nitrogen monoxide and of nitrite with deoxyhaemocyanin and methaemocyanin of Helix pomatia.

Deoxyhaemocyanin, treated with NO under strictly anaerobic conditions, yielded methaemocyanin and N2O in a fast reaction. In a further slow reaction this methaemocyanin lost its triplet electron paramagnetic resonance (EPR) signal at g = 4 and yielded a nitrosyl derivative with a characteristic g = 2 Cu(II) EPR signal, indicating the binding of a single NO per copper pair. Thus under strictly anaerobic conditions deoxyhaemocyanin and methaemocyanin, treated with NO, gave the same derivative as shown by circular dichroism and EPR spectra. Methaemocyanin yielded, moreover, reversibly a nitrite derivative, characterized by a triplet signal at g = 4 with 7 hyperfine lines.     

Influence of BLK polymorphisms on the risk of rheumatoid arthritis

B cell lymphocyte kinase (BLK) encodes a member of the Src kinase family and thus may influence the proliferation and differentiation of cells. A single nucleotide polymorphism (SNP) located in the first intron of BLK has shown that the risk C allele of rs2248932 is associated with lower levels of messenger RNA expression of BLK. We hypothesized that this polymorphism may contribute to rheumatoid arthritis (RA) susceptibility. We studied BLK rs2248932 T/C gene polymorphisms in 329 patients with RA and 697 controls in a Chinese population. Genotyping was done using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). When the BLK rs2248932 TT homozygote genotype was used as the reference group, the CC genotype was associated with a significantly increased risk of RA. In the recessive model, when the BLK rs2248932 TT/TC genotypes were used as the reference group, the CC homozygote genotype was associated with a significantly increased susceptibility to RA. In stratification analyses, a significantly increased risk for RA associated with the BLK rs2248932 CC genotype was evident among younger patients, CRP-negative patients and anti-CCP-positive patients compared with the BLK rs2248932 TT/TC genotype. The risk was also significantly evident among RF-positive patients, patients with lower ESR levels, patients with lower or higher DAS28 score and patients with a lower functional class. These findings suggested that the functional SNP BLK rs2248932 T/C variant allele was associated with RA development. However, our results were obtained from a moderate-sized sample, and therefore this is a preliminary conclusion. Validation in a larger study from a more diverse ethnic population is needed to confirm these findings.     

Influence of genotype-dependent effects of covariates on the outcome of segregation analysis of the body mass index.

Several recent studies of the body mass index (BMI) have provided support for a recessive major gene influencing heaviness in humans. Segregation analysis of the BMI was carried out recently in a series of randomly sampled French-Canadian families to determine whether we could replicate the major gene finding by using a residual phenotype adjusted for the effects of age and sex. The best model included a recessive major effect for high BMI values with residual familial resemblance; however, Mendelian transmission could not be confirmed, and the no-transmission hypothesis (where all the tau's are constrained to be equal) was not rejected. Considering that the BMI is a complex phenotype affected by many factors and that there are known variations in body composition during growth and aging, we undertook a reanalysis of the data, using a model that allowed the estimation of genotype-specific age and gender effects. New tests on the transmission parameters satisfy the criteria for interfering Mendelian segregation. The results suggest that individuals with the "high" recessive genotype show the greatest degree of heaviness at birth, with a subsequent trend toward lower values throughout life, while individuals with the dominant "normal" genotypes show no appreciable trends with age. In addition, the "high" genotype appears to confer a greater degree of heaviness in females as compared with males. These results, along with other observations from the data, suggest that, while a recessive single gene influence may be discernible, the phenotypic expression of the BMI is likely to be complicated by genotype x environment interactions and, possibly, by the action of other loci. Further, the data also are consistent with the hypothesis that modifying factors may include the adoption of a more prudent life-style by individuals genetically predisposed to heaviness and a secular increase in the incidence, prevalence, and potency of environmentally based triggers leading to a higher penetrance of the "heavy" genotype in the young.     

On the mechanism of non-endosomial peptide-mediated cellular delivery of nucleic acids

Recently, we described a new strategy for the delivery of nucleic acids into mammalian cells, based on an amphipathic peptide of 27 residues called MPG, which was designed on the basis of a hydrophobic domain derived from a fusion sequence associated with a nuclear localization sequence and separated by a linker. This peptide carrier constitutes a powerful tool for the delivery of nucleic acids in cultured cells, without requiring any covalent coupling. We have examined the conformational states of MPG in its free form and complexed with a cargo, as well as its ability to interact with phospholipids, and have investigated the structural consequences of these interactions. In spite of its similarity to the similarly designed cell-penetrating peptide Pep-1, MPG behaves significantly differently from the conformational point of view. Circular dichroism (CD) analysis reveals a transition from a nonstructured to a beta-sheet conformation upon interaction with phospholipids. We propose that the membrane crossing process involves formation of a transient transmembrane pore-like structure. Partial conformational change of MPG is associated with formation of a complex with its cargo, and an increase in sheet content occurs upon association with the cell membrane.     

Epidemiological evaluation of the use of genetics to improve the predictive value of disease risk factors.

The prevention of common diseases relies on identifying risk factors and implementing intervention in high-risk groups. Nevertheless, most known risk factors have low positive predictive value (PPV) and low population-attributable fraction (PAF) for diseases (e.g., cholesterol and coronary heart disease). With advancing genetic technology, it will be possible to refine the risk-factor approach to target intervention to individuals with risk factors who also carry disease-susceptibility allele(s). We provide an epidemiological approach to assess the impact of genetic testing on the PPV and PAF associated with risk factors. Under plausible models of interaction between a risk factor and a genotype, we derive values of PPV and PAF associated with the joint effects of a risk factor and a genotype. The use of genetic testing can markedly increase the PPV of a risk factor. PPV increases with increasing genotype-risk factor interaction and increasing marginal relative risk associated with the factor, but it is inversely proportional to the prevalences of the genotype and the factor. For example, for a disease with lifetime risk of 1%, if all the risk-factor effect is confined to individuals with a susceptible genotype, a risk factor with 10% prevalence and disease relative risk of 2 in the population will have a disease PPV of 1.8%, but it will have a PPV of 91.8% among persons with a genotype of 1% prevalence. On the other hand, genetic testing and restriction of preventive measures to those susceptible may decrease the PAF of the risk factor, especially at low prevalences of the risk factor and genotype.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of protein and mannoprotein antigens of Candida albicans of relevance for the serodiagnosis of invasive candidiasis.

Antigens from Candida albicans blastoconidia and germ tubes were identified by two-dimensional electrophoresis and Western blotting and characterized by microsequencing, reactivity with concanavalin A, and a panel of human sera. Antigens identified included a polydispersed area in the acidic high-molecular-mass regions of blastoconidium and germ-tube extracts, and 16 antigens varying in molecular masses and isoelectric points (pIs). The majority of the detected antigens, especially those in the polydispersed region, showed mannosyl groups, as determined by concanavalin A reactivity. Antibodies present in sera from patients with invasive candidiasis showed high reactivity with a number of antigens not detected with sera from blood donors. Eight of the 16 antigens could be identified by reactivity with monoclonal antibodies or by microsequencing. Five antigens showed homology with five enzymes previously described as antigens in C. albicans: enolase, phosphoglycerate kinase, malate dehydrogenase, and two isoforms of the fructose biphosphate aldolase. However, to our knowledge, this is the first report of the immunogenic activity of a kexin precursor, a mitochondrial complex I chaperone, and a diacylglycerol kinase catalytic domain from C. albicans. Antigens described in this study may be of potential interest for the serodiagnosis of invasive candidiasis.     

On the mechanism of non-endosomial peptide-mediated cellular delivery of nucleic acids

Recently, we described a new strategy for the delivery of nucleic acids into mammalian cells, based on an amphipathic peptide of 27 residues called MPG, which was designed on the basis of a hydrophobic domain derived from a fusion sequence associated with a nuclear localization sequence and separated by a linker. This peptide carrier constitutes a powerful tool for the delivery of nucleic acids in cultured cells, without requiring any covalent coupling. We have examined the conformational states of MPG in its free form and complexed with a cargo, as well as its ability to interact with phospholipids, and have investigated the structural consequences of these interactions. In spite of its similarity to the similarly designed cell-penetrating peptide Pep-1, MPG behaves significantly differently from the conformational point of view. Circular dichroism (CD) analysis reveals a transition from a nonstructured to a β-sheet conformation upon interaction with phospholipids. We propose that the membrane crossing process involves formation of a transient transmembrane pore-like structure. Partial conformational change of MPG is associated with formation of a complex with its cargo, and an increase in sheet content occurs upon association with the cell membrane.     

Kinetics of reduction of the primary donor of photosystem II. Influence of pH in various preparations

The kinetics of reduction of P-680, oxidized by a flash, have been measured with chloroplasts treated with Tris, hydroxylamine or cholate, with PS-II particles from Phormidium (Tris-treated) and with subchloroplast particles prepared with digitonin or Triton. In all cases the electron transfer from D1 to P-680+ has similar rates, influenced similarly by pH and D1 has a one-electron capacity.     

Growth of flagellar filaments of Escherichia coli is independent of filament length

Bacterial flagellar filaments grow at their distal ends, from flagellin that travels through a central channel ～2 nm in diameter. The flagellin is extruded from the cytoplasm by a pump powered by a proton motive force (PMF). We measured filament growth in cells near the mid-exponential-phase with flagellin bearing a specific cysteine-for-serine substitution, allowing filaments to be labeled with sulfhydryl-specific fluorescent dyes. We labeled filaments first with a green maleimide dye and then, following an additional period of growth, with a red maleimide dye. The contour lengths of the green and red segments were measured. The average lengths of red segments (～2.3 μm) were the same regardless of the lengths of the green segments from which they grew (ranging from less than 1 to more than 9 μm in length). Thus, flagellar filaments do not grow at a rate that decreases exponentially with length, as formerly supposed. If flagellar filaments were broken by viscous shear, the broken filaments continued to grow. Identical results were obtained whether flagellin was expressed from fliC on the chromosome under the control of its native promoter or on a plasmid under the control of the arabinose promoter.