Pedersen fetuin contains three adipogenic factors with distinct biochemical characteristics.

Crude Pedersen fetuin, derived from fetal bovine serum, contains adipogenic activity. Biochemical characterization was undertaken by following the differentiation of the 1246 adipogenic cell line. The present paper provides evidence that crude fetuin contains distinct proteins with adipogenic activity. By molecular sieve fractionation using Sephacryl S-300, the majority of adipogenic activity eluted in two distinct peaks, FI (molecular weight greater than 669 kDa) and FII (molecular weight ranging from 445 and 232 kDa). In addition a minor activity was found in a third peak, FIII (molecular weight around 69 kDa). Partial purification and biochemical characterization indicate that FI and FII are two distinct factors. FI has a PI higher than 9.4, is destroyed by alkaline treatment, and is stable when treated with acid. FII has a PI lower than 4.0, is alkali stable, but is destroyed completely by treatment with acid. Moreover, our data show that adipogenic factors are distinct from another protein alpha 2 macroglobulin known to be found in crude Pedersen fetuin. These results suggest that serum contains two large molecular weight proteins bearing adipogenic activity which could play an important role in the control of the adipose differentiation process.     

Cyclic 3':5'-nucleotide phosphodiesterase determined in various human tissues by DEAE-cellulose chromatography.

Tissue extracts from human heart, lung, liver, kidney, skeletal muscle and cerebrum displayed at least 3 distinct cyclic 3':5'-nucleotide phosphodieterase (EC 3.1.4.17) activity peaks (FI, FII, FIII) on DEAE-cellulose chromatography and various properties of these forms were compared in each tissue. FI eluted at about 0.08 M sodium acetate, hydrolyzed cyclic GMP more rapidly than it did cyclic AMP, and cyclic GMP hydrolysis by FI in most tissues was enhanced by a protein activator in the presence of CaCl2. As only high concentrations of cyclic AMP inhibited cyclic GMP hydrolytic activity of FI, the enzyme probably has a low affinity for cyclic AMP. FII eluted at about 0.2 M sodium acetate, hydrolyzed both nucleotides at equal rates, and substrate affinities were relatively low. Cyclic GMP hydrolysis by FII was also stimulated by addition of a protein activator in the presence of CaCl2 and cyclic AMP hydrolysis in this fraction was accelerated by a micromolar fraction of cyclic GMP. FII eluted at about 0.35 M hydrolyzed cyclic AMP preferentially and was insensitive to protein activator. These two cyclic nucleotides act as mutual inhibitors of the hydrolysis in this fraction. Ratio of the cyclic GMP to cyclic AMP hydrolysis was in the order FI, FII, FIII. Four activity peaks were eluted from the cerebral extract and enzymes from this tissue exhibited much the same properties as observed in the other tissues examined herein.     

Acacia senegal gum: continuum of molecular species differing by their protein to sugar ratio, molecular weight, and charges

The main chemical and physical features of the Acacia senegal exudate gum and its molecular fractions isolated by chromatographies were determined using a wide variety of methods. Three main molecular fractions were isolated after hydrophobic interaction chromatography (HIC) and biochemical analyses confirmed the presence of an arabinogalactan-peptide (FI), an arabinogalactan-protein (FII), and a glycoprotein (FIII) fraction as described commonly in the literature. Further purification of FIII using size exclusion chromatography revealed three distinct populations. A wide molecular weight distribution within each population with the presence of at least two distinct molecular species per population was identified by high performance size exclusion chromatography coupled to on line multi-angle laser light scattering (HPSEC-MALLS). In addition, both sugars content (neutral and uronic acids) and UV profiles revealed that FIII was composed of a continuum of molecular species differing both by their protein-to-sugar ratio and molecular weight. FI and FII had average molecular weight M(w) of 2.86 x 10(5) and 1.86 x 10(6) g.mol(-1), respectively, and a low polydispersity index (M(w)()/M(n) approximately 1.3). The three populations identified in FIII after HIC separation had M(w) of 2.67 x 10(6), 7.76 x 10(5), and 2.95 x 10(5) g.mol(-1) and very low polydispersity indexes (1.13, 1.04, and 1.01). Estimation of the polypeptide backbone length in the three fractions gave 43, 2253, and 4443 amino acid residues, respectively, hydroxyproline (Hyp) and serine being the most prominent residues within FI and FII, Hyp and Asx (asparagine + aspartic acid) within FIII. Secondary structure prediction from circular dichroism data resulted in polyproline II, beta-sheet, and random coil structures for FII and FIII, whereas no secondary structure was identified in FI. The existence of exposed tryptophanyl residues to the solvent was noticed by fluorescence in FII and FIII, tryptophan residues being absent from FI. In addition, 8-5' non cyclic diferulic acid was identified to be covalently linked to carbohydrate moieties of FII. Infrared spectroscopy identified the different vibrations of saccharidic and peptidic bonds with absorbance amplitudes in agreement with sugar and protein elementary analyses. Titration measurements in order to evaluate the number of charges on total Acacia gum and its molecular fractions revealed that 100% of charges came from polysaccharidic moieties (i.e., glucuronic acids) in FI. Charges coming from polysaccharidic moieties were of 91.3% and 37.9% for FII and FIII, respectively, the remaining 8.7% and 62.1% charges in FII and FIII molecular fractions coming from the polypeptidic backbone.     

Effect of Candida albicans cell wall components on the adhesion of the fungus to human and murine vaginal mucosa

In this study, cell walls from Candida albicans were separated and chitin was isolated from these cell walls. A chitin soluble extract (CSE) prepared from the chitin inhibited in vitro adhesion of C. albicans to human epithelial vaginal cells (VEC), and blocked in vivo attachment to murine vaginal mucosa, thereby preventing candidal infection in these animals. These findings suggest that the CSE acts as an adhesin-like substance.Fractionation of CSE yielded two fractions: FI and FII, of which only FI exhibited inhibitory activity. Chemical analysis of CSE and its two fractions revealed that CSE contains over 70% of proteins, most of which were found in the non-active fraction. In addition, 3% of amino-sugars were found in the FI active fraction. Lipids were also detected in the unfractionated CSE and in both fractions.Experiments to further characterize the component(s) in the CSE inhibiting the attachment of C. albicans are in progress in our laboratory.     

Purification and Characterization of Chlorophyllase from Alga (Phaeodactylum tricornutum) by Preparative Isoelectric Focusing

Chlorophyllase from a diatom alga (Phaeodactylum tricornutum) was obtained and the partially purified extract has been further purified using preparative isoelectric focusing on a Rotofor cell. Three fractions, FI, FII, and FIII, were separated from the Rotofor cell and salt and ampholytes were removed to give fractions FI′, FII′, and FIII′, respectively. Enzyme fractions FI′, FII′, and FIII′, respectively. Enzyme fractions FI′, FII′, and FIII′ showed specific activities of 15.2 × 10^?4, 226.7 ×10^?4 and 33.8 × 10^?4 µmol/mg protein/min, respectively. Most of the enzyme activity (84%) was in fraction FII′. The optimum pH for chlorophyllase activity was 8.0 for FI′ and 8.5 for both FII′ and FIII′. Apparent K_m values for enzyme fractions FI′, FII′, and FIII′ were 2.1nM, 2.3nM, and 2.0 nM, respectively. Enzyme fractions FII′ and FIII′ showed higher chlorophyllase activity towards the partially purified chlorophyll when it was compared to that with the crude chlorophyll as well as with both chlorophylls a and b. However, the enzyme fraction FI′ had higher activity towards the crude chlorophyll when it was compared to that with both chlorophylls a and b, but with a preference for chlorophyll a over chlorophyll b. The inhibitory effect of diisopropyl flurophosphate (DIFP) on chlorophyllase activity demonstrates a noncompetitive inhibitor kinetics with K_i values of 1.29mM, 2.14mM, and 0.71mM for FI′. FII′, and FIII′, respectively.     

Characterization of gastric mucosal membranes. X. Immunological studies of gastric (H+ + K+)-ATPase

Gastric mucosal homogenates from hog were fractionated by differential and density gradient centrifugation and free-flow electrophoresis. The two major membrane fractions (FI and FII) thus obtained are distinct both enzymically and in terms of transport reactivity. This heterogenicity extends to their antigenic activity. Purified antibodies which were raised against the K+-ATPase-containing H+ transport fraction FI were of two types: inhibitory and non-inhibitory. Inhibitory antibodies reduced the K+-ATPase activity by approximately 80% and the K+-p-nitro-phenylphosphatase activity by approximately 40% in a concentration-dependent manner, while the small Mg++-dependent component of the enzyme activity was unaffected. Antibodies inhibiting the K+-ATPase also inhibited H+ transport. These antibodies did not cross-react with the other major membrane fraction isolated by free- flow electrophoresis, FII, and gave a single band on rocket immunoelectrophoresis. Antibodies against this FII fraction also did not react with the K+-ATPase and were heterogeneous, giving at least four bands with rocket immunoelectrophoresis and inhibiting both the 5'- nucleotidase and Mg++-ATPase of this fraction. Immunofluorescent staining of tissue sections showed that the FI was derived from the parietal cell of gastric tissue and was localized to the supranuclear area of the cell. Staining of isolated rat gastric cell suspensions by FI antibodies confirmed the selectivity of the antibody and showed a polar, plasma membrane localization. FII antibodies also largely stained the parietal cells in tissue sections. In the 16 hog tissues tested, FI antibodies cross-reacted only with gastric fundus, thyroid and weakly with thymus. Immunoelectronmicroscopy showed that FI antibodies reacted strongly with the secretory membrane at the apical cell surface of the parietal cells and at the secretory canaliculi, weakly with the apical surface of the zymogen cell, and not with the basal-lateral surface of the cells. Thus, the protontranslocating ATPase is localized in the parietal cells and in the region postulated to be the site of acid secretion.     

An antigen capture assay for the measurement of serum clusterin concentrations

We have developed and validated a robust antigen capture assay for the measurement of serum clusterin. Increased clusterin expression, and alterations in serum clusterin levels have been associated with a number of disease states. In particular, clusterin has been shown to be associated with tissue regression and apoptosis in the rat ventral prostate in response to androgen ablation or administration of anti-androgens. The object of this study was to determine if changes in human serum clusterin can be used as a diagnostic or prognostic marker to monitor the response to hormonal therapy in patients with prostate cancer, and to determine if clusterin concentrations increase with the progression towards androgen independence. The antigen capture assay was used for an extensive analysis of human serum clusterin concentration in fasting males, and to determine if there is any relationship between clusterin and age or cholesterol levels. The average clusterin level in serum is 101+/-42 microg/ml (n=96). There is no correlation to age or serum cholesterol levels. Analysis of serum clusterin levels in patients with newly diagnosed prostate cancer (n=5), hormone responsive tumors (n=5), and hormone refractory disease (n=5), demonstrates that no significant changes in serum clusterin levels accompany the progression of prostatic disease, or response to hormone therapy.     

Clusterin interacts with SCLIP (SCG10-like protein) and promotes neurite outgrowth of PC12 cells

Clusterin has been known as a chaperone-like molecule capable of interacting with various proteins. In this study, we show that clusterin interacts with the microtubule-destabilizing stathmin family protein SCLIP by GST pull-down and co-immunoprecipitation assays. Interestingly, SCLIP interacts with 80 kDa mature form of clusterin in the cytosolic fraction of PC12 cells permeabilized by low concentration of a weak nonionic detergent digitonin, but not with intracellular variants of clusterin known as binding isoforms of Ku70 or TGF-beta receptors. Both clusterin and SCLIP are co-localized at the perinuclear region and growth cone of PC12 cells. In addition, we show that the minimal domains for the interaction are mapped to the C-terminal valine-rich region (367-447) of clusterin and the N-terminal palmitoylation and membrane attachment site (1-34) of SCLIP. Finally, we demonstrate that ectopic expression of clusterin in PC12 cells elongates neurite-formation triggered by NGF and induces spontaneous neurite outgrowth even in the absence of NGF. Taken together, these results suggest that the clusterin interacts with SCLIP and the interaction may act as an important modulator during neuronal differentiation.     

Structural analysis of clusterin and its subunits in ram rete testis fluid

Clusterin is a protein present in the rete testis fluid of the ram that elicits aggregation of erythrocytes and Sertoli cells in vitro. In view of its possible biologic function in relation to cell-cell interaction in the testis, we isolated this protein from ram rete testis fluid using sequential high-performance liquid chromatography columns and performed a detailed physicochemical characterization. This protein consists of two molecular variants designated form I and form II clusterin. Each form of clusterin consists of two subunits with an apparent molecular weight of 40,000. It is of note that the two subunits have no homology in their N-terminal amino acid sequences. However, the N-terminal amino acid pairs of the two subunits derived for the two forms of clusterin are identical. Using o-phthalaldehyde to block the Lys residue at the fourth amino acid pair from the N-terminus which leaves the Pro residue free for subsequent Edman degradation, we have deduced the N-terminal sequence of each of the two subunits for form I clusterin. Comparison of the NH2-terminal sequences of the two subunits of clusterin with the release 10.0 of the protein sequence data base of the Protein Identification Resource indicated no homology between either of the subunits of clusterin and any of the known proteins in the data base. A highly specific radioimmunoassay developed for clusterin was used to measure its concentrations in the fluids of the rete testis and cauda epididymis. Since a significant amount of immunoreactive clusterin was found in serum, the protein was partially purified from this source by immunoaffinity chromatography. Immunoreactive serum clusterin was smaller than the testicular clusterin (Mr 37,000 vs 40,000), but both proteins share common epitopes as demonstrated by radioimmunoassay and immunoblots. However, serum clusterin does not possess the biologic activity of the testicular clusterin in that it does not elicit cell aggregation in vitro. It is of note that deglycosylation of testicular clusterin can also eliminate this in vitro biologic activity, suggesting that the serum clusterin might be a deglycosylated form of the testicular protein and the carbohydrate core plays an important role in determining the cell aggregation activity. Studies on the distribution of this protein in the reproductive compartment indicate that it is highly concentrated in the rete testis and the cauda epididymal fluids. This suggests that this protein might have some important functions in the reproductive tract.     

Separation of Antioxidant-Rich Alternanthera Sessilis Red Extracts by Sephadex LH-20 and Identification of Polyphenols Using HPLC-QToF-MS/MS

This study aimed to fractionate Alternanthera sessilis Red (ASR) crude extracts and determine their antioxidant activities as well as the related active components in the whole plant. ASR was extracted with water and ethanol, and further separated using a Sephadex LH-20 column. Following the assessments of the polyphenolic contents and antioxidant activities of crude extracts (H₂O_ASR and EtOH_ASR) and fractions, a HPLC-QToF analysis was performed on the crude extracts and selected fractions (H₂O_ASR FII and EtOH_ASR FII). Three water fractions (H₂O_ASR FI, FII and FIII) and four ethanolic fractions (EtOH_ASR FI, FII, FIII and FIV) were derived from their crude extracts, respectively. EtOH_ASR FII exhibited the greatest total phenolic content (120.41 mg GAE/g fraction), total flavonoid content (223.07 mg RE/g fraction), and antioxidant activities (DPPH IC₅₀=159.43 μg/mL; FRAP=1.93 mmol Fe²⁺/g fraction; TEAC=0.90 mmol TE/g fraction). Correlation analysis showed significant (p<0.01) positive correlations between both TPC (r=0.748–0.970) and TFC (r=0.686–0.949) with antioxidant activities in the crude extracts and fractions. Flavonoids were the major compounds in the four selected samples tentatively identified using HPLC-QToF-MS/MS, with the highest number of 30 polyphenol compounds detected in the most active fraction, EtOH_ASR FII.     

Optimization of conditions for protease production by Chryseobacterium taeanense TKU001

A protease-producing bacterium was isolated and identified as Chryseobacterium taeanense TKU001. An extracellular metalloprotease with novel properties of solvent- and surfactant-stable was purified from the culture supernatant of C. taeanense TKU001 with shrimp shell wastes as the sole carbon/nitrogen source. The optimized condition for protease production was found when the culture was shaken at 37 degrees C for 3 days in 50 mL of medium containing 0.5% shrimp shell powder (SSP) (w/v), 0.1% K2HPO4, and 0.05% MgSO4.7H2O. Two extracellular proteases (FI and FII) were purified and characterized, and their molecular weights, pH and thermal stabilities were determined. The molecular masses of TKU001 protease FI and FII determined by SDS-PAGE and gel filtration were approximately 41 kDa and 75 kDa, respectively. The optimum pH, optimum temperature, pH stability, and thermal stability of TKU001 protease FI were 8, 60 degrees C, pH 6-9, and 60 degrees C, respectively. The optimum pH, optimum temperature, pH stability, and thermal stability of TKU001 protease FII were 7, 60 degrees C, pH 7-9, and 50 degrees C, respectively. TKU001 protease FI and FII were both inhibited completely by EDTA, indicating that the TKU001 protease FI and FII were metalloproteases. TKU001 protease FI and FII retained more than 75% of its original protease activity after preincubation for 10 days at 4 degrees C in the presence of 25% most tested organic solvents. Additionally, the TKU001 protease FI retained 79%, 80%, and 110% of its original activity in the presence of 2% Tween 20, 2% Tween 40, and 2% Triton X-100, respectively. However, at the same condition, the activity of TKU001 protease FII retained 100%, 100%, and 121% of its original activity, respectively. This is the first report of C. taeanense being able to use shrimp shell wastes as the sole carbon/nitrogen source for proteases production. The novelties of the TKU001 protease include its high stability to the solvents and surfactants. These unique properties make it an ideal choice for application in detergent formulations and enzymatic peptide synthesis.     

Relation between Aggregation Behavior of Glutenin and Its Polypeptide Composition

It was found in the previous studies that the quality of flour is closely related to the aggregation behavior of gluten separated from it, and that difference of aggregation behavior of gluten was mainly due to the nature of glutenin contained in it. In the present paper, aggregation behaviors of glutenins were investigated in relation to their polypeptide compositions with ten kinds of flours. Component polypeptides of glutenin were fractionated into three fractions, FI, FII and FIII, by gel filtration, and their polypeptide compositions and aggregation behaviors were investigated. The ratio of the three fractions was different among the ten glutenins, and a correlation was found between τ₁₀/C values, a parameter for aggregation reaction, of glutenins and their FI and FII contents. τ₁₀/C decreased with increasing content of FI and with decreasing content of FII. Therefore, the aggregation behavior of gluten depends at least partly on the contents of FI and FII in glutenin.     

Serological studies on Aspergillus fumigatus

Summary 1) Extract was obtained by repeated cryolysis of acetone-dried mycelia ofAsp. fumigatus killed by formalin. High titer immune serum could be easily obtained by immunizing rabbits with this extract.2) FI antigen prepared from mycelial extract by alcoholic precipitation exhibited species-specificity when examined by the precipitin test.3) FII, prepared from FI by chloroform-butanol treatment, was no better than FI in antigenic activity.4) FI could be used as antigen in the skin test with rabbits infected withAsp. fumigatus, when the final reading was based on the redness (larger than 5 mm in diameter) 36 hours after the injection of antigen.     

Adenosine-3':5'-monophosphate-dependent and plasma-membrane-associated protein kinase from bovine corpus luteum. Solubilization and properties of solubilized enzyme.

The solubilization of plasma membrane fractions FI and FII associated protein kinases has been attempted using monovalent salts of high ionic strength and various detergent treatments. Extraction of FI and FII plasma membranes with high ionic strength salt solutions did not release more than 20% of the protein kinase activity. Similarly, monovalent salts released little adenosine 3':5'-monophosphate (cyclic AMP) binding activity, but after extraction binding capacity of cyclic [3H]AMP to plasma membranes was increased about 150-200%. Triton X-100 was a better solubilizing agent that Lubrol WX or deoxycholate. In addition to solubilization, 0.1% Triton X-100 also stimulated the protein kinase activity 150-200%. The properties of Triton X-100 solubilized FI and FII and purified cytosol KII were characterized with respect to protein substrate specificity, effect of cyclic AMP, cyclic nucleotide specificity, effects of divalent metal ion and gonadotropins. Upon sucrose density gradient centrifugation, FI solubilized protein kinase and cyclic AMP binding activities co-sedimented with a sedimentation coefficient of 6.3 S. The FII solubilized protein kinase sedimented as two components with sedimentation coefficients of 7.7 S and 5.5 S. The cyclic AMP binding activity also sedimented as two components with sedimentation coefficient 6.7 S and 5.5 S. Cyclic AMP caused dissociation of solubilized protein kinase from FI into a single catalytic (4.8 S) and two cyclic AMP binding subunits (8.1 S and 6.7 S). FII solubilized enzyme was dissociated into one catalytic (4.8 S) and one cyclic AMP binding subunit (6.3 S). Fractionation of FI and FII solubilized enzymes on DEAE-cellulose column chromatography resolved them each into two peaks Ia, Ib and IIa, IIb, respectively. Peaks Ib and IIb were more sensitive to cyclic AMP STIMULATION THAN Ia and IIa peaks. From these studies it is concluded that the plasma-membrane associated and cytosol protein kinases have similar catalytic properties but differ in some of their physical properties.     

Endorphins in human cerebrospinal fluid

Opiate activity in CSF samples drawn from patients with suspected intracranial hydrodynamic dysfunction has been fractionated on Sephadex G-10 and separated by column electrophoresis in agarose suspension. From the Sephadex G-10 chromatography two receptor active fractions (FI and FII) were recovered. Both FI and FII were further resolved by the electrophoresis. FI separated into at least four components and FII into two components. The study also includes a comparison of the endorphin concentrations in CSF (samples drawn from healthy volunteers) measured by receptorassay with those detected by radioimmunoassay of beta-endorphin, [Met]enkephalin and dynorphin, respectively. The data obtained indicated negligible quantities of the radioimmunoassayable endorphins in the total CSF opiate activity.     

Donor-age-related changes in the morphology, growth potential, and collagen biosynthesis in rat fibroblast subpopulations in vitro

Primary explant, mass and clonal fibroblast cultures established from BN rat skin and lungs were used to examine changes in cell behaviour associated with aging. Three distinct fibroblast cell types, i.e. FI, FII and FIII, could be identified on the basis of their morphological and proliferative properties. They could also be distinguished from each other by the amount and type of collagen they synthesized in clonal cultures. FI cells are diploid, spindle shaped and highly proliferative, and they synthesize low levels of type-I and -III collagen. Epithelioid FII cells are also diploid, proliferate slowly, and exhibited elevated collagen synthesis as compared to FI cells. FIII fibroblasts are large, stellate, tetraploid cells that proliferate more slowly than the other types but synthesize large amounts of collagen. In comparison to FI cells, the level of type-III-collagen synthesis is slightly elevated in FIII fibroblasts. In primary explant, mass and clonal cultures, the relative proportions of FI, FII and FIII cells were found to change as a function of the age of the donor animal. The increasing predominance of FIII cells in mass cultures of fibroblasts obtained from donors of increasing ages was consistent with the increased level of collagen synthesis in these cultures. Our observations indicate that the differentiation of normal BN rat fibroblasts occurs via a three-phase process. We discuss the age-related changes in the relative abundance of FI, FII and FIII cells in vivo in the context of alterations in fibroblast replacement and changes in connective tissue that occur during aging.     

Adenosine 3'':5''-cyclic monophosphate-dependent and plasma-membrane-associated protein kinase(s) from bovine corpus luteum.

Plasma-membrane fractions FI and FII isolated from bovine corpus luteum by discontinuous sucrose-density-gradient centrifugation, at sucrose-density interfaces of 1.14/1.16 and 1.16/1.18 respectively, contained membrane-associated protein kinases that phosphorylated both the structural proteins of membranes as well as exogenously added protein substrates. Both fractions were characterized with respect to endogenous and exogenous protein substrate specificity, pH-dependence, effect of bivalent metal ions and sensitivity toward cyclic nucleotides. These membrane-associated kinases showed an optimum pH of 6.0 and had an absolute requirement for bivalent metal ions such as Mg2+, Mn2+, or Co2+ that cannot be replaced by Ca2+. Both the activities were stimulated two- to four-fold by cyclic AMP in vitro with an apparent Km of 83 and 50 nM for fractions FI and FII respectively. Other cyclic 3':5'-nucleotides were effective only at higher concentrations, but even the most effective, cyclic IMP, showed a stimulation nearly an order of magnitude lower than that of cyclic AMP. In contrast, stimulation by cyclic dTMP and cyclic dAMP was very weak. Cyclic AMP showed no significant effect on the apparent Km value of both enzymes for histone and MgCl2 but it somewhat decreased the Km value for ATP. Nucleoside triphosphates like GTP, CTP and UTP inhibited the transfer of [32P]Pi from [gamma-32P]ATP into mixed histone catalysed by membrane-associated kinases either in the presence or in the absence of cyclic AMP. In addition to protein kinases, these membrane fractions also possessed cyclic AMP-binding activities. The apparent association constant (Kalpha) for cyclic AMP binding was 1.0 X 10(10) and 2.6 X 10(10) M for FI and FII membrane fractions respectively.     

Compositional Difference of Wheat Flour Glutens in Relation to Their Aggregation Behaviors

Comparison of the aggregation behavior of gluten was made by following the turbidity change of gluten suspension with four kinds of glutens prepared from flours different in flour quality. The four kinds of glutens showed different behaviors suggesting a relation to the flour quality. From the observation of the aggregation behavior of separated glutensis and gliadins and also of reconstituted glutens, it was concluded that the aggregation behavior of gluten is determined mainly by the nature of glutenin it contains. To investigate the difference of compositions of glutenins, component polypeptides of glutenin were fractionated by gel filtration into three fractions FI, FII and FIII after reduction and cyanoethylation of glutenin The glutenin from a strong flour was rich in FII, while the glutenin from a weak flour in FI. The observation of the aggregation behaviors of FI, FII and FIII suggested that the aggregation behavior of glutenin depends on the contents of these fractions.     

Genetics of clusterin isoform expression and Alzheimer's disease risk

The minor allele of rs11136000 within CLU is strongly associated with reduced Alzheimer's disease (AD) risk. The mechanism underlying this association is unclear. Here, we report that CLU1 and CLU2 are the two primary CLU isoforms in human brain; CLU1 and CLU2 share exons 2-9 but differ in exon 1 and proximal promoters. The expression of both CLU1 and CLU2 was increased in individuals with significant AD neuropathology. However, only CLU1 was associated with the rs11136000 genotype, with the minor "protective" rs11136000T allele being associated with increased CLU1 expression. Since CLU1 and CLU2 are predicted to encode intracellular and secreted proteins, respectively, we compared their expression; for both CLU1 and CLU2 transfected cells, clusterin is present in the secretory pathway, accumulates in the extracellular media, and is similar in size to clusterin in human brain. Overall, we interpret these results as indicating that the AD-protective minor rs11136000T allele is associated with increased CLU1 expression. Since CLU1 and CLU2 appear to produce similar proteins and are increased in AD, the AD-protection afforded by the rs11136000T allele may reflect increased soluble clusterin throughout life.     

Genotyping by REA-PFGE of Listeria monocytogenes isolated from clinical, environmental and food samples

Listeria monocytogenes strains isolated from clinical food and environmental samples were genotyped by Restriction Enzyme Analysis with Pulsed Field Gel Electrophoresis (REA-PFGE) using ApaI and AscI enzymes according to PulseNet Europe procedure. Analysis of DNA fragments profiles obtained by AscI digestion demonstrated presence of 62 REA-PFGE profiles grouped in 2 lineages (FI, FII). Diversity of strains source among both lineages was observed. Statistical analysis showed, that strains isolated from clinical samples more frequently are included to lineage FI, then lineage FII. Non-clinical strains were more frequently included to lineage FII. Combined analysis of REA-PFGE profiles for ApaI and AscI enzymes showed 8 unique pulsotypes characteristic for two or more L. monocytogenes isolates. Moreover researched L. monocytogenes strains were analyzed by multiplex-PCR according Doumith et al methodology. PCR-group 4B was most frequent among strains isolated from clinical samples. Correlation between PCR-group and pulsotype was observed only in few cases.