Comparative amino acid sequence analysis of Thermotoga maritima β-glucosidase (BglA) deduced from the nucleotide sequence of the gene indicates distant relationship between β-glucosidases of the BGA family and other families of β-1,4-glycosyl hydrolases

The primary structure of the bglA gene region encoding a -glucosidase of Thermotoga maritima strain MSB8 was determined. The bglA gene has the potential to code for a polypeptide of 446 amino acids with a predicted molecular mass of 51545 Da. The T, maritima -glucosidase (BglA) was overexpressed in E. coli at a level comprising approximately 15–20% of soluble cellular protein. Based on its amino acid sequence, as deduced from the nucleotide sequence of the gene, BglA can be classified as a broad-specificity -glucosidase and as a member of the -glucosidase family BGA, in agreement with the results of enzymatic characterization of the recombinant protein. Comparative sequence analysis revealed distant amino acid sequence similarities between BGA family -glucosidases, a -xylosidase, -1,4-glycanases of the enzyme family F (mostly xylanases), and other families of -1,4-glycosyl hydrolases. This result indicates that BGA -glucosidases may comprise one enzyme family within a large enzyme order of retaining -glycosyl hydrolases, and that the members of these enzyme groups may be inter-related at the level of active site architecture and perhaps even on the level of overall three-dimensional fold.     

13Cα and 13Cβ chemical shifts as a tool to delineate β-hairpin structures in peptides

Unravelling the factors that contribute to the formation and the stability of -sheet structure in peptides is a subject of great current interest. A -hairpin, the smallest -sheet motif, consists of two antiparallel hydrogen-bonded -strands linked by a loop region. We have performed a statistical analysis on protein -hairpins showing that the most abundant types of -hairpins, 2:2, 3:5 and 4:4, have characteristic patterns of ¹³C and ¹³C conformational shifts, as expected on the basis of their and angles. This fact strongly supports the potential value of ¹³C and ¹³C conformational shifts as a means to identify -hairpin motifs in peptides. Their usefulness was confirmed by analysing the patterns of ¹³C and ¹³C conformational shifts in 13 short peptides, 10–15 residues long, that adopt -hairpin structures in aqueous solution. Furthermore, we have investigated their potential as a method to quantify -hairpin populations in peptides.     

Synergistic effect of BMP-2 and ascorbate on the phenotypic expression of osteoblastic MC3T3-E1 cells

Of TGF- superfamily proteins, BMP-2 enhanced alkaline phosphatase (ALP) activity in cultured osteoblastic cells, MC3T3-E1, to the same level promoted by ascorbate, whereas TGF-s (1, 2, 3) reduced ALP activity and altered cell morphology under the same conditions. Activin appeared to have no distinct effect on ALP synthesis. Ascorbate dependent increase in ALP synthesis was markedly stimulated in the presence of BMP-2. The synergistic effect of ascorbate on ALP synthesis was replaced by type I collagen coated on the culture dish. However, BMP-2 appeared not to bind to type I collagen. These findings indicate that BMP-2 acts directly on osteoblastic cells through its receptors and collagenous matrix can neither recruit BMP-2 nor modulate directly the action of BMP-2 in the pericellular area. Treatment of cells grown in ascorbate media with TGF-s decreased rapidly the cellular ALP activity indicating that TGF-s direct cells to the differentiated stage.     

Localization of Transforming Growth Factor β in Association with Neuromuscular Junctions in Adult Human Muscle

Transforming growth factors- 1, 2, and 3 are known for their regulatory function in embryogenesis, fibrogenesis, and tissue repair of different cell types. A trophic function of TGF- subclasses for motoneurons has been shown in vitro. TGF- 1 is a potent survival factor for cultured embryonic rat motoneurons. In addition, TGF- 1 stimulates proliferation of rat Schwann cells. Recently, TGF- 2 has been reported to be associated with the subsynaptic nuclei of mature rat neuromuscular junctions. In this study, we investigated the expression of TGF- 1, 2, and 3 at neuromuscular junctions in skeletal muscle of 11 adults without neuromuscular disease. On muscle biopsies, neuromuscular junctions were depicted by acetylcholine esterase reaction and acetylcholine receptor antibodies. TGF- 1, 2, and 3 were stained immunohistochemically with monoclonal antibodies. Some muscle fibers showed low levels of inhomogeneous immunoreactivity for both TGF- 1 and TGF- 3. Intense immunoreactivity of TGF- 1 and 3 was shown at the postsynaptic area of neuromuscular junctions. TGF- 2 was expressed in the same subcellular distribution, but less strongly. In conclusion, the colocalization of TGF- with neuromuscular junctions may suggest a significant function in neuromuscular communication.     

Synthesis of disaccharide derivatives employing β-N-acetyl- d-hexosaminidase, β-d-galactosidase and β-d-glucuronidase

-N-Acetyl-d-hexosaminidase from Aspergillus oryzae catalysed the stereo- and regiospecific formation of the 6-O-benzylated disaccharide derivatives GalNAc1-3(6- OBn)Gal-SEt and GlcNAc1-3(6-OBn)Gal-SEt, which were obtained in transglycosylation reactions employing ethyl 6- O-benzyl-1-thio--d-galactopyranoside as acceptor. Preparative amounts of the chitobiose derivative GlcNAc1- 3GlcNAc-OPhNO2-p was prepared as well. - N-Acetyl-d-hexosaminidase from bovine testes catalysed the specific synthesis of GlcNAc1-3(6-OBn)GlcNH2-SEt and GalNAc1-3(6-OBn)GlcNH2-SEt, employing ethyl 2-amino-6-O-benzyl-2-deoxy-1-thio--d-glucopyranoside as acceptor. -d-Glucuronidase from E. coli was found to catalyse the formation of GlcA1-3(6-OBn)GlcNH2- SEt employing the same acceptor.     

Single mid-chain GlcNAcβ1-6Galβ1-4R sequences of linear oligosaccharides are resistant to endo-β-galactosidase ofBacteroides fragilis

Endo--galactosidase (EC 3.2.1.103) ofBacteroides fragilis, at 250 mU ml^–1, did not cleave the internal galactosidic linkage of the linear radiolabelled trisaccharide GlcNAc1-6Gal1-4GlcNAc, or those of the tetrasaccharides Gal1-4GlcNAc1-6Gal1-4GlcNAc and Gal1-4GlcNAc1-6Gal1-4Glc. The isomeric glycans which contained the GlcNAc1-3Gal1-4GlcNAc/Glc sequence were readily cleaved.Abbreviations GlcNAc 2-acetamido-2-deoxy-d-glucose - Lact lactose - MT maltotriose - MTet maltotetraose - R _MTet chromatographic migration rate in relation to that of maltotetraose     

Interactions of Aβ(1–40) with Glycerophosphocholine and Intact Erythrocyte Membranes: Fluorescence and Circular Dichroism Studies

Deposition of amyloid peptide in human brain in the form of senile plaques is a neuropathological hallmark of Alzheimers disease (AD). Levels of a phospholipid breakdown product, glycerophosphocholine (GPC), also increase in AD brain. The effect of GPC on amyloid (1–40) peptide (A) aggregation in PBS buffer was investigated by circular dichroism and fluoresence spectroscopy; interactions of A and GPC with the intact erythrocyte membrane was examined by fluoresence spectroscopy. Fluorescamine labeled A studies indicate GPC enhances A aggregation. CD spectroscopy reveals that A in the presence of GPC adopts 14% more -sheet structure than does A alone. Fluorescamine anisotropy measurements show that GPC and A interact in the phospholipid head-group region of the erythrocyte membrane. In summary, both soluble A and GPC insert into the phospholipid head-group region of the membrane where they interact leading to -sheet formation in soluble A which enhances A aggregation.     

Primary structure of the hemoglobin beta-chain of rose-ringed parakeet (Psittacula krameri)

The primary structure of Rose-ringed Parakeet hemoglobin -chain was established, completing the analysis of this hemoglobin. Comparisons with other avian -chains show variations smaller than those for the corresponding -chains. There are 11 amino acid exchanges in relationship to the only other characterized psittaciform -chain, and a total of 35 positions are affected by differences among all avian -chains analyzed (versus 61 for the -chains). At three positions, the Psittacula -chain has residues unique to this species. Three ₁₁ contacts are modified, by substitutions at positions 51, 116, and 125.     

Enzymatic syntheses of GlcNAcβ1-2Man and Galβ1-4GlcNAcβ1-2Man as components of complex type sugar chains

GlcNAc1-2Man and GlcNAc1-6Man were synthesized using the reverse hydrolysis activity of -N-acetylglucosaminidase from both jack beans and Bacillus circulans. In turn, Gal1-4GlcNAc1-2Man and Gal1-4GlcNAc1-6Man were synthesized regioselectively using the transglycosylation activity of -galactosidase from Diplococcus pneumoniae and B. circulans, respectively. These di- and trisaccharides are important components of complex type sugar chains and will be used as intermediates in our synthetic studies. Abbreviations: pNp--GlcNAc, p-nitrophenyl 2-acetamido-2-deoxy--D-glucopyranoside; pNp--Gal, p-nitrophenyl -D-galacto-pyranoside     

Transfection of rabbit articular chondrocytes by the calcium phosphate procedure: Improvement of efficiency and reproducibility

The understanding of cartilage disorders relies on the possibility of studying mechanisms which monitor the regulation of matrix protein genes through introducing efficiently and in a reproducible manner these genes, or their regulatory regions, into cells. To this end, we attempted to improve the transfection efficiency of rabbit articular chondrocytes by the calcium phosphate procedure. Transfection efficiencies were assessed by measuring the expression of the Lac Z reporter gene encoding -galactosidase using anin situ staining (X-gal staining) and an enzymatic assay (-galactosidase assay).Results revealed that addition of 4 U ml^–1 of hyaluronidase before and during transfection increases by 2 to 4-fold the transfection efficiency of rabbit articular chondrocytes. Furthermore, we demonstrated that the use of a giant calcium phosphate DNA coprecipitate gives a higher transfection efficiency and much more reproducible results than those obtained with classical small volumes of precipitates.Abbreviations CPRG Chlorophenol red--D-galactopyranoside - EDTA Ethylenediaminetetraacetic acid - HEPES N-2-Hydroxyethylpiperazine-N-2-ethanesulfonic acid - HBS Hepes buffered saline - RAC Rabbit articular chondrocytes - X-gal 5-bromo-4-chloro-3-indolyl--D-galactopyranoside     

Restricted T cell receptor V-β and J-β usage in T cells from interleukin-2-cultured lymphocytes of ovarian and renal carcinomas

Tumour-infiltrating lymphocytes (TIL) are often observed in human tumours and their presence has been correlated with a better prognosis. It has been suggested that TIL are enriched for tumour-specific cytotoxic cells, and TIL activated and expanded in vitro by interleukin-2 (IL-2) are currently used in the therapy of human cancer. We have studied the T cell repertoire in IL-2-expanded TIL cells from patients with ovarian and renal carcinoma using T-cell-receptor-V--specific monoclonal antibodies and a polymerase-chain-reaction-based Southern blot technique for analysis of J- usage. In TIL lines derived from three of nine patients with ovarian carcinomas and from two of eight patients with renal carcinomas, selective usage of the V-6 or V-5 T-cell receptor gene products was found. The majority of the cells were CD4⁺, with up to 40% of the T cells utilizing the same V- gene. T-cell lines derived from peripheral blood lymphocytes from patients or healthy donors contained normal levels of V- subsets. Only moderate levels of V-6⁺ T cells were detected from freshly isolated TIL and the increase of this subpopulation appeared as a result of in vitro culture. The level of clonal restriction, as measured by the usage of J- gene segments within the V-5 or V-6 families, was analysed using a recently developed technique based on the polymerase chain reaction. Evidence for restricted J- usage was detected only in TIL expanded in vitro, while this was not the case in freshly isolated tumour-derived lymphocytes or T cell lines obtained from peripheral blood lymphocytes. The presence of a population with biased T cell receptor expression in cells derived from tumour tissue could be explained by their activation in vivo as a result of contact with tumour antigens and should be taken into consideration when discussing the therapeutic efficiency of IL-2-expanded TIL.     

Activities ofβ-glucanases andβ-glucosidases during blastospore formation inSaccharomycopsis fibuligera

Summary The activities of three glycosidases, -glucosidase and (1,3)- and (1,6)-glucanases have been monitored during growth and blastospore formation inSaccharomycopsis fibuligera. The assays were carried out on the cell-free culture and in a cell-free extract and a wall autolysate preparation from the growing cells. In complex medium containing 1% glucose an increase in the level of all three enzymes was associated with the transition from mycelium to blastospores. When the level of glucose was increased to 5% blastospore formation was repressed and the level of -glucanases only increased at the end of the fermentation. The -glucosidase activity increased during the growth phase. In a defined medium in which slow growth in a wholly yeast-like form was observed, growth was not associated with a high level of -glucanase activity.     

Tail-to-tail orientation of the Atlantic salmon alpha- and beta-globin genes

We report the cloning of a cDNA and two corresponding -globin genes of the Atlantic salmon (Salmo salar L.) as well as two genes for -globins. Nucleotide sequence analysis of the cDNA shows that the predicted -globin peptide comprises 148 amino acids with a calculated molecular mass of 16,127 Da and an overall amino acid similarity of 40–50% to higher vertebrates and 60–90% to fish sequences. The study of the genomic organization of - and -globin genes shows that, as is the case in Xenopus, the salmon genes are adjacent. Two sets of linked - and -globin genes were isolated and restriction-enzyme polymorphisms indicate that they belong to two distinct loci, possibly as a result of the salmon tetraploidy. In each locus the - and -globin genes are oriented 3 to 3 relative to each other with the RNA coding sequences located on opposite DNA strands. This is the first evidence for this type of arrangement found for globin genes. Moreover, while the linkage found in salmon and Xenopus supports the hypothesis of an initial tandem duplication of a globin ancestor gene, our results raise the question of the actual original orientation of the duplicated genes. Correspondence to: F. Gannon     

Benzamidine as a spectroscopic probe for the primary specificity subsite of trypsin-like serine proteinases

Summary Formation and dissociation of the benzamidine: -trypsin adduct is accompanied by reversible spectral changes in the ultraviolet region (between 230 and 300 nm). The pH-independent difference extinction coefficient of the adduct (benzamidine: -trypsin complex minus the free proteinase) is 1.75 mM^–1 cm^–1 at 248 nm. This signal can be used in studies of inhibitor and substrate binding by rapid kinetic techniques. Therefore, following the spectral changes associated with the displacement of benzamidine from the primary specificity subsite, the kinetics of the -trypsin: BPTI complex formation were investigated between pH 2.9 and 7.6 (I = 0.1 M) at 21 ± 0.5 °C. Under all the experimental conditions the -trypsin: BPTI complex formation, examined by benzamidine displacement experiments, may be described in terms of a simple competition event. On the other hand, the very same reaction followed by displacement of another spectroscopic probe, proflavine, appears to involve the ternary proflavine: -trypsin:BPTI adduct (7). The difference between the kinetic processes of -trypsin: BPTI complex formation, observed by using benzamidine and proflavine as reaction indicators, suggests that the two dye molecules bind at non-coincident regions of the proteinase active center. The advantages in using benzamidine as a sensitive probe specific for the S₁ subsite of the recognition center of trypsin-like proteinases, as compared to proflavine, are emphasized.Abbreviations BPTI bovine basic pancreatic trypsin inhibitor (Kunitz inhibitor) - pNGB p-nitrophenyl-p-guanidinobenzoate - NaDodSO₄ sodium dodecyl sulfate     

Primary structure of two nonallelicβ-globin chains from DBA/2 mice

The amino acid sequences of the major and minor globin chains from DBA/2 mice have been determined. This information is of interest because DBA/2 mice are the strain of origin for most murine erythroleukemia lines. The primary structure of DBA/2 globins agrees completely with that predicted from the coding properties of BALB/c globin genes. This identity does not support a rapid evolutionary divergence in inbred mouse strains, at least at these loci in these strains.This work was supported in part by National Institutes of Health Grants AM26956, HL24908, and AM14923 and by the Department of Energy under Contract DE-AC05-840R21400 with Martin Marietta Systems. The SUNY Buffalo Sequencing Facility was supported by a grant from the James Cummings Foundation and National Institutes of Health Grants RR05400 and RR07066.     

Evolution of parallel β/α-barrel enzyme family lightened by structural data on starch-processing enzymes

The parallel /-barrel domain consisting of eight parallel -sheets surrounded by eight -helices has been currently identified in crystal structures of more than 20 enzymes. This type of protein folding motif makes it possible to catalyze various biochemical reactions on a variety of substrates (i.e., it seems to be robust enough so that different enzymatic functionalities could be designed on it). In spite of many efforts aimed at elucidation of evolutionary history of the present-day /-barrels, a challenging question remains unanswered: How has the parallel /-barrel fold arisen? Although the complete sequence comparison of all /-barrel amino acid sequences is not yet available, several sequence similarities have been revealed by using the highly conserved regions of -amylase as structural templates. Since many starch-processing enzymes adopt the parallel /-barrel structure these enzymes might be useful in the search for evolutionary relationships of the whole parallel eight-folded /-barrel enzyme family.     

β-Carotene production by Flavobacterium multivorum in the presence of inorganic salts and urea

Flavobacterium multivorum, a non-fermenting Gram-negative bacteria, normally produces zeaxanthin (3R, 3 R-, -carotene-3, 3 diol) as its main carotenoid. The effect of supplementation of various inorganic salts and urea on the growth, total carotenoid production, and proportion of -carotene (, -carotene), -cryptoxanthin (, -caroten-3-ol), and zeaxanthin produced by F. multivorum was investigated. Urea and several salts, such as calcium chloride, ammonium chloride, lithium chloride, and sodium carbonate, improved total carotenoid production by 1.5- to 2.0-fold. Urea and sodium carbonate had an unexpectedly strong positive effect on -carotene production at the expense of zeaxanthin formation. The effect was found to be independent of incubation time, and -carotene represented 70% (w/w) of the total carotenoid content. The cumulative effect of urea and sodium carbonate was further studied using response surface methodology. An optimum medium was found to contain 4,000 and 4,070 mg l^–1 urea and sodium carbonate, respectively. The maximum -carotene level was 7.85 g ml^–1 culture broth, which represented 80% (w/w) of the total carotenoid produced. Optimization resulted in 77- and 88-fold improvements in the volumetric and specific -carotene levels, respectively, accompanied by a simultaneous decrease in the zeaxanthin level as compared to the control medium. The carotenoid production profile in the optimized medium indicated that -carotene was produced maximally during the late exponential phase at 0.41 g ml^–1 h^–1. It is possible that this organism could be an excellent commercial source of either -carotene or zeaxanthin, depending on initial culture conditions.     

Biochemical and immunological responses of hairy cell leukemia patients to interferonβ

Summary Ten hairy-cell leukemia patients were treated with interferon (IFN-) at a dose rate of 2 × 10⁶ IU/m² × 5 days for 4 weeks (induction therapy) and, thereafter, at the same dose three times a week for 11 months (maintenance therapy). The effect of this treatment on serum neopterin, 2-microglobulin, (2–5)oligoadenylate [(2–5)A _n ] levels, intracellular (2–5)A _n values and human Mx protein synthesis was analysed. There were significant rises in serum neopterin and (2–5)A _n levels during both induction and maintenance, whereas 2-microglobulin levels rose only during induction. Rises in intracellular (2–5)A _n were documented mainly during induction, but they were not significantly higher than pretherapy values. IFN provoked an increase in human Mx protein synthesis over the entire induction — maintenance period, but was only significantly higher than baseline during induction. All markers proved useful for monitoring the effects of IFN dose schedules, but were not predictive of clinical outcome. Natural killer activity and IFN production, which were initially defective, followed a different trend from that of the other factors studied, in that increases were documented only late in the course of therapy when the disease was already in remission.     

Binding of Amyloid Beta-Protein to Intracellular Brain Proteins in Rat and Human

Amyloid beta-protein (A), in its soluble form, is known to bind several circulatory proteins such as apolipoprotein (apo) E, apo J and transthyretin. However, the binding of A to intracellular proteins has not been studied. We have developed an overlay assay to study A binding to intracellular brain proteins. The supernatants from both rat and human brains were found to contain several proteins that bind to A 1–40 and A 1–42. No major difference was observed in the A binding-proteins from brain supernatants of patients with Alzheimer's disease and normal age-matched controls. Binding studies using shorter amyloid beta-peptides and competitive overlay assays showed that the binding site of A to brain proteins resides between 12–28 amino acid sequence of A. The presence of several intracellular A-binding (AB) proteins suggests that these proteins may either protect A from its fibrillization or alternatively promote A polymerization. Identification of these proteins and their binding affinities for A are needed to assess their potential role in the pathogenesis of Alzheimer's disease.     

Comparison of the apoptotic processes induced by the oxysterols 7β-hydroxycholesterol and cholesterol-5β,6β-epoxide

Oxysterols have been shown to induce apoptosis in a variety of cell lines. The mechanism of oxysterol-induced apoptosis is mainly known at the post-mitochondrial level. The aim of the present study was to compare the pathway of apoptosis induced by the oxysterols 7-hydroxycholesterol (7-OH) and cholesterol-5,6-epoxide (-epoxide) in U937 cells. To this end, we employed a range of inhibitors of apoptosis; a broad-spectrum caspase inhibitor, a specific caspase-3 inhibitor and an inhibitor of cytochromec release and the antioxidants; trolox, ebselen and resveratrol. The three inhibitors of apoptosis prevented cell death induced by 7-OH; however, in -epoxide-treated cells, the inhibitor of cytochromec release did not protect against apoptosis. The cellular antioxidant glutathione was depleted in 7-OH-treated cells but not in cells incubated with -epoxide. Trolox, a water-soluble synthetic analogue of -tocopherol, prevented 7-OH-induced apoptosis but did not protect against cell death induced by -epoxide. Ebselen and resveratrol did not protect U937 cells against apoptosis induced by either 7-OH or -epoxide. Our results suggest that differences occur in the pathways of apoptosis induced by 7-OH and -epoxide in U937 cells.