Production of a novel syrup containing neofructo-oligosaccharides by the cells of Penicillium citrinum

A novel syrup containing neofructo-oligosaccharides was produced from sucrose (Brix 70) by whole cells of Penicillium citrinum. The efficiency of fructo-oligosaccharides production was more than 55% and those of the main carbohydrate components, 1-kestose (Fruf 21Fruf 21 Glc), nystose (Fruf 21Fruf 21 Fruf 21 Glc) and neokestose (Fruf 26 Glc12 Fruf), were 22, 14 and 11%, respectively.     

Presence of hybridizing DNA sequences homologous to bovine acidic and basicβ-crystallins in all classes of vertebrates

Summary The eye lens-crystallins in cow and chicken are encoded by a family of at least six genes. In order to assess the distribution of the corresponding genes among other vertebrates we hybridized -crystallin sequences (A2, A3/A1, A4, B1, B2, B3), isolated from a bovine lens cDNA library, to Southern blots on whichEcoR1-digested chromosomal DNA was blotted from different vertebrate species. These included human, chimpanzee, calf, rat, pigeon, duck, monitor lizard, toad, trout, and lamprey. Positive hybridization signals were found in the representatives of virtually all classes of vertebrates. The basic B-crystallins gave hybridization signals in more species than the acidic A ones. In monitor lizard and toad the weakest hybridization signals for basic crystallin probes were found. For acidic crystallin probes the distribution pattern was more simple; among cold-blooded vertebrates a signal for A2 was found in trout and lamprey, for A4 in trout, and for A3/A1 only in toad. The results demonstrate that the duplications leading to the -crystallin gene family occurred before or during the earliest stages of vertebrate evolution.     

Ellagic acid toxicity and interaction with benzo[a]pyrene and benzo[a]pyrene 7,8-dihydrodiol in human bronchial epithelial cells

Ellagic acid, a plant phenol present in various foods consumed by humans, has been reported to have both anti-mutagenic and anti-carcinogenic potential. To evaluate the potential anti-carcinogenic property of ellagic acid, we tested its effects on the toxicity of ben-zo[a]pyrene and benzo[a]pyrene, 7,8-dihydrodiol and binding of benzo[a]yrene to DNA in cultured human bronchial epithelial cells. The toxicity of ellagic acid itself for human bronchial epithelial cells was also determined. Using a colony-forming efficiency assay, it was found that a nontoxic concentration of ellagic acid (5 g/ml) enhanced the toxicity of benzo[a]pyrene.7,8-dihydrodiol in human bronchial epithelial cells. In contrast, ellagic acid at concentrations of l.5 and 3.0 g/ml inhibited binding of benzo[a]pyrenemetabolites to DNA in these cells. An explanation for the potentiating effect of ellagic acid on the toxicity of benzo[a]pyrene, 7,8-dihydrodiol will require further investigation into the possible mechanisms of interaction between these two compounds.Abbreviations B[a]P benzo[a]pyrene - B[a]P 7,8-DHD (±)trans-7,8-dihydro-7,8-dihydroxybenzo[a]pyrene - B[a]PDE-1 (±)-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene - B[a]PDE-2 (±) 7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene - B[a]PDE-1:dG N²-]10{7,8,9-dihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene]yl}:deoxyguanosine - B[a]PDE-2:dG NZ-{10-[7,8,9-trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene]yl}:deoxyguanosine - CFE colony forming efficiency - EA ellagic acid - HBE human bronchial epithelial     

Peptide des β-Alanins als Ersatz für die freie Aminosäure bei pantothensäurebedürftigen Hefezellen und ihr Verhalten gegenüber dem β-Alanin-Antagonisten Asparagin

Zusammenfassung Pantothensäurebedürftige Hefezellen können ihren Bedarf an diesem Vitamin nicht allein aus -Alanin decken, sondern auch aus Benzoyl--Alanin, -Alanyl-d,l-Norleucin und -Alanyl-l-Histidin. Der Antagonist Asparagin hemmt die Verwertung dieser Peptide genauso wie diejenige der freien Aminosäure. Durch höhere Konzentrationen an -Alanin oder -Alanyl-d,l-Norleucin läßt sich die Hemmwirkung nicht allein kompensieren, es kommt sogar zu einer Förderung des Hefewachstums. Der Antagonist wird dann zum Synergisten.

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Summary The -alanine containing peptides benzoyl--alanine, -alanyl-d,l-norleucine and -alanyl-l-histidine can substitute for the amino acid -alanine in a pantothenic acid requiring yeast. Asparagine, an antagonist of -alanine, affects these peptides in a similar manner. In combination with an overdose of -alanine or -alanyl-d,l-norleucine, asparagine is no longer an antagonist but becomes a synergist.

     

Polymorphism of human liver alcohol dehydrogenase: Identification of ADH2 2-1 and ADH2 2-2 phenotypes in the Japanese by isoelectric focusing

Liver homogenate-supernatants from most Japanese exhibit an atypical pH optimum for ethanol oxidation at pH 8.8 instead of 10.5, the typical pH-activity optimum. It has been proposed that atypical livers contain alcohol dehydrogenase isozymes with ₂ subunits while typical livers contain isozymes with ₁ subunits, both produced by the ADH ₂ gene. Because it is difficult to differentiate the atypical ADH₂ 2-2 phenotype from the ADH₂ 2-1 phenotype by starch gel electrophoresis, an agarose isoelectric focusing procedure was developed that clearly separated the atypical Japanese livers into two groups, A1 and A2. The isozymes in A1 and A2 livers were purified. Type A1 livers contained a single isozyme with an atypical pH-rate profile; it was designated ₂₂. Three isozymes were isolated from A2 livers, two of which corresponded to ₁₁ and ₂₂. A third, absent from the typical and the atypical A1 livers, had an intermediate mobility; it was designated ₂₁. Type A1 livers are, therefore, the homozygous ADH₂ 2-2 phenotype, and type A2 livers, the heterozygous ADH₂ 2-1 phenotype. The ADH₂ 2-2 phenotype was found in 53% of 194 Japanese livers, and the ADH₂ 2-1 phenotype, in 31%. Accordingly, the frequency of ADH ₂ ² was 0.68.This study was supported by U.S. Public Health Service Grant AA 02342.     

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.     

Structure of a new nonasaccharide isolated from human milk: VI2Fuc,V4Fuc,III3Fuc-p-lacto-N-hexaose

The structure of a new nonasaccharide isolated from human milk has been investigated. By using methylation analysis, FAB-MS and¹H-and¹³C-NMR spectroscopy as basic methods of structural investigation, this oligosaccharide was identified as VI²--Fuc,V⁴-Fuc,III³--Fuc-p-lacto-n-hexaose: Fuc1-2Gal1-3[Fuc1-4]GlcNAc1-3Gal1-4[Fuc1-3]GlcNAc1-3Gal1-4Glc.Abbreviations COSY correlation spectroscope - DP degree of polymerisation - FAB-MS fast atom bombardment-mass spectrometry - HPLC high performance liquid chromatography - NMR nuclear magnetic resonance - GLC gas-liquid chromatography     

Large scale preparation of PA-oligosaccharides from glycoproteins using an improved extraction method

We have developed a new method for the large scale preparation of pyridylaminated (PA-) oligosaccharides from glycoproteins. Phenol/chloroform extration was adapted for the removal of protein and excess 2-aminopyridine, improving the efficiency of preparation. From a 2.5 g sample of human apo-transferrin, 25–30 mol of agalacto biantennary PA-oligosaccharide could be obtained. By increasing the concentration of PA-oligosaccharide substrate, we were able to detect a very low level ofN-acetylglucosaminlytransferase IV activity in CHO cell extracts.Abbreviations PA 2-aminopyridine - SDS sodium dodecyl sulfate - GlcNAc N-acetylglucosamine - GnT N-acetylglucosaminyltransferase - Gn,Gn-bi-PA GlcNAc1-2Man1-3(GlcNAc1-2Man1-6)Man1-4GlcNAc1-4GlcNAc-2-aminopyridine - Gn,Gn,Gn-tri-PA GlcNAc1-2(GlcNAc1-4)Man1-3(GlcNAc1-2Man1-6)Man1-4GlcNAc1-4GlcNAc-2-aminopyridine - Gn,Gn,Gn-trí-PA GlcNAc1-2Man1-3({GlcNAc1-2(GlcNAc1-6)Man1-6})Man1-4GlcNac1-4GlcNAc-2-aminopyridine - Gn,(Gn),Gn-bi-PA GlcNAc1-2Man1-3(GlcNAc1-4)(GlcNAc1-2Man1-6)Man1-4GlcNAc1-4GlcNAc-2-aminopyridine     

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.     

Bacillus subtilis bacteriophages SP beta c1 is a deletion mutant of SP beta

Summary The restriction fragment patterns of two mutant forms of the temperate Bacillus subtilis bacteriophage SP have been examined. The DNA of a heat-inducible mutant, SPc2, which has a molecular size of 128 kilobases (kb), yields the same restriction pattern as the wild type SPc⁺ DNA. The DNA of a clear-plaque mutant, SPc1, has a molecular size of 117 kb, and is deleted for an 11 kb region of phage DNA. Neither SPc1 nor SPc2 DNA is cleaved by the endonuclease HaeIII.     

Role of βγ-Dimers of GTP-Binding Proteins in Processes of Hormonal Signal Transduction

This review summarizes and analyzes data on structural and functional peculiarities of - and -subunits of heterotrimeric G proteins and their participation in -dimer in processes of hormonal signal transduction to the cell. The molecular mechanisms are discussed, which are responsible for formation of the -dimer complex and its interaction with the wide spectrum of signal and structural proteins, such as G protein -subunit, serpentine type receptors, kinases of G protein-coupled receptors, adenylyl cyclase, phospholipase C2, phosphatidyl-inositol-3-kinase, Raf-kinase, potassium and calcium channels, microtubular and RGS-proteins. The characteristics of - and -subunits are given for living organisms of different phylogenetic levels (lower eukaryotes, fungi, plants, vertebrates, and invertebrates). It is concluded that the -dimer is the most important regulatory link in various signal systems to carry out switch from one signal cascade to another and thereby to determine the final cellular response to the signal impulse.     

Variability of bacterial gene-directed enzyme production in human genetically deficient cells

Summary Human -galactosidase-deficient skin fibroblasts from a patient with generalized gangliosidosis (GM₁-gangliosidosis type I) were treated with phage plac DNA, coding for Escherichia coli -galactosidase (-D-galactoside galactohydrolase, EC 3.2.1.23). New -galactosidase activity detected in cell extracts of phage DNA-treated GM₁-gangliosidosis fibroblasts continued to vary considerably from one experiment to another. It behaved like the E. coli z-gene product upon immunochemical and physicochemical investigation. In some experiments the antigenic behavior of resultant -galactosidase activity in plac DNA-treated cells resembled that of mutant E. coli -galactosidase. Among the factors and variables that may be responsible for the variation in the results obtained here and elsewhere, low physical binding between prokaryotic mRNA sequences and fibroblast ribosomal RNA could play a part connected with effective translation. This hypothesis is discussed under the aspect of a comparison of the ribosomal binding site of lac z mRNA with the 3-terminus of the eukaryotic 18s ribosomal RNA, which shows limited possibilities for base-pairing interactions.More extensive possibilities for forming Watson-Crick base pairs between their initiation site and the eukaryotic ribosomal binding site exist for other prokaryotic messengers, such as those of Q-replicase, f 1-coat protein, or UDPG-4-epimerase.     

Modulation of cytokine responses of murine CD8+ αβ intestinal intraepithelial lymphocytes by IL-4 and IL-12

The immune responses of the intestine mucosa feature the noninflammatory type, such as IgA production and oral tolerance. Th2 type cytokines have been implicated in the induction of these noninflammatory responses. In the present study, cytokine responses of CD8+ and CD4+ TCR+ intestinal intraepithelial lymphocyte ( iIEL) subsets to TCR stimulation under the influence of IL-12, IL-4, or CD28 costimulation were examined. IL-12 enhanced production of IL-10 and IFN- by the CD8+ iIEL significantly but only marginally affected the CD8+ subset, whereas IL-4 induced IL-4, IL-5, and IL-10 production and augmented TGF- production by both subsets. CD28 costimulation induced production of Th2 cytokines by CD4+ iIEL in the absence of exogenous IL-4. Unlike lymph node CD4+ cells, the CD28 costimulation-induced Th2 differentiation of CD4+ iIEL was not inhibited by IFN-. These results demonstrate active cytokine production by CD4+, CD8+, as well as CD8+ iIEL. The Th2-skewed cytokine profile of CD8+ iIEL and the IFN--resistance of Th2 differentiation of the CD4+ iIEL suggest that both iIEL subsets contribute to the induction of noninflammatory mucosal immune responses.     

UDP-GlcNAc: Galβ3GalNAc-Mucin: (GlcNAc → GalNAc) β6-N-Acetylglucosaminyltransferase and UDP-GlcNAc: Galβ3(GlcNAcβ6) GalNAc-Mucin (GlcNAc → Gal)β3-N-Acetylglucosaminyltransferase from Swine Trachea Epithelium

Summary Two specific -N-acetylglucosaminyltransferases involved in the branching and elongation of mucin oligosaccharide chains, namely, a 1,6 N-acetylglucosaminylsaminyltransferase that transfers N-acetylglucosamine from UDP-N-acetylglucosamine to Gal3GalNAc-Mucin to yield Gal3(GlcNAc6)GalNAc-Mucin and a 3-N-acetylglucosaminyl transferase that transfers N-acetylglucosamine from UDP-N-acetylglucosamine to Gal3(GlcNAC6)GalNAc-mucin to yield GlcNAc3Gal3 (GlcNAc6)GalNAc-Mucin were purified from the microsomal fraction of swine trachea epithelium. The 1,6-N-acetylglucosaminyltransferase was purified about 21,800-fold by procedures which included affinity chromatography on DEAE columns containing bound asialo Cowper's gland mucin glycoprotein with Gal1,3GalNAc side chains. The apparent molecular weight estimated by gel filtration was found to be about 60 Kd. The purified enzyme showed a high specificity for Gal1,3GalNAc chains and the most active substrates were mucin glycoproteins containing these chains. The apparent Km of the 6-glucosaminyltrans-ferase for Cowper's gland mucin glycoprotein containing Gal1,3GalNAc chains was 0.53 µM; for UDP-N-acetylglucosamine, 12 µM; and for Gal 1,3GalNAc NO₂ø, 4 mM. The activity of the 6-glucosaminyltransferase was dependent on the extent of glycosylation of the Gal3GalNAc chains in Cowper's gland mucin glycoprotein.The best substrate for the partially purified 3-Glucosaminyltransferase was Cowper's gland mucin glycoprotein containing Gal1,3(GlcNAc6)GalNAc side chains. This enzyme showed little or no activity with intact sialylated Cowper's gland mucin glycoprotein or derivatives of this glycoprotein containing GalNAc or Gal1,3GalNAc side chains.The radioactive oligosaccharides formed by these enzymes in large scale reaction mixtures were released from the mucin glycoproteins by treatment with alkaline borohydride, isolated by gel filtration on Bio-Gel P-6 and characterized by methylation analysis and sequential digestion with exoglycosidases. The oligosaccharide products formed by the 6- and 3-glucosaminyltransferases were shown to be Gal3(GlcNAC6) GalNAc and GlcNAc3 Gal3(GlcNAC6)GalNAc respectively.Taken collectively, these results demonstrate that swine trachea epithelium contains two specific N-acetylglucosaminyltransferases which catalyze the initial branching and elongation reactions involved in the synthesis of O-linked oligosaccharide chains in respiratory mucin glycoproteins. The first enzyme a 6-glucosaminyltransferase converts Gal3GalNAc chains in mucin glycoproteins to Gal3(GlcNAc6)GalNAc chains. This product is the substrate for a second 3-glucosaminyltransferase which converts the Gal3(GlcNAc6)GalNAc chains to GlcNAc3Gal(GlcNAc6)GalNAc chains in the glycoprotein. The 3-glucosaminyltransferase did not utilize Gal3GalNAc chains as a substrate and this results in an ordered sequence of addition of N-acetylglucosamine residues to growing oligosaccharide chains in tracheal mucin glycoproteins.Abbreviations NeuNAc N-acetylneuraminic acid - GalNAcol N-acetylgalactosaminitol - CGMG Cowper's gland mucin glycoprotein - GalNAc-CGMG Cowper's gland mucin glycoprotein containing GalNAc side chains O-glycosidically linked to serine or threonine - Gal3GalNAc-CGMC Cowper's gland mucin glycoprotein containing Gal3GalNAc side chains - MES 2-(N-morpholino) Ethane Sulfonic acid - PBS Phosphate Buffered Saline     

Identification and characterization of the functional alpha origin of DNA replication of the R6K plasmid and its relatedness to the R6K beta and gamma origins

Summary The functional R6K origin is composed of two DNA elements, one of 580 bp carrying the origin sequences and the other of 277 bp containing the seven 22 bp direct repeats previosly identified as also required for and origin activity. These two genetic elements are separated by approximately 3,000 bp of R6K sequences which are dispensable for origin activity. The function of the origin depends on the presence in cis of the 580 bp and the 277 bp fragments and requires that they be oriented as in the intact R6K. Activation of the origin depends on the R6K replication initiation protein .Within the 580 bp of the origin, there is a sequence of 98 bp which appears as an inverted repeat of 96 bp in the replicon. Deletion of the 96 bp or 98 bp results in inactivation of the and the origins respectively. These long repeats are palindromic and it is suggested that these may serve as the recognition signals for initiation of DNA replication in the and the origins of R6K. DNA homology analysis performed on , and origin sequences, also reveals 10–23 bp sequences in the and the origins that are related to the family of 22 bp direct repeats in the origin which were shown previously to be binding sites for the protein.     

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.     

α-and β-Glycosidases in maize roots

Four glycosidases were analyzed in 10 mm apical segments prepared from growing roots (15 mm) of Zea mays L. The pH optima were found to be 5.8 for -glucosidase, 4.4 for -galactosidase, 6.4 for -glucosidase and 6.0 for -galactosidase. The -glucosidase showed 4-fold higher activity than the -galactosidase. The distribution of the -glucosidase activity was signifcantly different from that of the -galactosidase, -glucosidase and -galactosidase.Abbreviations -Glu -glucosidase - -Gal -galactosidase - -Glu -glucosidase - -Gal -galactosidase     

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.