Comparative study of the production of extracellular β-glucosidase by four different strains of Aspergillus using submerged fermentation

Four strains of Aspergillus (Aspergillus niger CDBB-H-176, A. niger CDBB-H-175, A. niger ATCC 9642, and Aspergillus terreus CDBB-H-194) were used to produce extracellular β-glucosidase. Using an orthogonal experimental design (L₉), we optimized the parameters of culture medium to maximize the activity of β-glucosidase. The optimal conditions (same for the four strains) were as follows: temperature, 30°C; pH, 6.0; orbital agitation, 200?rpm; concentration of sucrose, 0.5% (w/v). The most productive strain was A. niger CDBB-H-175, with a yield of 701.2?U/mL. In a second stage, we optimized (L₁₈) the concentration of nutrients in the culture medium to determine whether this modification would increase the production of β-glucosidase. The optimal conditions for A. niger CDBB-H-175 were as follows (%, w/v): NaNO₃, 0.3; KCl, 0.3; KH₂PO₄, 0.15; NH₄NO₃, 0.1; NH₄H₂PO₄, 0.1; MgSO₄?·?7H₂O, 0.05; yeast extract, 0.1. The production of β-glucosidase under these conditions was 1207.9?U/mL. Enzymatic assays were used to characterize the enzyme; the optimum temperature and pH of β-glucosidase produced by the four selected micro-organisms were found to be 65°C and 5.0, respectively. We determined the Michaelis–Menten constants (K_m) only for A. niger CDBB-H-175 and CDBB-H-176; the values were 2.7 and 2.2?mM, respectively.     

Production of aromatic d-amino acids from α-keto acids and ammonia by coupling of four enzyme reactions

A multi-enzyme system composed of glutamate racemase, thermostable d-amino acid aminotransferase, glutamate dehydrogenase and formate dehydrogenase was employed for the production of aromatic d-amino acids, d-phenylalanine and d-tyrosine, from the corresponding α-keto acids, phenylpyruvate and hydroxyphenylpyruvate, respectively. The optimal concentration of ammonium formate for the production of these d-amino acids was found in the range of 0.25–1.0 M. The optimal concentration of α-keto acid was determined to be 50 mM, above which the productivity greatly decreased. To keep the concentration of α-keto acid around this concentration, α-keto acid was intermittently fed into the multi-enzyme system during the production period. By running the multi-enzyme system for 35 h, 48 g l⁻¹ of d-phenylalanine and 60 g l⁻¹ of d-tyrosine were produced with 100% of optical purity from the equimolar amounts of phenylpyruvate and hydroxyphenylpyruvate, respectively. The production levels of both aromatic d-amino acids were demonstrated to be dependent on the stability of glutamate racemase.     

Mass spectrometric analysis of rat hemoglobin by FAB-overlapping: Primary structure of the α-major and of four β constitutive chains

• 1.1. The globin chain components of Sprague-Dawley rat hemoglobin were obtained by reverse-phase HPLC which showed the presence of two α-chain and four β-chains.
• 2.2. The accurate molecular weight of each globin chain was determined by means of electrospray mass spectrometry. Extensive mass spectrometric analysis on several enzymatic digests by fast atom bombardment mass spectrometry (FAB-overlapping) meant to determine the complete sequence of the α-major and of the four β-globins.
• 3.3. The primary structure of the α-major globin was found in agreement with literature data (Garrick et al., 1975 Biochem. J.149, 245–258; Chua et al., 1987).
• 4.4. Sequence analysis of the four β -globin chains showed that amino acid differences are restricted to two protein portions: the region 22–25 and 123–125, the remaining portions of the molecule being unchanged in the four globins. Furthermore, all the amino acid replacements correspond to single point DNA mutations and (with the exception of the substitution Asp 22 → Asn in the β₂-globin) involve uncharged substitutions.

     

Mediation by calcium/calmodulin-dependent protein kinase II of suppression of GABA_A receptors by NMDA

Using nystatin-perforated whole-cell recording configuration, the modulatory effect of N-methyl-D-aspartate (NMDA) on γ-aminobutyric acid (GABA)-activated whole-cell currents was investigated in neurons freshly dissociated from the rat sacral dorsal commissural nucleus (SDCN). The results showed that: (i) NMDA suppressed GABA- and muscimol (Mus)-activated currents (IGABA and IMUS), respectively in the Mg2 -free external solution containing 1 μmol/L glycine at a holding potential (VH) of -40 mV in SDCN neurons. The selective NMDA receptor antagonist, D-2-amino-5-phosphonovaleric acid (APV, 100 μmol/L), inhibited the NMDA-evoked currents and blocked the NMDA-induced suppression of IGABA; (ii) when the neurons were incubated in a Ca2 -free bath or pre-loaded with a membrane-permeable Ca2 chelator, BAPTA AM (10 nmol/L), the inhibitory effect of NMDA on IGABA disappeared. Cd2 (10 μmol/L) or La3 (30 μmol/L), the non-selective blockers of voltage-dependent calcium channels, did not affect the suppressio     

Control of lymphocyte infiltration of lung tumors in mice by host’s genes: mapping of four Lynf (lymphocyte infiltration) loci

Tumor infiltration by lymphocytes is essential for cell-mediated immune elimination of tumors in experimental systems and in immunotherapy of cancer. Presence of lymphocytes in several human cancers has been associated with a better prognosis. We present evidence that individual propensity to tumor infiltration is genetically controlled. Infiltrating lymphocytes are present in 50% of lung tumors in O20/A mice, but in only 10% of lung tumors in OcB-9/Dem mice. This difference has been consistent in experiments conducted over 8 years in two different animal facilities. To test whether this strain difference is controlled genetically, we analyzed the presence of infiltrating lymphocytes in N-ethyl-N-nitroso-urea (ENU) induced lung tumors in (O20 × OcB-9) F₂ hybrids. We mapped four genetic loci, Lynf1 (Lymphocyte infiltration 1), Lynf2, Lynf3, and Lynf4 that significantly modify the presence and intensity of intra-tumoral infiltrates containing CD4⁺ and CD8⁺ T lymphocytes. These loci appear to be distinct from the genes encoding the molecules that are presently implicated in lymphocyte infiltration. Our findings open a novel approach for the assessment of individual propensity for tumor infiltration by genotyping the genes of the host that influence this process using DNA from any normal tissue. Such prediction of probability of tumor infiltration in individual cancer patients could help considerably to assess their prognosis and to decide about the application and the type of immunotherapy.     

Which cervical sampler? A comparison of four methods

Four cytology sampling methods were compared in 1063 patients referred for colposcopy with a recent abnormal smear. A dyskaryotic smear of any grade was considered a positive result, though comparisons were limited to cases with a subsequent biopsy confirming CINII or III. There were no differences between the abilities of any of the four methods to detect higher grades of CIN (χ²₃=4.603, P >0.20). the presence or absence of endocervical cells in a smear was not significantly associated with any variation in success rate (χ²₁=0.959, P >0.30). the joint analysis of the four methods and the presence/absence of endocervical cells also showed no significant effects (χ²₇=12.768, 0.1 > P >0.05). In the latter analysis the trend towards a conventional level of significance was accounted for by the Aylesbury spatula giving a relatively high success rate when endocervical cells were present. the suggestion of advantage for the Aylesbury spatula merits further investigation.     

Mapping of18-26S rDNA loci in four species of the genus Paris by fluorescence in situ hybridization（FISH）

18-26S rDNA loci were mapped on chromosomes in four species of Par is,and the num-ber and position of rDNA sites in these species were compared f or analysis of the distribution of the sites. All the plants were diploids,and t he genome consisted of five chromosomes,A,B,C,D and E. （1）P. polyphylla var. yunnanensis,2n=10=6m+4t. Two18-26S rDNA loci were de-tected on the short arms o f C and D chromosomes;（2）P. forrestii,2n=10=6m+4t. One locus was detected on th e long arm of B chromosome,and also two loci on the short arms of C and D chromosomes;（3）P. axialis. 2n=10=6m（2sat）+4t（2sat）+1-2B. Two loci were detected o n the short arms of C and D chromosomes. One locus was detected in the cell with t wo B-chromosomes（B）,but none was detected in that with only one B chromosome, indicating that rRNA gene existed on B chromsome,and an unequal division occurr ed during mitotic cycle of B-chromosomes. （4）P. daliensis,2n=10=4m+2sm+2st+2t. O ne locus was detected on the short arm of D chromo-some. The signals of18-26S rD NA appeared not only in the second constriction but also in the other regions of chromosome. It is noteworthy that one locus was detected in the terminal region o n the short arm of C chromosome in all the four species studied.     

Enhancement of the mutagenicities of N-methyl-N′-nitro-N-nitrosoguanidine and N-methyl-N-nitrosourea by glucose

The mutagenicity of N-methyl-N′-nitro-N-nitrosoguanidine to Salmonella typhimurium hisG46 was enhanced by pre-incubating the chemical with bacteria in sodium phosphate buffer. Addition of glucose (to 15 mM) to the pre-incubation mixture further enhanced the mutagenicity. Pre-incubation with glucose also increased the mutagenicity of N-methyl-N-nitrosourea. Fructose, galactose, pyruvate and succinate also enhanced the mutagenicity of N-methyl-N′-nitro-N-nitrosoguanidine. The effect of glucose was observed with S. typhimurium strains hisG46, TA1975, TA1950, TA1535 and TA100.     

Unsegmented annelids? Possible origins of four lophotrochozoan worm taxa

In traditional classification schemes, the Annelida consists of the Polychaeta and the Clitellata (the latter including the Oligochaeta and Hirudinida). However, recent analyses suggest that annelids are much more diverse than traditionally believed, and that polychaetes are paraphyletic. Specifically, some lesser-known taxa (previously regarded as separate phyla) appear to fall within the annelid radiation. Abundant molecular, developmental, and morphological data show that the Siboglinidae, which includes the formerly recognized Pogonophora and Vestimentifera, are derived annelids; recent data from the Elongation Factor-1α (EF-1α) gene also suggest that echiurids are of annelid ancestry. Further, the phylogenetic origins of two other lesser-known groups of marine worms, the Myzostomida and Sipuncula, have recently been called into question. Whereas some authors advocate annelid affinities, others argue that these taxa do not fall within the annelid radiation. With advances in our understanding of annelid phylogeny, our perceptions of body plan evolution within the Metazoa are changing. The evolution of segmentation probably is more plastic than traditionally believed. However, as our understanding of organismal evolution is being revised, we are also forced to reconsider the specific characters being examined. Should segmentation be considered a developmental process or an ontological endpoint?     

Oxidation of Bovine β-Casein by Hypochlorite

We recently observed two 2,4-dinitrophenylhydrazine (DNPH)-reactive proteins of 40 and 120 kDa in the bronchoalveolar lavage fluids of rats exposed to >95% O₂ for 48 h. The N-terminal sequences of these proteins were both identical over 16 amino acids with rat β-casein, which, in addition to its more common association with milk, is produced by cytotoxic T-lymphocytes, and has been found to have proinflammatory properties. Because of the inflammatory response that accompanies hyperoxic lung injury, we investigated the oxidation of bovine β-casein by HOCl. Following exposure to HOCl at 4°C for 15 min, derivatization with DNPH, washing, and digestion with trypsin, the resultant peptides were separated by reverse-phase HPLC. One peptide isolated from a peak absorbing at 365 nm was identified as AVP(Y*)PQR, corresponding to amino acids 177–183 of bovine β-casein. Analysis of the peptide by both electrospray and matrix assisted laser desorption ionization (MALDI) mass spectrometry identified a molecular ion MH⁺ of 1008.5 Da, which represents an increase of 178 Da from the calculated monoisotopic MH⁺ of the unmodified peptide of 830.45 Da. Daughter ion spectra of the doubly charged parent ion of the peptide further support the oxidation of the tyrosine to the quinone methide, with subsequent conversion to the corresponding hydrazone with DNPH. A second pair of products were identified as arising from oxidation of Y¹⁹³ within the tryptic peptide constituted by amino acids 184–202, and the corresponding chymotryptic cleavage side product, 191–202. Exposure of β-casein to increasing amounts of HOCl revealed that M and Y residues were the most susceptible, although bovine β-casein contains no C, and a single W, which would not be detected by our methods. The approach described in the present report can be used to evaluate the contributions of distinct mechanisms of oxidation in other experimental or pathological models. © 1997 Elsevier Science Inc.     

Inheritance and biochemical analysis of four electrophoretic variants of β-conglycinin from soybean

Summary Three genes which code for variant -conglycinin subunits were identified. Alleles Cgy ₁ ^S and Cgy ₂ ^S were codominant with Cgy ₁ and Cgy ₂ and produced and subunits, respectively, with reduced electrophoretic mobility. Allele Cgy ₃ ^D increased the mobility of at least one polypeptide in the subunit family and exhibited incomplete dominance. Gene loci Cgy ₂/Cgy ₂ ^S and Cgy ₃ ^D /cgy ₃ ^D were linked, whereas Cgy ₁/Cgy ₁ ^S / cgy ₁ segregated independently of the others. Techniques developed for purification of normal -conglycinin subunits were effective in purifying the altered subunits. Deglycosylated variant proteins from seeds containing the alleles Cgy ₁ ^S , Cgy ₂ ^S , or Cgy ₃ ^D also has altered mobility relative to deglycosylated normal proteins. Therefore, the altered subunits contained changes in their amino acid sequences rather than in their carbohydrate moieties. This interpretation is consistent with the observed codominant or incompletely dominant mode of inheritance for these alleles and suggests that each contains an altered nucleotide sequence in the structural gene. A fourth variant, which exhibited doublet and a electrophoretic bands, was inherited in a recessive fashion. Deglycosylated subunit proteins from this variant were identical in electrophoretic mobility to those of the deglycosylated normal protein. This suggests that the doublet phenotype resulted from an alteration in the carbohydrate moiety of these subunits. The gene or genes which condition this variant presumably are required for normal post-translational modification of the subunit carbohydrates and as such may be useful for investigating these events.Cooperative research of USDA-ARS and the Indiana Agric. Exp. Stn., Purdue Univ., West Lafayette, IN 47907, USA. Indiana Agric. Exp. Stn. Journal Article 10,323. Financial support from the American Soybean Research Foundation is gratefully acknowledged     

Biomass production by alders on four abandoned agricultural soils in Québec

The production of aboveground tissue of three alder species (Alnus crispa (Ait.) Pursh,A. rugosa (Du Roi) Spreng. andA. glutinosa (L) Gaertn.) on four sites ranged from 0.4 t ha^–1 yr^–1 to 4.0 t ha^–1 yr^–1 after four growing seasons. Large differences were observed among the four sites studied and among species. Soil nutrient levels affected the biomass production and foliar symptoms of P and Mg deficiency occurred withA. crispa andA. rugosa. Because of their poor aboveground biomass production (0.4–1.4 t ha^–1 yr^–1),A. crispa andA. rugosa should be used mainly as nurse trees. For its higher potential for biomass production (up to 4.0 t ha^–1 yr^–1), and its apparent higher ability to use P and Mg on deficient sites,A. glutinosa should be used preferably toA. crispa andA. rugosa for the production of biomass.     

The magic of four： induction of pluripotent stem cells from somatic cells by Oct4, Sox2, Myc and Klf4

The developmental process from a fertilized egg to agrown adult is programmed with remarkable accuracy.While the genetic information of the fertilized egg and itsdescendent somatic cells are the same, it is the selectiveexpressions of the same genome that give rise to the 200or so different cell types in an adult. The differentiatedstates of these adult cells are maintained epigenetically,presumably through the modification of chromatins and theassociated histones. In higher mammals, it was thought thatthe differentiation process is irreversible until the successfulcloning of Dolly [1]. By transferring a nucleus from a fullydifferentiated cell in the mammary gland, Wilmut and col-leagues were able to generate an exact replica of a highermammal, the Doily [1]. This work not only demonstratedthat the genome of differentiated cells can be reprogrammedinto an embryonic state and then to resume a full-fledgeddevelopmental process to generate a normal adult, but alsorejuvenated the field of animal cloning. The prospect thata somatic cell from a patient may be reprogrammed to the     

Activation of phosphates to substitution(II). A comparison of catalyses initiated directly by VO2+ and by oxidation of VO2+ by MnO4−

Vanadium(V)-induced hydrolyses of triphosphates in aqueous solutions were initiated in two ways: (1) oxidizing vanadium(IV)-polyphosphate complexes to produce metastable vanadium(V) complexes; (2) forming VO₂⁺-polyphosphate complexes by acidification of solutions of VO₄^3? and polyphosphate to yield equilibrium mixtures of V(V), polyphosphate, and their complexes. Hydrolysis rates for the complexes formed at 40°C ? T ? 25°C follow the order V₂PPP_i = 2(VPPP_i) ≌ (VO₂) ATP ? V(PPP_i)₂ ≌ PPP_i. The hydrolysis of (V(V))₂(PPP_i) was not very temperature sensitive; the activation enthalpy appears small and the activation entropy large and negative. Mechanistic studies reveal that requirements for the activated state in metal-ion-catalyzed hydrolysis of polyphosphates include monodentate polyphosphate ligated cis to H₂O or OH^? in the coordination sphere of the metal ion:

     

Inhibition of cholesterol esterases by Δ1-tetrahydrocannabinol

Δ¹-Tetrahydrocannabinol was found to inhibit the action of esterases derived from rat adrenal and luteinized ovary on exogenous cholesteryl palmitate. The drug was effective at a dose of 3.2μM causing greater than 30% inhibition; at 16μM almost complete inhibition occured. These findings are similar to those we have recently reported with mouse Leydig cells (1) showing that this is an effect common to steroidogenic tissues and raising the possibility that a variety of endocrine effects of this drug may be due to direct action on these tissues.