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101.
Calvet S Clapés P Vigo JP Xaus N Jorba X Mas RM Torres JL Valencia G Serralheiro ML Cabral JM Empis JM 《Biotechnology and bioengineering》1992,39(5):539-549
The influence of five different N-terminal protecting groups (For, Ac, Boc, Z, and Fmoc) and reaction conditions (temperature and dimethylformamide content) on the alpha-chymotrypsin-catalyzed synthesis of the dipeptide derivative X-Phe-Leu-NH(2) was studied. Groups such as For, Ac, Boc, and Z always rendered good peptide yields (82% to 85%) at low reaction temperatures and DMF concentrations, which depended on the N-alpha protection choice. Boc and Z were the most reactive N-alpha groups and, in addition, the most suitable for peptide synthesis. On the other hand, the use of empirical design methodologies allowed, with minimal experimentation and by multiple regression, to deduce an equation, which correlates the logarithm of the first order kinetic constant (log k') with reaction temperature, DMF concentration, and hydrophobicity (log P values) of the different protecting groups. The predictive value of the equation was tested by comparing the performance of another protective group, such as Aloc, with the performance predicted by said equation. Experimental and calculated k' values were found to be in good agreement. 相似文献
102.
The aquatic fungus Blastocladiella emersonii provides a system for studying the regulation of expression of regulatory (R) and catalytic (C) subunits of cAMP-dependent protein kinase (PKA). Blastocladiella cells contain a single PKA with properties very similar to type II kinases of mammalian tissues. During development cAMP-dependent protein kinase activity and its associated cAMP-binding activity change drastically. We have previously shown that the increase in cAMP-binding activity during sporulation is due to de novo synthesis of R subunit and to an increase in the translatable mRNA coding for R (Marques et al., Eur. J. Biochem. 178, 803, 1989). In the present work we have continued these studies to investigate the mechanism by which the changes in the level of kinase activity take place. The C subunit of Blastocladiella has been purified; antiserum has been raised against it and used to determine amounts of C subunit throughout the fungus' life cycle. A sharp increase in C subunit content occurs during sporulation and peaks at the zoospore stage. Northern blot analyses, using Blastocladiella C and R cDNA probes, have shown that the levels of C and R mRNAs parallel their intracellular protein concentrations. These results indicate a coordinate pretranslational control for C and R subunit expression during differentiation in Blastocladiella. 相似文献
103.
L. Oliveira 《Planta》1992,188(3):279-288
Germination of aplanospores in Vaucheria longicaulis Hoppaugh var. macounii Blum proceeds through three stages of development. Stage I begins with the initiation of germination and lasts approx. 2 h. During this stage germinating filaments grow at an accelerated rate (266 ± 12 m · h–1). Stage II is characterized by a sharp decline in the growth rate of germinating filaments (96 ± 4 m · h–1) and lasts 4 h. This is followed, during the next 4 h, by a recovery in the growth rate (168 ± 8 m · h–1) of germinating filaments, stage III. Growth rates stabilize and remain unchanged during subsequent development (Oliveira and Fitch, 1988, J. Submicrosc. Cytol. Pathol. 20, 397–406). The Ca2+-influx modulators LaCl3, nifedipine and methyl 1,4-dihydro-2,6-dimethyl-3-nitro-4 (2-trifluoromethylphenyl)-pyridine-5-carboxylate (Bay K-8644), the ionophore calcimycin (A23187), the intracellular Ca2+-release antagonist 8-N-N'-(diethylamino)-octyl-3,4, 5-trimethoxybenzoate (TMB-8), the Ca2+-uptake inhibitor ruthenium red and the phosphoinositide-cycle modulators LiCl and myo-inositol show that the events required for the initiation are distinct from those required for the completion of each stage of germination. These studies in conjunction with microinjection of germinating filaments with inositol 1,4,5-trisphosphate, the natural ligand for Ca2+ release from Ca-storing organelles (endoplasmic reticulum, vacuole), and treatment with chlorotetracycline (CTC), to visualize the distribution of membrane-bound Ca2+ reveal that both the initiation and completion of each stage of germination are controlled by Ca2+ signals which are restricted to well-defined time intervals and are modulated by the origin (source) of Ca2+.Abbreviations BAPTA
1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid
- Bay K-8644
methyl 1,4-dihydro-2,6-dimethyl-3-nitro-4(2-trifluoromethylphenyl)-pyridine-5-carboxylate
- CTC
chlorotetracycline
- InsP3
inositol 1,4,5-trisphosphate
- RR
ruthenium red
- TMB-8
8-N-N-(diethylamino)-octyl-3,4,5-trimethoxybenzoate
The author wishes to express his gratitude to the technical group of the Immunocytochemistry Unit for their help with the microinjection studies. This work was supported by a grant from the Natural Sciences and Engineering Research Council of Canada (grant A-7844). 相似文献
104.
K Fischer-Stenger A W Updegrove G A Cabral 《Proceedings of the Society for Experimental Biology and Medicine. Society for Experimental Biology and Medicine (New York, N.Y.)》1992,200(3):422-430
The purpose of this study was to examine the effect of delta 9-tetrahydrocannabinol (delta 9-THC), the major psychoactive component of marijuana, on T lymphocyte functional competence against herpes simplex virus Type 1 (HSV1) infection. Spleen cells from C3H/HeJ (H-2k) mice primed with HSV1 and exposed to delta 9-THC were examined for anti-HSV1 cytolytic T lymphocyte (CTL) activity. Flow cytometry was used to determine whether delta 9-THC altered T cytotoxic (Lyt-2+) and T helper (L3T4+) lymphocyte numbers or cell ratios. Nomarski optics microscopy was used to determine whether effector lymphocytes from drug-treated mice were able to bind to virally infected L929 (H-2k) target cells. Cytotoxicity assays demonstrated that CTL from mice exposed to delta 9-THC were deficient in anti-HSV1 cytolytic activity. delta 9-THC in vivo treatment had little effect on the number of T lymphocytes expressing the Lyt-2 or L3T4 antigens. Nomarski optics microscopy revealed that the CTL from the drug-treated mice were able to bind specifically to the HSV1-infected targets. However, delta 9-THC in vivo exposure affected CTL cytoplasmic polarization toward the virus-infected target cell. CTL granule reorientation toward the effector cell-target cell interface following cell conjugation occurred at a lower frequency in co-cultures containing CTL from drug-treated mice. These results suggest that delta 9-THC elicits dysfunction in CTL by altering effector cell-target cell postconjugation events. 相似文献
105.
J. A. L. Santos J. M. S. Cabral C. L. Cooney 《Bioprocess and biosystems engineering》1992,7(5):205-211
This work describes the recovery of an extracellular alkaline protease from fermentation broths of a Bacillus sp ATCC 21536, at pH=10.0 using ultrafiltration (MWCO 100,000) and microfiltration (0.1 m) membranes in hollow fiber devices. The influence of membrane pore size and polymeric material and membrane filtration performance was studied. High protein recoveries and high average flux rates were obtained with polysulfone membranes. A decrease of concentration polarization was obtained, simultaneously with enhancement of filtration flux rate and enzyme recovery by using submicron sized charged particles. These polymers lead to flocculation and adsorption of whole cells and soluble factors from the fermentation broth. The best results were obtaiend by combination of cationic (0.1%) and anionic (0.04%) polymers. 相似文献
106.
H B Nader M A Porcionatto I L Tersariol M A Pinhal F W Oliveira C T Moraes C P Dietrich 《The Journal of biological chemistry》1990,265(28):16807-16813
The purification of two heparitinases and a heparinase, in high yields from Flavobacterium heparinum was achieved by a combination of molecular sieving and cation-exchange chromatography. Heparinase acts upon N-sulfated glucosaminido-L-iduronic acid linkages of heparin. Substitution of N-sulfate by N-acetyl groups renders the heparin molecule resistant to degradation by the enzyme. Heparitinase I acts on N-acetylated or N-sulfated glucosaminido-glucuronic acid linkages of the heparan sulfate. Sulfate groups at the 6-position of the glucosamine moiety of the heparan sulfate chains seem to be impeditive for heparitinase I action. Heparitinase II acts upon heparan sulfate producing disulfated, N-sulfated and N-acetylated-6-sulfated disaccharides, and small amounts of N-acetylated disaccharide. These and other results suggest that heparitinase II acts preferentially upon N,6-sulfated glucosaminido-glucuronic acid linkages. The total degradation of heparan sulfate is only achieved by the combined action of both heparitinases. The 13C NMR spectra of the disaccharides formed from heparan sulfate and a heparin oligosaccharide formed by the action of the heparitinases are in accordance to the proposed mode of action of the enzymes. Comparative studies of the enzymes with the commercially available heparinase and heparitinase are described. 相似文献
107.
Dulce E. Oliveira Ana Lúcia C. Santos Neto Anita D. Panek 《Analytical biochemistry》1981,113(1):188-192
A quantitative in situ assay of yeast α-glucosidase involving permeabilization of the cells by freezing and thawing is described. The assay was applied to different strains in different physiological states and was shown to give results comparable to those obtained with total cell homogenates. The primary advantage of the in situ assay was the possibility of analyzing a large number of samples from the same culture during a growth curve using a very reduced cell mass. 相似文献
108.
Wafa H. Cabrera Olga M. Ibanez Silvio L. Oliveira Osvaldo A. Sant'Anna Maria Siqueira Denise Mouton Guido Biozzi 《Immunogenetics》1982,16(6):583-592
The effect of the selective breeding of mice for high or low antibody production to complex immunogens is largely nonspecific, since it modifies the responsiveness of high (H) and low (L) lines to many antigens unrelated to the selection antigen. However, the nonspecific effect of the polygenic control operating in these lines is not a general feature. For example, the group of genes in selection IV, carried out for responsiveness to somatic antigen of Salmonella, does not modify the responses to sheep erythrocytes (SE). Despite equivalent responses in H and L mice of selection IV, a large variability was found in individual responses of F2 interline hybrids, which demonstrates the presence of alleles with high or low effect on responses to SE. A selective breeding (Selection IV-A) was therefore initiated from this F2 population for responsiveness to SE. A progressive interline divergence occurred during the first seven generations of selection; the interline separation was due to polygenic regulation (about four independent loci from a preliminary estimate).Equivalent responses to the s antigen of Salmonella are observed in the two lines. This constitutes additional evidence for distinct polygenic regulation of responses to SE and to somatic antigen. Moreover, the pattern of responses to several unrelated antigens (nonspecific effect) also differs between Selections IV and IV-A.Abbreviations H
high responder lines
- L
low responder lines
- s
somatic antigen of Salmonella
- f
flagellar antigen of Salmonella
- R
response to selection
- S
selection differential
- F0
foundation population
- h2
heritability (realized)
- RGG
rabbit gamma globulin
- CE
chicken erythrocyte
- HE
human erythrocyte
- PE
pigeon erythrocyte
- SE
sheep erythrocyte 相似文献
109.
Glucoamylase (exo-1,4-α-d-glucosidase, EC 3.2.3.1) was coupled to several porous silica matrices by an improved metal-link/chelation process using alkylamine derivatives of titanium(IV)-activated supports. In order to select the titanium activation procedure which gave stable enzyme preparations, long-term stability tests were performed. The immobilized glucoamylase preparations, in which the carrier was activated to dryness with a 15% w/v TiCl4 solution, displayed very stable behaviour, with half-lives of ~60 days. The optimum operating conditions were determined for these preparations. There are significant differences between the behaviour of the immobilized enzyme and the free enzyme. The apparent Km increased on immobilization due to diffusional resistances. The pH optimum for the immobilized preparation showed a slight shift to acid pH relative to that of the soluble enzyme. Also, the optimum temperature descreased to 60°C after immobilization. In order to test Michaelis-Menten kinetics at high degrees of conversion, time-course analysis of soluble starch hydrolysis was performed. It was observed that simple Michaelis-Menten kinetics are not applicable to the free/immobilized glucoamylase-starch system at high degrees of conversion. 相似文献
110.
Female Agus rats developed hepatic porphyria at a much faster rate than female Porton-Wistar rats when fed a diet containing 0.01% of hexachlorobenzene (HCB). They also showed a greater inhibition of liver uroporphyrinogen decarboxylase [EC 4.1.1.37] activity and a marked stimulation of 5-aminolaevulinate synthetase [EC 2.3.1.37]. The difference between the two strains could not be correlated with differences in the liver concentrations of HCB. However, control Agus rats were found to possess significantly higher levels of total non-haem iron in their livers than the Porton animals. This was particularly apparent after 24 h of starvation and is further evidence for the involvement of iron in the pathogenesis of HCB-induced porphyria. The posterior lobes of the livers from the Agus rats given HCB became porphyric more slowly than the remainder with less severe inhibition of uroporphyrinogen decarboxylase. In contrast to their increased susceptibility to HCB, the Agus rats were less susceptible to another prophyrogenic agent, 3,5-diethoxycarbonyl-1,4-dihydrocollidine. 相似文献