A partially purified enzyme (carbocyclase) from the flavedo of Citrus limonum formed α-pinene, β-pinene, limonene, and γ-terpinene from geranyl pyrophosphate (GPP) and neryl pyrophosphate. The maximum specific activities obtained were 7.0 and 3.6 nmol/ min/mg, respectively. Cross-inhibition by the two substrates were observed and the ability to utilize neryl pyrophosphate was almost completely lost with aging. Citronellyl pyrophosphate and dimethylallyl pyrophosphate were the most effective inhibitors of carbocyclase. Isopentenyl pyrophosphate, the monophosphate esters of nerol and geraniol, as well as inorganic pyrophosphate were much less effective inhibitors. The enzyme had an absolute requirement for Mn2+. It could be replaced with about 2% effectiveness by Mg2+ and Co2+. Kinetic studies showed that the observed reaction rate correlates with the calculated concentration of the GPP (Mn2+)2 species. Previous evidence with nonenzymatic reactions and the results presented support the view that the mechanism of carbocyclase may be the intramolecular analog of prenyltransferase. 相似文献
The pg1 gene from the filamentous fungus Aspergillus kawachii, which codifies for an acid polygalacturonase, was cloned into the pYES2 expression vector, giving rise to the pYES2:pg1ΔI construct. Engineered Saccharomyces cerevisiae, transformed with pYES2:pg1ΔI construct, both expressed and exported an active polygalacturonase with a MW of ~60 kDa and an isoelectric point of 3.7, similar to those reported for the wild-type enzyme. The recombinant enzyme has the ability to hydrolyze polygalacturonic acid at pH 2.5. Heterologous PG1 production was studied under controlled conditions in batch and fed-batch systems. A simultaneous addition of glucose and galactose was found to be the most suitable feeding strategy assayed, resulting in a final PG1 production of 50 U/ml. The production process proposed in this study could be applied for the industrial production of a novel and useful polygalacturonase. 相似文献
Chagas' disease or American trypanosomiasis is a parasitic zoonosis which constitutes and important public health problem in most of the Latin American countries. According to the development of socio-political events in the world, it is possible at present to speak of rural-periurban Chagas' disease and urban Chagas' disease. Rural-periurban Chagas' disease. In its endemo-enzootic condition it is distributed in vast areas from Mexico in the north and Argentina and Chile in the south. It is calculated that the population at risk is about 90 million persons, not less than 16-18 million are Trypanosoma cruzi infected and approximately 38% of these present or have presented pathology caused by the parasite. Organs most frequently affected: heart, esophagus and colon. The corresponding biological vectors are hematophagus triatomid bugs, with greater than 100 species synantropic (st) or sylvatic (sv), existing between parallels 41 N. and 46 S., but only about 36, which have been found infected, have some relationship with man because their adaptation to human dwelling. The human parasitose is less extended due to the fact that the vectors of the region are predominantly sv. The known reservoirs are more than 180 species of terrestrial mammals: domestic, st and sv. Man is possibly the most important. Some available relevant epidemiological information is summarized as follows: Additionally, some autochthonous cases of T. cruzi human infection have been registered in the United States, Trinidad-Tobago, Guyana and Belize. Moreover, infected vectors and/or sv reservoirs have been observed in almost a dozen of Caribbean countries. Urban Chagas' disease. As a consequence of possible better salaries and many other motivations, in the last decades there have been significant and constant migrations from rural to urban areas in many Latin American countries. This situation has facilitated the dissemination of T. cruzi infection through infected reservoirs--mostly humans--and/or passively transported infected vectors. In most of the cases these rural-urban migrations occur in chagasic endemic areas within a same country or in neighbouring ones; in others, the migration can involve countries where Chagas' disease does not exist, transmission being via blood transfusion or placental. According to some estimates, with a mean rate of 1.5% chagasic infected blood donors the minimum risk of T. cruzi transmission is nearly 12.5-25.0% when the volumen of transfused blood is 500 ml.(ABSTRACT TRUNCATED AT 400 WORDS) 相似文献
Seriola lalandi is an economically important species that is globally distributed in temperate and subtropical marine waters. Aquaculture production of this species has had problems associated with intensive fish farming, such as disease outbreaks or nutritional deficiencies causing high mortality. Intestinal microbiota are involved in many processes that benefit a host, such as disease control, stimulation of the immune response, and the promotion of nutrient metabolism. The aim of this study is to evaluate the in vitro probiotic properties of bacteria isolated from the intestinal content of wild Seriola lalandi. The probiotic potential was evaluated in terms of (i) the antimicrobial activity against vibrios causing outbreaks in farmed fish; (ii) the ability to stimulate genes related to an innate immune response in fish; and (iii) antibiotic resistance. Nineteen isolates identified as Pseudomonas, Shewanella, Psychrobacter, and Acinetobacter showed antimicrobial activity and significant relative expression of cytokines, serum amyloid A protein (SAA), hepcidin, and lysozyme. A positive correlation was observed between the levels of expression and the bacterial load after 24 h of exposure. Pseudomonas isolates showed a level of antibiotic resistance. In conclusion, isolates of the genera Shewanella, Psychrobacter, and Acinetobacter could serve as potential probiotics in S. lalandi culture.
Plant Cell, Tissue and Organ Culture (PCTOC) - A biotechnological system for the production of plants biomass and phenylpropanoids of maqui was developed in photomixotrophic TIBs. The in vitro... 相似文献
CmPI-II is a Kazal-type tight-binding inhibitor isolated from the Caribbean snail Cenchritis muricatus. This inhibitor has an unusual specificity in the Kazal family, as it can inhibit subtilisin A (SUBTA), elastases and trypsin. An alanine in CmPI-II P1 site could avoid trypsin inhibition while improving/maintaining SUBTA and elastases inhibition. Thus, an alanine mutant of this position (rCmPI-II R12A) was obtained by site-directed mutagenesis. The gene cmpiR12A was expressed in P. pastoris KM71H yeast. The recombinant protein (rCmPI-II R12A) was purified by the combination of two ionic exchange chromatography (1:cationic, 2 anionic) followed by and size exclusion chromatography. The N-terminal sequence obtained as well as the experimental molecular weight allowed verifying the identity of the recombinant protein, while the correct folding was confirmed by CD experiments. rCmPI-II R12A shows a slightly increase in potency against SUBTA and elastases. The alanine substitution at P1 site on CmPI-II abolishes the trypsin inhibition, confirming the relevance of an arginine residue at P1 site in CmPI-II for trypsin inhibition and leading to a molecule with more potentialities in biomedicine. 相似文献
Regulation of eukaryotic cell cycle progression requires sequential activation and inactivation of cyclin-dependent kinases (CDKs). Activation of the cyclin B-cdc2 kinase complex is a pivotal step in mitotic initiation and the tyrosine kinase Wee1 is a key regulator of cell cycle sequence during G2/M transition and inhibits mitotic entry by phosphorylating the inhibitory tyrosine 15 on the cdc2 M-phase-inducing kinase. Wee1 degradation is essential for the exit from the G2 phase. In trypanosomatids, little is known about the genes that regulate cyclin B-cdc2 complexes at the G2/M transition of their cell cycle. Although canonical tyrosine kinases are absent in the genome of trypanosomatids, phosphorylation on protein tyrosine residues has been reported in Trypanosoma brucei. Here, we characterized a Wee1-like protein kinase gene from T. brucei. Expression of TbWee1 in a Schizosaccharomyces pombe strain null for Wee1 inhibited cell division and caused cell elongation. This demonstrates the lengthening of G2, which provided cells with extra time to grow before dividing. The Wee1-like protein kinase was expressed in the procyclic and bloodstream proliferative slender forms of T. brucei and the role of Wee1 in cell cycle progression was analyzed by generating RNA interference cell lines. In the procyclic form of T. brucei, the knock-down of TbWee1 expression by RNAi led to inhibition of parasite growth. Abnormal phenotypes showing an increase in the percentage of cells with 1N0K, 0N1K and 2N1K were observed in these RNAi cell lines. Using parasites with a synchronized cell cycle, we demonstrated that TbWee1 is linked to the G2/M phase. We also showed that TbWee1 is an essential gene necessary for proper cell cycle progression and parasite growth in T. brucei. Our results provide evidence for the existence of a functional Wee1 in T. brucei with a potential role in cell division at G2/M. 相似文献
The renin-angiotensin system expressed in adipose tissue has been implicated in the modulation of adipocyte formation, glucose metabolism, triglyceride accumulation, lipolysis, and the onset of the adverse metabolic consequences of obesity. As we investigated angiotensin II signal transduction mechanisms in human preadipose cells, an interplay of extracellular-signal-regulated kinases 1 and 2 (ERK1,2) and Akt/PKB became evident. Angiotensin II caused attenuation of phosphorylated Akt (p-Akt), at serine 473; the p-Akt/Akt ratio decreased to 0.5±0.2-fold the control value without angiotensin II (p<0.001). Here we report that the reduction of phosphorylated Akt associates with ERK1,2 activities. In the absence of angiotensin II, inhibition of ERK1,2 activation with U0126 or PD98059 resulted in a 2.1±0.5 (p<0.001) and 1.4±0.2-fold (p<0.05) increase in the p-Akt/Akt ratio, respectively. In addition, partial knockdown of ERK1 protein expression by the short hairpin RNA technique also raised phosphorylated Akt in these cells (the p-Akt/Akt ratio was 1.5±0.1-fold the corresponding control; p<0.05). Furthermore, inhibition of ERK1,2 activation with U0126 prevented the reduction of p-Akt/Akt by angiotensin II. An analogous effect was found on the phosphorylation status of Akt downstream effectors, the forkhead box (Fox) proteins O1 and O4. Altogether, these results indicate that angiotensin II signaling in human preadipose cells involves an ERK1,2-dependent attenuation of Akt activity, whose impact on the biological functions under its regulation is not fully understood. 相似文献