Effect of the Medium Dielectric Strength on the Activity of Alpha Chymotrypsin

The rates of hydrolysis of TrEE, TEE, and ATEE¹ by α-chymotrypsin were determined in media of variable dielectric strength. Many substances which modify the dielectric constant of the medium, exert additional specific effects on the reaction rate, noticeable at more or less elevated concentrations. Notwithstanding, it is possible to differentiate the dielectric and specific effects by comparing the rates in solvents of distinct nature at relatively low concentrations. Thus, the effect of varying the dielectric strength could be studied within wider ranges (ΔD = 20 with TrEE and ca. 28 with ATEE) than in the previous study of trypsin (ΔD = 12). The dielectric effect on α-chymotrypsin is the opposite of that observed with trypsin. In both cases there is a linear relationship between the logarithm of the rate of hydrolysis and the reciprocal of the dielectric constant. The slope is negative with α-chymotrypsin and positive with trypsin. According to expressions relating the dielectric constant to the rate in non-enzymatic reactions, the behavior of α-chymotrypsin is like that of a negative ion, while trypsin behaves as a positive ion. The enzyme activity appears to depend upon the arrangement of charges in the enzyme and substrate molecules, rather than on the presence of certain atomic groupings in the substrate.     

ω-Aga IVA selectively inhibits the calcium-dependent fraction of the evoked release of [3H]GABA from synaptosomes

The effect of -Aga IVA, a P-type Ca²⁺ channel blocker, on the release of the inhibitory neurotransmitter GABA and on the elevation of Ca_i induced by depolarization was investigated in [³H]GABA and fura-2 preloaded mouse brain synaptosomes, respectively. Two strategies (i.e. 20 mM external K⁺ and veratridine) that depolarize by different mechanisms the preparation were used. High K⁺ elevates Ca_i and induces [³H]GABA release in the absence of external Na⁺ and in the presence of TTX, conditions that abolish veratridine induced responses. The effect of -Aga IVA on the Ca²⁺ and Na⁺ dependent fractions of the depolarization evoked release of [³H]GABA were separately investigated in synaptosomes depolarized with high K⁺ in the absence of extermal Na⁺ and with veratridine in the absence of external Ca²⁺, respectively. The Ca²⁺ dependent fraction of the evoked release of [³H]GABA and the elevation of Ca²⁺ induced by high K⁺ are markedly inhibited (about 50%) in synaptosomes exposed to -Aga IVA (300 nM) for 3 min before depolarization, whereas the Na⁺ dependent, Ca²⁺ independent carrier mediated release of [³H]GABA induced by veratridine, which is sensitive to verapamil and amiloride, is not modified by -Aga IVA. Our results indicate that an -Aga IVA sensitive type of Ca²⁺ channel is highly involved in GABA exocytosis.     

A role for calcium/calmodulin kinase(s) in the regulation of GABA exocytosis

A possible role for protein kinases in the regulation of GABA exocytosis in nerve endings was investigated. The effect on the release of the radioactive neurotransmitter ([³H]GABA) from mouse brain synaptosomes of several protein kinase inhibitors was estimated after treatment with 37 mM K⁺ in the absence of external Na⁺, a condition under which [³H]GABA release is completely Ca²⁺ dependent. Among the inhibitors one group inhibit the kinases by binding to the catalytic site (i.e. staurosporine and H7) and others (TFP, sphingosine and W7) act on the regulatory site of protein kinases. The compounds of the second group, which are reported to inhibit calmodulin dependent events and the increase in cytosolic Ca²⁺ (Ca _i ) induced by high K⁺ depolarization, were the most efficient inhibitors of [³H]GABA release. The selective inhibitor of CaMPK II, KN-62, also markedly diminished [³H]GABA release as well as the increase in Ca _i induced by high K⁺. The kinase inhibitors from the first group that are unable to diminish the increase in Ca _i induced by high K⁺ were also less efficient inhibitors of [³H]GABA release even at high concentrations. The present results indicate that at the doses tested all the drugs inhibit to some extent the release of the Ca²⁺ dependent fraction of [³H]GABA perhaps by inhibiting a CaMPK II mediated phosphorylation step triggered by depolarization and facilitated by the elevation of Ca _i . In addition, the second group of antagonists and KN-62 inhibit the elevation of Ca _i to high K⁺ thus exhibiting a higher efficiency on [³H]GABA release than the first group of antagonists.     

Biolistic transformation of chrysanthemum with the nucleocapsid gene of tomato spotted wilt virus

In vitro regeneration and biolistic transformation procedures were developed for several commercial chrysanthemum Dendranthema grandiflora Tzvelev, syn. Chrysanthemum morifolium Ramat. cultivars using leaf and stem explants. Studies on the effect of several growth regulators and kanamycin on chrysanthemum regeneration were conducted, and a step-wise procedure to optimize kanamycin selection and recovery of transgenic plants was developed. A population of putative transformed chrysanthemum plants cvs. Blush, Dark Bronze Charm, Iridon, and Tara, was obtained after bombardment with tungsten microprojectiles coated with the binary plasmid pBIN19 containing the nucleocapsid (N) gene of tomato spotted wilt virus (TSWV) and the marker gene neomycin phosphotransferase (NPT II). PCR analysis of 82 putative transgenic plants selected on kanamycin indicated that the majority of the lines (89%) were transformed and contained both genes (71%). However, some transgenic lines contained only one of the genes: either the NPT II (15%) or the TSWV (N) gene (14%). Southern blot analysis on selected transgenic lines confirmed the integration of the TSWV (N) gene into the chrysanthemum genome. These results demonstrate the development of an efficient procedure to transfer genetic material into the chrysanthemum genome and selectively regenerate transgenic chrysanthemum plants at frequencies higher than previously reported.     

Silencing porcine genes significantly reduces human-anti-pig cytotoxicity profiles: an alternative to direct complement regulation

The future of solid organ transplantation is challenged by an increasing shortage of available allografts. Xenotransplantation of genetically modified porcine organs offers an answer to this problem. Strategies of genetic modification have ‘humanized’ the porcine model towards clinical relevance. Most notably, these approaches have aimed at either antigen reduction or human transgene expression. The object of this study was to evaluate the relative effects of both antigen reduction and direct complement regulation on the human-anti-porcine complement dependent cytotoxicity response. Genetically modified animals were created through CRISPR/Cas9-directed mutation and human transgene delivery. Pigs doubly deficient in GGTA1 and CMAH genes were compared to pigs of the same background that expressed a human complement regulatory protein (hCRP). A third animal was made deficient in GGTA1, CMAH and B4GalNT2 gene expression. Cells from these animals were subjected to measures of human antibody binding and antibody-mediated complement-dependent cytotoxicity by flow cytometry. Human IgG and IgM antibody binding was unchanged between the double knockout and the transgenic hCRP double knockout pig. IgG and IgM binding was reduced by 49.1 and 43.2 % respectively by silencing the B4GalNT2 gene. Compared to the double knockout, human anti-porcine cytotoxicity was reduced by 8 % with the addition of a hCRP (p = .032); It was reduced by 21 % with silencing the B4GalNT2 gene (p = .012). Conclusions: Silencing the GGTA1, CMAH and B4GalNT2 genes in pigs achieved a significant antigen reduction. Changing the porcine carbohydrate profile effectively mediates human antibody-mediated complement dependent cytoxicity.     

Newly created ponds complement natural waterbodies for restoration of macroinvertebrate assemblages

相似文献   

A review through recovery,purification and identification of genipin

Genipin is a secoiridoid that may be obtained directly from Genipa americana and after geniposide hydrolysis from Gardenia jasminoides fruits. It is well known that genipin is a high added value product which has a great potential for its application in different fields, such as medicine and food industry. A lack of information and research about the development of novel methods for the environmental friendly recovery and purification of genipin exists, focused on the green recovery of genipin through emerging technologies that lead to the sustainable extraction. In the present review, the available methods for recovery of genipin and geniposide are enlisted and described, as well as the reaction mechanism of genipin, and the developed methods for genipin and geniposide identification and quantification based on HPLC analysis. Moreover, this work reveals the scarcity of available identification methods for genipin, that should be taken into account to fulfill the recovery and quantification process of genipin.