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1.
Papain, a cysteine protease isolated from the latex of Carica papaya, is known to undergo irreversible thermal unfolding. In this study, we found that thermal unfolding of papain is accompanied by a simultaneous self-assembly process where this protein is observed to aggregate above 50°C. The extent of aggregation increased with increasing protein concentration from 3–40 μM. The aggregation was confirmed by enhanced turbidity, light scattering intensity, 1-anilino-8-naphthalene sulfonate (ANS) fluorescence intensity and by transmission electron microscopy. Furthermore, we noted that post-micellar concentration of sodium dodecyl sulfate (SDS) remarkably suppresses the thermal aggregation of papain. Far-UV circular dichroism studies revealed that SDS significantly enhances α-helical content of the protein and also tends to prevent its unfolding, and thus inhibits aggregation. Additionally, papain showed maximal activity at 65°C in neutral buffer. However, in the presence of 6 mM SDS (above its critical micellar concentration), the enzyme lost activity by about 10-fold. Thus, promoting the helical propensity of the protein does not appear to be a suitable strategy to overcome the aggregation related problems of industrially important proteins such as papain, which are not only required to be protected against aggregation but also need to remain functionally active in the presence of aggregation inhibitors.  相似文献   
2.
Stored grains are subject to deterioration and losses through various factors, but mainly insects and fungi. Various techniques are employed to detect stored product pests; however, there is an urgent need for an industrial-scale on-line detection technique. Near-infrared hyperspectroscopic imaging and soft X-rays have shown the potential for real-time application. These techniques are particularly effective for detecting internal infestations of stored grains. The digital images of the scanned objects are analyzed for various spectral and image features using statistical techniques such as complex multivariate tools. Classification accuracies as high as 80–100 % have been achieved for various pest and grain combinations. Dual-energy X-rays have been shown to detect the concealed eggs of stored product insect pests. The main threats to stored cereals come from Aspergillus spp., Penicillium spp., and Fusarium spp., which may produce mycotoxins. These imaging techniques have shown good results in the detection of fungal infections of stored grain.  相似文献   
3.
Soybean exhibits markedly reduced growth and yields under flooding stress. To determine the functional roles of four soybean proteins in post-flooding recovery, the organ/stress specificity and time-dependency of their enzymatic activities were analyzed. Peroxidase activity decreased in root and hypocotyl exposed to flooding and cold stresses, but increased during the post-stress recovery period. In contrast, its activity increased in both root and hypocotyl under drought stress. Acid phosphatase activity was suppressed in root treated with flooding and cold stresses, and slightly increased during the recovery period; however, the opposite profile was observed in hypocotyl. In response to drought stress, it did not change in root, but was decreased in hypocotyl. Beta-ketoacyl reductase activity did not change in root under flooding conditions, but was decreased in hypocotyl, although the activity increased slightly during the recovery period. In addition, it was decreased in both organs under drought and cold stresses, but again increased during the recovery period. Nucleotidylyl transferase activity was increased in root under flooding and drought stresses, but was decreased in hypocotyl. It was decreased in response to cold stress, but exhibited a slight increase during the recovery period. Furthermore, the treatment with jasmonate and salicylate suppressed the activities of peroxidase and acid phosphatase in root and hypocotyl under flooding stress; however, the activity of acid phosphatase increased during the recovery period. Nucleotidylyl transferase activity in root was also elevated by treatment with jasmonate, but gradually decreased during the recovery period. These results suggest that jasmonate-induced changes in nucleotidylyl transferase activity may facilitate soybean root recovery after flooding stress.  相似文献   
4.
Hereditary nonpolyposis colorectal cancer (HNPCC) is a common autosomal dominant cancer-susceptibility condition characterized by early onset colorectal cancer. Germ-line mutations in one of four DNA mismatch repair (MMR) genes, hMSH2, hMLH1, hPMS1, or hPMS2, are known to cause HNPCC. Although many mutations in these genes have been found in HNPCC kindreds complying with the so-called Amsterdam criteria, little is known about the involvement of these genes in families not satisfying these criteria but showing clear-cut familial clustering of colorectal cancer and other cancers. Here, we applied denaturing gradient-gel electrophoresis to screen for hMSH2 and hMLH1 mutations in two sets of HNPCC families, one set comprising families strictly complying with the Amsterdam criteria and another set in which at least one of the criteria was not satisfied. Interestingly, hMSH2 and hMLH1 mutations were found in 49% of the kindreds fully complying with the Amsterdam criteria, whereas a disease-causing mutation could be identified in only 8% of the families in which the criteria were not satisfied fully. In correspondence with these findings, 4 of 6 colorectal tumors from patients belonging to kindreds meeting the criteria showed microsatellite instability, whereas only 3 of 11 tumors from the other set of families demonstrated this instability. Although the number of tumors included in the study admittedly is small, the frequencies of mutations in the MMR genes show obvious differences between the two clinical sets of families. These results also emphasize the practical importance of the Amsterdam criteria, which provide a valid clinical subdivision between families, on the basis of their chance of carrying an hMSH2 or an hMLH1 mutation, and which bear important consequences for genetic testing and counseling and for the management of colorectal cancer families.  相似文献   
5.
Subunit-specific antibodies to all the γ subunit isoforms described in mammalian brain (γ1, γ2S, γL, and γ3) have been made. The proportion of GABAA receptors containing each γ subunit isoform in various brain regions has been determined by quantitative immunoprecipitation. In all tested regions of the rat brain, the γ1, and γ3 subunits are present in considerable smaller proportion of GABAA receptor than the γ2 subunit. Immunocytochemistry shows that γ1 immunoreactivity concentrates in the stratum oriens and stratum radiatum of the CA1 region of the hippocampus. In the dentate gyrus, γ1 immunoreactivity concentrates on the outer 2/3 of the molecular layer coinciding with the localization of the axospinous synapses of the perforant pathway. In contrast, γ3 immunoreactivity concentrates on the basket cells and other GABAergic local circuit neurons of the hilus. These cells are also rich in γ2S. In the cerebellu, γ1 immunolabeling was localized on the Bergmann glia. The γ2S and γ2L subunits are differentially expressed in various brain regions. Thus the γ2S is highly expressed in the olfactory bulb and hippocampus whereas the γ2L is very abundant in inferior colliculus and cerebellum, particularly in Purkinje cells, as immunocytochemistry, in situ hybridization and immunoprecipitation techniques have revealed. The γ2S and γ2L coexist in some brain areas and cell types. Moreover, the γ2S and γ2L subunits can coexist in the same GABAA receptor pentamer. We have shown that this is the case in some GABAA receptors expressed in cerebellar granule cells. These GABAA receptors also have α and β subunits forming the pentamer. Immunoblots have shown that the rat γ1, γ2S, γ2L and γ3 subunits are peptides of 47, 45, 47 and 44 kDa respectively. Results also indicate that there are aging-related changes in the expression of the γ2S and γ2L subunits in various brain regions which suggest the existence of aging-related changes in the subunit composition of the GABAA receptors which in turn might lead to changes in receptor pharmacology. The results obtained with the various γ subunit isoforms are discussed in terms of the high molecular and binding heterogeneity of the native GABAA receptors in brain. Special issue dedicated to Dr. Kinya Kuriyama  相似文献   
6.
Abstract Isolation and identification of allatostatin 4 (AST 4) from crude brain extracts of female Diploptera punctata are described. Synthetic analogues of allatostatin 4 truncated from the N-terminal were evaluated to gain some knowledge of structure-activity correlation. AST 4 reversibly inhibits the biosynthesis of JH in vitro . AST 4 has the following sequence: Asp-Arg-Leu-Tyr-Ser-Phe-Gly-Leu-NH2. This neurohormone (AST 4) has no sequence similarity with any known neuropeptide from other organisms. Synthetic AST 4 as well as truncation fragments, including two with the five and six amino acids terminal residues deleted showed in vitro activity distinguishable from that of the native allatostatin.  相似文献   
7.
Most small multicopy plasmids of Gram-positive bacteria and many in Gram-negative bacteria replicate by a rolling-circle (RC) mechanism. The replication initiator proteins encoded by the RC plasmids and single-stranded bacteriophages of Escherichia coli have origin-specific nicking-closing activities that are required for the initiation and termination of RC replication. We have investigated the sequence requirements for termination of RC replication of plasmid pT181. The initiator nick site is located in the loop of a hairpin region (IRII) within the pT181 origin of replication. By mutational analysis, we have found that several nucleotides within the stem of IRII which are critical for the initiation activity are dispensable for termination of replication. We also demonstrate that nucleotides in the right arm of IRII, but not the left arm, are absolutely required for termination of RC replication. We have also identified specific nucleotides in IRII that are critical for its termination activity. The sequence of the right arm of the hairpin must be located downstream of the initiator nick site for termination, suggesting that termination requires a specific orientation of the initiator protein at the origin.  相似文献   
8.
The origin of replication of plasmid pT181 is nicked by the plasmid-encoded RepC protein. The free 3'-hydroxyl end at the nick is presumably used as primer for leading strand DNA synthesis. In vitro replication of pT181 was found to generate single-stranded DNA in addition to the supercoiled, double-stranded DNA. The single-stranded DNA was circular and corresponded to the pT181 leading strand. Recombinant plasmids were constructed that contain two pT181 origins of replication in either direct or inverted orientation. In vitro replication of the plasmid carrying two origins in direct orientation was shown to generate circular, single-stranded DNA that corresponded to initiation of replication at one origin sequence and termination at the other origin. These results demonstrate that the origin of pT181 leading strand DNA replication also serves as the site for termination of replication. Interestingly, the presence of two origins in inverted orientation resulted in initiation of replication at one origin and stalling of the replisome at the other origin. These results suggest that RepC can reinitiate replication at the second origin by nicking partially replicated, relaxed DNA. These data are consistent with the replication of pT181 by a rolling circle mechanism and indicate that single-stranded DNA is an intermediate in pT181 replication.  相似文献   
9.
10.
Heavy Metals Induce Lipid Peroxidation and Affect Antioxidants in Wheat Leaves   总被引:10,自引:0,他引:10  
The possible role of Zn and Cr as catalytic inducers of free radicals in wheat leaves was investigated. Treatment of excess heavy metals decreased the chlorophyll and carotenoid content in wheat leaves with the increase in time of excision. A sharp increase in proline accumulation was marked with the increase in metal concentration. Total peroxide content and lipid peroxidation measured as malondialdehyde content showed uniform increase under metal treatment in excised leaves. At almost all concentrations, catalase, guaiacol peroxidase and superoxide dismutase activities decreased with a minor increase in the earlier days of excision. Though glutathione content decreased ascorbate content showed significant increase in wheat leaves under heavy metal treatment. This revised version was published online in July 2006 with corrections to the Cover Date.  相似文献   
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