Proceedings of the National Science Council, Republic of China. Part B, Life sciences最新文献

The exact mechanisms involved in the postmortem meat tenderization process and the nature of changes associated with improvement in tenderness are complex and not fully understood. Based on the relevant evidence thus far obtained, the focus of this review is on clarifying the factors affecting meat tenderness, particularly the toughening and tenderness phases, possible endogenous proteinases involved in meat tenderization and how these proteinases contribute to meat tenderization. Of the different biochemical and ultrastructural changes occurring in the meat tenderization process, myofibril disruption at the Z-disk and contractile proteins are discussed in detail. This myofibril disruption can perhaps be ascribed to the synergistic action of calcium-dependent proteinases (both mu- and m-calpains) and lysosomal proteinases, especially the cathepsins B and L.

Digitalis-like immunoreactive factors (DLIF) are special types of steroids with lactone rings in their structures. Clinically, this type of compound can be used as medicine for heart failure; thus, the elevated endogenous DLIF found under certain pathological conditions are interferent substances in digoxin immunoassay. Endogenous DLIF with biological and immunological properties similar to cardiotonic drugs, such as digoxin, have been found in several tissues and body fluids of animals and humans. Since these endogenous Na+, K(+)-ATPase inhibitors can be considered hormones in nature, immunoassays must be selected detection of them to achieve the required sensitivity and specificity. In this study, we used three sets of in-house formulated immunoassays for DLIF and ouabain-like factors (OLF) detection. Using a polyclonal antibody-based ouabain enzyme immunoassay, the mean +/- S.E.M. of OLF in the sera of 10 healthy individuals were determined to be (9.1 +/- 0.9) x 10(-11) M. Using a monoclonal antibody-based ouabain enzyme immunoassay, the mean +/- S.E.M of OLF in the sera of 10 healthy individuals was (8.2 +/- 1.2) x 10(-11) M while using a antibody fragment Fab-based enzyme immunoassay for digoxin, the mean +/- S.E.M of DLIF in 11 healthy individuals was (4.0 +/- 1.2) x 10(-10) M. In conclusion, our immunological data indicate that DLIFs are normal constituents of human blood. Although DLIF is the major component, coexistence of OLF with DLIF in healthy individuals can not be excluded.

Exposure of NIH 3T3 cells to 375 mM ethanol at 37 degrees C for 20 min could induce a rapid increase in the protein level and cellular activity of oncogenic proline-directed protein kinase FA/glycogen synthase kinase-3 alpha (PDPK FA/GSK-3 alpha), up to approximately 300% of the control level, in a time- and concentration-dependent manner. The maximal inductive effect on PDPK FA/GSK-3 alpha also occurred within 40 min when cells were treated with only 100 mM ethanol. Similarly, exposure of NIH 3T3 cells to 100 microM cadmium for 2 h could induce a rapid increase in the protein level and cellular activity of PDPK FA/GSK-3 alpha, up to approximately 250% of the control level, in a time- and concentration-dependent manner. The maximal inductive effect on this kinase reached within 3 h when cells were treated with only 50 microM cadmium. The results demonstrate that PDPK FA/GSK-3 alpha may not represent a constitutively active/mitogen-inactivated protein kinase as previously conceived. Taken together with the previous report that PDPK FA/GSK-3 alpha is a heat-inducible protein kinase, the results further demonstrate that PDPK FA/GSK-3 alpha may represent a typical cellular stresses-inducible protein kinase subject to early induction by heat, ethanol and cadmium.

The inhibitory effect of human epidermoid cancer cells A431 caused by conjugate toxin containing transforming growth factor (TGF-alpha) and ricin A was studied. TGF-alpha is a protein with 50 amino acids that specifically binds and stimulates phosphorylation of cell surface epidermal growth factor receptor (EGFR) and, subsequently, triggers cell proliferation. TGF-alpha as a ligand for EGFR is internalized upon binding and decomposed within lysosome. Lectin ricin is contained in the castor bean plant. The lectin consists of two subunits, ricin A and B. Toxic ricin A binds to ribosome and inhibits protein synthesis of target cells. In view of the abundance of EGFR in human cancer cells, the receptor-mediated endocytosis with the conjugate toxin composed of TGF-alpha and ricin A was synthesized, purified and tested for growth inhibition in both normal and tumor cells. The cytotoxicity of the conjugate was studied within the range of 10(-12) and 10(-8) M and IC50 was found to be 10(-10) M for human A431 epidermoid cells that over-express EGFR. Compared to A431 cells, the brain metastatic variant of human Non-Small Cell Lung Cancer (NSCLC) H226Br squamous cells showed a reduced inhibitory effect. On the other hand, no inhibitory effect was found with other NSCLC cells studied and normal human lung cells because of the fewer available EGF binding sites on the surface of the cells. These results indicated that the amount of the available EGFR contributes to the cytotoxic effect on human cancer cells, thereby demonstrating involvement of the receptor-mediated endocytosis of the conjugate. The result from 12labeled EGF-mediated competition assay further demonstrated the specific inhibition activity conferred by TGF-alpha and ricin A conjugation. Due to poor recovery of the chemical conjugation, modification in the form of a recombinant toxin is needed for further in-depth studies.

Human mitochondrial DNA (mtDNA) is a multi-copy extra-chromosomal genetic element, which is exposed to a high steady-state level of reactive oxygen species and free radicals generated by the respiratory chain in mitochondria. Thus, it is much more vulnerable to oxidative damage and mutation than is nuclear DNA. In the past decade, more than two-dozen mutations of mtDNA have been observed in the somatic tissues of aged individuals. Among them, the 4,977 bp and 7,436 bp deletions and the A3243G and A8344G point mutations frequently occur and accumulate exponentially with age in muscle and other human tissues. These mtDNA mutations occur alone or co-exist in old human tissues at relatively low levels (< 5%). Aside from mutation, oxidative damage to mtDNA also increases in an age-dependent manner in human tissues. On the other hand, more than a hundred mtDNA mutations have been detected in patients with mitochondrial myopathy and encephalomyopathy. The mutant mtDNA often coexists with the wild-type mtDNA in affected tissues (a condition termed heteroplasmy). Usually the clinical severity of the disease is correlated with the proportion of the mutate mtDNA in the target tissues (usually > 80%). The threshold of the mutant mtDNA which is required to elicit clinical symptoms varies with different mutations. At the same level, large-scale deletions usually cause much more severe pathologies than do point mutations. The pattern of distribution of the mutant mtDNA and the energy demand of the target tissues are important factors in determining the pathological outcome of the mutation. The mutant mtDNA is usually widely distributed in the body tissues of the patient, thereby leading to multi-system disorders, which are frequently seen in mitochondrial diseases. Although a majority of the pathogenic point mutations are maternally transmitted, large-scale deletions of mtDNA are mostly sporadic. In addition, tandem duplication and depletion of mtDNA have also been found in the muscle and other affected tissues of elderly subjects and some patients with mitochondrial myopathy. Moreover, recent work in our laboratory has shown that oxidative damage to DNA in affected tissues is significantly higher than that in normal tissues. It is now established that mutation and oxidative damage of mtDNA are contributory factors to aging and that at high levels, they cause a fall of ATP supply below the threshold of energy needed by affected tissues in patients with mitochondrial diseases. These advances have laid the foundation for the development of biomedical gerontology and mitochondrial medicine.

Cucumber mosaic virus (CMV) is an icosahedrion plant virus and contains three different single-stranded positive sense genomic RNAs. The very 3' ends of each of the genomic RNAs can fold into a tRNA-like structure. Based on the structural analysis of the 3' tRNA-like structure of the brome mosaic virus (BMV), we superimposed and redrew the 3' tRNA-like structure of CMV. We homogenized virus infected or healthy tobacco leaves with polytron and carried out low speed centrifugation twice and ultra-centrifugation three times to get detergent solubilized membrane bound fractions. We accidentally found that these fractions were enriched with a host-encoded RNA-dependent RNA polymerase (RdRp) activity. Similar activity could also be found in other plants tested. Alternately, the membrane bound fraction could be simply precipitated by low speed centrifugation (3,000 g) and high speed ultra-centrifugation (40,000 g). The pellet was then suspended in a detergent-containing buffer, after which 25%-55% glycerol gradient fractionation was performed. Activity was tested through the incorporation of [alpha-32P]UTP using endogenous CMV RNAs as templates on each fraction collected. It was found that most of the fractions contained the viral-encoded RNA-dependent RNA polymerase. The products of RdRp reaction were found to have a double-stranded from through further analysis of the RNase protection assay.

The specific growth rates of Brochothrix thermosphacta, Listeria monocytogenes, Yersinia enterocolitica, Bacillus cereus, Escherichia coli O157:H7, Salmonella spp., Staphylococcus aureus and Clostridium perfringens at various temperatures were taken from the Food MicroModel database, and the data sets of specific growth rate versus temperature were fitted using the multiplicative model (r = a Td, r = specific growth rate; T = temperature; a, d = regression parameters). The exponential d-value derived from microbial growth at suboptimum temperatures reflected the effectiveness of temperature in enhancing growth. A microorganism with a large d-value exhibited a large increment of growth rate as temperature increased. The d-value of a microorganism was related to the temperature range for growth. The temperature range for the growth of psychrotrophs was usually narrow for B. thermosphacta and Y. enterocolitica; hence the d-values of these two psychrotrophs were close to 1 whereas d-values of mesophiles, such as B. cereus, E. coli O157:H7, Salmonella spp., and S. aureus, were 2.31-2.90, and the d-value of C. perfringens, a thermophile, was 3.29. The values of parameter a of the model were affected by extra salt added into cultures. For all the strains mentioned above, the a-values decreased when the cultures contained higher levels of salt. The lowering of the a-value implied that the influence of temperature on the growth rate in the model was reduced. The change of the d-value was dependent on the capability of the microorganism to overcome the obstacle to growth and was affected by the composition of the nutrients and by inhibitory factors in the culture. The influence of environmental factors on the d-value was also found in Chinese sausages. The d-value of a dominant spoilage strain of Enterococcus sp. derived from sausages was 0.833 whereas the d-value derived from MRS cultures was 2.36. In refrigerated foods which usually contained some preservatives and were stored at low temperature, the d-value of psychrotrophic spoilage bacteria was around 1. In this case, the linear model could be a reasonable choice for predicting the proliferation of spoilage bacteria.

Modulation of protein phosphorylation activities by insulin was investigated in glioma and normal glial cells. Insulin suppressed the in vitro protein phosphorylation of glioma cells in a dose-dependent manner while it stimulated that of meningiomas, neurilemmomas and glial cells. Although gliomas and glial cells contained different species of tyrosyl phosphoproteins before treatment, they expressed similar kinds of tyrosyl phosphoproteins in response to insulin. Insulin increased the activities of casein kinase II and total protein kinase C (PKC) in glioma and normal glial cells. The membrane-bound PKC activity in U373-MG cells was elevated by insulin. The PKC isozymes, including subtypes alpha, beta, delta, epsilon and gamma, were detected in gliomas, but few were found in glial cells. Insulin down regulated the cytosolic PKC-gamma and the membrane-bound PKC-epsilon proteins in gliomas. These results indicate that an altered insulin signaling pathway exists in human gliomas, which might involve differential regulation of PKC isozymes.

The correlation of polymorphic DNA haplotype of the alpha-L-iduronidase (IDUA) gene and IDUA activity in Chinese subjects was investigated. Genomic DNA extracted from 85 randomly sampled normal individuals was used to amplify fragments containing the polymorphic change site A8, Q33H (exon 1), R105Q (exon 3), A361T (exon 8), or V454I (exon 9). The PCR amplified products were analyzed by means of restriction fragment length polymorphism (RFLP) or allele specific oligonucleotide (ASO) hybridization. Leukocytes were isolated from the above 85 samples, and their IDUA activities were determined. A wide range of IDUA activity (50-300 nmol/h/mg cell protein) with an average of 156 nmol/h/mg cell protein was observed. When the allele frequency was compared between individuals with IDUA activity below 90% or above 110% of the average, a bias toward the common allele "1" of Q33H (Gln33) was detected in individuals with higher IDUA activity. Conversely, the polymorphic allele "2" of R105Q (Gln105), A361T (Thr361), and V454I (Ile454) was found in the higher IDUA activity group. Linkage disequilibrium analysis of the haplotype data revealed strong nonrandom association among the polymorphic alleles of R105Q, A361T, and V454I. Of the haplotypes constructed by Q33H, R105Q, A361T, and V454I, a positive correlation between haplotype 1,2,2,2 (Gln33, Gln105, Thr361, Ile454) and IDUA activity was observed. The IDUA activity was found to increase with Gln105, Thr361, or Ile454 polymorphic changes by mutagenesis and expression of IDUA cDNA in COS-7 cells. By combining the positive effect of Gln105, Thr361, and Ile454 in one cDNA construct, it may be possible to produce a high activity IDUA protein for MPS I enzyme replacement therapy.

The effects of a combination of dexamethasone (DEX, an inhibitor of inducible nitric oxide synthase (iNOS) synthesis) and aminoguanidine (AG, an inhibitor of iNOS activity) on the anesthetized rat treated with endotoxin (lipopolysaccharide, LPS) were examined. In addition, we investigated whether a complete inhibition of nitric oxide (NO) formation caused by LPS prevents the hypotension, restores the vascular hyporeactivity to normal and improves the survival rate. The combination of DEX (3 mg/kg at 30 min prior to LPS) plus AG (15 mg/kg at 2 h after LPS) inhibited the overproduction of nitrate (an indicator of NO) and prevented the development of delayed hypotension, but further enhanced tachycardia in rats treated with LPS for 6 h. In addition, the vascular hyporeactivity to norepinephrine (NE) was, however, only partially restored in endotoxemic rats treated with DEX plus AG. During the experimental period, the survival rate of LPS-rats treated with DEX plus AG was also improved when compared to that of rats treated with LPS only. The beneficial effects of the combined therapy with DEX plus AG on rats with endotoxemia, suggesting that (i) combined treatment of animals with DEX plus AG may reduce some of the detrimental effects of LPS and (ii) NO only partially contributes to the vascular hyporeactivity in endotoxic shock.