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

Iron is a vital element in life. However, it may participate in diverse pathological processes by catalyzing the formation of reactive oxygen free radicals. During the past decade, considerable evidence has supported the role of oxidative stress in the development of atherosclerosis and related cardiovascular diseases. The oxidation of low-density lipoprotein (LDL) and lipid is believed to be one of the crucial events leading to plaque formation in vasculature. It has been hypothesized that iron-mediated oxidation is involved in this process. In favor of this idea, several epidemiological studies have shown that the level of body iron stores is positively correlated with the incidence of coronary heart disease in human populations. However, some studies have yielded conflicting results. Recently, studies conducted in our laboratory and others have demonstrated that iron deposition is prominent in human atherosclerotic lesions. The iron deposits appear to colocalize with ceroid, which is an end product of extensively oxidized lipid and protein complex, in lesions, providing histological evidence to support the iron hypothesis. Additional experiments in animals have further revealed that the severity of atherosclerosis can be markedly influenced by iron overload or deficiency. Collectively, these data provide a strong pathological basis to support the detrimental role of iron in vascular damage and progression of the disease.

Anserine and carnosine found in animal skeletal muscle are capable of inhibiting the catalysis of lipid oxidation by heme and non-heme iron. A demineralization technique and a proteolytic enzyme (papain) were used in this research in order to reduce the levels of proxidants while maintaining high levels of anserine and camosine in poultry (chicken, duck and turkey) meat extracts. Undemineralized poultry meat extracts contained larger amounts of anserine, camosine, heme and non-heme iron (p < 0.05) than did demineralized poultry meat extracts. Both undemineralized and demineralized breast meat extracts of chicken, duck and turkey contained higher concentrations of anserine and camosine, but lower amounts of heme and non-heme iron than did thigh meat extracts. In chicken, duck and turkey meat (breast and thigh) extracts (undemineralized and demineralized), the anserine concentrations were greater (p < 0.05) than the camosine concentrations. The hydrogen-donating ability of undemineralized and demineralized chicken breast meat extracts was not significantly different (p > 0.05): however, demineralized chicken breast meat extracts showed higher (p < 0.05) ferrous chelating ability than did undemineralized meat extracts. The concentrations of anserine, camosine, heme and non-heme iron in chicken breast meat extracts increased (p < 0.05) with the addition of papain (1%) to the meat mixture before extraction. Heme and non-heme iron in the chicken breast meat extracts increased as the reaction time for papain increased from 30 to 120 min, but the concentrations of anserine and camosine were not significantly affected by the longer reaction time for papain. The hydrogen-donating ability and ferrous chelating ability of demineralized chicken breast meat extracts were not significantly affected by papain. The ratios of carnosine/anserine were very specific in the chicken, duck and turkey meat extracts (breast and thigh); and the turkey meat extracts had lower (p < 0.05) camosine/anserine ratios than did the chicken and duck meat extracts. The camosine/anserine ratios of undemineralized and demineralized poultry meat extracts were not significantly different (p > 0.05). This suggests that the carnosine/anserine ratios of undemineralized chicken (0.62 - 0.80), duck (0.75 - 0.77) and turkey (0.15 - 0.16) meat extracts could be used to estimate the single meat species in uncooked or cooked meat products.

DIDS (4, 4'-diisothiocyanostilbene-2, 2'-disulfonic acid) has been recognized as an anion channel blocker. In this study, we demonstrated that DIDS significantly enhanced the production of free radicals in mouse peritoneal neutrophils. By means of a luminol-chemiluminescence (LCL) monitoring system, DIDS markedly increased LCL which could be suppressed by SOD, sodium azide (NaN3), EGTA and BAPTA-AM and only slightly inhibited by staurosporine (STP). Depletion of the endoplasmic reticulum (ER)-Ca2+ store by means of thapsigargin (TG) had no effects on DIDS-enhanced LCL, but DIDS significantly increased the amount of intracellular free calcium as monitored by means of fura-2 staining. These results indicate that DIDS may enhance free radical production mediated by Ca2+ release from the mitochondria. Both phorbol-12-myristate-13-acetate (PMA) and DIDS can induce increased translocation of p47-phox of the neutrophil to the membrane fraction, which is inhibited by STP pretreatment. Since free radical generation could reduce the cytoplasmic pH (pHi), we further examined whether DIDS was capable of inducing intracellular acidification. The result indicated that DIDS certainly lowered the pHi which was also suppressed by pretreatment with either NaN3 or NaCN, but not by diphenyleneiodonium (DPI). These findings lead us to propose a working hypothesis that DIDS mainly induces superoxide production accompanied by decreasing pHi mediated through a Ca2+ -dependent effect on the mitochondria rather than on NADPH oxidase. Using the lipophilic fluorescent dye DiOC6(3), we showed that DIDS decreased the transitional mitochondrial membrane potential. NaN3, but not STP or pyrrolidine dithiocarbamate (PDTC), antagonized DIDS in the course of decreasing the mitochondrial membrane potential. Taken together, all of these findings imply a possible role of anion channels of the mitochondria in modulating free radical production and intracellular acidification of neutrophils through alteration of the mitochondrial transition membrane potential and Ca2+ -release.

A mutant strain of E. coli EP1 harbouring pGL-5 was employed to develop a process for producing penicillin G acylase (PGA). In comparison with different carbon sources in the medium, it was found that the specific levels of PGA activity obtained in the glucose medium were the lowest. which was likely due to catabolic repression. Phenylacetic acid (PAA) was previously reported to be an regulatory inducer for PGA production, whereas in this study, the addition of PAA repressed both cell growth and enzyme expression. In a fed-batch culture, the increase of specific PGA activity followed the pattern of the cell concentration during the early to middle cell growth phase. With application of pure oxygen aeration and an appropriate medium design, the cell concentration reached 162 (g wet weight/l), which was 2.4 times higher compared to that of the original operation, and a specific PGA activity of 37 (IU/g wet weight) was achieved after 12 h of cultivation.

Isokinetic exercise has been commonly used in knee rehabilitation, conditioning and research in the past two decades. Although many investigators have used various experimental and theoretical approaches to study the muscle and joint force involved in isokinetic knee extension and flexion exercises, only a few of these studies have actually distinguished between the tibiofemoral joint forces and muscle forces. Therefore, the objective of this study was to specify, via an eletromyography(EMG)-driven muscle force model of the knee, the magnitude of the tibiofemoral joint and muscle forces acting during isokinetic knee extension and flexion exercises. Fifteen subjects ranging from 21 to 36 years of age volunteered to participate in this study. A Kin Com exercise machine (Chattecx Corporation, Chattanooga, TN, U.S.A.) was used as the loading device. An EMG-driven muscle force model was used to predict muscle forces, and a biomechanical model was used to analyze two knee joint constraint forces; compression and shear force. The methods used in this study were shown to be valid and reliable (r > 0.84 andp < 0.05). The effects on the tibiofemoral joint force during knee isokinetic exercises were compared with several functional activities that were investigated by earlier researchers. The muscle forces generated during knee isokinetic exercise were also obtained. Based on the findings obtained in this study, several therapeutic justifications for knee rehabilitation are proposed.

Rice plants were treated with 50 microM copper sulfate to induce the synthesis of phytochelatins by means of a series of enzymatic reactions, including that of photochelatin synthase. Phytochelatin synthase extracted from 3-week-old rice seedlings was purified through a series of steps including precipitation with ice-chilled acetone, QAE A-50 anion exchange column, Amicon XM-50 ultrafiltration and Polybuffer Exchange (PBE) 94 chromato-focusing. This enzyme had a molecular mass of about 100 kDa with an isoelectric point of 4.0. The temperature and pH optima of this enzyme were 55 degrees C and pH 7.5, respectively. The enzyme was thermal tolerable and unstable under refrigeration at 4 or -20 degrees C. Cadmium was the most effective stimulator, followed by lead, copper, silver, cobalt and other divalent cations. Calcium and magnesium had no effect.

Plaque pH measurements can be used to detect individual's caries susceptibility and the cariogenic potential of ingested foods. However, repeated measurements with a touch electrode at a single site may affect the metabolic state of the dental plaque and result in changes of its inherent pH values. The purposes of this study were to evaluate the influence of repeated measurements of resting plaque pH values and to determine the pH values at different interdental sites. Eleven dental students participated in the study. The dental plaque was built up after thorough oral hygiene care. The subjects were then instructed to maintain a normal diet but to refrain from any oral hygiene care for the next 48 hours. Measurement of plaque pH was performed with an antimony electrode at 6 interproximal sites, including central spaces between the upper and between the lower central incisors, and the area between the second premolar and the first molar in each quadrant. At each tested site, measurements were taken 5 times at 0, 10, 30, 45 and 60 min. The results showed that the plaque pH became slightly alkaline at all the tested locations after repeated measurements. However, only the lower incisor area showed a significant change in pH values. Interdental plaque on the lower arch showed higher pH values than did that on the upper arch. There was no significant difference between the pH values on the right and left corresponding sites as determined using the Wilcoxon signed ranks test.

The unique cytokeratin K19 specifically expresses in simple epithelial cells, basal cells of non-keratinized stratified squamous epithelium, epidermal cells during the embryonic stage and squamous carcinoma cells, but it is not expressed in adult epidermis. Interestingly, when epidermal cells are cultured in vitro, K19 is re-expressed in the supra-basal layer. K19 expression was used as a marker for epidermal cell growth and differentiation. In order to clarify the temporal and spatial sequential expression in cultured keratinocyte, two-stage human keratinocyte culture systems were used to examine K19 expression in keratinocytes in a proliferation and differentiation stages through immunoblotting and immunohistochemistry assay. According to our results, K19 was not expressed in cultured human keratinocytes in the proliferation stage but was re-expressed in keratinocytes three days after the cultured medium was changed to a differentiation medium. Immunohistochemical observation revealed that K19 was persistently expressed in the supra-basal layer of cultured keratinocytes during first three weeks of culturing, but none was detectable in the basal cell layer. When keratinocytes were cultured with an "inserted cultured dish," K19 was persistently expressed in all layers of keratinocytes nourished by medium both from an inner chamber and an outer chamber. The different expression of K19 in these two different culture systems seemed to indicate that down regulation of K19 expression in keratinocyte was related to the direction of medium supply.

In this review, we attempt to demonstrate that reverse micelles are simple artificial systems that mimic many life systems from cell division to the creation of an enzyme catalytic mechanism. For a membranous enzyme like placental alkaline phosphatase, the kinetic properties observed in reverse micelles might represent those found under physiological conditions. The reverse micellar system, consisting of a positively charged surfactant, mimics a detoxification enzyme glutathione transferase. We propose a novel island-in-oil-lake reverse micellar model for the glutathione transferase that can account for almost all the catalytic properties of this enzyme. Reverse micelles may provide an excellent model system in investigating the reaction mechanism of other detoxification enzymes.

One of the mechanisms contributing to antibody diversity is created by the association of different heavy and light chains. The combinability of heavy and light chains has been studied previously in two systems: in vitro chain recombination and hybrid hybridoma. Here, a novel in vivo chain combination assay system involving a heavy chain-loss variant, 26.4.1LL, producing two kappa light chains (L(DEX) and L(MPC)) different in size is described. In conjunction with DNA transfection, immunoprecipitation and SDS-PAGE, the structural basis of noncovalent interaction between heavy and light chains can be elucidated systematically by examining the relative association tendency of a heavy chain with two light chains. To demonstrate the usefulness of this system, three stably transfected 26.4.1LL cell lines expressing gamma2b heavy chains, designated as H(DEX), H(CHI) and H(ARS), respectively, with structural interrelated variable regions were generated: H(DEX) differs from H(CHI) only in framework regions whereas H(CHI) differs from H(ARS) in complementarity-determining regions. The relative amounts (R values) of L(DEX) and L(MPC) associated with the heavy chains H(DEX), H(CHI) and H(ARS) in the assembled immunoglobulin molecules were found to be 1.02, 0.64 and 0.05, respectively, suggesting that the complementarity-determining regions and framework regions contribute equally to the V(L)-V(H) interaction. This conclusion is consistent with previous observations based on calculation of the buried area in the V(L)-V(H) interface, thus demonstrating the usefulness of this system.