结核杆菌PEPCK表达及保护性免疫反应效果

目的研究结核杆菌磷酸烯醇型丙酮酸羧激酶(PEPCK)的表达,并观察此融合蛋白使机体产生的保护性免疫反应。方法将含有结核杆菌磷酸烯醇型丙酮酸羧激酶(pckA)基因的重组质粒转化大肠埃希菌HB₁₀₁,表达出融合蛋白PEPCK,并进行蛋白质印迹(Western blot)分析;选BALB/c小鼠60只,随机分为实验组和对照组。实验组每只小鼠用表达的PEPCK 10μg加弗氏不完全佐剂进行腹腔免疫注射,对照组小鼠仅用弗氏不完全佐剂注射。每隔2周免疫1次,共免疫3次。末次免疫2周后,分别取小鼠脾脏、血清,检测CD4⁺T细胞和CD8⁺、T细胞及各细胞因子。结果成功表达出融合蛋白PEPCK,并能够与小鼠抗卡介苗(BCG)血清反应;实验组小鼠的脾脏明显增大,粘连严重;CD4⁺T细胞增殖明显(73.5±3.69),CD4⁺/CD8⁺比值显著升高(5.1±0.98)(P<0.01);血清中γ干扰素(IFN-γ)、白介素12(IL-12)和α肿瘤坏死因子(TNF-α)均有不同程度的升高。结论PEPCK能够诱发小鼠产生细胞免疫和体液免疫反应,是很好的抗结核疫苗候选分子之一。     

Vitamin A analogs as tests for liver vitamin A status in the rat

Two chlorinated retinol analogs (Ro 11-0503 and Ro 11-8284) were assayed in rat serum and correlated to retinol in liver. Rats were fed a retinol-free diet to deplete their liver stores, then repleted with 0.5, 1.0, 3.0, and 10.0 mg retinyl palmitate/kg diet. Rats were given intraperitoneal injections of the analog then killed after an additional week on the diets. Analogs were measured by HPLC. The relative abundance of both analogs in serum was inversely correlated with the amount of retinyl palmitate in the liver. Serum analog concentrations may be useful as indications of liver retinol stores.     

Subcellular location of phosphoenolpyruvate carboxykinase in hepatocytes from fed and starved rats

To evaluate published indications that about 25% of the gluconeogenic enzyme, phosphoenolpyruvate carboxykinase (PEPCK), is located in mitochondria of adult rat liver, cell fractionations were conducted with hepatocytes isolated from rats that were fed ad libitum or starved for 2 days. Hepatocytes were exposed to digitonin for 10 s, and the released materials were separated from residual cell structures by centrifugation through a layer of brominated hydrocarbon. In addition to PEPCK, activities of 9 other enzymes were measured in the untreated cells and with good recovery in the two fractions obtained with digitonin treatment. By comparison with the release of marker enzymes for the cytosol and mitochondria, the subcellular distribution of PEPCK was determined. With cells from either fed or 2-day-starved rats, this enzyme was released exactly like lactate dehydrogenase and within 2-3% of phosphoglycerate kinase and pyruvate kinase. These results indicate that, even after induction by starvation, at least 97% of PEPCK activity is located in the cytosol of rat liver.     

Schedule of protein ingestion and circadian variations of glycogen phosphorylase, glycogen synthetase and phosphoenolpyruvate carboxykinase in rat liver.

The circadian rhythms of liver glycogen and hepatic activity of glycogen synthetase (GS), glycogen phosphorylase (GP) and phosphoenolpyruvate carboxykinase (PEPCK) were studied in adult male rats. The rats either received a mixed diet ad libitum (10% protein) or a protein meal (1.85 g protein) given at 09:00 or 21:00 hours, with free access to a protein-free diet (separately-fed). When the protein meal was ingested at 09:00 hours it was followed by a drop in liver glycogen and a persistent daylight increase in GP and PEPCK activities, this phenomenon being attenuated when proteins were ingested during darkness (21:00 hours). Moreover in the latter case, the circadian rhythm of liver glycogen was modified (glycogen accumulation occurring later) and the protein meal ingestion was followed after a transient decrease by a high and sustained GS activity during a long period (12 hours). The drop in the hepatic glycogen level and the unusually long daylight period of sustained GP and PEPCK activities in separately-fed rats consuming the protein meal at 09:00 hours suggests that, in this case, part of the ingested nitrogen could have been catabolized and used for gluconeogenesis, thus explaining our previous observation of lower nitrogen retention observed in this group of rats.     

Vitamin B-12-deficiency affects immunoglobulin production and cytokine levels in mice

To clarify the role of B-12 in the immunological function, serum C3, IgM, IgG, IgE contents, splenocytes expression of CD4, CD8, and CD4 positive intracellular IFN-gamma and IL-4 were examined in B-12-deficient mice, and the effect of the administration of CH3-B-12 was also studied. Serum C3, IgM and IgG contents were lower in B-12-deficient mice than in the control mice. On the other hand, serum IgE content was significantly higher in B-12-deficient mice, and the value in CH3-B-12 administered mice, administered CH3-B-12 to B-12-deficient mice for 48 h before the end of feeding period, showed a tendency to recovery. CD4+CD8- cells and CD4+CD8-/CD4-CD8+ ratio in splenocytes were significantly higher in B-12-deficient mice than in control mice. CD4+IFN-gamma+ cells was significantly lower in B-12-deficient mice than in control mice, and CD4+IL-4+ was significantly higher in B-12-deficient mice than in control mice. These results suggest that B-12-deficiency causes CD4+CD8-T cells shift from the T helper type 1 to the T helper type 2, which participate in the IgE production and elevates CD4+CD8-/CD4-CD8+ ratio. Thus, B-12 plays a role in maintaining the immune function in mice.     

Pyruvate carboxylase and phosphoenolpyruvate carboxykinase activity in developing rats: effect of manganese deficiency

The activities of two liver gluconeogenic enzymes, pyruvate carboxylase (PC) and phosphoenolpyruvate carboxykinase (PEPCK) as well as plasma glucose were measured in manganese-sufficient and manganese-deficient rats from birth to 30 d of age. Initial (d 0) PC activity was similar in the two groups. PC activity increased 1.5-fold in control pups and 2.4-fold in manganese-deficient pups from d 0 to 3 postnatally. PEPCK activity increased 1.5-fold in control pups and 2.2-fold in manganese-deficient pups from d 0 to 3. By d 8, the activity of PEPCK in manganese-deficient pups was 60% of control levels. Compared to control pups, plasma glucose concentration was lower in manganese-deficient pups on d 1 and 2, coinciding with a period of high neonatal mortality. These findings suggest that glucose homeostasis in the newborn may be compromised by manganese deficiency and may support the concept that changes in cellular manganese concentration may be important in the regulation of carbohydrate metabolism.     

Vitamin K status in the elderly

PURPOSE OF REVIEW: Poor vitamin K nutrition has recently been linked to several chronic diseases associated with abnormal calcification, which affect many elderly. To understand the impact of vitamin K nutrition on healthy aging it is necessary to assess both the determinants and the adequacy of vitamin K nutritional status of the elderly. RECENT FINDINGS: Overall, elderly persons consume more vitamin K than young adults. However, a subgroup of the elderly population does not meet the current recommended dietary intakes for this nutrient. The first meta-analysis evaluating the data on the role of vitamin K and bone health concluded that increased intakes of vitamin K are warranted to reduce bone loss and fracture risk among the elderly. Recent studies suggest that nondietary determinants of vitamin K status need to be factored into any discussion on the adequacy of nutritional status of the elderly. One promising area of research is the interrelationship between estrogen and vitamin K. SUMMARY: Evidence is emerging to support recommendations to increase intakes of vitamin K among the elderly to reduce bone loss and fracture risk. Much more research is required, however, to identify nondietary determinants of vitamin K status, and their impact on the elderly.     

Vitamin A utilization status in chronic alcoholic patients

The utilization status of vitamin A (retinol) (treated with oral retinol - 2500 I.U. daily (=250 micrograms) x 5 days - "OROVITE -7", Bencard, England) in 25 patients (M = 23, F = 2; mean age +/- S.D. = 43.88 +/- 12.67; range = 28-70 years), 3 out of 25 patients (12%) were found to be deficient in the vitamin and during treatment further improvement of the blood levels of the vitamin was observed in all except one elderly male patient (age 61 years) and the mean levels on admission (661.04 micrograms/l) was also slightly improved after treatment (662.84 micrograms/l). Night blindness, alcoholic liver disease and hypogonadism are commonly seen in chronic alcoholic patients. Falling plasma levels of the vitamin indicate exhaustion of its hepatic storage. It is therefore suggested that chronic alcoholics should be given vitamin A supplementation along with other polyvitamins during conventional detoxification therapy for ethanol withdrawal syndrome in order to prevent dangerous manifestations of hypovitaminosis A, such as night blindness, cancer, hypogonadism and alcoholic liver disease.     

Vitamin A status of young children in Southern Brazil

A comprehensive survey was carried out to assess the vitamin A status of preschool children of poor migrant families in the periurban population of Ribeirao Preto, a typical agricultural town in the sugarcane and coffee region of the State of Sao Paulo in Southern Brazil. The intake of vitamin A and carotenoids from the rice and bean based diet of these children is considered low and appears to influence blood concentrations and liver reserves of this vitamin. With respect to plasma vitamin A, 1.8% of the children had a deficient level (less than 10 micrograms%), whereas 48.8% of the children had a low level (less than 20 micrograms%). Most children with inadequate plasma vitamin A (less than 20 micrograms%) responded positively to a massive dose of 200,000 IU vitamin A, suggesting that these children may be at risk of having low liver stores of vitamin A. Rose Bengal staining test and rapid dark adaptation time did not indicate definite signs of conjunctival xerosis and night blindness among these children. No ocular evidence of hypovitaminosis A was found in the children studied, but marginal or inadequate vitamin A status appears to be a common public health problem among young children in this region of Brazil.     

Vitamin A status of children in Sri Lanka.

A representative country-wide rural nutrition status survey determined the extent and distribution of vitamin A deficiency in Sri Lanka in children 6 through 71 months of age. Trained paramedical personnel recorded the presence or absence of selected ophthalmological signs and symptoms associated with vitamin A deficiency in 13,450 children. The results of the country-wide clinical survey indicate that a vitamin A deficiency problem of public health importance may exist in two of 15 health areas. Serum vitamin A levels were determined on 346 survey children from two of 15 health areas and compared with clinical findings for these areas. The lowest mean serum vitamin A, 26.3 microgram/100 ml, occurred in children with clinical eye findings. A high prevalence of clinical eye findings, 34%, and the low mean serum vitamin A value, 28.2 microgram/100 ml, were found in the group of chronically undernourished children--children who are less than 90% of their expected height for age. The survey results enabled planned redirection of the distribution of vitamin A capsules to preschool children in Sri Lanka to areas shown to have the highest prevalences of ophthalmological signs and symptoms and/or the highest prevalence of chronic undernutrition.     

Vitamin A status of preterm infants during infancy

Plasma retinol and retinol-binding protein (RBP) were measured in 67 enterally fed preterm infants (750-1398 g) at 33 +/- 2 wk postconceptional age (PCA), and at regular intervals during infancy. Retinol and RBP declined by 35 +/- 2 wk PCA and remained low at 38 wk after discharge, with the infants fed a term-infant formula. At 38 +/- 2 wk PCA, 48% (32 of 67) of these infants had plasma retinol concentrations less than 0.35 mumol/L. Mean retinol and RBP rose over the next 7 mo, but large numbers of infants (26 of 59 at 48 wk, 10 of 61 at 57 wk) had hyporetinolemia (0.35-0.67 mumol/L). Plasma RBP leveled off at 57 +/- 2 wk PCA and remained low (less than 0.95 mumol/L) in many infants throughout the first year of life. Lower plasma retinol and RBP concentrations at 33 and 38 wk correlated with longer periods of intravenous nutrition. At 57 and 69 wk, lower retinol and RBP correlated with higher birth order. Suboptimal vitamin A status may occur for many months after preterm infants are discharged from the hospital.     

孕期高蛋白饲料对子代大鼠脂肪酸合成酶和磷酸烯醇丙酮酸羧激酶的影响

目的探讨孕期膳食对子代肥胖和肥胖相关基因脂肪酸合成酶(FAS)和磷酸烯醇丙酮酸羧激酶(PEPCK)表达的影响。方法将Wistar孕鼠随机分为2组,分别食用标准饲料和高蛋白质饲料。所产雄性子代分为对照组和高蛋白质组。交叉哺乳,断乳后用标准饲料喂养至成年时,从对照组中随机抽取一部分大鼠(高脂对照组)与高蛋白质组的大鼠均喂饲高脂饲料,高脂诱导肥胖至实验结束。分期处死大鼠取材,应用荧光定量PCR法检测褐色脂肪中FAS及PEPCK mRNA的表达丰度。结果孕期高蛋白质饲料可降低子代断乳及高脂诱导肥胖后的体重及肥胖率(P<0.05);高蛋白质组褐色脂肪组织中PEPCK mRNA的表达持续低于对照值和高脂对照组,而FAS mRNA在诱导肥胖前、后分别低于对照组和高脂对照组。结论孕期适当增加蛋白质摄入,可通过程序性降低子代FAS和PEPCK基因的表达,预防子代成年高脂诱导肥胖的发生。