Characterization and purification of membrane-associated phosphatidylinositol-4-phosphate kinase from human red blood cells

The membrane-bound form of phosphatidylinositol-4-phosphate (PtdInsP) kinase was purified 4,300-fold from human red blood cells to a specific activity of 117 nmol min-1 mg-1. Although this enzyme copurified with red blood cell membranes, it was solubilized by high salt extraction in the absence of detergent indicating that it is a peripheral membrane protein. The major protein seen in the most purified preparation migrated at 53,000 daltons on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The major PtdInsP kinase activity in this preparation was also coincident with this 53,000-dalton band upon renaturation of activity from SDS-PAGE. To test further whether the 53,000-dalton protein contained PtdInsP kinase activity, antibodies were prepared against the gel-purified 53,000-dalton protein. This antiserum was able to precipitate both the 53,000-dalton peptide and PtdInsP kinase activity from red blood cell membranes. The apparent size of the native enzyme in the most purified preparation was determined to be 150,000 +/- 25,000 daltons by gel filtration. This PtdInsP kinase activity was at least 100-fold more active in phosphorylating PtdInsP than phosphatidylinositol and was easily separated from the red cell membrane phosphatidylinositol kinase by salt extraction. Analysis of the reaction product, phosphatidylinositol 4,5-bisphosphate, indicates that the enzyme phosphorylates phosphatidylinositol 4-phosphate specifically at the 5'-hydroxyl of the inositol ring. The apparent Km for ATP was 2 microM, and the concentrations of Mg2+ and Mn2+ giving half-maximal activity were 2 and 0.2 mM, respectively. Mg2+ supported 3-fold higher activity than Mn2+ at optimal concentrations. The enzymatic activity was inhibited by its product, phosphatidylinositol 4,5-bisphosphate and enhanced by phosphatidylserine.     

Sucrose synthase in rice plants : growth-associated changes in tissue specific distributions

Different parts of the rice (Oryza sativa L.) plant at different growth stages were analyzed for sucrose synthase (SS) by enzyme activity assay and enzyme-linked immunosorbent assay directly on the extracts or on the eluates from a gel filtration column. On a dry matter basis, the amount of soluble protein and SS activity decreased significantly, but the amount of enzyme protein changed little in growing leaves. In the grain, the SS activity was the highest at the early ripening stage and decreased later, but the amount of SS protein increased with the increase in maturity. In the root, a low activity of SS was detectable only in the tillering but not in other stages. Immunoblotting of SS protein extracted from different parts of rice showed two bands. Elution patterns of crude extracts from a gel filtration column showed the presence of several types of SS protein. Among them, two to three types with larger elution volumes had the SS activity but others with smaller elution volumes (considered as the aggregated forms) had no activity. The SS purified from different parts of the plant showed similar but distinctly different electrophoretic mobilities in a native gel. It has been concluded that different isozymes are expressed in different tissues at different growth stages.     

Drugs of abuse--opiates.

Treating opiate-dependent patients can be difficult for many physicians because the patients'' life-styles, values, and beliefs differ from those of the physicians. Primary care physicians, however, are often involved in the treatment of the medical complications of opiate abuse, and physicians must often manage a patient''s opiate dependence until appropriate referral to a drug abuse treatment program can be arranged. Treatment is guided by an understanding of the patient''s addictive disease, for which there are specific diagnostic criteria, and an understanding of the pharmacology of opiates of abuse and the medications used in treating opiate dependence. The opiate agonist, methadone, is useful for both detoxification and maintenance. The opiate antagonist, naloxone, is the treatment of choice for opiate overdose, and naltrexone, also an opiate antagonist, is a useful adjunct in subgroups of opiate-dependent patients for preventing relapse. New medications for the treatment of opiate dependence are being developed.     

Purification and characterization of a novel nucleoside phosphorylase from a Klebsiella sp. and its use in the enzymatic production of adenine arabinoside

An adenosine-assimilating bacterium, Klebsiella sp. strain LF1202, inducibly formed a novel nucleoside phosphorylase which acted on both purine and pyrimidine nucleosides when the cells were cultured in medium containing adenosine as a sole source of carbon and nitrogen. The enzyme was purified (approximately 83-fold, with a 17% activity yield) to the homogeneous state by polyacrylamide gel electrophoresis. The molecular weight of the purified enzyme was calculated to be 125,000 by gel filtration of Sephadex G-200 column chromatography, although the enzyme migrated as a single protein band with a molecular weight of 25,000 on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis; thus, it was thought to consist of five identical subunits. Besides purine nucleosides (adenosine, inosine, and guanosine), the purified enzyme also acted on pyrimidine nucleosides such as uridine, 2'-deoxyuridine, and thymidine. The purified enzyme catalyzed the synthesis of adenine arabinoside, a selective antiviral pharmaceutic agent, from uridine arabinoside and adenine.     

High frequency differences between high and low voltage electrocorticograms in the ovine fetus

Dawes (1986) has stated that, "The difference between high and low voltage activity depends solely on the presence in the latter of higher amplitude oscillations with relatively low frequency superimposed on the low voltage components as shown by spectral analysis". We have tested the constancy of the high frequency section of the power spectrum of the electrocorticogram in 7 near-term sheep fetuses. Under sterile conditions we implanted biparietal electrodes in the dura and a ground lead subcutaneously on the scalp. Five days after the surgery, with the animal standing quietly in the laboratory, we acquired the fetal electrocorticograms. Data were acquired during several high and low voltage electrocorticographic cycles in each animal. Two hundred power spectra were obtained during high and low voltage fetal electrocortical activity and statistically analyzed by paired t-test to discern differences in power between the high and low voltage pattern at each frequency (n = 7). We found that at all frequencies between 4 and 12 cycles/s the high voltage electrocorticogram had significantly more power than the low voltage electrocorticogram (P less than 0.05). This is in accordance with the established literature. We also observed that from 17 through 24 cycles/s the low voltage electrocorticogram is significantly higher than the high voltage electrocorticogram (P less than 0.05). In this frequency range the power of the high voltage expressed as a percentage of the power of the low voltage were respectively, 80, 74, 71, 66, 64, 64, 67, 64. These differences are of considerable magnitude and may be physiologically important.     

西双版纳热带季节雨林植物种类多样性的一种研究方法

一、方法1.样地的选择样地分别选取热带干性季节雨林的典型代表——以箭毒木(Antiaris toxicaria)、龙果(Pouteria grandifolia)为标志的群落,以千果榄仁(Terminalia myriocarpa)、番龙眼     

“引黄济青”输水沿线水生生物分布与蓄水库富营养化关系的研究

“引黄济青”是国家“七五”重点工程,由黄河下游博兴县境内打渔张闸引水,在渠首沉沙后,再经惠民、东营、潍坊和青岛的10个县区约253km的明渠输送,到达唠山县棘洪滩蓄水库蓄存,实现向青岛市日供水3.0×10~5m~3。明渠输水过程中,来白沿线的营养物质会污染水体,加之输水在库中滞留时间长,会不会发生富营养化,这是社会普遍关注的问题。     

稀土元素对甜菜种子萌发,幼苗生长及块根产量的影响

本文研究了稀土元素对甜菜的生物效应。结果表明,稀土元素可显著地提高种子萌发率,促进幼苗生长,增加幼苗根系活力、叶片净光合速率与块根产量,并明显地降低了甜菜叶片细胞膜在低温下的透性,即提高了叶片的抗寒力。     

Genetic analysis of two tomato mutants affected in the regulation of iron metabolism

Iron is one of the most important micronutrients for plants. Like other organisms, plants have developed active mechanisms for the acquisition of sufficient iron from the soil. Nevertheless, very little is known about the genetic mechanisms that control the active uptake. In tomato, two spontaneously derived mutants are available, which are defective in key steps that control this process. The recessive mutationchloronerva (chln) affects a gene which controls the synthesis of the non-protein amino acid nicotianamine (NA), a key component in the iron physiology of plants. The root system of the recessive mutantfer is unable to induce any of the characteristic responses to iron deficiency and iron uptake is thus completely blocked. We present a characterization of the double mutant, showing that thefer gene is epistatic over thechln gene and thus very likely to be one of the major genetic elements controlling iron physiology in tomato. In order to gain access to these two genes at the molecular level, both mutants were precisely mapped onto the high density RFLP map of tomato. Thechln gene is located on chromosome 1 and thefer gene is on chromosome 6 of tomato. Using this high-resolution map, a chromosome walk has been started to isolate thefer gene by map-based cloning. The isolation of thefer gene will provide new insights into the molecular mechanisms of iron uptake control in plants.