Sequence Determinants of GLUT1 Oligomerization: ANALYSIS BY HOMOLOGY-SCANNING MUTAGENESIS*

The human blood-brain barrier glucose transport protein (GLUT1) forms homodimers and homotetramers in detergent micelles and in cell membranes, where the GLUT1 oligomeric state determines GLUT1 transport behavior. GLUT1 and the neuronal glucose transporter GLUT3 do not form heterocomplexes in human embryonic kidney 293 (HEK293) cells as judged by co-immunoprecipitation assays. Using homology-scanning mutagenesis in which GLUT1 domains are substituted with equivalent GLUT3 domains and vice versa, we show that GLUT1 transmembrane helix 9 (TM9) is necessary for optimal association of GLUT1-GLUT3 chimeras with parental GLUT1 in HEK cells. GLUT1 TMs 2, 5, 8, and 11 also contribute to a less abundant heterocomplex. Cell surface GLUT1 and GLUT3 containing GLUT1 TM9 are 4-fold more catalytically active than GLUT3 and GLUT1 containing GLUT3 TM9. GLUT1 and GLUT3 display allosteric transport behavior. Size exclusion chromatography of detergent solubilized, purified GLUT1 resolves GLUT1/lipid/detergent micelles as 6- and 10-nm Stokes radius particles, which correspond to GLUT1 dimers and tetramers, respectively. Studies with GLUTs expressed in and solubilized from HEK cells show that HEK cell GLUT1 resolves as 6- and 10-nm Stokes radius particles, whereas GLUT3 resolves as a 6-nm particle. Substitution of GLUT3 TM9 with GLUT1 TM9 causes chimeric GLUT3 to resolve as 6- and 10-nm Stokes radius particles. Substitution of GLUT1 TM9 with GLUT3 TM9 causes chimeric GLUT1 to resolve as a mixture of 6- and 4-nm particles. We discuss these findings in the context of determinants of GLUT oligomeric structure and transport function.     

Analysis of protein-mediated 3-O-methylglucose transport in rat erythrocytes: rejection of the alternating conformation carrier model for sugar transport

3-O-Methylglucose (3OMG) transport in rat erythrocytes (RBCs) is mediated by a low-capacity, facilitated diffusion-type process. This study examines whether the characteristics of sugar transport in rat RBCs are consistent with the predictions of two diametric, theoretical mechanisms for sugar transport. The one-site carrier describes a transport mechanism in which sugar influx and efflux substrate binding sites are mutually exclusive. The two-site carrier describes a transport mechanism in which sugar influx and efflux substrate binding sites can exist simultaneously but may interact in a cooperative fashion when occupied by substrate. Michaelis and velocity parameters for saturable 3OMG transport in rat erythrocytes at 24 degrees C were obtained from initial rate measurements of 3OMG transport. The results are incompatible with the predictions of the one-site carrier but are consistent with the predictions of a symmetric two-site carrier, displaying negligible cooperativity between substrate binding sites. This allows reduction of the two-site carrier transport equations to a form containing fewer constants than the one-site carrier equations without limiting their predictive success. While the available evidence does not prove that rat erythrocyte sugar transport is mediated by a two-site mechanism, we conclude that adoption of the formally more complex one-site model for sugar transport in rat erythrocytes is unnecessary and unwarranted. Counterflow experiments have also been performed in which the time course of radiolabeled 3OMG uptake is measured in cells containing saturating levels of 3OMG. The results of these experiments are consistent with the hypothesis [Naftalin et al. (1985) Biochim. Biophys. Acta 820, 235-249] that exchange of sugar between intracellular compartments (cell water and hemoglobin) can be rate limiting for transport under certain conditions.     

正常中国人中枢运动系统传导时间的测定

本文应用高电压、低输出阻抗刺激器,经皮给予大脑皮层和脊髓电刺激(BSPES),同时在上肢鱼际(Thenar)和下肢胫骨前肌(Muscle tibialis anterior)上记录诱发肌肉动作电位,测定了64名正常健康中国人(男:46;女:18)的中枢运动系统传导时间。受试者年龄为20—67岁,身高为156—185cm。刺激大脑皮层出现反应的潜伏期与刺激脊髓出现反应的潜伏期之差为中枢运动传导时间(CMCT)。实验测得鱼际的 CMCT 为6.69±1.48ms;胫骨前肌的 CMCT 为12.90±1.59ms。经统计学处理证明,CMCT 与左右侧肢体、性别、年龄及身高无关。说明 CMCT 是无损伤测定与评价中枢运动系统功能的较精确的一种客观指标。本文根据所测数据,计算出脊髓内运动传导速度为71.34±10.89 m/s,与文献报道的锥体束传导速度50—70 m/s 相近。因此,CMCT 反映了锥体束的传导时间。     

Identification of the native form of chicken gizzard myosin light chain kinase with the aid of monoclonal antibodies

Examination, by immunoblotting, of myosin light chain kinase-containing fractions obtained during purification of the enzyme from chicken gizzard has shown that a single species (Mr = 136,000) exists in the muscle and that this enzyme is degraded, primarily to a 130,000-dalton fragment, during purification. These conclusions were confirmed by phosphorylation of the different species of myosin light chain kinase by the isolated catalytic subunit of cyclic AMP-dependent protein kinase.     

Superovulation of beef heifers with Folltropin: A new FSH preparation containing reduced LH activity

The optimum superovulatory dose of Folltropin was determined and compared with a standard 28 mg dose of FSH-P in beef heifers. In Experiment 1, mean numbers of corpora lutea (CL) did not differ among the groups treated with 10, 20, 30 or 40 mg Folltropin or FSH-P, and the mean CL number was reduced (P<0.05) only in the 5 mg Folltropin group. Mean numbers of ova/embryos recovered, fertilized and transferable were greater (P<0.05) for the 10, 20 and 30 mg Folltropin groups than for the 5 mg group. The 40 mg Folltropin group and the FSH-P group were intermediate. The percentage of fertilized and transferable embryos did not differ over the dosages used in this experiment. In Experiment 2, mean numbers of CL were greater for the 9, 18 and 36 mg Folltropin groups than for the 4.5 mg group, with the 9 mg group being lower than the 36 mg group (P<0.05). The 18 mg group was intermediate and did not differ. Mean numbers of ova/embryos recovered and fertilized ova were greater for the 9, 18 and 36 mg groups (P<0.05) than for the 4.5 mg group. The percent of fertilized and mean number and percentage of transferable embryos did not differ among treatments. We conclude that Folltropin may be a satisfactory superovulatory replacement for FSH-P and that a dose of 18 to 20 mg Folltropin may be within the optimum superovulatory dosage range for beef heifers. Dosages of Folltropin of more than twice the optimum did not result in deterioration of ova/embryo quality.     

酪氨酸对大鼠子宫胞浆雌,孕激素受体的影响

采用放射受体分析法,测定了动情周期不同阶段及去卵巢大鼠子宫胞浆雌二醇和孕酮受体含量,并观察了子宫腔内注射酪、丝、苏三种氨基酸对子宫胞浆雌。醇、孕酮受体含量的影响。结果表明：（１）Ｌ－酪氨酸对动情前期、动情期、间情期大鼠子宫胞浆雌二醇和孕酮受体都具有明显的降低作用。（２）Ｌ－酪氨酸也降低去卵巢大鼠子宫胞浆雌二醇和孕酮含量,即这一作用不是通过影响卵巢激素分泌实现的。（３）Ｌ－苏氨酸仅可降低动情期和间情期大鼠子宫胞浆孕酮受体含量,而对相应周期雌二醇受体没有明显作用。（４）Ｌ－丝氨酸和Ｌ－苏氨酸对去卵巢大鼠子宫胞浆雌二醇和孕酮受体均无影响。     

乙烯消减盐渍胁迫对苜蓿种子萌发的抑制作用

在无盐条件下,外源乙烯对苜蓿种子萌发有促进作用,但对最终发芽率无影响。盐渍严重抑制苜蓿种子萌发,加入１～５０μｌ／Ｌ（ｖ／ｖ）外源乙烯或０．１～５．０ｍｍｏｌ／Ｌ１－氨基环丙烷－１－羧酸（ＡＣＣ）或５～１００ｍｇ／Ｌ（ｗ／ｖ）乙烯利（ＥＴＨ）均能极显著地减轻ＮａＣｌ对苜蓿种子萌发的抑制作用。激动素（ＫＴ）也有类似作用,并能促进萌发种子的乙烯产生,它与ＡＣＣ一起使用,则对种子萌发和乙烯产生均显示加成作用。在ＮａＣｌ胁迫下,应用乙烯和乙烯利虽有利萌发,但幼芽鲜重和下胚轴长度明显低于无盐对照。     

Identification of a Genetic Locus in Pseudomonas aureofaciens Involved in Fungal Inhibition

In iron-rich conditions, Pseudomonas aureofaciens PA147-2 produces an antibiotic-like compound that inhibits the growth of a plant fungal pathogen, Aphanomyces euteiches. To contribute to the potential use of PA147-2 as a biocontrol organism, we report the identification of a genetic locus important for antibiotic biosynthesis. Mutants defective for fungal inhibition (Af^-) were generated by Tn5 mutagenesis. Southern hybridization of total DNAs from three Af^- mutants indicated that loss of fungal inhibition was due to a single Tn5 insertion in each mutant. Restriction mapping of the mutation points showed that in two mutants the Tn5 insertions were in the same 16.0-kb EcoRI fragment and were separated by 2.1 kb. A genomic library of PA147-2 was constructed and screened by using a region of DNA flanking the Tn5 insertion in one mutant (PA109) as a probe to recover complementing cosmids. Three cosmids containing a 16.0-kb EcoRI fragment complementary to the two mutants were recovered. Allele replacement by homologous recombination with putative complementing cosmids restored one mutant to antifungal activity against A. euteiches. Southern analysis of the complemented mutants confirmed that allele replacement had occurred between cosmid DNA and Tn5. The wild-type 16.0-kb EcoRI fragment was cloned from the cosmid and complemented the two mutants to antifungal activity. An antifungal compound was isolated from PA147-2 grown on solid medium. Antifungal activity correlated to a peak on high-pressure liquid chromatography analysis. Under the same growth and extraction conditions, the antifungal activity seen in PA147-2 was absent in two Af^- mutants. Furthermore, absence of an antifungal compound in each mutant correlated to the absence of the wild-type “antifungal” peak on high-pressure liquid chromatography analysis.