3H-azidopine photoaffinity labeling of high molecular weight proteins in chloroquine resistant falciparum malaria

Using 3H-azidopine, we have succeeded in labeling proteins from chloroquine resistant (CR) human falciparum malaria parasites in the molecular weight range of 155-170 kd. Vinblastine does not compete, but azidopine blocks the labeling using 3H-azidopine. Relatively little or no labeling of the 155-170 kd protein is seen in the chloroquine sensitive strain using 3H-azidopine. Further competition can be seen with nicardipine and reserpine (71%) respectively and verapamil (61%), chloroquine (48%), quinacrine (56%), trifluoperazine (32%) and chlorpromazine (33%). We speculate that this may be the glycoprotein responsible for the resistance to chloroquine in falciparum malaria.     

Carbonyldiphosphonate, a selective inhibitor of mammalian DNA polymerase delta

Twenty-three pyrophosphate analogues were screened as inhibitors of proliferating cell nuclear antigen independent DNA polymerase delta (pol delta) derived from calf thymus. Carbonyldiphosphonate (COMDP), also known as alpha-oxomethylenediphosphonate, inhibited pol delta with a potency (Ki = 1.8 microM) 20 times greater than that displayed for DNA polymerase alpha (pol alpha) derived from the same tissue. Characterization of the mechanism of inhibition of pol delta indicated that COMDP competed with the dNTP specified by the template and was not competitive with the template-primer. In the case of pol alpha, COMDP did not compete with either the dNTP or the polynucleotide substrate. COMDP inhibited the 3'----5' exonuclease activity of pol delta weakly, displaying an IC50 greater than 1 mM.     

Binding of sulfonamide and acetamide to the active-site Zn2+ in carbonic anhydrase: a theoretical study

Self-consistent field molecular orbital (SCF MO) calculations at both 4-31G and STO-3G levels have been used to examine the binding conformations of sulfonamide and acetamide compounds to the active site of carbonic anhydrase. The results are as follows: (1) sulfonamide binds to the Zn2+ ion in its deprotonated form through the sulfonamide nitrogen to the fourth coordination site of the metal ion; (2) acetamide as neutral species binds to the basic form of the enzyme through the carbonyl oxygen to the fifth coordination site of the metal ion; and (3) the acetamidate ion binds to the acid form of the enzyme through the amide nitrogen to form a tetracoordinated metal complex with three histidine ligands. Analysis of the effects of individual active-site residues on the binding conformations of these inhibitors suggests that metal alone favors bidentate coordination of sulfonamidate and acetamidate complexes and that electron donation from three histidine ligands to the metal ion determines the formation of a tetracoordinated metal complex, which is further stabilized by the presence of Thr 199, as it receives one hydrogen bond from the sulfonamide NH- or from the acetamide NH- and donates a backbone NH hydrogen bond to a sulfonamide oxygen. The calculated binding conformation of sulfonamide and the hydrogen-bonding interactions between sulfonamide and the enzyme are consistent with the X-ray diffraction study of the AMSulf-HCA II complex. However, no X-ray structures are available for amide-HCA II complexes.(ABSTRACT TRUNCATED AT 250 WORDS)     

A cytoplasmic thyroid hormone binding protein: characterization using monoclonal antibodies

We have previously purified a cellular thyroid hormone binding protein (p58) from a human carcinoma cell line [Kitagawa, S., Obata, T., Hasumura, S., Pastan, I., & Cheng, S.-y. (1987) J. Biol. Chem. 262, 3903-3908]. In the present study, the binding characteristics, the molecular properties, and subcellular localization of p58 were further characterized. Binding of the purified p58 to thyroid hormones was examined. Analysis of binding data indicates that p58 binds to 3,3',5-triiodo-L-thyronine (T3) with a Kd of 24.3 +/- 0.3 nM and n = 0.71. p58 binds to L-thyroxine similarly as to T3. However, D-T3 and reverse-T3 bind to p58 with an affinity 4- and 20-fold less than that of T3, respectively. By use of the purified p58 as an immunogen, two hybridomas, J11 and J12, secreting monoclonal antibodies to p58 were isolated; both antibodies belong to the IgG1K subclass. J12 recognizes p58 from human, monkey, dog, hamster, and rat, but not mouse. J11 exhibits a similar species specificity except that it does not react with p58 from hamster. With these antibodies, p58 was found to be not posttranslationally modified by glycosylation, sulfation, or phosphorylation. It has a cellular degradation rate t1/2 congruent to 2.1 h. Immunocytochemical studies indicate that p58 is located in the nonmembranous cytoplasm (cytosol). These results are consistent with subcellular fractionation studies which show that greater than 95% of J11 and J12 reactivity and T3 binding activity can be found in the 110,000g supernatant.     

Albumin — vitamin D-binding protein haplotypes in Asian-Pacific populations

Summary We have determined the various haplotypic combinations between alleles as well as restriction fragment length polymorphisms of two linked genetic markers, albumin and vitamin D-binding protein or group-specific component, in a number of Asian-Pacific populations. Using the partial maximum likelihood method, we constructed a phylogenetic network from the haplotype frequencies to assess relationships among the populations sampled. No systematic linkage disequilibrium was detected between most of the combinations, suggesting a lack of operation of any selection pressure at the two loci. The phylogenetic analysis confirmed the known interrelationships among various populations in the Asian-Pacific region. The Australian aborigines clustered closely with the non-Austronesian-speaking highlanders from Papua New Guinea, as expected. Similarly, the Austronesian-speaking Polynesians, Micronesians, and the Southeast Asians branched off together as a separate group. The position of the Austronesian-speaking Tolais from New Britain with respect to other populations from the Southwest Pacific was anomalous. The Tolais revealed a strong affinity with the Australian aborigines, which is inexplicable. The populations from China formed a tight cluster with other populations from the Asian-Pacific region. Genetic interrelationships of these populations with the white Australians were remote, which is in accordance with the known affinities of various human racial groups.     

Interactions of chiral molecules with an (r)-n-(3,5-dinitrobenzoyl) phenylglycine HPLC stationary phase

Forty different chiral molecules were studied by liquid chromatography with a Pirkle-type, (R)-N-(3,5-dinitrobenzoyl) phenylglycine (DNBPG), chiral stationary phase column. The dramatic effect of a small molecular change on chiral recognition was demonstrated using DL-amino acid derivatives. The inductive effect on chiral recognition was also studied using trifluoro-, trichloro-, dichloro-, monochloroacetyl, and acetyl derivatives of four different chiral amines. The study of the enantiomer separation of 11 different crown ethers of 2,2′-binaphthyldiyl showed that the rigidity of the chiral center can be an additional parameter in chiral recognition for the DNBPG phase but not for a β-cyclodextrin bonded chiral phase. It is apparent from this study that steric effects, inductive effects, and molecular rigidity play important roles in chiral recognition with DNBPG chiral stationary phases.     

Sex determination in the dioecious Melandrium

Summary Melandrium album (2n=24), a dioecious species with heteromorphic sex chromosomes (XY, males and XX, females), has a strong genetic commitment for sex determination. We report here a procedure for obtaining haploid plants from cultured anthers and show that genotype, pollen stage, cold treatment and certain culture media components are essential for a reproducible yield of embryos. Our procedure increased the number of responsive anthers and not the number of responsive microspores per anther. Most likely, our experimental system allows the recovery of competent microspores, and this on a medium containing either an auxin or a cytokinin. All of the 36 anther-derived plants tested expressed a female phenotypic sex instead of the theoretical one male one female ratio. When analysed cytologically, the plants exhibited the corresponding female genetic sex (one or two X chromosomes).     

烟草抗黑胫病突变体的细胞筛选

经实验我们成功地建立了在细胞水平上筛选烟草抗黑胫病突变体的筛选体系。该体系的主要内容为:γ-射线500—2000拉德诱变高度感病品种的花药后用50—80％的黑胫病菌粗毒素为选择压力,筛选出抗毒素花粉植株,用离体叶片法测定选出抗病植株,再从后代鉴定中选出抗病性能够稳定遗传的突变系。γ-射线及高浓度毒素处理均能得到抗病植株。选自感病品种的花粉植株中约有9—50％是真正抗病的。这些抗病植株中有一部分的抗病性能够稳定遗传。用该法已从感病优质品种小黄金1025及乔庄黑苗中选出6个突变系。并自N.C.628(抗)×小黄金1025(感)及N.C.628(抗)×庆胜2号(感)的F_1花粉植株中选出4个抗病系。所有的抗病系经3—4代后均表现出稳定抗性。其中一个突变体(R400)的抗性似由不完全显性多基因控制。