In vitro studies on methyl mercury distribution in human blood

Studies comparing the methyl mercury (mHg) content of maternal and newborn blood have shown increased levels in the newborn. This has been attributed to facilitated transplacental diffusion because of high fetal hematocrit (Hct). This study shows the converse, that the diffusion of mHg diminishes progressively with increasing Hct. The diffusion of m203Hg across a Millipore membrane (0.45 microns) separating compartments A and B of a diffusion cell was studied. When both compartments contained saline or plasma alone, equilibration from A to B occurred in 5 h. Introduction of human red blood cells (RBC) in saline (Hct 20%) into B resulted in a twofold increase in diffusion of mHg when compared to saline alone. Increasing Hct in saline in compartment B resulted in a progressive decrease in diffusion (r = -0.95, P less than 0.001). The presence of RBC in plasma (Hct 20%) in B resulted in a 70% decrease in diffusion; with increasing Hct, diffusion was further reduced (r = -0.95, P less than 0.001). Direct addition of mHg to RBC in saline resulted in 98% RBC uptake. Increasing concentrations of plasma (at a constant Hct) resulted in a progressive decrease in RBC uptake. In undiluted plasma at Hct 14%, RBC uptake of mHg was 35%. Plasma electrophoresis showed that much of the mHg was associated with a high-molecular-weight lipoprotein fraction. Plasma components appear to be important in the distribution of mHg in blood, and may be a factor in the relatively higher blood levels in the fetus.     

Identification of domain required for catalytic activity of auxilin in supporting clathrin uncoating by Hsc70

During clathrin-mediated endocytosis Hsc70, supported by the J-domain protein auxilin, uncoats clathrin-coated vesicles. Auxilin contains both a clathrin-binding domain and a J-domain that binds Hsc70, and it has been suggested that these two domains are both necessary and sufficient for auxilin activity. To test this hypothesis, we created a chimeric protein consisting of the J-domain of auxilin linked to the clathrin-binding domain of the assembly protein AP180. This chimera supported uncoating, but unlike auxilin it acted stoichiometrically rather than catalytically because, like Hsc70, it remained associated with the uncoated clathrin. This observation supports our proposal that Hsc70 chaperones uncoated clathrin by inducing formation of a stable Hsc70-clathrin-AP complex. It also shows that Hsc70 acts by dissociating individual clathrin triskelions rather than cooperatively destabilizing clathrin-coated vesicles. Because the chimera lacks the C-terminal subdomain of the auxilin clathrin-binding domain, it seemed possible that this subdomain is required for auxilin to act catalytically, and indeed its deletion caused auxilin to act stoichiometrically. In contrast, deletion of the N-terminal subdomain weakened auxilin-clathrin binding and prevented auxilin from polymerizing clathrin. Therefore the C-terminal subdomain of the clathrin-binding domain of auxilin is required for auxilin to act catalytically, whereas the N-terminal subdomain strengthens auxilin-clathrin binding.     

The nesprins are giant actin-binding proteins,orthologous to Drosophila melanogaster muscle protein MSP-300

Nesprin-1 and nesprin-2 (also known as Syne-1 and Syne-2,) are large ( approximately 3300-residue) vertebrate proteins associated with emerin and lamin A at the nuclear envelope of muscle cells and other cell types. We show that the previously described nesprins are short isoforms of giant proteins comprising an actin-binding amino-terminus connected to a carboxy-terminal klarsicht-related transmembrane domain by a massive ( approximately 6000-8000 amino acid) spectrin-like rod domain, making full-length nesprin-1, at one megadalton, the largest non-titin protein hitherto described in humans. We find that MSP-300, a 7000-residue Drosophila melanogaster protein whose disruption results in defects of muscle development, corresponds to the N-terminal two-thirds of the Drosophila nesprin ortholog. A nesprin-like protein is also encoded by the nematode genome. Moreover, we demonstrate that the larger isoforms of nesprin-1, like MSP-300, are localized to the sarcomeric Z-line of both skeletal and cardiac muscle. The recognition that a characteristic muscle-specific mutant phenotype in the fly results from a disruption of its nesprin ortholog reinforces the candidacy of the human proteins for involvement in genetic diseases of skeletal and cardiac muscle.     

Role of cyclin G-associated kinase in uncoating clathrin-coated vesicles from non-neuronal cells

Auxilin is a brain-specific DnaJ homolog that is required for Hsc70 to dissociate clathrin from bovine brain clathrin-coated vesicles. However, Hsc70 is also involved in uncoating clathrin-coated vesicles formed at the plasma membrane of non-neuronal cells suggesting that an auxilin homolog may be required for uncoating in these cells. One candidate is cyclin G-associated kinase (GAK), a 150-kDa protein expressed ubiquitously in various tissues. GAK has a C-terminal domain with high sequence similarity to auxilin; like auxilin this C-terminal domain consists of three subdomains, an N-terminal tensin-like domain, a clathrin-binding domain, and a C-terminal J-domain. Western blot analysis shows that GAK is present in rat liver, bovine testes, and bovine brain clathrin-coated vesicles. More importantly, liver clathrin-coated vesicles, which contain GAK but not auxilin, are uncoated by Hsc70, suggesting that GAK acts as an auxilin homolog in non-neuronal cells. In support of this view, the clathrin-binding domain of GAK alone induces clathrin polymerization into baskets and the combined clathrin-binding domain and J-domain of GAK supports uncoating of AP180-clathrin baskets by Hsc70 at pH 7 and induces Hsc70 binding to clathrin baskets at pH 6. Immunolocalization studies suggest that GAK is a cytosolic protein that is concentrated in the perinuclear region; it appears to be highly associated with the trans-Golgi where the budding of clathrin-coated vesicles occurs. We propose that GAK is a required cofactor for the uncoating of clathrin-coated vesicles by Hsc70 in non-neuronal cells.     

Effect of the petite mutation on maltose and alpha-methylgucoside fermentation inSaccharomyces cerevisiae.

Summary Several hundred petite mutants were isolated from yeast strains of different genotype to examine the effect of the petite mutation on maltose and alpha-methylglucoside fermentation. In most cases petite mutants isolated retain the ability to ferment maltose and alpha-methylglucoside, although at a slower rate. In one strain (1403-7A), however, the ability to ferment alpha-methylglucoside is completely lost in all petite mutants isolated from this strain. It is suggested that mitochondrial factors may be involved in the utilization of alpha-methylglucoside in strain 1403-7A.     

Identification of a novel genetic element in Escherichia coli K-12.

Induction of the SOS repair processes of Escherichia coli K-12 caused a 14.4-kilobase species of circular deoxyribonucleic acid, called element e14, to be excised from the chromosome. To aid further characterization of this species, an 11.6-kilobase segment of e14 was inserted into the HindIII site of plasmid pBR313. To map e14 on the E. coli K-12 chromosome, the recombinant plasmid, pAG2, was used to transform a polA recipient, an event which required integration of pAG2 into the recipient chromosome. This recombinational event was dependent upon the region of homology between the incoming plasmid and the chromosome, as no transformants were scored when either a strain cured of the element was the recipient or pBR313 was the transforming deoxyribonucleic acid. Using these transformants, we have shown that e14 maps between the purB and pyrC loci near min 25. Several strains of E. coli K-12 were found to contain e14; however, one strain, Ymel trpA36, did not. In addition, e14 was found to be absent in both E. coli B/5 and E. coli C. The approach to mapping developed for this work could be used to map other fragments of E. coli deoxyribonucleic acid which have no known phenotype.