Evidence that erythroid-type glucose transporter intrinsic activity is modulated by cadmium treatment of mouse 3T3-L1 cells

Previous studies suggest that regulation of hexose uptake in Chinese hamster ovary fibroblasts can occur by alterations in glucose transporter intrinsic activity without changes in cell surface transporter number (Harrison, S. A., Buxton, J. M., Helgerson, A. L., MacDonald, R. G., Chlapowski, F. J., Carruthers, A., and Czech, M. P. (1990) J. Biol. Chem. 265, 5793-5801). We tested this hypothesis using 3T3-L1 fibroblasts and adipocytes which exhibit 5-6-fold increases in 2-deoxyglucose or 3-O-methylglucose uptake when exposed to low micromolar concentrations of cadmium for 18 h. Cadmium treatment decreased the apparent Km of 3T3-L1 fibroblasts for 3-O-methylglucose influx from approximately 28 to 9 mM and increased the apparent Vmax by 2-3-fold. These fibroblasts lack the skeletal muscle/adipocyte-type (GLUT4) transporter and showed only a small increase in total cellular immunoreactive HepG2 type (GLUT1) transporter in response to cadmium. Furthermore, cell surface GLUT1 levels did not change in 3T3-L1 fibroblasts exposed to cadmium, as assessed by the binding to intact cells of an antibody which recognizes an extracellular GLUT1 epitope. Insulin enhanced 2-deoxyglucose uptake 2-fold in 3T3-L1 fibroblasts, but did not further stimulate cadmium-activated transport rates. In contrast, insulin stimulated hexose transport 15-fold in 3T3-L1 adipocytes, which express both GLUT1 and GLUT4 proteins, and this effect was fully additive with the 5-fold effect of cadmium. Cadmium had little or no effect on immunoreactive GLUT1 or GLUT4 in isolated 3T3-L1 adipocyte plasma membranes. In contrast, insulin action led to marked recruitment (3-fold) of GLUT4 to the plasma membrane fraction in adipocytes treated with or without cadmium. Taken together, these data are consistent with the hypothesis that cadmium-activated sugar uptake is catalyzed by GLUT1, whereas insulin-stimulated sugar uptake is catalyzed predominantly by GLUT4 in 3T3-L1 adipocytes. Furthermore, the data suggest that the GLUT1 transporter can undergo significant increases in intrinsic catalytic activity in response to cadmium treatment of 3T3-L1 fibroblasts and adipocytes.     

Rat procathepsin B. Proteolytic processing to the mature form in vitro.

Expression of rat procathepsin B in yeast led to the secretion of both the latent and mature forms of the enzyme. Culture in the presence of a cysteine proteinase inhibitor prevented this processing. We have expressed and purified a mutant form of rat procathepsin B whose active-site cysteine residue has been changed to a serine, and which also lacks the glycosylation site in the mature region of the protein. This non-active mutant protein was secreted essentially in an unprocessed form. The purified protein has been incubated with a variety of proteinases, and results indicate that cathepsins D and L, as well as mature cathepsin B itself, can produce a processed (single-chain) form of cathepsin B from this precursor. Amino-terminal sequencing of these processed forms has revealed that they are all elongated by a few residues with respect to the mature form found in vivo. The action of a combination of cathepsin B with dipeptidylpeptidase I produced a single-chain form of cathepsin B with the correct amino terminus. This work has also shown that the processing of procathepsin B to a single-chain form can be an autocatalytic process, in at least an intermolecular manner.     

Rates of DNA change and phylogeny from the DNA sequences of the alcohol dehydrogenase gene for five closely related species of Hawaiian Drosophila

The sequence of 1.6 kb of DNA surrounding the alcohol dehydrogenase (Adh) gene from five species of the Planitibia subgroup of the Hawaiian picture-winged Drosophila, with estimated divergence times of 0.4-5.1 Myr, has been determined. The gene trees which were found by using the sequence divergence from different regions of the sequences are generally in accord with the phylogeny proposed for these species when chromosomal inversions and island of origin are used. One of the species (D. picticornis) appears to be more distant from the other species in this group than they are from a member of the Grimshawi group (D. affinidisjuncta) which is chromosomally more distant. Two of the species (D. differens and D. plantibia) show heterogeneity in the nucleotide changes in the Adh coding region, heterogeneity which is interpreted to be due to a gene conversion or recombination after hybridization between the two species. The minimal rate of nucleotide substitution of synonymous nucleotides and of nontranscribed nucleotides downstream from the coding region is estimated as 1.5 x 10(-8) and 1.1 x 10(-8) substitutions/nucleotide/year, respectively. This rate is two to three times the maximal rate estimated for mammalian synonymous substitutions.     

Chromosomal assignment of the human genes coding for the major proteins of the desmosome junction, desmoglein DGI (DSG), desmocollins DGII/III (DSC), desmoplakins DPI/II (DSP), and plakoglobin DPIII (JUP)

We have established PCR assays for the genes coding for the major proteins of the desmosome type of cell junction, the desmosomal cadherins DGI (desmoglein) and DGII/III (desmocollins), and the plaque proteins DPI/II (desmoplakin) and DPIII (plakoglobin) and used them to test human-mouse and human-rat somatic cell hybrids with different contents of human chromosomes. From these data we were able to assign DGI to chromosome 18 (DSG), DGII/III to chromosome 9p (DSC), DPI/II to chromosome 6p21-ter(DSP), and DPIII to chromosome 7 (JUP).     

Transmembrane molecular assemblies regulated by the greater cadherin family

The cadherin family of cell-cell adhesion molecules is turning out to be much more diverse than previously thought, with members involved in several kinds of intercellular junctions. The adhesive specificity and cytoskeletal interaction of these members varies. Their cytoplasmic terminals are specialized for binding several families of 'mediator' proteins which interconnect to the actin or intermediate filament systems. These multi-molecular complexes have roles not only in cell-cell adhesion, but also in intracellular signalling of developmental information.     

Plastoquinol diffusion in linear photosynthetic electron transport

The diffusion of plastoquinol and its binding to the cytochrome bf complex, which occurs during linear photosynthetic electron transport and is analogous to reaction sequences found in most energy-converting membranes, has been studied in intact thylakoid membranes. The flash-induced electron transfer between the laterally separated photosystems II and photosystems I was measured by following the sigmoidal reduction kinetics of P-700⁺ after previous oxidation of the intersystem electron carriers. The amount of flash-induced plastoquinol produced at photosystem II was (a) reduced by inhibition with dichlorophenyl-dimethylurea and (b) increased by giving a second saturating flash. These signals were simulated by a new model which combines a deterministic simulation of reaction kinetics with a Monte Carlo approach to the diffusion of plastoquinol, taking into account the known structural features of the thylakoid membrane. The plastoquinol molecules were assumed to be oxidized by either a diffusion-limited or a nondiffusion-limited step in a collisional mechanism or after binding to the cytochrome bf complex. The model was able to account for the experimental observations with a nondiffusion-limited collisional mechanism or with a binding mechanism, giving minimum values for the diffusion coefficient of plastoquinol of 2 × 10^-8 cm²s^-1 and 3 × 10^-7 cm²s^-1, respectively.     

Distribution of podolactone-type plant growth inhibitors in the coniferae

Plant growth inhibitors of the podolactone-type have been detected by bioassay in ten further species of Podocarpus. The most active extracts in P. elatus were from root tips, root cortex and very young leaves. Fifty-seven other conifers were examined for this type of activity. It is present in Cephalotaxus harringtonia where it is probably due to the presence of harringtonolide, which, like momilactone B from rice husks, shows podolactone-type inhibition of the growth of etiolated dwarf pea hooks.     

Population characteristics and the distribution of general medical practitioners.

By applying the logic of the Resource Allocation Working Party to the analysis of the distribution of general medical practitioners, the relevant Family Practitioner Committee (FPC) populations were weighted according to known patterns of use related to specific characteristics--namely, age, sex, marital state, and socioeconomic group. Comparative weightings were also calculated using standardised mortality ratios. Adjusting the populations to take account of differential use has relatively little impact on national variations in list sizes but an appreciable effect on particular FPCs, notably East and West Sussex, Dorset, and the Isle of Wight. Inequalities in the distribution of general practitioners are increased considerably, however, if figures taking account of the inflation of list sizes and cross-boundary flows are used. To formulate and monitor policy about the distribution of general practitioners more sensitive measures of population and its likely demand for services must be developed.     

Complete tyrosine assignments in the high field 1H nuclear magnetic resonance spectrum of the bovine pancreatic trypsin inhibitor.

The low-field portions of the 250-MHz 1H nuclear magnetic resonance (NMR) specra of native and chemically modified bovine basic pancreatic trypsin inhibitor (BPTI) have been studied as a function of pH over the range pH 5-13. Resonances associated with the 16 protons of the aromatic rings of the four BPTI tyrosines have been located and assigned to specific tyrosyl residues. Titrations of pH yielded pK's for tyrosines-10, -21, -23, and -35 of 10.4, 11.0, 11.7, and 11.1, respectively. The resonances associated with the nitrotyrosine-10 protons of mononitrated BPTI and the nitrotyrosine-10 and -21 protons of dinitrated BPTI have been similarly located, assigned and titrated yielding pK's for nitrotyrosine-10 and -21 of 6.6 and 6.4, respectively. The high-field NMR spectrum indicates that the aromatic ring of tyrosine-35 rotates less than 160 times per second at 25 degrees for pH's in the range 5-9.