Biomedical subjects

K Varadaraj

Publications and source records attributed to K Varadaraj.

10 recordsLinked to original sources

PubMed2001

Optical dysfunction of the crystalline lens in aquaporin-0-deficient mice.

Aquaporin-0 (AQP0), a water transport channel protein, is the major intrinsic protein (MIP) of lens fiber cell plasma membranes. Mice deficient in the gene for AQP0 (Aqp0, Mip) were generated from a library of gene trap embryo stem cells. Sequence analysis showed that the gene trap vector had inserted into the first exon of Aqp0, causing a null mutation as verified by RNA blotting and immunochemistry. At 3 wk of age (postnatal day 21), lenses from null mice (Aqp0(-/-)) contained polymorphic opacities, whereas lenses from heterozygous mice (Aqp0(+/-)) were transparent and did not develop frank opacities until approximately 24 wk of age. Osmotic water permeability values for Aqp0(+/-) and Aqp0(-/-) lenses were reduced to approximately 46% and approximately 20% of wild-type values, respectively, and the focusing power of Aqp0(+/-) lenses was significantly lower than that of wild type. These findings show that heterozygous loss of AQP0 is sufficient to trigger cataractogenesis in mice and suggest that this MIP is required for optimal focusing of the crystalline lens.

Age Factors↗

PubMed2001

Site-directed mutations in the transmembrane domain M3 of human connexin37 alter channel conductance and gating.

Connexin37 (Cx37) is expressed principally in endothelial cells. We have introduced individual point mutations (Cx37-V156D or Cx37-K162E) in the putative pore lining segment M3 of a polymorphic human Cx37 (Cx37-S319) and expressed them in N2A and RIN cells. RT-PCR and immunofluorescence microscopy were used to confirm the expression of the proteins. Stably transfected cells were subjected to electrophysiological studies. Experiments were performed on cell pairs using the dual whole cell patch-clamp method. Single channel records showed that both mutants display a variety of conductive states (Cx37-V156D, 47-250 pS; Cx37-K162E, 58-342 pS) in contrast to the typical high conductance of 340-375 pS and subconductive state of 60-80 pS reported for Cx37-S319. Analysis of the macroscopic data for Cx37-K162E revealed a broadened Vo indicating the influence of the mutation on voltage gating. Our data indicate that substitution of a conserved residue with a charged residue could cause changes in the main state and/or in the size of the pore. It is possible that these particular residues in the M3 domain interact electrostatistically with several of the other domains in the Cx37 protein.

Animals↗

PubMed2000

Functional expression and biophysical properties of polymorphic variants of the human gap junction protein connexin37.

Connexin37 (Cx37) forms gap junction channels between endothelial cells, and two polymorphic Cx37 variants (Cx37-S319 and Cx37-P319) have been identified with a possible link to atherosclerosis. We studied the gap junction channel properties of these hCx37 polymorphs by expression in stably transfected communication-deficient cells (N2A and RIN). We also expressed a third, truncated variant (Cx37-fs254Delta293) and Cx37 constructs containing epitope tags added to their amino or carboxyl termini. All Cx37 constructs were produced by the transfected cells as demonstrated by RT-PCR and immunoblotting and trafficked to appositional surfaces between cells as demonstrated by immunofluorescence microscopy. Dual whole cell patch-clamping studies demonstrated that Cx37-P319, Cx37-S319, and Cx37-fs254Delta293 had large unitary conductances ( approximately 300 pS). However, addition of an amino terminal T7 tag (T7-Cx37-fs254Delta293) produced a single channel conductance of 120-145 pS with a 24-30 pS residual state. Moreover, the kinetics of the voltage-dependent decline in junctional current for T7-Cx37-fs254Delta293 were significantly slower than for the wild type, implying a destabilization of the transition state. These data suggest that the amino terminus of Cx37 plays a significant role in gating as well as conductance. The carboxyl terminal tail has lesser influence on unitary conductance and inactivation kinetics.

Amino Acid Sequence↗

PubMed1999

The role of MIP in lens fiber cell membrane transport.

MIP has been hypothesized to be a gap junction protein, a membrane ion channel, a membrane water channel and a facilitator of glycerol transport and metabolism. These possible roles have been indirectly suggested by the localization of MIP in lens gap junctional plaques and the properties of MIP when reconstituted into artificial membranes or exogenously expressed in oocytes. We have examined lens fiber cells to see if these functions are present and whether they are affected by a mutation of MIP found in CatFr mouse lens. Of these five hypothesized functions, only one, the role of water channel, appears to be true of fiber cells in situ. Based on the rate of volume change of vesicles placed in a hypertonic solution, fiber cell membrane lipids have a low water permeability (pH2O) on the order of 1 micron/sec whereas normal fiber cell membrane pH2O was 17 micron/sec frog, 32 micron/sec rabbit and 43 micron/sec mouse. CatFr mouse lens fiber cell pH2O was reduced by 13 micron/sec for heterozygous and 30 micron/sec for homozygous mutants when compared to wild type. Lastly, when expressed in oocytes, the pH2O conferred by MIP is not sensitive to Hg2+ whereas that of CHIP28 (AQP1) is blocked by Hg2+. The fiber cell membrane pH2O was also not sensitive to Hg2+ whereas lens epithelial cell pH2O (136 micron/sec in rabbit) was blocked by Hg2+. With regard to the other hypothesized roles, fiber cell membrane or lipid vesicles had a glycerol permeability on the order of 1 nm/sec, an order of magnitude less than that conferred by MIP when expressed in oocytes. Impedance studies were employed to determine gap junctional coupling and fiber cell membrane conductance in wild-type and heterozygous CatFr mouse lenses. There was no detectable difference in either coupling or conductance between the wild-type and the mutant lenses.

Animals↗

PubMed1999

A three-state model for connexin37 gating kinetics.

The gating behavior of human connexin 37 (hCx37) is unaffected by the nature of the bathing monovalent (for Na, K, Rb). It is modified by [Mg] in the millimolar range. For fitting the kinetics, we propose a simple extension to three states of the canonical 2-state model of the hemichannel. The extra closed state allows for some immobilization of a hemichannel at high transjunctional voltages. The model is reasonably efficient at fitting data at various voltage protocols. Interpreting the fits of the data at different [Mg] is consistent with a binding site for Mg.

Biophysical Phenomena↗

PubMed1998

Effects of lens major intrinsic protein on glycerol permeability and metabolism.

Lens Major Intrinsic Protein (MIP) is a member of a family of membrane transport proteins including the Aquaporins and bacterial glycerol transporters. When expressed in Xenopus oocytes, MIP increased both glycerol permeability and the activity of glycerol kinase. Glycerol permeability (pGly) was 2.3 +/- 0.23 x 10(-6) cm sec-1 with MIP vs. 0.92 +/- 0.086 x 10(-6) cm sec-1 in control oocytes. The pGly of MIP was independent of concentration from 5 x 10(-5) to 5 x 10(-2) m, had a low temperature dependence, and was inhibited approximately 90%, 80% and 50% by 1.0 mM Hg++, 0.2 mM DIDS (diisothiocyanodisulfonic stilbene), and 0.1 mm Cu++, respectively. MIP-enhanced glycerol phosphorylation, resulting in increased incorporation of glycerol into lipids. This could arise from an increase in the total activity of glycerol kinase, or from an increase in its affinity for glycerol. Based on methods we present to distinguish these mechanisms, MIP increased the maximum rate of phosphorylation by glycerol kinase (0.12 +/- 0.03 vs. 0.06 +/- 0.01 pmol min-1 cell-1) without changing the binding of glycerol to the kinase (KM approximately 10 micron).

Animals↗

PubMed1997

Molecular characterization of four members of the alpha-tubulin gene family of the Bermuda land crab Gecarcinus lateralis.

Four alpha-tubulin isoforms recovered from a cDNA library from regenerating limb buds of the Bermuda land crab Gecarcinus lateralis have been characterized. Two clones (alpha 1 and alpha 2) contained complete coding sequences with start and stop codons; the other two clones were partial, lacking 5' ends. The four isoforms showed high homology in their coding sequences but rather low homology in their non-coding regions. Identity between the nucleotide sequences of alpha 1 and alpha 2 was 83.4%; between their predicted amino acid sequences it was 88.9%. The inferred number of amino acid residues for both alpha 1 and alpha 2 was 451, and their calculated molecular weights were 58.29 and 58.45 kDa, respectively. The greatest divergence in the predicted crab alpha-tubulin proteins occurred near the carboxy terminus, as in alpha-tubulins of other organisms. When compared with other species; nucleotide sequences of all four clones showed highest homology to alpha-tubulin genes of an insect (Drosophila melanogaster), while their predicted amino acid sequences were most highly homologous to an alpha-tubulin of a mammal (Rattus norvegicus). Southern blots revealed a total of five to seven alpha-tubulin genes encoded in the G. lateralis genome. Northern blots showed single bands of approximately 2.2 kb with an alpha 1-tubulin probe and 1.9 kb with an alpha 2-tubulin probe. mRNA levels of both tubulin isoforms appeared to be independently regulated at different stages of the intermolt cycle in both epidermis and limb buds. Western blots of 1D gels of proteins from epidermis, limb buds, or claw muscle showed tissue- and stage-specific changes in tubulin content; similar analyses on blots of 2D gels revealed differences in the number of alpha-tubulin isoforms that were expressed.

Amino Acid Sequence↗

PubMed1996

Actin-encoding cDNAs and gene expression during the intermolt cycle of the Bermuda land crab Gecarcinus lateralis.

Two actin-encoding cDNAs (act1 and act2) from Gecarcinus lateralis have been sequenced or partially sequenced and the corresponding proteins deduced. The act1 cDNA has a complete ORF; the act2 cDNA lacks most of the 5' end of the coding region. The nucleotide (nt) sequences of both clones are very similar to act sequences of many organisms, the most closely related being from another arthropod, the silkmoth Bombyx mori. The proteins Act1 and Act2 are more similar to vertebrate cytoplasmic actin isoforms (beta-actins) than to vertebrate muscle actins (alpha-actins); they are also more similar to animal actins than to those of fungi or plants. Codon usage is strongly biased toward C or G in the third position. The deduced number of amino acid (aa) residues and calculated Mr for Act1 are 376 aa and 41.94 kDa, respectively. The deduced aa sequence of Act1 is very similar to those of muscle actins of B. mori and Drosophila melanogaster. Southern blots indicated seven to eleven act genes in the crab genome. Northern blots probed with a segment from the 3' UTR of act1 showed a single band of approx. 1.6 kb in poly(A)+ mRNAs from epidermis, limb bud or claw muscle and in total RNAs from ovary and gill, and two bands of approx. 1.6 and 1.8 kb in total RNA from midgut gland. Western blots of one-dimensional gels of proteins from the four layers of the exoskeleton, epidermis, limb buds and claw muscle were probed with a monoclonal Ab against chicken gizzard actin; tissue- and stage-specific changes in actin content were observed. The presence of several isoforms, and differences in their number and occurrence at various stages of the intermolt cycle, were detected on Western blots of two-dimensional gels.

Actins↗

PubMed1994

Denaturants or cosolvents improve the specificity of PCR amplification of a G + C-rich DNA using genetically engineered DNA polymerases.

We describe conditions that improve the specificity of amplification of a G + C-rich (57% G + C) DNA by PCR. Under standard conditions a 368-bp segment of the approx. 2.1-kb repeat unit of a satellite DNA that accounts for approx. 3% of the genome of the Bermuda land crab, Gecarcinus lateralis, was not amplified specifically. To establish optimal conditions for amplification of the segment of the G + C-rich satellite, we used two genetically engineered enzymes, AmpliTaq DNA polymerase and AmpliTaq DNA polymerase, Stoffel fragment (SF), and a number of denaturants or co-solvents. In the absence of denaturants or co-solvents, amplified products of both enzymes contained non-specific bands upon gel electrophoresis. Addition of certain denaturants or co-solvents to PCR mixtures resulted in the production of the single specific band of the expected size. Reagents that improved specificity of the amplified product were formamide, glycerol, DMSO, Tween-20 and NP-40; on the other hand, urea, ethanol and 1-methyl-2-pyrrolidone (NMP) inhibited amplification. Of the two enzymes, SF was more specific and efficient. The products of AmpliTaq DNA polymerase included one or more extra bands, even in the presence of denaturants or co-solvents, except for glycerol or DMSO.

Base Sequence↗

PubMed1994

Cytoplasmic localization of transcripts of a complex G+C-rich crab satellite DNA.

The primary sequence and higher order structures of a G+C-rich satellite DNA of the Bermuda land crab Gecarcinus lateralis have been described previously. The repeat unit of the satellite is approximately 2.1 kb. In exploring a possible function for this satellite, we asked whether it is transcribed. As a probe for transcripts, we used a segment of DNA amplified from a 368 bp EcoRI fragment from the very highly conserved 3' end of the satellite DNA. During polymerase chain reaction (PCR) amplification, the probe was simultaneously either radiolabeled or biotinylated. Tissue- and stage-specific transcripts were observed when blots of poly(A)+ mRNAs recovered from polysomes isolated from crab tissues [including midgut gland (hepatopancreas), limb bud, and claw muscle] were probed with the satellite DNA fragment. The presence of satellite transcripts in polysomal mRNAs is strong evidence that the transcripts had reached the cytoplasm. To corroborate the presence of transcripts in the cytoplasm, we investigated in situ hybridization of satellite probes with RNAs in tissue sections. Biotinylated satellite DNA probes were applied to sections of midgut gland, limb bud papilla, ovary, or testis of anecdysial crabs. Retention of RNAs in tissue sections was improved by UV-irradiation prior to hybridization. Transcripts were abundant in the cytoplasm of all tissues except testis. Sections of crab midgut gland treated with RNase A prior to hybridization and sections of mouse pancreatic tumor served as controls; neither showed any signals with the probe.

Animals↗