Rat lens beta-crystallins are internally duplicated and homologous to gamma-crystallins

The nucleotide sequence of two cloned rat lens beta-crystallin cDNAs pRL beta B3-2 and pRL beta B1-3 has been determined. pRL beta B3-2 contains the complete coding information for a beta-crystallin, designated beta B3, of 210 amino acid residues. pRL beta B1-3 is incomplete at its 5' end; the 5' codogenic information which is not present in this cDNA clone was deduced from the cloned gene. pRL beta B1-3 codes for a beta-crystallin polypeptide, designated beta B1, whose full length is 247 amino acid residues. Considerable sequence homology is noted between the amino- and carboxy-terminal halves of each protein. The two rat beta-crystallins show a substantial sequence homology with each other (60%) as well as with the published sequences of rat gamma-crystallin (37%) and bovine and murine beta-crystallins (55 and 45%). All these proteins have a two-domain structure which, like the bovine gamma II-crystallin, might be folded into four remarkably similar protein motifs. Our data further indicate that the beta-crystallins can be subdivided into two groups which are evolutionarily related. Both groups are, although more distantly, also related to the gamma-crystallins.     

Transport of amino acids and ammonium in mycelium of Agaricus bisporus.

Mycelium of Agaricus bisporus took up methylamine (MA), glutamate, glutamine and arginine by high-affinity transport systems following Michaelis-Menten kinetics. The activities of these systems were influenced by the nitrogen source used for mycelial growth. Moreover, MA, glutamate and glutamine uptakes were derepressed by nitrogen starvation, whereas arginine uptake was repressed. The two ammonium-specific transport systems with different affinities and capacities were inhibited by NH(+)(4), with a K(i) of 3.7 microM for the high-velocity system. The K(m) values for glutamate, glutamine and arginine transport were 124, 151 and 32 microM, respectively. Inhibition of arginine uptake by lysine and histidine showed that they are competitive inhibitors. MA, glutamate and glutamine uptake was inversely proportional to the intracellular NH(+)(4) concentration. Moreover, increase of the intracellular NH(+)(4) level caused by PPT (DL-phosphinotricin) resulted in an immediate cessation of MA, glutamine and glutamate uptake. It seems that the intracellular NH(+)(4) concentration regulates its own influx by feedback-inhibition of the uptake system and probably also its efflux which becomes apparent when mycelium is grown on protein. Addition of extracellular NH(+)(4) did not inhibit glutamine uptake, suggesting that NH(+)(4) and glutamine are equally preferred nitrogen sources. The physiological importance of these uptake systems for the utilization of nitrogen compounds by A. bisporus is discussed.     

Exon skipping in the sterol 27-hydroxylase gene leads to cerebrotendinous xanthomatosis

We report a new mutation in the sterol 27-hydroxylase (CYP 27) gene in a Dutch family with cerebrotendinous xanthomatosis: a G→A transition in the splice donor site in intron 4. This mutation leads to skipping of exon 4, resulting in a loss of 66 amino acids in the CYP 27 enzyme molecule. Received: 15 March 1997 / Accepted: 26 March 1997     

Altered fatty acid composition in regulatory mutants of Streptomyces cinnamonensis

Abstract cDNA-RNA liquid hybridization analysis was used to compare the RNA sequence homology between two members of the Nudaurelia β virus family, Trichoplusia ni virus ( T.ni V) and Dasychira pudibunda virus ( D.p V). Heterologous hybridization experiments demonstrated that these viruses shared little sequence homology. Using oligo(dT) chromatography and oligo(dT)_12–18 as a primer for cDNA synthesis it was shown that neither T.ni V nor D.p V RNA genomes possess a poly(A) tract at the 3' end.     

Immunotherapy of lymphoid and nonlymphoid tumors with monoclonal anti-Lyt-1 antibodies

Monoclonal antibodies against the T cell differentiation antigen Lyt-1 were effective in the therapy of transplanted mouse tumors. A possible mechanism whereby anti-Lyt-1 antibodies directly bind and affect the tumor cells was excluded by the following findings: a) growth of lymphoid and nonlymphoid tumors (which lack Lyt-1 antigen) was affected by anti-Lyt-1 antibodies; and b) the curative effect of passively administered anti-Lyt-1 anti-bodies was abrogated in mice depleted of T cells, supporting a mechanism whereby host Lyt-1+ cells were involved in tumor therapy. Treatment with anti-Lyt-1 antibodies was not accompanied by depletion of Lyt-1+ cells from lymphoid organs, indicating that the administered antibodies altered Lyt-1+ cell functions without affecting their frequency. In view of the in vitro enhancing effects of anti-Lyt-1 antibodies on a variety of immune responses (including lymphokine secretion and generation of cytotoxic T cell), it is suggested that the potentiation of Lyt-1+ cell activity by passively administered anti-Lyt-1 antibodies results in tumor rejection.     

Cell surface heparan sulfate proteoglycans from human vascular endothelial cells. Core protein characterization and antithrombin III binding properties.

Human aortic endothelial cells (HAEC) and human umbilical vein endothelial cells (HUVEC) were labeled with 35SO(4)2- for 48 h. The membrane-associated proteoglycans were solubilized from these monolayers with detergent and purified by ion-exchange chromatography on Mono Q, incorporation in liposomes, and gel filtration. The liposome-intercalated proteoglycans were 125I-iodinated and treated with heparitinase before SDS-polyacrylamide gel electrophoresis. Radio-labeled proteins with apparent molecular masses of 130, 60, 46, 35, and 30 kDa (HAEC) and 180, 130, 62, 43, and 35 kDa (HUVEC) were detected by autoradiography. Further characterization by affinity chromatography on immobilized monoclonal antibodies and by Northern blot analysis provided evidence for the expression of syndecan, glypican, and fibroglycan in human endothelial cells. Most of the heparan sulfate which accumulated in the subendothelial matrix was implanted on a 400-kDa core protein. This protein was immunologically related to perlecan and bound to fibronectin. Binding studies on immobilized antithrombin III suggested that all membrane-associated heparan sulfate proteoglycan forms had the capacity to bind to antithrombin III but that high affinity binding was more typical for glypican. Most of the proteoglycans isolated from the extracellular matrix also bound only with low affinity to antithrombin III. These results imply that glypican may specifically contribute to the antithrombotic properties of the vascular wall.