Crystallization of and preliminary X-ray data for the plasma retinol-binding protein

Crystals of the human and rabbit plasma retinol-binding proteins have been grown from solutions of polyethylene glycol 6000 and CdCl2. Two crystal forms have been observed for the human protein, while the rabbit protein has only crystallized in one form which is isomorphous with one of the human serum retinol-binding protein crystals. The crystals differ in their morphologies, but are both in space group P212121 and have similar unit cell sizes (a = 45.9, b = 53.3, c = 72.0 A and a = 45.7, b = 48.7, and c = 76.5 A). The crystals diffract to approximately 2.0 A resolution. In both cases there is 1 molecule/asymmetric unit.     

Effect of vagotomy and thoracic sympathectomy on responses of the monkey to water immersion

Cardiopulmonary stretch receptors have been implicated as part of a reflex mechanism linking changes in blood volume to changes in renal excretion. Experiments were performed to determine whether total denervation of these receptors by combined cervical vagotomy and thoracic sympathectomy affects the renal responses of the monkey to head-out water immersion, a maneuver that translocates blood to the thorax and elicits an increase in renal salt and water excretion. Macaca fascicularis monkeys first underwent chronic bilateral thoracic sympathectomy or sham denervation performed in two stages a week apart. One to two weeks later, they were anesthetized with pentobarbital sodium, and the sympathectomized animals underwent bilateral cervical vagotomy. Control renal function did not differ between the two groups. Immersion of 90-min duration increased central venous and mean arterial pressures by similar amounts in both groups, but heart rate increased only in the sham-denervated animals. Denervation did not affect the magnitudes or delay the times of onset of the increases in urine flow, absolute and fractional sodium excretion, and osmolar and free water clearances occurring with immersion. These results demonstrate that in the anesthetized monkey cardiopulmonary receptors are not necessary for eliciting the renal responses to immersion.     

Production of phenylalanine ammonia-lyase (PAL): isolation and evaluation of yeast strains suitable for commercial production of l-phenylalanine

Summary Phenylalanine Ammonia-Lyase (PAL) containing microorganisms were isolated from a wide variety of natural habitats. The best 21 strains to emerge from the primary screen were screened for PAL activities in both directions using l-phenylalanine and t-cinnamate substrates. Twelve of the latter strains were compared for total cell production and PAL activity and 7 isolates were chosen for examination of the extent of PAL induction in various media. On the basis of these screens, isolate SPA 10 (identified as Rhodotorula rubra) was selected for further optimization. Growth was optimal at 28° C and pH 5.0, although cellular PAL activity was shown to be higher at sub-optimal temperatures (36° C) and pH (8.0) for growth. Synthesis of PAL was repressed when grown in the presence of various sugars and NH ₄ ⁺ ions. Manipulation of fermentation conditions enabled PAL synthesis to occur at maximum biomass levels, upon glucose exhaustion. PAL was rapidly inactivated within cells shortly after maximum synthesis was attained: feeding of d,l-isoleucine and low concentrations of d,l-phenylalanine, and shifting of fermentation temperature conferred catalyst stability for fermentations over 100 h. These results demonstrate the suitability and superiority of isolate SPA 10 for the commercial production of l-phenylalanine from trans-cinnamic acid.     

Influence of transposable elements on the structure and function of the A1 gene of Zea mays

The structure of the A1 gene of Zea mays was determined by sequencing cDNA and genomic clones. The gene is composed of four exons and three short introns. The 40.1-kd A1 protein is an NADPH-dependent reductase. Germinal derivatives of the mutable a1-m1 allele with either recessive or wild-type phenotype have been isolated. Sequence analysis of these revertant alleles indicates that frame-shift mutations abolish A1 gene function, whereas one additional amino acid within the protein sequence still allows wild-type gene expression. The presence of a second, promoter-like structure, upstream of the functional A1 gene promoter is discussed with respect to its possible involvement in differential expression of the A1 gene. The structure of the a1-m2 8004, 3456 and 4412 alleles, featuring distinguishable phenotypes in the presence of Spm(En), was also determined. In all alleles the 1080-bp-long inhibitor (I) element is located 15 bp upstream of the CAAT box of the A1 gene promoter. The unusual response of a1-m2 alleles to trans-active signals of the Spm(En) element is discussed with respect to the position of the I inserts. Also presented are data on the structure and insertion sites of transposable elements determined by cloning and sequencing of the mutable a1 alleles a1-mpapu, a1-mr 102 and a1-ml.     

Insulin causes insulin-receptor internalization in human erythrocyte ghosts.

The effect of incubation with insulin on insulin-receptor internalization by erythrocyte ghosts was investigated. The number of surface insulin receptors decreased by 30-40% after incubation of ghosts with insulin. Total insulin-receptor binding to solubilized ghosts was the same in insulin-incubated and control ghosts, whereas insulin binding to an internal vesicular fraction was substantially increased in insulin-incubated ghosts. Our findings suggest that erythrocyte-ghost insulin receptors are internalized to a vesicular compartment in response to incubation with insulin.     

Determination of the range in binding-site densities of rat skin heparin chains with high binding affinities for antithrombin.

Rat skin heparin proteoglycans vary markedly in the proportions of their constituent polysaccharide chains that have high binding affinity for antithrombin. As the proportion of such chains in a proteoglycan rises, their degree of affinity for antithrombin also increases [Horner (1987) Biochem. J. 244, 693-698]. The antithrombin-binding-site densities of such chains have now been determined, by measuring heparin-induced enhancement of the intrinsic fluorescence of antithrombin and by chemical analysis for the disaccharide sequence glucuronosyl-N-sulphoglucosaminyl (3,6-di-O-sulphate), which is unique to this site in heparin [Lindahl, Bäckström, Thunberg & Leder (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 6551-6555]. Antithrombin-binding-site density ranged from one to five sites per chain.     

Prostaglandins as reducing agents: a model of adenylate cyclase activation?

It has been suggested that adenylate cyclase activation involves reduction of a disulfide linkage. Prostaglandin E1 (PGE1), prostaglandin E2 (PGE2), prostaglandin I2 (PGI2) and prostaglandin F2 alpha (PGF2 alpha) were tested for their ability to act as reducing agents with either cytochrome c, or the disulfide 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), the latter with a catalytic amount of ferric chloride. PGE1, PGE2, and PGI2 significantly reduced cytochrome c while PGF2 alpha did not. PGE1, PGE2 and PGI2 reduced DTNB while PGF2 alpha did not. The results are consistent with the postulate that prostaglandins which are effective in activating adenylate cyclase can act as reducing agents and might be involved in reductive activation of adenylate cyclase.     

Class II genes of the human major histocompatibility complex. Organization and evolutionary relationship of the DR beta genes

The genes of the polymorphic HLA-DR molecules are located within the human major histocompatibility complex. We have studied the HLA-DR genes of an HLA homozygous individual typed to be DR4, Dw4, and DRw53. Fourteen cosmid and phage clones from genomic libraries were isolated and grouped into three clusters comprising a total of 165 kilobases. These clusters contain four DR beta genes. Nucleotide sequence determination showed that two of the genes encode beta chains that carry the DR4 and DRw53 specificities, respectively, while the other two genes are presumably pseudogenes. Comparisons of the nucleotide sequences of all four DR beta genes of the DR4 haplotype show that the genes are extensively similar, approximately 90% in both exons and introns. All four genes are equally similar to each other. These observations are consistent with the notion that the genes arose by duplications that were followed by homogenization through gene conversion. The existence of more than one DR beta gene homologue but only a single DR alpha gene homologue in mouse, rabbit, and cattle suggests that the DR beta gene duplications occurred at or early during mammalian speciation.