The FER gene is evolutionarily conserved and encodes a widely expressed member of the FPS/FES protein-tyrosine kinase family.

We have recently isolated human and rat cDNAs (designated FER and flk, respectively) which encode nonreceptor protein-tyrosine kinases which are very similar to one another and related in sequence and domain structure to the c-fps/fes gene product. We show that FER and flk are human and rat counterparts of an evolutionarily conserved gene, hereafter termed FER regardless of species. The human and rat FER genes encode a widely expressed 94-kilodalton protein-tyrosine kinase which is antigenically related to the fps/fes protein-tyrosine kinase. The structural and antigenic similarities between the FER and fps/fes proteins suggest that they are members of a new family of nonreceptor protein-tyrosine kinases.     

DNA-binding domain of human c-Myc produced in Escherichia coli.

We have identified the domain of the human c-myc protein (c-Myc) produced in Escherichia coli that is responsible for the ability of the protein to bind sequence-nonspecific DNA. Using analysis of binding of DNA by proteins transferred to nitrocellulose, DNA-cellulose chromatography, and a nitrocellulose filter binding assay, we examined the binding properties of c-Myc peptides generated by cyanogen bromide cleavage, of mutant c-Myc, and of proteins that fuse portions of c-Myc to staphylococcal protein A. The results of these analyses indicated that c-Myc amino acids 265 to 318 were responsible for DNA binding and that other regions of the protein (including a highly conserved basic region and a region containing the leucine zipper motif) were not required. Some mutant c-Mycs that did not bind DNA maintained rat embryo cell-cotransforming activity, which indicated that the c-Myc property of in vitro DNA binding was not essential for this activity. These mutants, however, were unable to transform established rat fibroblasts (Rat-1a cells) that were susceptible to transformation by wild-type c-Myc, although this lack of activity may not have been due to their inability to bind DNA.     

Progesterone in uterine and arterial tissue and in jugular and uterine venous plasma of sheep

Concentrations of progesterone in uterine and arterial tissue and in uterine and jugular venous plasma were determined. Blood was collected on Days 4 and 9 postestrus from the jugular vein and the first and last venous branches draining each uterine cornu; uterine tissue and arteries were subsequently collected. Progesterone was greater (p less than 0.05) in the cranial third than in the middle or caudal thirds of the uterine horn adjacent to the corpus luteum (CL)-bearing ovary or in any third of the contralateral horn. Progesterone in uterine arterial segments adjacent to the CL-bearing ovary was higher (p less than 0.05) than in contralateral segments. Progesterone was higher (p less than 0.05) in blood from the first venous branch of the cranial third of the uterine cornu adjacent to the ovary with the CL, than in the last branch of the caudal third, or contralateral horn, or in jugular blood. When oviductal veins were resected on Day 9 postestrus, progesterone in the first vein draining the cranial third of the uterine cornu adjacent to the CL-containing ovary was not different (p greater than 0.05) 48 h after resection than in the same vessel in the opposite horn or in jugular blood. We concluded that progesterone and other ovarian products may be delivered to the uterus locally.     

Urea hydrolysis by immobilized urease in a fixed-bed reactor: analysis and kinetic parameter estimation

Urea hydrolysis by urease immobilized onto ion exchange resins in a fixed-bed reactor has been studied. A modified Michaelis-Menten rate expression is used to describe the pH-dependent, substrate- and product-inhibited kinetics. Ionic equilibria of product and buffer species are included to account for pH changes generated by reaction. An isothermal, heterogeneous plug-flow reactor model has been developed. An effectiveness factor is used to describe the reaction-diffusion process within the particle phase. The procedure for covalent immobilization of urease onto macroporous cation exchangers is described. Urea conversion data are used to estimate kinetic parameters by a simplex optimization method. The best-fitted parameters are then used to predict the outlet conversions and pH values for systems with various inlet pH values, inlet urea and ammonia concentrations, buffers, particle sizes, and spacetimes. Very good agreement is obtained between experimental data and model predictions. This immobilized urease system exhibits quite different kinetic behavior from soluble urease because the pH near the enzyme active sites is different from that of the pore fluid. This effect results in a shift of the optimal pH value of the V(max) (pH) curve from 6.6 (soluble urease) to ca. 7.6 in dialysate solution, and ca. pH 8.0 in 20mM phosphate buffer. The reactor model is especially useful for estimating intrinsic kinetic parameters of immobilized enzymes and for designing urea removal columns.     

South western blot mapping: a procedure for simultaneous characterization of DNA binding proteins and their specific genomic DNA target sites

A method called "South Western blot mapping" for rapid characterization of both DNA binding proteins and their specific sites on genomic DNA is described. Proteins are separated on a sodium dodecyl sulfate (SDS) polyacrylamide gel, renatured by removing SDS in the presence of urea, and blotted onto nitrocellulose by diffusion. The genomic DNA region of interest is digested by restriction enzymes selected to produce fragments of appropriate but different sizes, which are subsequently end-labeled and allowed to bind to the separated proteins. The specifically bound DNA is eluted from each individual protein-DNA complex and analyzed by acrylamide gel electrophoresis. Evidence that tissue-specific DNA binding proteins may be detected by this technique is presented. Moreover, their sequence-specific binding allows the purification of the corresponding selectively bound DNA fragments and may improve protein-mediated cloning of DNA regulatory sequences.     

Conformations of the central transforming region (Ile 55-Met 67) of the p21 protein and their relationship to activation of the protein

The GTP-binding p21 protein, encoded by the ras-oncogene, becomes transforming if amino acid substitutions are made at critical positions in the polypeptide chain, e.g., at Gly 12, Gly 13, Ala 59, Gln 61 and Glu 63. Most of these substitutions occur in two phosphate-binding loop regions, Tyr 4-Thr 20, herein designated as segment 1, and Ile 55-Met 67, herein designated, as segment 2. These two segments are homologous to two corresponding regions in the two purine nucleotide binding proteins, bacterial elongation factor (EF-tu) (Val 12-Thr 28 corresponds to segment 1; His 78-Ile 92 corresponds to segment 2) and adenylate kinase (ADK) (Lys 9-Cys 25 corresponds to segment 1 and Tyr 95-Arg 107 corresponds to segment 2). We find that the conformations of the segment 1 region in the p21 protein, EF-tu and ADK are similar to one another and that the conformation of the segment 2 region of EF-tu is superimposable on that of segment 2 of ADK. Furthermore, the relative position of the two segments in EF-tu is strikingly similar to that of the two segments in ADK. In the originally proposed X-ray structure for the p21 protein, the conformation of segment 2 in the p21 protein is not similar to that found for the other two proteins, and its disposition relative to segment 1 and the remainder of the protein is also different from that observed for the other two proteins.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evolutionary conservation of the human homologue of the yeast cell cycle control gene cdc2 and assignment of Cd2 to chromosome 10

Summary The human homologue of the fission yeast Schizosaccharomyces pombe cell cycle control gene cdc2 has been assigned to chromosome 10. DNA hybridization reveals that this gene is highly conserved in vertebrates. The human CDC2 gene probe detects a simple two-allele polymorphism in Taq1-digested DNA.     

Human esterase D gene: complete cDNA sequence,genomic structure,and application in the genetic diagnosis of human retinoblastoma

Summary The gene encoding human esterase D (EsD), a member of the nonspecific esterase family, is a useful genetic marker for retinoblastoma (RB) and Wilson's disease. Previously we identified a cDNA clone from this gene and determined its chromosomal location. In this report, we present the complete cDNA sequence of the human EsD gene. A long open reading frame encoded a predicted protein of 282 amino acids with molecular weight of 30 kD. A computer-assisted search of a protein sequence data base revealed homology with two other esterases, acetylcholinesterase of Torpedo and esterase-6 of Drosophila. Homologous region were centered around presumptive active sites, suggesting that the catalytic domains of the esterases are conserved during evolution. Three genomic clones of this gene were also isolated and characterized by restriction mapping. At least ten exons were distributed over a 35-kb (kilobase pair) region; each exon contained an average of 100 basepairs (bp). A polymorphic site for Apa I, located within an intron of the esterase D gene, can be used to identify chromosome 13 carrying defective RB alleles within retinoblastoma families.     

Studies on the submicrosomal fractions of bovine oligodendroglia: Lipid composition and glycolipid biosynthesis

Oligodendroglia were isolated from bovine brain, and a crude, microsomal fraction obtained from cell homogenates was subfractionated into myelin (MP), plasma membranes (PM), Golgi (GF), smooth (SER) and rough (RER) endoplasmic membranes using discontinuous-sucrose gradient centrifugation. The submicrosomal fractions were characterized by ultrastructural examination and analysis of the specific organelle markers. The myelin and plasma membrane rich fractions contained characteristically the highest amounts of the lipid with lower mole percentages of total phospholipids and phosphatidylcholine, and higher concentrations of phosphatidylethanolamine (+plasmalogens), cholesterol and galactolipids. Considerable amounts of the typical myelin galactolipids (galacto-cerebrosides, sulfatides and monogalactosyl diglycerides) were also found in the Golgi fraction (GF). The GF fraction had the greatest enrichment of glycolipid-forming galactosyltransferases, and the distribution of these enzymes correlated well with that of the Golgi marker enzymes. The results give evidence that intracellular Golgi apparatus of oligodendroglia is rich in the myelin-specific lipids, and suggest its involvement in the synthesis and processing of myelin lipids.     

Effect of dietary protein on cholesterol homeostasis in diabetic rats

Normal and streptozotocin (STZ)-diabetic rats were studied in order to examine the effects of altering the type of dietary protein on cholesterol homeostasis. Rats were fed a non-purified or a purified diet containing either casein or soybean protein. The results obtained on the specific aspects of lipid metabolism were remarkably similar in control rats fed the non-purified (Purina Lab Chow) diet or the purified diet with the soybean protein. However, most of the findings obtained with the above two groups were different from those obtained with rats fed the purified diet containing casein. In the latter group, plasma cholesterol was elevated following a 15-day feeding period as compared to the other two dietary groups. The excess plasma cholesterol in the casein-fed group was found in two lipoprotein fractions with densities of 1.023-1.045 g/ml and 1.045-1.086 g/ml, respectively. The latter lipoprotein fraction was also enriched with apolipoprotein E. The casein-fed animals also showed a lower fractional rate of plasma cholesterol esterification and an abnormal accumulation of cholesterol in the body despite inhibition of cholesterol synthesis in the liver and in the intestines. Twelve to 15 days after the induction of diabetes, plasma cholesterol increased to a similar extent in the rats on all three diets. However, the distribution of cholesterol among the lipoprotein fractions was markedly different. The percentage of cholesterol in fractions of d less than 1.086 g/ml was increased while that carried in the fraction of d 1.086-1.161 g/ml decreased in the rats fed the nonpurified diet and the casein diet. In contrast, there was no change in the distribution of lipoprotein cholesterol between the diabetic and the control rats fed the soybean protein diet. The hepatic synthesis of cholesterol was unaltered in diabetic rats fed the nonpurified diet and the purified diet with soybean protein, but was increased 2.4-fold in diabetic rats fed casein. Intestinal cholesterol synthesis was increased in all three dietary groups. The increase was highest in the rats fed casein and lowest in rats fed soybean protein. The rate of sterol synthesis in the kidneys was not significantly affected by the diet or diabetes. In all three dietary groups diabetes led to an abnormal accumulation of cholesterol in the body. This accumulation was highest in the casein-fed rats and lowest in those fed the soybean protein diet. The cholesterol content of the kidneys was markedly increased by dietary casein.(ABSTRACT TRUNCATED AT 400 WORDS)