Association of polymorphisms in the HLA-B region with extended haplotypes

Genomic probes from the HLA-B region of the major histocompatibility complex (MHC) were used to study the association of restriction fragment length polymorphisms (RFLPs) with various MHC alleles, complotypes, and extended haplotypes. The two DNA probes, M20A and R5A, were derived from previously cloned cosmids and are located 38 and 110 kilobases (kb) centromeric to HLa-B, respectively. Five different RFLP variants occuring in five different haplotypic combinations were detected within a panel of 40 homozygous-typing cells and cells from 21 families using Bst EII. In two informative families with HLA-B/DR recombinations the inheritance of the RFLP variants was consistent with their mapping between HLA-B and complotypes. The R5A/M20A haplotypic pattern 6.5 kb/3.0 kb (A) had a normal Caucasian frequency of approximately 0.43 and was found in all independent examples of the extended haplotypes [HLA-B8,SC01,DR3], [HLA-B18,F1C30, DR3], [HLa-Bw62,SC33,Dr4], [HLa-B44,SC30,Dr4], and [HLA-Bw47,FC91,0DR7]. The patterns of 6.9 kb/ 3.0 kb (B), 6.5 kb/4.7 kb (C), 1.45 kb/3.0 kb (D), and 6.9 kb/4.7 kb (E) had normal Caucasian frequencies of approximately 0.23, 0.15, 0.15, and 0.04 and were found on all independent examples of [HLA-B38,SC21, DR4], [HLA-Bw57,SC61,DR7], [HLA-B7,SC31,DR2], and [HLA-B44,FC31,DR7], respectively. Individual complotypes or HLA-B alleles which were markers of extended haplotypes showed variable associations. For example, HLA-B7 and the complotype SC31 were associated with all R5A/M20A RFLP haplotypes except haplotype E, although [HLA-B7,SC31,DR7] was associated exclusively with haplotype D. HLA-B27, not known to be part of an extended haplotype, was suprisingly exclusively associated with the 6.5 kb/4.7 kb or C haplotypic pattern in all five instances tested. These findings support the concept of regional conservation of DNA on independent examples of extended haplotypes. The results also further characterize these haplotypes.     

Determination of epithelial half-somites in skeletal morphogenesis.

The segmental body plan of vertebrates arises from the metameric organization of the paraxial mesoderm into somites. Each mesodermal somite is subdivided into at least two distinct domains: rostral and caudal. The segmental pattern of dorsal root ganglia, sympathetic ganglia and nerves is imposed by differential properties of either somitic domain. In the present work, we have extended these studies by investigating the contribution of rostral or caudal-half somites to vertebral development using grafts of multiple somite halves. In both rostral and caudal somitic implants, the grafted mesoderm dissociates normally into sclerotome and dermomyotome, and the sclerotome further develops into vertebrae. However, the morphogenetic capabilities of each somitic half differ. The pedicle of the vertebral arch is almost continuous in caudal half-somite grafts and is virtually absent in rostral half-somite implants. Similarly, the intervertebral disk is present in rostral half-somite chimeras, and much reduced or virtually absent in caudal somite chimeras. Thus, only the caudal half cells are committed to give rise to the vertebral pedicle, and only the rostral half cells are committed to give rise to the fibrocartilage of the intervertebral disk. Each vertebra is therefore composed of a pedicle-containing area, apparently formed by the caudal half-somite, followed by a pedicle-free zone, the intervertebral foramen, derived from the rostral somite. These data directly support the hypothesis of resegmentation, in which vertebrae arise by fusion of the caudal and rostral halves of two consecutive somites.     

Respiratory activity of alginate-encapsulated Pseudomonas fluorescens cells introduced into soil

Summary Alginate-entrapped cells of Pseudomonas fluorescens were introduced into soil microcosms to evaluate their respiratory activity (O₂ consumption and CO₂ evolution) and survival during a 14-day incubation period at 20°C. Alginate-entrapped cells and cells resuspended in sterile distilled water and introduced into sterile soil exhibited relatively similar O₂ consumption/CO₂ evolution and survival over the 14-day period. The same treatments in non-sterile soil exhibited lower respiratory activity and a population density decrease of about 2.0 Log. cfu/g after 14 days. Alginate-entrapped bacterial cells may be a useful method for introducing genetically-engineered and non-engineered bacterial strains into the soil environment.     

Human mast cell proteases hydrolyze neurotensin, kinetensin and Leu5-enkephalin.

Purified mast cell carboxypeptidase cleaved the C-terminal leucines from Leu5-enkephalin (Leu-ENK), neurotensin (NT), and kinetensin (KT), with Km values of 36, 16, and 15 microM, and kcat values of 44, 51, and 53 s-1, respectively. To better predict potential in vivo hydrolysis products generated by mast cell proteases, these peptides were incubated with released skin mast cell supernatants. Leu5-enkephalin was hydrolyzed only by carboxypeptidase. Kinetensin was cleaved by tryptase, chymase, and carboxypeptidase to yield KT(1-3), KT(1-7), KT(1-8), KT(4-7), and KT(4-8), the last two peptides by the concerted action of two of the proteases. NT(1-11) and NT(1-12) were generated from neurotensin by chymase and carboxypeptidase, respectively.     

Distribution of opioidergic,sympathetic and neuropeptide Y-positive nerves in the sphincter of Oddi and biliary tree of the monkey,Macaca fascicularis

Summary The opioidergic, sympathetic and neuropeptide Y-positive innervation of the sphincter of Oddi (common bile duct sphincter and pancreatic duct sphincter), as well as other segments of the extrahepatic biliary tree was studied in the monkey by use of immunohistochemistry. Methionine-enkephalin-positive nerves were seen to innervate the smooth muscle of all portions of the sphincter of Oddi and also local ganglion cells. No methionine-enkephalin-positive nerves could be detected in the common bile duct, pancreatic duct or gallbladder. Tyrosine hydroxylase-positive nerves occurred between smooth muscle bundles and also ran to local ganglion cells as well as along the common bile duct. Neuropeptide Y-positive nerves were observed within smooth muscle of the sphincter of Oddi (all portions), common bile duct, pancreatic duct and gallbladder. No evidence of any differential innervation of the pancreatic duct and common bile duct sphincters could be detected with these markers.     

Isolation and partial characterization of an ion channel protein from human sperm membranes

Human sperm cells were fractionated and plasma membrane proteins were separated by molecular gel sieving chromatography (Sephacryl S-200 followed by HPLC). A pore-forming protein was extracted from sperm cell membranes. The partially purified protein migrated with Mr 100,000-110,000, as determined by molecular sieving gel chromatography, and with a Mr 90,000 when analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions. The channel activity was also extracted with Triton X-114, suggesting a hydrophobic nature for this protein. This protein was incorporated into planar lipid bilayers, resulting in the formation of voltage-dependent ion channels. Single channel fluctuations of 130 pS/unit in 0.1 M NaCl were resolved; however, channels preferentially aggregated in triplets having an open state life-time that persisted for several seconds. The channels studied here were more selective for monovalent cations than anions, but also showed some permeability to anions and larger electrolytes, suggesting a large functional pore diameter. The role of this sperm channel in normal sperm physiology and/or fertilization is presently unclear.     

Effects of atrial natriuretic factor on urinary concentration of catecholamines and renin secretion in dogs

This study evaluated the effects of synthetic atrial natriuretic factor (ANF) on renal hemodynamics, urinary excretion of electrolytes, norepinephrine (NE), and dopamine (DA); and renal production of renin in anesthetized dogs. Following a bolus (1 micrograms/kg body weight) and infusion (0.1 microgram/kg/min) for 30 min, there was significant increase in urine flow (220 +/- 41%), glomerular filtration rate (72 +/- 14%), and urinary sodium excretion (170 +/- 34%). There was a decrease in renin secretory rate and the concentration ratio of urine NE to DA following ANF was decreased (p less than 0.05). These data suggest that ANF decreases renal production of NE and renin.     

The distribution of cell surface proteins on spreading cells. Comparison of theory with experiment.

Bretscher (1983) has shown that on uniformly spread giant HeLa cells, the receptors for low density lipoprotein (LDL) and transferrin are concentrated toward the periphery of the cells. To explain these nonuniform distributions, he proposed that on giant HeLa cells, recycling receptors return to the cell surface at the cell's leading edge. Since the distribution of coated pits on these cells is uniform, Bretscher and Thomson (1983) proposed that there is a bulk membrane flow toward the cell centers. Here we present a mathematical model that allows us to predict the distribution of cell surface proteins on a thin circular cell, when exocytosis occurs at the cell periphery and endocytosis occurs uniformly over the cell surface. We show that on such a cell, a bulk membrane flow will be generated, whose average velocity is zero at the cell center and increases linearly with the distance from the cell center. Our model predicts that proteins that aggregate in coated pits will have concentrations that are maximal at the cell periphery. We fit our theory to the data of Bretscher and Thomson (1983) on the distribution of ferritin receptors for the following cases: the receptors move by diffusion alone; they move by bulk membrane flow alone; they move by a combination of diffusion and bulk membrane flow. From our fits we show that tau m greater than 3.5 tau p, where tau m and tau p are the lifetimes of the membrane and the ferritin receptor on the cell surface, and that tau pD less than 6.9 X 10(-7) cm2, where D is the ferritin receptor diffusion coefficient. Surprisingly, we obtain the best fits to the data when we neglect membrane flow. Our model predicts that for proteins that are excluded from coated pits, the protein concentration will be Gaussian, being maximal at the cell center and decreasing with the distance from the cell center. If on giant HeLa cells a protein with such a distribution could be found, it would strongly support Bretcher's proposal that there is an inward membrane flow.     

Immunochemical studies on the interaction between synthetic glycoconjugates and alpha-L-fucosyl binding lectins

Evonymus europaea lectin precipitated with alpha DGal(1----3) beta DGal(1----4)beta DGlcNAc-bovine serum albumin (BSA), alpha LFuc(1----2)beta DGal(1----3)beta DGlcNAc-BSA, alpha LFuc(1----2)beta DGal(1----4)DGlcNAc, and alpha DGal(1----3)[alpha LFuc(1----2)]beta DGal-BSA. However, the lectin neither precipitated with alpha LFuc(1----2)-beta DGal-BSA, alpha DGal(1----3)beta DGal-BSA, or beta DGal(1----4)beta DGlcNAc-BSA nor agglutinated erythrocytes of Oh phenotype having multiple terminal beta DGal(1----4)beta DGlcNAc residues. These results indicate that the minimal structural requirement for glycoprotein precipitation or cell agglutination by the lectin includes any of the three trisaccharides (fucosylated or nonfucosylated) derived from the blood group B tetrasaccharide. The monosaccharides linked to the beta-D-galactosyl residue in the blood group B tetrasaccharide, namely, alpha-D-galactose, alpha-L-fucose, and N-acetyl-beta-D-glucosamine, participate almost equally in binding to the lectin in as much as removal of any one of these sugars reduces the inhibiting potency of the resulting trisaccharide. alpha LFuc(1----2)beta DGal(1----3)beta DGlcNAc-BSA (H type 1) and alpha LFuc(1----2)beta DGal(1----4)beta DGlcNAc (H type 2) were precipitated to the same extent. The E. europaea lectin neither precipitated alpha DGal(1----4)-beta DGal(1----4)beta DGlcNAc-BSA, Lea-BSA, Leb-BSA, or beta DGlcNAc(1----4)[alpha LFuc(1----6)]beta DGlcNAc-BSA nor agglutinated Oh,Lea and Oh,Leb erythrocytes, demonstrating that terminal D-galactose linked alpha-(1----4) to subterminal beta-D-galactose, or alpha-L-fucose linked to N-acetylglucosamine, prevents lectin binding. Corey-Pauling-Koltun molecular models, built on the basis of data from 1H NMR and hard-sphere exo-anomeric (HSEA) calculations provided by Lemieux and co-workers [Lemieux, R. U., Bock, K., Delbaere, L. T. J., Koto, S., & Rao, V. S. (1980) Can. J. Chem. 58, 631-653], show that these alpha-D-galactosyl and alpha-L-fucosyl groups act to sterically hinder lectin binding to these oligosaccharides; these observations also suggest that the lectin binds to the beta-side of these oligosaccharides. These sides, on both blood group H type 1 and blood group H type 2 oligosaccharides, provide a similar contour which can fully account for their equal reactivity with E. europaea lectin. The only difference found between Lotus and Ulex I lectins in precipitating ability was that only Lotus precipitated with beta DGlcNAc(1----4)[alpha LFuc(1----6)]beta DGlcNAc-BSA.(ABSTRACT TRUNCATED AT 400 WORDS)