Physical mapping of deletion breakpoints in patients with X-linked ichthyosis: evidence for clustering of distal and proximal breakpoints

Previous studies have shown that approximately 80% of patients with X-linked ichthyosis have a total deletion of the steroid sulphatase (STS) locus which lies in Xp22.3-Xpter. We show by Southern analysis that a common core of sequences are absent in 78.6% of our cases, suggesting that the deletion breakpoints may be highly clustered. To characterize the region in more detail a long-range physical map of over 3 megabases (Mb) surrounding the STS locus was constructed using pulse-field gel electrophoresis. The map enabled the order of sequences tel-SI19-GMGXY3-[STS,GMGXY19]-GMGX9-[dic56 ,SIII2]-cen and the localization of the deletion breakpoints to be established. In ten cases the pulse-field evidence supports the clustering of breakpoints and indicates a deletion size of 2 Mb in most patients. Five CpG islands have been positioned around the STS locus and may be associated with other loci in the region involved in mental retardation and Kallman's syndrome. The map will be instrumental in an attempt to isolate and characterize the deletion breakpoints and to access other genes located in the region.     

Processing, secretion, and biological properties of a novel growth factor of the fibroblast growth factor family with oncogenic potential.

We recently reported that the protein encoded in a novel human oncogene isolated from Kaposi sarcoma DNA was a growth factor with significant homology to basic and acidic fibroblast growth factors (FGFs). To study the properties of this growth factor (referred to as K-FGF) and the mechanism by which the K-fgf oncogene transforms cells, we have studied the production and processing of K-FGF in COS-1 cells transfected with a plasmid encoding the K-fgf cDNA. The results show that, unlike basic and acidic FGFs, the K-FGF protein is cleaved after a signal peptide, glycosylated, and efficiently secreted as a mature protein of 176 or 175 amino acids. Inhibition of glycosylation impaired secretion, and the stability of the secreted K-FGF was greatly enhanced by the presence of heparin in the cultured medium. We have used the conditioned medium from transfected COS-1 cells to test K-FGF biological activity. Similar to basic FGF, the K-FGF protein was mitogenic for fibroblasts and endothelial cells and induced the growth of NIH 3T3 mouse cells in serum-free medium. Accordingly, K-fgf-transformed NIH 3T3 cells grew in serum-free medium, consistent with an autocrine mechanism of growth. We have also expressed the protein encoded in the K-fgf protooncogene in COS-1 cells, and it was indistinguishable in its molecular weight, glycosylation, secretion, and biological activity from K-FGF. Taken together, these results suggest that the mechanism of activation of this oncogene is due to overexpression rather than to mutations in the coding sequences.     

Synthesis of active Olisthodiscus luteus ribulose-1,5-bisphosphate carboxylase in Escherichia coli

The ribulose-1,5-bisphosphate carboxylase (Rubisco) large- and small-subunit genes are encoded on the chloroplast genome of the eukaryotic chromophytic alga Olisthodiscus luteus. Northern blot experiments indicate that both genes are co-transcribed into a single (>6 kb) mRNA molecule. Clones from the O. luteus rbc gene region were constructed with deleted 5 non-coding regions and placed under control of the lac promoter, resulting in the expression of high levels of O. luteus Rubisco large and small subunits in Escherichia coli. Sucrose gradient centrifugation of soluble extracts fractionated a minute amount of carboxylase activity that cosedimented with native hexadecameric O. luteus Rubisco. Most of the large subunit synthesized in E. coli appeared insoluble or formed an aggregate with the small subunit possessing an altered charge: mass ratio compared to the native holoenzyme. The presence in O. luteus of a polypeptide that has an identical molecular mass and cross reacts with antiserum generated against pea large-subunit binding protein may indicate that a protein of similar function is required for Rubisco assembly in O. luteus.     

Regulation of Brain and Cerebrospinal Fluid Calcium by Brain Barrier Membranes Following Vitamin D-Related Chronic Hypo- and Hypercalcemia in Rats

Male Fischer-344 rats, 21 days old, were fed diets containing 0 (LOD), 2,200 (CONT), or 440,000 (HID) international units of vitamin D3 per kilogram for 12 weeks. [Ca] was measured in plasma, CSF, brain, and choroid plexus. In addition, 45Ca and 36Cl transfer coefficients (KCa and KCl) for uptake from blood into CSF and brain were determined. Although plasma ionized [Ca]s in LOD and HID rats were 50% and 136%, respectively, of values in CONT animals, CSF and brain [Ca]s ranged from only 85% to 110% of respective CONT values. Choroid plexus [Ca] was increased by 37% after HID diet, but was decreased only 10% after LOD. KCa values at CSF, parietal cortex, and pons-medulla were negatively correlated with plasma ionized [Ca], whereas KCl values at CSF and brain were not different between the diet groups. The findings demonstrate that central nervous system [Ca] is maintained during chronic hypo- or hypercalcemia by saturable transport of Ca at brain barrier membranes. This transport does not seem to involve modulation by 1,25-dihydroxyvitamin D3.     

Cloning of hemA from Rhizobium sp. NGR234 and symbiotic phenotype of a gene-directed mutant in diverse legume genera

Summary The hemA gene which encodes -aminolaevulinic acid synthase (ALAS), was cloned and characterized from the broad host-range Rhizobium strain NGR234. A cosmid, identified by hybridization with the cloned gene of R. meliloti and complementation of an R. meliloti hemA mutant, was subcloned to yield a 5.5 kb fragment containing the entire NGR234 gene. A physical-genetic map was made and the interposon was introduced into a single EcoRI site which bisects the gene. The mutated gene was homogenotized into NGR234 to generate a hemA mutant, with a view to evaluating the role of rhizobial bacteroid ALAS activity for a wide variety of legume symbioses. The mutant strain formed an ineffective (Fix^–) symbiosis with all tested host plants. These included tropical legumes that produce either indeterminate (Leucaena) or determinate (Desmodium, Macroptilium, Lablab, Vigna) root nodules.Abbreviations ALA -aminolaevulinic acid - ALAS aminolaevulinic acid synthase - Lb leghaemoglobin - Lb-haem haem moiety of leghaemoglobin     

Production and Characterization of Monoclonal Antibodies to Wall-Localized Peroxidases from Corn Seedlings

A library of 22 hybridomas, which make antibodies to soluble wall antigens from the coleoptiles and primary leaves of etiolated corn (Zea mays L.) seedlings, was raised and cloned three times by limit dilution to assure monoclonal growth and stability. Two of these hybridomas made immunoglobulin G antibodies, designated mWP3 and mWP19, which both effectively immunoprecipitated peroxidase activity from crude and partially purified preparations of wall peroxidases. Direct peroxidase-binding assays revealed that both antibodies bound enzymes with peroxidase activity. As judged by immunoblot analyses, mWP3 recognized a M_r 98,000 wall peroxidase with an isoelectric point near 4.2, and mWP19 recognized a M_r 58,000 wall peroxidase. Immunogold localization studies showed both peroxidases are predominately in cell walls.     

An influx of extracelluar calcium is required for initiation of the human sperm acrosome reaction induced by human follicular fluid

The role of Ca²⁺ in the human sperm acrosome reaction was investigated using the fluorescent calcium indicator fura-2. Previous experiments have shown that a Sephadex G-75 column fraction of human follicular fluid can stimulate the human sperm acrosome reaction [Suarez SS, Wolf DP, Meizel S (1986): Gamete Res 14:107–121]. Using fura-2, we demonstrated that this Sephadex G-75 fraction also stimulates a rapid, transient increase in intracellular free Ca²⁺. This Ca²⁺ transient is blocked either by chelation of extracellular calcium or by addition of the Ca²⁺ antagonist La³⁺. We have also been able to stimulate the acrosome reaction in human sperm without significant loss of motility, using the divalent cation ionophore ionomycin. Acrosome reactions stimulated by whole follicular fluid, the G-75 fraction, or ionomycin are all blocked by removal of extracellular Ca²⁺. These results strongly suggest that an influx of extracellular Ca²⁺ is responsible for intiating the acrosome reaction in human sperm treated with human follicular fluid. This is the first demonstration in mammalian sperm that a potentially physiological stimulus can cause an increase in intracellular Ca²⁺ concomitant with the acrosome reaction.     

Evidence for neuropeptide Y mediation of eating produced by food deprivation and for a variant of the Y1 receptor mediating this peptide's effect.

Neuropeptide Y (NPY) elicits eating when injected directly into the paraventricular nucleus (PVN) or perifornical hypothalamus (PFH). To identify the essential regions of the NPY molecule and the relative contributions of Y1 and Y2 receptors, the eating stimulatory potency of NPY was compared to that of its fragments, analogues, and agonists when injected into the PVN or PFH of satiated rats. Additionally, antisera to NPY was injected into the cerebral ventricles (ICV) to determine whether passive immunization suppresses the eating produced by mild food deprivation. Tests with NPY fragments revealed that NPY(2-36) was surprisingly potent, nearly three times more so than intact NPY. In contrast, fragments with further N-terminal deletions were progressively less effective or ineffective, as was the free acid form of NPY. Collectively, this suggests that both N- and C-terminal regions of NPY participate in the stimulation of eating. Tests with agonists revealed that the putative Y1 agonist [Pro34]NPY elicited a strong dose-dependent feeding response, while the putative Y2 agonist, C2-NPY, had only a small effect at the highest doses. Although this suggests mediation by Y1 receptors, the uncharacteristically high potency of NPY(2-36) may additionally suggest that the receptor subtype underlying feeding is distinct from that mediating other responses. Additional results revealed that ICV injection of antisera to NPY, which should inactivate endogenous NPY, produced a concentration-dependent suppression of eating induced by mild food deprivation. This finding, along with published work demonstrating enhanced levels of hypothalamic NPY in food-deprived rats, suggests that endogenous NPY mediates the eating produced by deprivation.     

The osmotic/calcium stress theory of brain damage: Are free radicals involved?

This overview presents data showing that glucose use increases and that excitatory amino acids (i.e., glutamate, aspartate), taurine and ascorbate increase in the extracellular fluid during seizures. During the cellular hyperactive state taurine appears to serve as an osmoregulator and ascorbate may serve as either an antioxidant or as a pro-oxidant. Finally, a unifying hypothesis is given for seizure-induced brain damage. This unifying hypothesis states that during seizures there is a release of excitatory amino acids which act on glutamatergic receptors, increasing neuronal activity and thereby increasing glucose use. This hyperactivity of cells causes an influx, of calcium (i.e. calcium stress) and water movements (i.e., osmotic stress) into the cells that culminate in brain damage mediated by reactive oxygen species.Special issue dedicated to Dr. Frederick E. Samson