Ligand requirements for Ca2+ binding to EGF-like domains.

Site-specific mutagenesis studies of the first epidermal growth factor-like (EGF-like) domain of human clotting factor IX suggest that the calcium-binding site present in this domain (dissociation constant Kd = 1.8 mM at pH 7.5 and ionic strength I = 0.15) involved the carboxylate residues Asp47, Asp49 and Asp64. To further characterize the ligands required for calcium binding to EGF-like domains, two new mutations, Asp47----Asn and Asp49----Asn, were introduced into the domain by peptide synthesis. 1H-NMR spectroscopy was used to obtain the dissociation constants for calcium binding to these mutations. Calcium binding to the Asp49----Asn modified domain is only mildly affected (Kd = 6 mM, I = 0.15), whereas binding to the Asp47----Asn modified domain is severely reduced (Kd = 42 mM, I = 0.15). From these data, it is proposed that the anionic oxygen atoms of the side chains of residues 47 and 64 are essential for calcium binding, whereas the side chain ligand for calcium at residue 49 can be a carboxyamide oxygen. As a control, the introduction of the modification Glu78----Asp in a region of the domain not believed to be involved in calcium binding had very little effect on the Kd for calcium (Kd = 2.6 mM, I = 0.15). Finally, the effect of an Asp47----Gly substitution found in the natural haemophilia B mutant, factor IXAlabama, was investigated. This peptide has a markedly reduced affinity for calcium (Kd = 37 mM, I = 0.15), suggesting that the defect in factor IXAlabama is due to impaired calcium binding to its first EGF-like domain.     

Temporal and spatial correlation of fertilization current, calcium waves and cytoplasmic contraction in eggs of Ciona intestinalis

Eggs of the ascidian Ciona intestinalis were loaded with the calcium indicator fura-2 via whole-cell clamp electrodes and changes in cytoplasmic calcium and cell currents were monitored during fertilization either in separate eggs or simultaneously in the same egg. The first indication of egg activation was the fertilization current; which reached peak values around 1 nA after 30 s. A wave of elevated calcium was detectable between 5 s and 30 s (mean = 21 s) after the start of the fertilization current. This wave spread across the egg increasing cytoplasmic calcium levels to at least 10 microM. When the fertilization current and calcium wave were complete and cytoplasmic calcium levels were decreasing to prefertilization levels, a cortical contraction wave spread across the egg surface. In eggs showing normal fertilization current, the calcium wave and the contraction wave were in the same direction. A region of elevated calcium persisted at the animal pole. Changing cytoplasmic calcium levels locally by local application of ionophore A23187 caused a contraction wave originating at the site of ionophore application. Increasing cytoplasmic calcium uniformly by facilitating calcium entry through voltage-regulated channels did not result in a contraction wave.     

Haemophilia B (sixth edition): a database of point mutations and short additions and deletions.

The sixth edition of the haemophilia B database lists in easily accessible form all known factor IX mutations due to small changes (base substitutions and short additions and/or deletions of <30 bp) identified in haemophilia B patients. The 1380 patient entries are ordered by the nucleotide number of their mutation. Where known, details are given on factor IX activity, factor IX antigen in circulation and origin of mutation. References to published mutations are given and the laboratories generating the data are indicated.     

Nerve growth factor prevents both neuroretinal programmed cell death and capillary pathology in experimental diabetes.

BACKGROUND: Chronic diabetes causes structural changes in the retinal capillaries of nearly all patients with a disease duration of more than 15 years. Acellular occluded vessels cause hypoxia, which stimulates sight-threatening abnormal angiogenesis in 50% of all type I diabetic patients. The mechanism by which diabetes produces acellular retinal capillaries is unknown. MATERIALS AND METHODS: In this study, evidence of programmed cell death (PCD) was sought in the retinas of early diabetic rats, and the effect of nerve growth factor (NGF) on PCD and capillary morphology was evaluated. RESULTS: Diabetes induced PCD primarily in retinal ganglion cells (RGC) and Muller cells. This was associated with a transdifferentiation of Muller cells into an injury-associated glial fibrillary acidic protein (GFAP)-expressing phenotype, and an up-regulation of the low-affinity NGF receptor p75NGFR on both RGC and Muller cells. NGF treatment of diabetic rats prevented both early PCD in RGC and Muller cells, and the development of pericyte loss and acellular occluded capillaries. CONCLUSIONS: These data provide new insight into the mechanism of diabetic retinal vascular damage, and suggest that NGF or other neurotrophic factors may have potential as therapeutic agents for the prevention of human diabetic retinopathy.     

The nucleoside sequence of tyrosine tRNA from Bacillus stearothermophilus.

The nucleotide sequence of tRNATyr from B. stearothermophilus has been determined: pG-G-A-G-G-G-G-s4U-A-G-C-G-A-A-G-U-Gm-G-C-U-A-A-m1A-C-G-C-G-G-C-G-G-A-C-U-Q-U-A-ms2i6A-A-psi-C-C-G-C-U-C-C-C-U-U-U-G-G-G-U-U-C-G-G-C-G-G-T-psi-C-G-A-A-U-C-C-G-U-C-C-C-C-C-U-C-C-A-C-C-AOH. A combination of classical fingerprinting methods, partial nuclease P1 digestion and two-dimensional homochromatography and a rapid "read off" sequencing gel technique were used to establish the complete nucleotide sequence.     

A pseudogene structure in 5S DNA of Xenopus laevis

The 5S DNA of Xenopus laevis, coding for oocyte-type 5S RNA, consists of many copies of a tandemly repeated unit of about 700 base pairs. Each unit contains a "pseudogene" in addition to the gene. The pseudogene has been partly sequenced and appears to be an almost perfect repeat of 101 residues of the gene. The order of components in the repeat unit is (5') long spacer--gene--linker--pseudogene (3') in the "+" strand (or H strand) of the DNA. The possible function of the pseudogene is discussed.     

The nucleotide sequence of oocyte 5S DNA in Xenopus laevis. II. The GC-rich region

The primary sequence of the GC-rich half of the repeating unit in X. laevis 5S DNA has been determined in both a single plasmid-cloned repeating unit and in the total population of repeatig units. The GC-rich half of the repeating unit contains a single long duplication of 174 nucleotides. The duplicated segment commences 73 nucleotides preceding the 5' end of the gene and terminates at nucleotide 101 of the gene. The duplicated portion of the gene, termed the pseudogene, differs by 10 nucleotides from the corresponding portion of the gene, and the remaining duplicated sequence of 73 nucleotides differs by 13 nucleotides. The plasmid-cloned repeating unit differs from the dominant sequence in the total population repeating units by 6 nucleotides in the GC-rich region. Evidence is provided that most of the CpG dinucleotides in 5S DNA are at least partially methylated.     

Altered cellular interactions between endothelial cells and nonenzymatically glucosylated laminin/type IV collagen.

Laminin and type IV collagen are two major basement membrane glycoproteins. In previous studies it has been shown that nonenzymatic glucosylation induces structural alterations of these macromolecules and also reduces their ability to self-associate. In the present study, endothelial cells were tested for their ability to adhere and spread on nonenzymatically glucosylated laminin and type IV collagen. Adhesion and spreading were reduced when glucosylated macromolecules were used as substrates. Glucosylation-induced changes in adhesion and spreading may be an important initial event signaling other phenotypic modifications of cells in the microvasculature and may be a crucial factor in order to understand the pathogenesis of diabetic microangiopathy at the molecular level.