Two naturally occurring mutations at the first and second bases of codon aspartic acid 156 in the proposed catalytic triad of human lipoprotein lipase. In vivo evidence that aspartic acid 156 is essential for catalysis.

We are studying naturally occurring mutations in the gene for lipoprotein lipase (LPL) to advance our knowledge about the structure/function relationships for this enzyme. We and others have previously described 11 mutations in human LPL gene and until now none of these directly involves any of the residues in the proposed Asp156-His241-Ser132 catalytic triad. Here we report two separate probands who are deficient in LPL activity and have three different LPL gene haplotypes, suggesting three distinct mutations. Using polymerase chain reaction cloning and DNA sequencing we have identified that proband 1 is a compound heterozygote for a G----A transition at nucleotide 721, resulting in a substitution of asparagine for aspartic acid at residue 156, and a T----A transversion, resulting in a substitution of serine for cysteine at residues 216. Proband 2 is homozygous for an A----G base change at nucleotide 722, leading to a substitution of glycine for aspartic acid at residue 156. The presence of these mutations in the patients and available family members was confirmed by restriction analysis of polymerase chain reaction-amplified DNA. In vitro site-directed mutagenesis and subsequent expression in COS cells have confirmed that all three mutations result in catalytically defective LPL. The two naturally occurring mutations, which both alter the same aspartic acid residue in the proposed Asp156-His241-Ser132 catalytic triad of human LPL, indicate that Asp156 plays a significant role in LPL catalysis. The Cys216----Ser mutation destroys a conserved disulfide bridge that is apparently critical for maintaining LPL structure and function.     

Do plants tap SOS signals from their infested neighbours?

Ecologist have not been able to show unambigous evidence for the involvement of plant-to-plant signal transfer in the defence strategies of plants. However, phytopathologists and plant physiologists recently demonstrated that resistance in undamaged plants can be elicited by volatiles of plant origin. Now that empirical evidence is accumulating, there is every reason to ask why plants use the available information on the infestion status of their neighbours and to assess the fitness advantages associated with the tuning of their defence. The debate on the ecological and evolutionary significance of interplant communication needs to be revived.     

Effect of 2,3-diphosphoglycerate on the Bohr effect of human adult hemoglobin

The effect of 2,3-diphosphoglycerate (DPG) on the Bohr effect of human hemoglobin has been studied by means of hydrogen ion titration techniques. The results indicate a) that both the acid and the alkaline Bohr effect are equally affected, b) that the DPG binding to deoxyhemoglobin (Hb) is much stronger than to carboxyhemoglobin (HbCO) and c) that Hb binds effectively one DPG molecule. The effect on the Bohr effect can roughly be described by assuming that upon binding two groups per tetramer change their pK from 6.8 to 7.8 and two others from 6.8 to 5.8. These groups very probably are the imidazole groups of the two histidines H21 (143)β and the two phosphate groups of DPG (second dissociation). From the experiments a value for the dissociation constant K of the Hb-DPG complex of about 10⁻⁵ M⁻¹ could be estimated at pH 6.2 and pH 7.5.     

Species-specific sperm adhesion in sea urchins. A quantitative investigation of bindin- mediated egg agglutination

Bindin, a protein component of the acrosomal vesicle of sea urchin sperm, has been isolated from Arbacia punctulata and strongylocentrous purpuratus. Using this isolated bindin, we have devised a quantitative assay for bindin-mediated egg agglutination and compared the agglutination of bindin eggs from A. puntulata and S. purpuratus. Bindin- mediated agglutination is species –specific in both species, although a measurable degree of heterotypic interaction is observed. Homotypic bindin-egg interactions differ significantly from heterotypic interactions both in the extent of agglutination and the size of the resulting aggregates. We also provide direct evidence that bindin particles agglutinate eggs by adhering to the surfaces of adjacent eggs. Although the A. punctulata bindin preparation displays the same functional properties and consists of one major polypeptide of the same apparent molecular weight as S. purpuratus bindin, its morphology is very different. Unlike the spherical aggregates observed with S. purpuratus bindin, A punctulata bindin exists as lamellar vesicles and binds significant amounts of phospholipids and Triton X-100, suggesting that it may be tightly associated with the acrosomal membrane. Having defined a number of the basic parameters of bindin-mediated agglutination, we examined the effect of a number of saccharides and glycopeptides on bindin-mediated egg agglutination. Carbohydrate-containing components derived from the egg cell surface by proteolysis were found to inhibit bindin-mediated egg agglutination at low concentrations, but this inhibition is not species specific.     

Influence of environmental temperature on mitochondrial membranes

Mitochondrial phospholipids from goldfish lateral line muscle were analysed with respect to polar and apolar groups. Groups of 20 goldfish, acclimated to 5, 20 and 30°C, were used. Temperature-induced shifts of both polar and apolar groups of the mitochondrial phospholipids were observed. The fatty acid composition of mitochondrial phospholipids is characterized by a large amount of polyenoic acids, dominated by docosahexaenoic acid and by octadecadienoic acid. At the higher acclimation temperatures, a significant decrease in docosahexaenoic acid is found. However, the resultant effect of environmental temperature on the degree of unsaturation is small, in contrast to the marked effect on mean chain length. Pronounced changes in the molar ratio of phosphatidylcholine and phosphatidylethanolamine are seen; a decrease in mitochondrial phosphatidylcholine is observed at low acclimation temperature, which is compensated for by a nearly equal increase in phosphatidylethanolamine. The main phospholipids are, apparently, phosphatidylcholine, phosphatidylethanolamine and cardiolipin, comprising 90% of the total pool of 12 species. It is found that the anionic nature of the phospholipids is increased at low acclimation temperatures. We discuss this effect and its probable importance in the stabilization of the surface potential of the mitochondrial membranes.     

Effects of cycloheximide on a cytoplasmic factor initiating meiotic maturation in Xenopus oocytes

Oocytes induced to undergo meiotic maturation by progesterone possess a cytoplasmic activity that causes germinal vesicle breakdown (GVBD). The cytoplasmic factor postulated to be responsible for this activity is designated as the maturation promoting factor (MPF). The activity of MPF was assayed by injecting cytoplasm into fully-grown oocytes to induce GVBD. It was found that maturing oocyte cytoplasm possesses MPF activity before GVBD begins. Treatment of progesterone stimulated oocytes with cycloheximide, either applied externally or injected, inhibited the appearance of MPF in the cytoplasm as well as GVBD when the inhibitor treatment was initiated before the cytoplasm exhibited MPF activity. In contrast, the same treatment did not inhibit GVBD when it was applied to oocytes after the cytoplasm possessed MPF activity. Furthermore, cycloheximide treatment of recipient oocytes did not inhibit the induction of GVBD by injected cytoplasm containing MPF. Cytoplasm of oocytes injected with MPF subsequently possessed MPF activity as high as that of the original donor cytoplasm in spite of its extensive dilution. This suggests that amplification of MPF took place in the recipient. Cycloheximide treatment did not inhibit the amplification of MPF. It was concluded that cycloheximide inhibits only the initial phase of induction of MPF activity, but neither its amplification nor its action on the nucleus that causes GVBD. From these results, a hypothesis concerning the cytoplasmic mechanism for the induction of GVBD has been proposed.     

Destabilization of secondary structure in 16S ribosomal RNA by dimethylation of two adjacent adenosines.

Fragments comprising the 49 nucleotides from the 3'-end have been purified from 16S ribosomal RNA of wild-type Escherichia coli and from a kasugamycin-resistant mutant that specifically lacks dimethylation of two adjacent adenines near the 3'-terminus. These fragments, obtained after treatment of ribosomes in vitro with the bacteriocin cloacin DF13, were used to study the effect of the methyl groups on the temperature dependent unfolding of double-stranded regions. Both fragments contain at least 3 independent melting transitions, of which the one with the highest Tm corresponds with the unfolding of a nine-basepair long central hairpin. Dimethylation of the adenines in the loop of this hairpin lowers the melting temperature (Tm) by approximately 2 degrees C at 0.2 M NaCl and by about 5 degrees C at 0.15 M NaCl. It is suggested that m6(2)Am6(2)A is more antagonistic to loop formation that ApA and that the function of the methyl groups is to help to destabilize the 3'-terminal hairpin in 16S rRNA in order to facilitate intermolecular interactions.     

The spin-state transition of the hemochrome non-equilibrium conformation in partially reduced human methemoglobin. A pulse-radiolysis study of aqueous-methanol solutions of methemoglobin.

The effect of external parameters on the relaxation process of the hemochrome-type non-equilibrium conformation in partially reduced methemoglobin has been investigated. The relaxation of the intermediate ferrous low-spin state to the high-spin equilibrium conformation of hemoglobin appears to be facilitated particularly by protons and phosphate ions. In addition to studying the spin-state transition in aquomethemoglobin we have also studied it in complexes of the heme group in methemoglobin with fluoride, azide and cyanide anions.     

Families with familial combined hyperlipidemia and families enriched for coronary artery disease share genetic determinants for the atherogenic lipoprotein phenotype.

Small, dense LDL particles consistently have been associated with hypertriglyceridemia, premature coronary artery disease (CAD), and familial combined hyperlipidemia (FCH). Previously, we have observed linkage of LDL particle size with four separate candidate-gene loci in a study of families enriched for CAD. These loci contain the genes for manganese superoxide dismutase (MnSOD), on chromosome 6q; for apolipoprotein AI-CIII-AIV, on chromosome 11q; for cholesteryl ester transfer protein (CETP) and lecithin:cholesterol acyltransferase (LCAT), on chromosome 16q; and for the LDL receptor (LDLR), on chromosome 19p. We have now tested whether these loci also contribute to LDL particle size in families ascertained for FCH. The members of 18 families (481 individuals) were typed for genetic markers at the four loci, and linkage to LDL particle size was assessed by nonparametric sib-pair linkage analysis. The presence of small, dense LDL (pattern B) was much more frequent in the FCH probands (39%) than in the spouse controls (4%). Evidence for linkage was observed at the MnSOD (P=.02), CETP/LCAT (P=.03), and apolipoprotein AI-CIII-AIV loci (P=.005) but not at the LDLR locus. We conclude that there is a genetically based association between FCH and small, dense LDL and that the genetic determinants for LDL particle size are shared, at least in part, among FCH families and the more general population at risk for CAD.     

Late thrombotic occlusion of a malapposed stent 10 months after intracoronary brachytherapy

We report a patient who received a stent following intracoronary 3-irradiation. Despite a good initial angiographic result, the stent appeared to be not fully expanded on intravascular ultrasound imaging at 6-month follow-up. Four months later, sudden thrombotic occlusion occurred shortly after aspirin cessation.