Enhanced clearance from plasma of low density lipoproteins containing a truncated apolipoprotein, apoB-89

Previously we have reported on a kindred with hypobetalipoproteinemia in which three sisters were found to be compound heterozygotes for two newly described truncated forms of apoB. apoB-40 and apoB-89. ApoB-89-containing low density lipoproteins (LDL) bound with increased affinity to cultured normal human fibroblasts and were internalized and degraded at increased rates, suggesting that the low plasma concentrations of apoB-89-LDL of the patients could be due to enhanced rates of clearance through LDL-receptors. To examine this hypothesis, apoB-89-LDL was isolated from the three study subjects and apoB-100-LDL from two control subjects. LDL was conjugated to the radioiodinated residualizing label, dilactitol tyramine (*I-DLT, containing either 125I or 131I). *I-DLT-apoB-89-LDL and *I-DLT-apoB-100-LDL were simultaneously injected into ear veins of rabbits. The clearance from plasma and hepatic accumulations of both radiolabeled LDL fractions were followed over 24 h. Fractional catabolic rates (FCR) of apoB-89-LDL were 0.105 +/- 0.012 h-1 compared to 0.054 +/- 0.007 h-1 for apoB-100-LDL. In agreement with the enhanced clearance from plasma, 1.72 to 1.87 times more *I-DLT-apoB-89-LDL than *I-DLT-apoB-100-LDL accumulated in the livers 24 h after injection. There was no significant difference in splenic accumulation, suggesting that LDL-receptors rather than scavenger receptors mediated the enhanced clearance of apoB-89-LDL. To assess further the importance of LDL-receptors, *I-DLT-apoB-89-LDL and *I-DLT-apoB-100-LDL were reductively methylated to inhibit their interactions with LDL-receptors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Unique Substrate Specificity of SplE Serine Protease from Staphylococcus aureus

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Identification of a 130-kDa albumin in tuatara (Sphenodon) and detection of a novel albumin polymorphism

Electrophoretic, immunochemical, and protein sequence analyses were performed on plasma albumin of the tuatara (Sphenodon), a rare reptile endemic to New Zealand. The analyses revealed that, unlike other terrestrial vertebrates, tuatara do not seem to possess a 60- to 75-kDa plasma albumin. The common form of plasma albumin in this genus has an apparent molecular mass of 130 kDa, making it by far the largest albumin reported for any terrestrial vertebrate. Starch gel electrophoresis of samples from tuatara on 24 of the 30 islands inhabited by this genus resolved two forms of the 130-kDa albumin (albumins A and C). A third albumin of approximately 170 kDa (albumin B), reflecting a novel alloalbuminemia, was found in tuatara in three geographically isolated populations. Albumin A appears to be restricted to populations at the southern extremity of the tuatara's distribution, while albumin C was found in all but four (southern) populations. Possible explanations for the origin and distribution of these albumins are discussed.     

Isolation of Aspergillus nidulans Mutants That Overcome brlA-Induced Growth Arrest

The Aspergillus nidulans brlA gene is a primary regulator of development-specific gene expression during conidiation. Forced activation of brlA in vegetative cells leads to inappropriate induction of conidiophore formation and causes growth to stop. In fact, when conidia containing a nutritionally inducible brlA gene fusion are placed on inducing medium, they fail to germinate. We used this phenotype to select 174 mutants that continue growing following such forced brlA activation. Forty-six of these mutants also produced abnormal developmental structures during air-induced conidiation as expected if the mutations resulted in an altered response to BrlA (designated sbr mutants for suppressors of brlA response). The predominant mutant class identified was defective in a known developmental regulatory gene, abaA. We also identified mutants with defects in the previously characterized early acting developmental regulatory genes flbB and flbD and in four previously undescribed loci designated sbrA-D. sbrA mutants represent the second largest group and are characterized by production of conidiophore stalks that lack a normal vesicle and form branching sterigmata that rarely make spores. Because abaA expression could not be detected in sbrA mutants following brlA activation we propose that sbrA functions as a developmental modifier, participating in brlA-dependent activation of other developmental regulators.     

Impaired platelet aggregation and sustained bleeding in mice lacking the fibrinogen motif bound by integrin alpha IIb beta 3.

Blood loss at sites of vascular rupture is controlled by the adhesion and aggregation of platelets and the formation of an insoluble fibrin matrix. Fibrinogen is considered to be critical in these processes by both providing an abundant dimeric ligand for alpha IIb beta 3-mediated platelet aggregation, and serving as the fundamental building block of the fibrin polymer. To establish an in vivo model system to examine in detail the importance of alpha IIb beta 3-fibrinogen interactions in platelet function, hemostasis, response to injury and vasoocclusive disease, and to test the prevailing hypothesis that the C-terminal segment of the fibrinogen gamma chain is essential for alpha IIb beta 3 binding, we have used gene-targeting technology in mice to eliminate the last five residues (QAGDV) from the gamma chain. Mice homozygous for the modified gamma chain gene (gamma delta 5/gamma delta 5) displayed a generally normal hematological profile, including normal platelet count, plasma fibrinogen level, clotting time and fibrin crosslinking. However, both gamma delta 5-fibrinogen binding to alpha IIb beta 3 and platelet aggregation were highly defective. Remarkably, another alpha IIb beta 3-dependent process, clot retraction, was unaffected by the gamma delta 5 mutation. Despite the preservation of clotting function, gamma delta 5/gamma delta 5 mice were unable to control blood loss following a surgical challenge and occasionally developed fatal neonatal bleeding events.     

Genetic variation in the genus Leiopelma and relationships to other primitive frogs

Allozyme variation was studied the three living species of Leiopelma. L. hamiltoni and L. archeyi are shown to be closely related to each other although L. hamiltoni is slightly more divergent relative to L. hochstetteri. This parallels previous cytoenetic data. The rarity and insularity of L. hamiltoni enables the calculation of a mutation rate based on genetic variance and population size. A mutation rate per generation of 2.7times 10^--⁶ is sufficient to account for the observed levels of variation. Six populations of L. hochstetteri show a pattern of genetic divergence that also closely parallels previously detected cytogenetic variation. L. hochstetteri is genetically distant from its congeneric species while all species of Leiopelma are at an extreme genetic distance from Ascaphus truei, the only other living amphicoelous fro At the limits of resolution of the allozme technique, Ascaphus clusters with the more morphologically advanced frogs, Discogiossus and Bombina, rather than with Leiopelma. Taken with other evidence, this supports recognition of two families, Leiopelmatidae anl Ascaphidae, with Leiopelma the probable sister group of all other frogs.     

La dormance embryonnaire chez Taxus baccata: Influence de la composition du milieu liquide sur I'induction de la germination Par

Embryo dormancy of Taxus baccata var. fastigiata is eliminated when cultured continuously in nutritive liquid medium. An equivalent percentage of germination is obtained when the embryos are transferred to agar medium after 8 days of liquid culture. There is no morphological development of the embryo during the period in the liquid medium. But we have ascertained that water-soluble germination inhibitors present in the embryo are leached out into the medium, permitting germination. Germination is totally absent when the embryos are cultured continuously in distilled water, alone or with minerals; incidental in sucrose solution; and maximal when the medium contains sucrose and Ca²⁺ or K⁺ ions. The extent of germination on agar medium depends upon the composition of the liquid medium in which the embryos are cultured for the initial 8 days. But this preliminary culture in the liquid medium does not always remove the endogenous inhibitors, irrespective of its composition. This can be achieved only in the presence of sucrose; and this process can be made more effective by the addition of Ca²⁺ ions.     

Facilitated Penetration of Amanitin—Albumin Conjugates into Hepatocytes after Coupling with Fluorescein

α-AMANITIN, a cyclic peptide of the toadstool Amanita phalloides^1,2, causes necrosis of liver and kidney cells, the first morphological lesions occurring in the nuclei^3,4. It acts by binding to RNA polymerase in eukaryotic .cells and inhibiting the enzyme^5–9. The hepatotoxicity of amanitin increases several times when it is conjugated to albumin, probably because of a slower rate of elimination of the toxin through the glomeruli^4,10. It is unlikely that the amanitin-albumin conjugate enters the hepatocyte by a mechanism involving its albumin moiety; it was therefore suggested¹¹ that penetration of the liver cells is consequent on binding of the amanitin group to the carrier involved in transport of this peptide. This led us to consider more generally the facilitation of penetration into cells by large molecules by means of binding to another molecule for which a carrier exists on the cell membrane^12,13.     

Thromboxane A2 biosynthesis in human disease

Thromboxane A2 (TxA2), the predominant cyclooxygenase product of human platelets, is a potent vasoconstrictor and platelet agonist. Although its biological properties are readily appreciable in vitro, it has been difficult to define its biological importance in vivo. To a large extent this reflected the problems associated with efforts to monitor biosynthesis of this eicosanoid and the lack of selective pharmacological probes that prevented the synthesis of TxA2 or antagonized its biological action in vivo. Recently the analysis of urinary metabolites of TxB2 has become simplified so that the methodology is readily applicable to clinical studies. This provides a noninvasive, time-integrated index of Tx biosynthesis. Although one cannot definitively establish a tissue of origin for metabolites measured in urine, indirect evidence suggests that urinary TxB2 derives primarily from the kidney whereas its dinor metabolite predominantly reflects platelet biosynthesis under physiological conditions. Although plasma concentrations of TxB2 are readily confounded by platelet activation ex vivo, the enzymatic metabolites formed from TxB2 have recently been identified and appear to bypass this problem. Combined analysis of long-lived (e.g., 11-dehydro-TxB2) and short-lived (e.g., 2,3-dinor-TxB2) metabolites in plasma promise to more accurately localize phasic increases in the biosynthesis of TxA2 and have been paralleled by the development of antagonists of the TxA2/prostaglandin endoperoxide receptor and their study of humans. The use of such specific probes in conditions characterized by abnormal biosynthesis of TxA2 promises to define the biological role of this mediator for humans.     

Acquisition of glucose by Rickettsia prowazekii through the nucleotide intermediate uridine 5''-diphosphoglucose.

The ability of Rickettsia prowazekii to transport potential sources of the glucose moiety of bacterial polysaccharides was determined. Transport was determined both by filtration assays and by centrifugation through nonaqueous layers. Uridine 5'-diphosphoglucose (UDPG) was transported, whereas glucose was not transported; the uptake of glucose phosphates, although greater than that for glucose, was markedly lower than the transport of UDPG. Furthermore, the activities of hexokinase and phosphoglucomutase, enzymes required for the metabolism of glucose and glucose 6-phosphate, were undetectable in rickettsial extracts. The uptake of UDPG had an extended time course and did not reach a plateau until 60 min. The maximum rate of uptake was 340 pmol/min per mg of protein, and the rate was half-maximal at a UDPG concentration of 220 microM. Measurement of true influx of UDPG was complicated by the low activity of this transport system and the metabolism of the UDPG. The uptake of labeled UDPG was markedly inhibited by a 10-fold excess of uridine monophosphate, uridine diphospho-N-acetylglucosamine, and uridine diphospho-N-acetylgalactosamine but not by a variety of other structurally related compounds.