Characterization of the heparin/heparan sulfate binding site of the natural cytotoxicity receptor NKp46

NKp46 is a member of a group of receptors collectively termed natural cytotoxicity receptors (NCRs) that are expressed by natural killer (NK) cells. NCRs are capable of mediating direct killing of tumor and virus-infected cells by NK cells. We have recently shown that NKp46 recognizes the heparan sulfate moieties of membranal heparan sulfate proteoglycans (HSPGs), thus enabling lysis of tumor cells by NK cells. In the current study, we further examined the residues in NKp46 that may be involved in heparan sulfate binding on tumor cells. On the basis of both the electrostatic potential map and comparison to the heparin binding site on human fibronectin, we predicted a continuous region containing the basic amino acids K133, R136, H139, R142, and K146 to be involved in NKp46 binding to heparan sulfate. Mutating these amino acids on NKp46D2 to noncharged amino acids retained its virus binding capacity but reduced its binding to tumor cells with a 10-100 fold lower K(D) when tested for direct binding to heparin. The minimal length of the heparin/heparan sulfate epitope recognized by NKp46 was eight saccharides as predicted from the structure and proven by testing heparin oligomers. Testing selectively monodesulfated heparin oligomers emphasized the specific contributions of O-sulfation, N-sulfation, and N-acetylation to epitope recognition by NKp46. The characterization of heparan sulfate binding region in NKp46 offers further insight into the identity of the ligands for NKp46 and the interaction of NK and cancers.     

Fusion-mediated microinjection of active amine and diamine oxidases into cultured cells: effect on protein and DNA synthesis in chick embryo fibroblasts and in glioma cells

Serum amine oxidase and/or porcine kidney diamine oxidase were trapped within reconstituted Sendai virus envelopes, and retained their activity. The trapped enzymes that were detected by radioimmunoblots were microinjected into cultured cells by fusion. When diamine oxidase was microinjected into cultured fibroblasts of chick or rat embryos, a temporary arrest in protein and DNA synthesis was observed. The inhibitory effect was more significant when both serum amine oxidase and kidney diamine oxidase were microinjected into those cultured cells. Fibroblasts of either chick or rat embryos transformed by Rous sarcoma virus were more susceptible to the injected enzymes than the normal cultures, showing a complete arrest in protein and DNA synthesis within 4 hours. Similar results were obtained by microinjecting diamine oxidase into cultured glioma cells. The injected enzyme catalyzed the oxidation of intracellular polyamines. The resulting oxidation product (hydrogen peroxide and aminoaldehydes) apparently caused the arrest in the synthesis of macromolecules.     

Reliability of reliability coefficients in the estimation of asymmetry

Although promising to provide insight into the interaction between genotype and environment, investigations into fluctuating asymmetry suffer from a lack of standardization in the reporting of measurement error. In the present paper we show, using both anthropometric and odonto-metric data, that the use of the reliability coefficient calculated for a bilateral measurement provides no indication of the reliability of the corresponding asymmetry estimate, because reliability of asymmetry depends on the relationship between measurement error and the difference between sides. Thus, we suggest that future investigations either provide reliability coefficients for asymmetry estimates specifically, or use methods that account for measurement error. © 1995 Wiley-Liss, Inc.     

Comparative skeletal features between Homo floresiensis and patients with primary growth hormone insensitivity (Laron Syndrome)

Comparison between the skeletal remains of Homo floresiensis and the auxological and roentgenological findings in a large Israeli cohort of patients with Laron Syndrome (LS, primary or classical GH insensitivity or resistance) revealed striking morphological similarities, including extremely small stature and reduced cranial volume. LS is an autosomal recessive disease caused by a molecular defect of the Growth Hormone (GH) receptor or in the post-receptor cascades. Epidemiological studies have shown that LS occurs more often in consanguineous families and isolates, and it has been described in several countries in South East Asia. It is our conclusion that the findings from the island of Flores, which were attributed to a new species of the genus Homo, may in fact represent a local, highly inbred, Homo sapiens population in whom a mutation for the GH receptor had occurred.     

A study of extracellular matrix-cell adhesion peptidic epitopes related to human serum amyloid A (SAA)

Serum amyloid A (SAA), an acute-phase protein, exists normally in the serum while complexed with high-density lipoprotein 3 (SAA- HDL3). Its levels increase markedly during inflammatory diseases. The pentapeptide Tyr-Ile-Gly-Ser-Asp (YIGSR-like) and the tripeptide Arg-Gly-Asn (RGD-like), related to the cell adhesion domains of laminin and fibronectin, respectively, exist in SAA within close proximity (YIGSDKYFHARGNY; amino acid residues 29–42). A structure-function study of linear and head- to-tail cyclic peptides, related to the amino acid residues 29–42 and 70–76 (GRGAEDS) of human SAA, was performed in order to evaluate their ability to inhibit adhesion of human T-lymphocytes to surfaces coated with extracellular matrix purified from bovine corneal cells.     

Lipoamide dehydrogenase deficiency due to a novel mutation in the interface domain.

An infant with a neurodegenerative disorder accompanied by lactic acidemia is described. In muscle homogenate, the activity of lipoamide dehydrogenase (LAD), the third catalytic subunit of pyruvate dehydrogenase complex (PDHc), alpha-ketoglutarate dehydrogenase complex (KGDHc), and branched-chain keto acid dehydrogenase complex was reduced to 15% of the control. The activity of PDHc was undetectable and the activity of KGDHc was 2% of the control mean. The immunoreactive LAD protein was reduced to about 10% of the control. Direct sequencing of LAD cDNA revealed only one mutation, substituting Asp for Val at position 479 of the precursor form. The mutation resides within the interface domain and likely perturbs stable dimerization. The phenotypic heterogeneity in LAD deficiency is not directly correlated with the residual LAD activity but rather with its impact on the multienzymatic complex activity.     

Tooth wear and dental pathology at the advent of agriculture: new evidence from the Levant

Differences in patterns of diet and subsistence through the analysis of dental pathology and tooth wear were studied in skeletal populations of Natufian hunter-gatherers (10,500-8300 BC) and Neolithic populations (8300-5500 BC, noncalibrated) from the southern Levant. 1,160 Natufians and 804 Neolithic teeth were examined for rate of attrition, caries, antemortem tooth loss, calculus, periapical lesions, and periodontal processes. While the Natufian people manifest a higher rate of dental attrition and periodontal disease (36.4% vs. 19%), Neolithic people show a higher rate of calculus. Both populations manifested low and similar rates of caries (6.4% in the Natufian vs. 6.7% in the Neolithic), periapical lesions (not over 1.5%), and antemortem tooth loss (3.7% vs. 4.5%, respectively). Molar wear pattern in the Neolithic is different than in the Natufian. The current study shows that the dental picture obtained from the two populations is multifactorial in nature, and not exclusively of dietary origin, i.e., the higher rate and unique pattern of attrition seen in the Natufian could result from a greater consumption of fibrous plants, the use of pestles and mortars (which introduce large quantities of stone-dust to the food), and/or the use of teeth as a "third hand." The two major conclusions of this study are: 1) The transition from hunting and gathering to a food-producing economy in the Levant did not promote changes in dental health, as previously believed. This generally indicates that the Natufians and Neolithic people of the Levant may have differed in their ecosystem management (i.e., gathering vs. growing grains), but not in the type of food consumed. 2) Changes in food-preparation techniques and nondietary usage of the teeth explain much of the variation in tooth condition in populations before and after the agricultural revolution.     

A new method for reconstitution of highly fusogenic sendai virus envelopes

A new way for reconstituting highly fusogenic Sendai virus envelopes is described. As opposed to previously described methods, in the present one the detergent (Triton X-100) is removed by direct addition of SM-2 Bio-beads to the detergent solubilized mixture of the viral phospholipids and glycoproteins, thus avoiding the long dialysis step. The vesicles obtained in the present work resemble, in their composition, size and features, envelopes of intact Sendai virus particles. The present method allows the enclosure of low and high molecular weight material within the reconstituted viral envelopes.