Distinct functional roles of the two intracellular phosphatase like domains of the receptor-linked protein tyrosine phosphatases LCA and LAR.

Protein tyrosine phosphorylation is regulated by both protein tyrosine kinases and protein tyrosine phosphatases (PTPases). Recently, the structures of a family of PTPases have been described. In order to study the structure-function relationships of receptor-linked PTPases, we analyzed the effects of deletion and point mutations within the cytoplasmic region of the receptor-linked PTPases, LCA and LAR. We show that the first of the two domains has enzyme activity by itself, and that one cysteine residue in the first domain of both LCA and LAR is absolutely required for activity. The second PTPase like domains do not have detectable catalytic activity using a variety of substrates, but sequences within the second domains influence substrate specificity. The functional significance of a stretch of 10 highly conserved amino acid residues surrounding the critical cysteine residue located in the first domain of LAR was assessed. At most positions, any substitution severely reduced enzyme activity, while missense mutations at the other positions tested could be tolerated to varying degrees depending on the amino acid substitution. It is suggested that this stretch of amino acids may be part of the catalytic center of PTPases.     

Structural diversity and evolution of human receptor-like protein tyrosine phosphatases.

Protein tyrosine phosphatases (PTPases), together with protein tyrosine kinases, regulate the tyrosine phosphorylation that controls cell activities and proliferation. Previously, it has been recognized that both cytosolic PTPases and membrane associated, receptor-like PTPases exist. In order to examine the structural diversity of receptor-like PTPases, we isolated human cDNA clones that cross-hybridized to a Drosophila PTPase cDNA clone, DPTP12, under non-stringent hybridization conditions. The cDNA clones thus isolated included LCA and six other novel receptor-like PTPases, named HPTP alpha, beta, gamma, delta, epsilon, and zeta. The cytoplasmic regions of HPTP alpha and epsilon are highly homologous, and are composed of two tandemly duplicated PTPase-like domains. The extracellular regions of HPTP alpha and epsilon are, respectively, 123 amino acids and 27 amino acids, and do not have obvious similarity to any known protein. The cytoplasmic region of HPTP beta contains only one PTPase domain. The extracellular region of HPTP beta, which is 1599 amino acids, is composed of 16 fibronectin type-III repeats. HPTP delta is very similar to leukocyte common antigen related molecule (LAR), in both the extracellular and cytoplasmic regions. Partial sequences of HPTP gamma and zeta indicate that they are highly homologous and contain two PTPase-like domains. The PTPase-like domains of HPTP alpha, beta and delta expressed in Escherichia coli had tyrosine phosphatase activities.     

Growth and development of biofeedback: A bibliographic update

Computerized literature searching techniques were used to examine publication patterns in the worldwide biofeedback literature. Searches were completed in the United States and in Japan for the years 1985 through 1987. The results were used to update the results of an earlier study (Hatch & Riley, 1985) that covered the years from 1964 through 1984. Publication growth curves were generated for several media, including scientific journal articles, books, doctoral dissertations, and popular magazine articles. Although publication of information about biofeedback remains active worldwide, there has been a declining trend in effect for the past several years. The American database grossly underestimated the number of Japanese biofeedback articles, and it is likely that the literatures of other countries outside of North America were similarly underestimated. Possible explanations for the various trends noted are discussed.These results were presented in part at the Twentieth Annual Meeting of the Association for Applied Psychophysiology and Biofeedback. We gratefully acknowledge the assistance of Margaret Cyr-Provost in preparing the data for analysis.     

Inhibitory effect of di- and tripeptidyl aldehydes on calpains and cathepsins

Eight different di- and tripeptidyl aldehyde derivatives, each having at its C-terminus an aldehyde analog of L-norleucine, L-methionine, or L-phenylalanine with a preceding L-leucine residue, were synthesized and tested for their inhibitory effects on several serine and cysteine endopeptidases. These compounds showed almost no inhibition of trypsin, and only weak inhibition of alpha-chymotrypsin and cathepsin H, while they exhibited marked inhibition of cathepsin B less than calpain II congruent to calpain I less than cathepsin L, being stronger in this order. The mode of inhibition of these cysteine proteinases was competitive for the peptide substrate used and inhibitor constants (Ki) were calculated from the Dixon plot. The best inhibitors found were: 4-phenyl-butyryl-Leu-Met-H for calpain I (Ki, 36 nM) and calpain II (Ki, 50 nM); acetyl-Leu-Leu-nLeu-H for cathepsin L (Ki, 0.5 nM); acetyl-Leu-Leu-Met-H for cathepsin B (Ki, 100 nM).     

Dimer Formation of a Cell-Cell Adhesion Protein, gp64 of the Cellular Slime Mold, Polysphondylium pallidum

The cellular slime mold, Polysphondylium pallidum, has two EDTA-resistanttypes of cell-cell adhesion. The major component of them hasbeen identified as a glycoprotein with a molecular mass of 64kDa on SDS-PAGE (referred to as gp64). We found that a substantialamount of the gp64 run as dimer, when gp64 was dissolved inSDS-sample buffer without 2-mercaptoethanol and then subjectedto electrophoresis. The occurrence of a homophilic dimer wasdemonstrated by analyzing the dimer-like band on a gel for itsamino acid sequence and amino acid composition. The dimer-likeband also was analyzed by three sorts of monoclonal antibodies,two of which recognize respectively a conforniational epitopeand a denatured epitope of the protein moiety of gp64. The dataindicate that the native conformation of gp64 is necessary fordimer formation. ²Present address: Institute of Immunological Science, HokkaidoUniversity, Sapporo, 060 Japan     

Estimation of the position and effect of a lethal factor locus on a molecular marker linkage map

In the mapping of DNA markers the distortion of segregation of marker genotypes is often observed, which may be caused by a lethal factor acting in filial generations derived from distant crosses. A method is presented for estimating the recombination values between a lethal factor locus and neighboring molecular markers, and the relative viability or fertilization ability of gametes or zygotes affected by the lethal factor in an F₂ population using the maximum likelihood method and the expectation conditional maximization (ECM) algorithm. Three selection models of gamete or zygote were considered, and the most likely one was determined by goodness of fit of the observed frequency of the phenotypes to the expected ones under the models. The method was applied to segregation data of molecular markers of an F₂ population consisting of 144 individuals derived from a cross between an Indica and a Japonica rice variety. The presence of a lethal factor locus (L) located on chromosome III that caused partial gametic selection in both the male and female sides was suggested. The locus L was tightly linked to RFLP marker number 23 of the RFLP linkage map of Saito et al. (1991a), and the fertilization chance of a male or female gamete possessing the lethal factor was, on average, 41.5% of that of the normal gamete.     

Gene transfer and expression in mouse preimplantation embryos by recombinant adenovirus vector

Replication-defective recombinant adenovirus, Adex4SRLacZL, was used as a vector for transferring exogenous genes in mouse zona pellucida-free eggs at the pronuclear stage. The vector contained the E. coli LacZ reporter gene under the control of the SRα promoter (SV40 early promoter-fused HTLV-I LTR), and the expression of the reporter gene was examined during preimplantation development in culture. Histochemical staining of the embryos for β-galactosidase activity showed that the exogenous LacZ gene as expressed in 98% of the embryos at the morula-blastocyst stages. As in the microinjection method, the exogenous genes could be pursued from the 2-cell stage. Neither apparent morphological changes nor cytotoxic effects were observed. Both the percentages of embryos expressing reporter genes and the rate of development to the blastocyst stage were higher in the adenovirus vector-treated embryos than in the microinjected ones. These results suggest that the adenovirus vector system is a useful tool in investigating the genetic control of early mammalian development. © 1995 wiley-Liss, Inc.     

Haptoglobin in Carnivora: a unique molecular structure in bear, cat and dog haptoglobins

Haptoglobin (Hp), a hemoglobin-binding protein in plasma, consists of α and β subunits and has a tetra-chain arrangement (β-α-α-β) connected by disulfide bridges in most mammals so far examined. Dog Hp has been reported to be unique compared with other Hps in respect that (1) the two αβ units are joined by a non-covalent interaction rather than a disulfide bridge and (2) the α chain has an oligosaccharide-binding sequence (Asn-X-Ser/Thr) and is glycosylated. To determine whether the unique structures of dog Hp are common in the Carnivora, we purified Hps from sera of bear and cat, and analyzed their subunit structure and partial amino acid sequences. The analyses by gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, under both reducing and non-reducing conditions, revealed that bear and cat Hps have similar subunit arrangements to dog Hp, suggesting the absence of a disulfide bridge between two α chains. This was confirmed by amino acid sequence analysis of the α chains: that is, Cys₁₅ participating in the inter-α chain disulfide bridge was replaced by Val in bear or Leu in cat and dog. Thus, the unique subunit arrangement of Hp reported in dog may be common in the Carnivora. In contrast to dog Hp, however, α chains of bear and cat Hps were found not to have the typical oligosaccharide binding sequence on their α chains and were not glycosylated.     

Cloning and sequence analysis of theMaackia amurensis haemagglutinin cDNA

Maackia amurensis haemagglutinin (MAH) is a leguminous lectin which preferentially binds to a cluster of sialylatedO-linked carbohydrate chains (Konami Y, Yamamoto K, Osawa T, Irimura T (1994)FEBS Lett 342:334–38). In the present study a 950 bp cDNA clone encoding MAH was isolated from a cDNA library constructed from germinatedMaackia amurensis seeds. From the nucleotide sequence, MAH was predicted to consist of 285 amino acid residues containing a signal peptide of 29 amino acids. The results also confirmed our previous findings from the amino acid sequence analysis, which indicated that two highly conserved amino acid residues in all other well-known leguminous lectins were replaced in MAH. These residues were lysine-105 and aspartic acid-135. The corresponding amino acid residues in other leguminous lectins were glycine and asparagine, respectively. These differences were due to the presence of nucleotides AAA and GAT in place of AAT/C and GGA/T.Abbreviations MAH Maackia amurensis haemagglutinin.