Sequences of cDNAs for human salivary and pancreatic α-amylases

The nucleotide sequences of the cloned human salivary and pancreatic α-amylase cDNAs correspond to the continuous mRNA sequences of 1768 and 1566 nucleotides, respectively. These include all of the amino acid coding regions. Salivary cDNA contains 200 bp in the 5′-noncoding region and 32 in the 3′-noncoding region. Pancreatic cDNA contains 3 and 27 bp of 5′- and 3′-noncoding regions, respectively. The nucleotide sequence humology of the two cDNAs is 96% in the coding region, and the predicted amino acid sequences are 94% homologous.Comparison of the sequences of human α-amylase cDNAs with those previously obtained for mouse α-amylase genes (Hagenbuchle et al., 1980; Schibler et al., 1982) showed the possibility of gene conversion between the two genes of human α-amylase.     

Requirement of caspase and p38MAPK activation in zinc-induced apoptosis in human leukemia HL-60 cells.

Zinc (Zn), an endogenous regulator of apoptosis, and has abilities both to induce apoptosis and inhibit the induction of apoptosis via the modulation of caspase activity. Due to the multifunctions of Zn, the intracellular Zn level is strictly regulated by a complex system in physiological and pathological conditions. The commitment of Zn to the regulation of apoptosis is not fully understood. In the present study, we investigated the role of intracellular Zn level in the induction of apoptosis in human leukemia cells (HL-60 cells) using a Zn ionophore [pyrithione (Py)]. Treatment of HL-60 cells with Zn for 6 h in the presence of Py (1 micro m) exhibited cytotoxicity in a Zn dose-dependent manner (25-200 micro m). Necrotic cells, assayed by trypan blue permeability, increased in number in a Zn dose-dependent fashion (50-100 micro m), but the appearance of apoptotic cells, assayed by formation of a DNA ladder and terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end-labeling method, peaked at 25 micro m, suggesting the dependence of intracellular Zn level on the execution of apoptosis. In fact, treatment with Py resulted in increases in intracellular Zn levels, and N,N,N',N'-tetrakis (2-pyridylmethyl)ethylenediamine, a cell-permeable Zn chelator, inhibited DNA ladder formation induced by Py/Zn treatment (1 micro m Py and 25 micro m Zn). Py/Zn treatment activated the caspases, as assessed by the proteolysis of poly(ADP-ribose) polymerase (PARP), which is a substrate of caspase, and activated p38 mitogen-activated protein kinase (p38MAPK), which is a transducer of apoptotic stimuli to the apparatus of the apoptosis execution. Z-Asp-CH2-DCB, a broad-spectrum inhibitor of caspase, attenuated proteolysis of PARP and DNA ladder formation by Py/Zn, indicating that apoptosis induced by Py/Zn is mediated by caspase activation. The p38MAPK-specific inhibitor SB203580 also inhibited induction of apoptosis by Py/Zn. Although SB203580 suppressed the proteolysis of PARP, Z-Asp-CH2-DCB did not inhibit the phosphorylation of p38MAPK, raising the possibility that apoptosis triggered by Py/Zn might be mediated by the p38MAPK/caspase pathway.     

Structure and function relationship of staphylocoagulase

The bacterial protein staphylocoagulase binds stoichiometrically to human prothrombin, resulting in a coagulant complex, staphylothrombin. The enzymatic properties of staphylothrombin differ from those of -thrombin in their substrate specificities toward natural and synthetic substrates, in addition to their interaction with protease inhibitors. In order to obtain information about the region of staphylocoagulase that interacts with human prothrombin, staphylocoagulase was cleaved by -chymotrypsin. Limited -chymotryptic cleavage of staphylocoagulase yielded three large fragments, of 43, 30, and 20 kD. The 43-kD fragment exhibited a high affinity for human prothrombin (K_d=1.7 nM), which is comparable to the affinity observed using intact staphylocoagulase (K_d=0.46 nM). A complex of the 43-kD fragment and prothrombin possessed both clotting and amidase activity essentially identical to that observed in a complex of intact staphylocoagulase and prothrombin. The 30-kD fragment exhibited weaker affinity for prothrombin (K_d=120 nM.) While clotting activity was not observed with a complex of this fragment and prothrombin, it nonetheless possessed a weak amidase activity. The 20-kD fragment was found only to bind to prothrombin. The NH₂-terminal sequence analyses of these fragments revealed that the 43-kD fragment constitutes the NH₂-terminal portion of staphylocoagulase, and contains the 30-kD and 20-kD fragments. It is therefore concluded that the functional region of staphylocoagulase for binding and activation of prothrombin is localized in the NH₂-terminal region of the intact protein. The 43-kD fragment contained 324 amino acids with a molecular weight of 38,098. The 43-kD fragment had an unusual amino acid composition based on a sequence in which the sum of Asp (28 residues), Asn (22), Glu (35), Gln (9), and Lys (52) residues accounted for more than 45% of the total. A comparison of the amino acid sequence of the 43-kD fragment with that of streptokinase did not reveal any obvious sequence homology. There was also no sequence homology with that of trypsin, -chymotrypsin, and elastase.This article was presented during the proceedings of the International Conference on Macromolecular Structure and Function, held at the National Defence Medical College, Tokorozawa, Japan, December 1985.     

Genotyping and sequence analysis of apolipoprotein E isoforms

Apolipoprotein E (apoE), a polymorphic plasma protein, is essential for catabolism of lipoproteins by receptor-mediated endocytosis. One of the apoE isoforms (E2) differs in its binding affinity to specific receptors and contributes to variations in lipoprotein metabolism. Diagnosis of apoE isoforms is done by isoelectric focusing, but it is hindered by various degrees of post-translational sialylation of the apoE protein. Electrophoretically silent structural variations may also escape detection by this technique. We describe a method for genotyping apoE based on hybridization of allele-specific oligonucleotides with enzymatically amplified genomic DNA, which permits unambiguous diagnosis of six common apoE phenotypes within 24 h. Among 100 E2 alleles present in 81 unrelated individuals genotyped by this technique, we found two rare structural mutants of apoE in addition to the common E2 form, E2(158Arg----Cys). Automated sequencing of amplified DNA identified the rare mutants as E2(136Arg----Ser) and E2(145Arg----Cys). The genotypic method may complement or even replace isoelectric focusing for routine determination of apoE phenotypes and for identification of rare structural variants.     

Genetic linkage between lipoprotein(a) phenotype and a DNA polymorphism in the plasminogen gene

Coronary heart disease risk correlates directly with plasma concentrations of lipoprotein(a) (Lp(a)), a low-density lipoprotein-like particle distinguished by the presence of the glycoprotein apolipoprotein(a) (apo(a)), which is bound to apolipoprotein B-100 (apoB-100) by disulfide bridges. Size isoforms of apo(a) are inherited as Mendelian codominant traits and are associated with variations in the plasma concentration of lipoprotein(a). Plasminogen and apo(a) show striking protein sequence homology, and their genes both map to chromosome 6q26-27. In a large family with early coronary heart disease and high plasma concentrations of Lp(a), we found tight linkage between apo(a) size isoforms and a DNA polymorphism in the plasminogen gene; plasma concentrations of Lp(a) also appeared to be related to genetic variation at the apo(a) locus. We found free recombination between the same phenotype and alleles of the apoB DNA polymorphism. This suggests that apo(a) size isoforms and plasma lipoprotein(a) concentrations are each determined by genetic variation at the apo(a) locus.     

Isolation and mapping of 88 new RFLP markers on human chromosome 8.

To obtain new RFLP markers for construction of a high-resolution map of human chromosome 8, a cosmid library was constructed from a somatic hybrid cell that contained chromosome 8 as the only human component in mouse genomic background. Eighty-eight new RFLP markers were isolated and characterized, and 71 of them were sublocalized to chromosomal bands by fluorescent in situ hybridization (FISH). Of these, 36 were localized to the short arm, 34 to the long arm, and 1 to the centromeric region. Five markers defined VNTR loci. This work represents the first extensive isolation and physical mapping of RFLP markers on human chromosome 8. These new markers will serve as useful resources for linkage mapping of loci for inherited diseases and for efforts to identify a putative tumor suppressor gene(s) on chromosome 8.     

Phototropic stimulation does not induce unequal distribution of indole-3-acetic acid in maize coleoptiles

Distribution of endogenous diffusible auxin into agar blocks from phototropically stimulated maize coleoptile tips was studied using a bioassay and a physicochemical assay, to clarify whether phototropism in maize coleoptiles involves a lateral gradient in the amount of auxin. At 50 min after the onset of phototropic stimulation, when the phototropic response was still developing, direct assay of the blocks with the Avena curvature test showed that the auxin activity in the blocks from the shaded half-tips was twice that of the lighted side, at both the first and second positive phototropic curvatures. However, physicochemical determination following purification showed that the amount of indole-3-acetic acid (IAA) was evenly distributed in the blocks from lighted and shaded coleoptile half-tips at both the first and second positive phototropic curvatures. The even distribution of the IAA was also confirmed with the Avena curvature test following purification by HPLC. These results indicate that phototropism in maize coleoptiles is not caused by a lateral gradient of IAA itself and thus cannot be described by the Cholodny-Went theory. Furthermore, the lower auxin activity in the blocks from the lighted half-tips suggests the presence of inhibitor(s) interfering with the action of auxin and their significant diffusion from unilaterally illuminated coleoptile tips.     

1H NMR studies of deuterated ribonuclease HI selectively labeled with protonated amino acids

Summary Two-dimensional (2D)¹H NMR experiments using deuterium labeling have been carried out to investigate the solution structure of ribonuclease HI (RNase HI) fromEscherichia coli (E. coli), which consists of 155 amino acids. To simplify the¹H NMR spectra, two fully deuterated enzymes bearing several prototed amino acids were prepared from an RNase HI overproducing strain ofE. coli grown in an almost fully deuterated medium. One enzyme was selectively labeled by protonated His, He. Val. and Leu. The other was labeled by only protonated His and Ile. The 2D¹H NMR spectra of these deuterated R Nase H1 proteins, selectively labeled with protonated amino acids, were much more simple than those of the normally protonated enzyme. The simplified spectra allowed unambiguous assignments of the resonance peaks and connectivities in COSY and NOESY for the side-chain protons. The spin-lattice relaxation times of the side-chain protons of the buried His residue of the deuterated enzyme became remarkably longer than that of the protonated enzyme. In contrast, the relaxation times of the side-chain protons of exposed His residues remained essentially unchanged.     

Synthesis of sex-specific forms of cytochrome P-450 in rat liver is transiently suppressed by hepatic monooxygenase inducers

The effects of phenobarbital (PB), 3-methylcholanthrene (MC), and alpha-naphthoflavone (alpha-NF) on the synthesis of drug-inducible forms of cytochrome P-450, P-450(PB-1), and P-450(MC-1), and sex-specific forms of cytochrome P-450, P-450(M-1), and P-450(F-1), in male and female rats were studied. Whereas P-450(PB-1) and P-450(MC-1) in liver microsomes were markedly induced in both sexes by treatment with PB and MC, respectively, the contents of P-450(M-1) and P-450(F-1) were significantly decreased by the treatments. alpha-NF, which is not a P-450 inducer, did not change the contents of sex-specific forms of cytochrome P-450. The translatable mRNAs of the P-450s were also determined by using an in vitro translation system. The mRNAs coding for P-450(PB-1) and P-450(MC-1) were increased by drug administrations. On the other hand, the mRNAs coding for P-450(M-1) and P-450(F-1) were transiently decreased by the drugs, and then returned to the normal levels. The time courses of the induction of the drug-inducible P-450s and the repression of the sex-specific P-450s showed no close correlation. alpha-NF had no effect on the synthesis of P-450(M-1) and P-450(F-1). We also found that the synthesis of P-450(M-1) in the livers of untreated rats showed no diurnal variations.     

Protein phylogeny of translation elongation factor EF-1α suggests microsporidians are extremely ancient eukaryotes

Partial regions of the mRNA encoding a major part of translation elongation factor 1 (EF-1) from a mitochondrion-lacking protozoan,Glugea plecoglossi, that belongs to microsporidians, were amplified by polymerase chain reaction (PCR) and their primary structures were analyzed. The deduced amino acid sequence was highly divergent from typical EF-1's of eukaryotes, although it clearly showed a eukaryotic feature when aligned with homologs of the three primary kingdoms. Maximum likelihood (ML) analyses on the basis of six different stochastic models of amino acid substitutions and a maximum parsimony (MP) analysis consistently suggest that among eukaryotic species being analyzed,G. plecoglossi is likely to represent the earliest offshoot of eukaryotes. Microsporidians might be the extremely ancient eukaryotes which have diverged before an occurrence of mitochondrial symbiosis. Sequence availability: The nucleotide sequence data reported here appear in the GSDB, DDBJ, EMBL, and NCBI databases with the accession number D32139