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Biomedical subjects

T Baba

Publications and source records attributed to T Baba.

At least 289 records · Page 16Linked to original sources
PubMed1993

Expression of modified cytochrome P450 2C10 (2C9) in Escherichia coli, purification, and reconstitution of catalytic activity.

The human cytochrome P450 (P450) 2C gene family is complex and heterologous expression methods are needed to facilitate the isolation of individual P450 proteins and the elucidation of their catalytic specificities. We prepared a series of constructs of P450 2C10 in the plasmid vector pCW, with modification of the 5' end of the coding sequence of the cDNA. Some were not expressed at all in Escherichia coli; two were expressed at levels of 5-20 nmol membrane-bound P450 (liter culture)-1--one (2C1028) with original codons 2-7 altered by substitution of the 5'-terminal sequence described by Barnes et al. (Barnes, H. J., Arlotto, M. P., and Waterman, M. R., Proc., Natl. Acad. Sci. USA 88, 5597-5601, 1991) and one (2C1029) with original codon 2 modified, codons 3-20 deleted, and alteration of the immediate downstream codons. In both cases the P450 2C10 proteins were found essentially only in the bacterial membranes. These proteins could be purified to a high degree by solubilization and a single DEAE chromatography step. Typical P450 Fe2+.CO absorption spectra were observed in the bacterial membranes and the purified preparations. The P450 2C1029 protein was found to have its N-terminal Met removed and the expected residues 2 (Ala)-24 were identified by amino acid sequence analysis. However, the other P450 (2C1028) was apparently blocked at the N-terminus. Three native P450 2C9/10 preparations isolated from human liver showed the expected sequences (beginning with Met) for at least the first 17 residues. The blocked N-terminus in the P450 2C1028 protein may be the result of the MALLLAVF sequence, which was also used in the expression of P450 3A4 and resulted in a blocked protein. Catalytic activities of P450 2C1028 and P450 2C1029 for tolbutamide hydroxylation were similar to those measured with purified liver P450 C29/10 in the presence of cytochrome b5, although the effect of cytochrome b5 did not always show the same pattern as with the isolated liver enzyme. The recombinant P450 2C10 enzymes did not catalyze (S)-mephenytoin 4'-hydroxylation.

Amino Acid Sequence↗
PubMed1993

Purification and characterization of a 38-kDa protein, sp38, with zona pellucida-binding property from porcine epididymal sperm.

A 38-kDa protein, sp38, was purified from the detergent extract of porcine epididymal sperm. Sp38 showed zona pellucida-binding properties similar to those of proacrosin. These two proteins specifically bound to the 90-kDa glycoprotein form of the zona pellucida components in a calcium-dependent manner. The binding of sp38 to the zona pellucida glycoprotein was inhibited by proacrosin. These findings suggest that the two proteins competitively interact with the zona pellucida during the early stage of fertilization.

Acrosin↗
PubMed1993

Antigenic protein specific for C3H strain mouse is a mitochondrial stress-70 protein.

Cells derived from C3H strain mouse produce an antigenic protein (CSA) specific for this strain [Kusakabe, M., et al. (1988) J. Cell Biol. 107, 257-265]. To examine the molecular basis of CSA, cDNA cloning of the antigenic protein was carried out. The deduced amino acid sequence demonstrates that CSA is the same protein as peptide-binding protein 74 (PBP74), a novel member of the stress-70 family. However, comparison of the PBP74/CSA sequences between C3H/HeN and BALB/c strain mice reveals the substitution of two amino acids in the substrate-recognition domain of the stress-70 protein. Western blot analysis indicates that one out of these two residues, arginine at residue 578 in the PBP74/CSA sequence of C3H mouse, contributes to the immunogenicity of CSA. Moreover, the subcellular localization of PBP74/CSA in mitochondria is also demonstrated by immunohistochemical analysis using anti-CSA monoclonal antibody. Thus, it is interesting that a genetic marker sequence in mice is located on the gene encoding a mitochondrial stress-70 protein.

Amino Acid Sequence↗
PubMed1993

Gene expression of mutant erythropoietin in hepatocellular carcinoma.

A case of hepatocellular carcinoma complicated by erythrocytosis showed an increased level of serum immunoreactive erythropoietin (EPO) and EPO bioactivity. RT-PCR (reverse transcriptase and polymerase chain reaction) of EPO mRNA extracted from a surgical specimen indicated high expression of EPO mRNA in the tumor tissue. The nucleotide sequences of PCR amplified regions of the EPO precursor mRNA in tumor tissue showed three differences to those of normal EPO cDNA. The deduced amino acid sequence of the coding region also showed three differences from that of normal EPO. The erythrocytosis improved and the high serum EPO immunoreactive and bioactive level decreased after resection of the tumor. This is the first demonstration of mutant EPO mRNA expression and bioactive mutant EPO protein in hepatocellular carcinoma tissue.

Aged↗
PubMed1993

Alteration of the structural properties of starch components by the lack of an isoform of starch branching enzyme in rice seeds.

This study describes the effect of starch-synthesizing enzymes on biosynthesis of storage starch in rice amylose-extender mutants, which contain branched D-glucans with abnormal structures. Western blot analysis indicated that two out of five amylose-extender mutant lines lacked an isoform of starch branching enzyme, termed RBE3, although the levels of granule-bound starch synthase and a major form of branching enzyme, RBE1, were normal in these two mutants. Proteins corresponding to the 87-kDa RBE3 molecule were present in the three other amylose-extender mutants as well as in the wild type. However, the level of branching enzyme activity significantly decreased in all amylose-extender mutants, suggesting that the 87-kDa proteins in these three mutants are inactive forms of RBE3. Therefore, we conclude that formation of the abnormal branched glucans in the amylose-extender mutant of rice is due to the lack of the RBE3 activity. The cDNA clones encoding RBE3 have been identified from a normal rice seed cDNA library in lambda gt11, using a synthetic oligonucleotide as a probe. The deduced amino acid sequence of RBE3 indicates that this protein is initially synthesized as a precursor of 825 amino acids, including a 65-residue transit peptide at the NH2 terminus. The sequences of the catalytic regions in amylolytic enzymes are highly conserved in the sequence of RBE3. Thus, the branching enzyme isoform belongs to a family of the amylolytic enzymes. RBE3 also shares a noticeable degree of sequence identity with RBE1, especially at the central portion of the protein molecule. However, RBE3 possesses an approximately 70-residue extra sequence at the NH2 terminus and lacks a COOH-terminal sequence of almost 50 residues as compared with RBE1. The structural differences at both termini may explain the distinct role in starch synthesis for RBE1 and RBE3.

1,4-alpha-Glucan Branching Enzyme↗
PubMed1993

Expression of modified human cytochrome P450 3A4 in Escherichia coli and purification and reconstitution of the enzyme.

A full-length human cytochrome P450 (P450) 3A4 cDNA clone and four derivatives in which the N-terminus was modified were inserted into a pCW vector and used to transform Escherichia coli DH5 alpha cells. Little expression was seen with the native sequence; the highest level of expression (range of 40-110 membrane-bound nmol P450 liter-1) was achieved with a construct (NF14) in which residues 3-12 were deleted. In all of the constructs P450 was found primarily in the membranes. The modified P450 3A4 (construct NF14) showed typical P450 hemoprotein spectra. The protein was purified to electrophoretic homogeneity in a five-step procedure [nominally 23 nmol P450 (mg protein)-1]. For most purposes it was found to be more practical to purify the modified P450 3A4 to approximately 70% homogeneity [nominally 15 nmol P450 (mg protein)-1] in a simple two-step process. The modified P450 3A4 (NF14) or P450 3A4 purified from human liver could be mixed with rabbit liver NADPH-P450 reductase to achieve catalytic activities nearly as high as those found in human liver microsomes (on a nmol P450 basis), but the optimal reconstitution conditions included not only a mixture of phosphatidylserine, L-alpha-dilauroyl- and L-alpha-dioleoyl-sn-glycero-3-phosphocholines, cholate, and cytochrome b5 suggested by others but also glutathione during the preincubation. Several other thiols were found not to substitute in this role. Good catalytic activity was seen for nifedipine oxidation, testosterone 6 beta-hydroxylation, and the 8,9-epoxidation and 3 alpha-hydroxylation of aflatoxin B1, reactions previously ascribed to the enzyme. These procedures provide a relatively convenient and reliable means of producing, purifying, and reconstituting a catalytically active and useful derivative of P450 3A4, a human P450 enzyme that has many roles in the oxidation of drugs and other xenobiotic chemicals.

Amino Acid Sequence↗
PubMed1993

The gene encoding a calcium-dependent protein kinase located near the sbe1 gene encoding starch branching enzyme I is specifically expressed in developing rice seeds.

A gene (spk) encoding a Ca(2+)-dependent protein kinase (SPK) is located in the region immediately upstream of the sbe1 gene encoding a starch branching enzyme. The spk gene is specifically expressed in developing seeds and its expression pattern is very similar to those of genes encoding starch-synthesizing enzymes such as sbe1 and waxy, seed lipid-synthesizing enzymes, as well as genes encoding seed storage proteins. A full-length spk cDNA was isolated from a cDNA library constructed from developing seeds. The deduced amino acid sequence showed that SPK has a high degree of homology to soybean and carrot Ca(2+)-dependent protein kinase, both of which contain calmodulin domains. The calmodulin domain, as well as the catalytic subdomain consensus regions of protein kinases are highly conserved in SPK. These results suggest that a tissue- and stage-specific protein kinase, SPK, is involved in the synthesis of seed storage compounds during seed development. They also strongly suggest that Ca2+ is required for seed development.

1,4-alpha-Glucan Branching Enzyme↗
PubMed1993

Exon/intron organization of the gene encoding the mouse epithelin/granulin precursor (acrogranin).

Mouse genomic clones encoding the epithelin/granulin gene and its 5'- and 3'-flanking regions have been isolated and sequenced. This gene was found to be a single-copy gene, and contained 13 exons interrupted by 12 introns. Eight out of the 12 introns are classified as phase 0, and are located within the central part of each of the tandem repeats in the amino acid sequence of the epithelin/granulin precursor. The first intron is unique because of the interruption of the 5'-untranslated region and its fairly large size (approximately 2.4 kbp). Consensus sequences for several of the potential regulatory elements are present in the 5'-flanking sequence, including a common CCAAT sequence.

Amino Acid Sequence↗
PubMed1993

Human myelomonocytic cell line THP-1 produces a novel growth-promoting factor with a wide target cell spectrum.

Conditioned medium from a human myelomonocytic cell line THP-1 promoted the growth of a wide variety of cell types, i.e., human and mouse myeloid cells (HL-60, U937, K562, and M1), mouse T-cells (EL-4), human B cells (Daudi and Raji), mouse mastocytoma cells (IC-2), human melanoma cells (A375-C6), mouse transformed fibroblast cells (L929), human lung fibroblast cells (TIG-1), and mouse bone marrow fibroblast/stromal-like cells. The growth-promoting activity was acid-labile. The activity was resistant to 50 degrees C for 5 min but completely lost in 5 min at 70 degrees C. The activity was resistant to treatment with trypsin but sensitive to chymotrypsin alpha, Pronase E, and proteinase K, indicating the proteinous nature of this activity. The activity was lost by dithiothreitol and 2-mercaptoethanol. Molecular weight (M(r) 50,000-70,000) was estimated by gel filtration-high performance liquid chromatography. After the sequential anion exchange, hydrophobic, and hydroxylapatite high performance liquid chromatography, the partially purified factor exhibited the same target cell spectrum as the conditioned medium.

Animals↗
PubMed1993

Acrogranin, an acrosomal cysteine-rich glycoprotein, is the precursor of the growth-modulating peptides, granulins, and epithelins, and is expressed in somatic as well as male germ cells.

Spermatogenesis is a unique system of differentiation involving cellular remodeling and the biogenesis of sperm-specific organelles. To study the biogenesis of one such organelle, the acrosome, we have been examining the gene expression, biosynthesis, and targeting of specific acrosomal proteins during mammalian spermatogenesis. An acrosomal marker that we recently purified and began characterizing is acrogranin, a 67,000-molecular-weight glycoprotein originally isolated from guinea pig testes. This glycoprotein is detected in pachytene spermatocytes and is found later in the acrosomes of developing spermatids and sperm. Immunoblotting of several tissues and immunofluorescent localization in frozen sections of guinea pig testes suggested that acrogranin was a germ cell-specific glycoprotein that was expressed meiotically and post-meiotically. However, Northern blot analysis demonstrated that the mRNA for acrogranin was ubiquitously expressed in all guinea pig and mouse tissues examined. Furthermore, the primary structures of guinea pig and mouse acrogranins, deduced from the cDNA sequences, reveal that this glycoprotein is a cysteine-rich molecule with a motif that is tandemly repeated seven times, very similar to that of the human epithelin/granulin precursor. We conclude that guinea pig and mouse acrogranins are homologues of the precursor of the human and rat epithelin/granulin peptides previously demonstrated to have growth-modulating properties.

Acrosome↗
PubMed1993

An endoplasmic reticulum protein, calreticulin, is transported into the acrosome of rat sperm.

Recently, we purified a Ca(2+)-binding protein from rat spermatogenic cells [Biochem. Biophys. Res. Commun. 176, 135-1364, 1991]. In the present study, this protein was identified as calreticulin, which is a resident protein of the endoplasmic reticulum (ER). Immunohistochemical studies revealed that calreticulin was present in the acrosome of both round spermatids and mature sperm. However, under immunoelectron microscopy, gold-particles were seen over other subcellular structures of spermatocytes, spermatids, and Sertoli cells. When the labeling density in subcellular structures of spermatids was analyzed, the acrosome was found to be most heavily labeled and the Golgi apparatus was second. The complete amino acid sequence of calreticulin, deduced from the cDNA sequence, shares a high degree of identity with that of the analogous mouse protein. The cDNA encoded a protein of 416 amino acids, including a 17-residue NH2-terminal signal sequence. The mature protein contains a KDEL sequence as an ER signal at the COOH terminus. Sperm calreticulin contained no glycosyl moiety. Northern blot analysis of RNAs from purified populations of rat spermatogenic cells indicated that the calreticulin mRNA was present in both pre- and postmeiotic cells. Immunoblot analysis of calreticulin during developmental stages showed that calreticulin was detected in the testis between the ages of 5 and 50 days. Furthermore, purified rat calreticulin contained two Ca(2+)-binding sites, a low affinity/high capacity site and a high affinity/low capacity site. These results suggest that calreticulin, which is not specific to testis, is closely associated with spermatogenesis of rats. This ER protein may be incorporated into the acrosomal vesicle via the Golgi apparatus, without glycosylation, during spermiogenesis, and may play an important role in the regulation of cell functions such as sperm motility and acrosome reaction.

Acrosome↗
PubMed1993

Molecular analysis of the gene encoding a rice starch branching enzyme.

The sequence of a rice gene encoding a starch branching enzyme (sbe1) shows extreme divergence from that of the rice gene, that is homologous to bacterial glycogen branching enzyme (sbe2). sbe1 is expressed abundantly and specifically in developing seeds and maximally in the middle stages of seed development. This expression pattern completely coincides with that of the waxy gene, which encodes a granule-bound starch synthase. Three G-box motifs and consensus promoter sequences are present in the 5' flanking region of sbe1. It encodes a putative transit peptide, which is required for transport into the amyloplast. A 2.2 kb intron (intron 2) precedes the border between the regions encoding the transit peptide and the mature protein, and contains a high G/C content with several repeated sequences in its 5' half. Although only a single copy of sbe1 is present in the rice genome, Southern analysis using intron 2 as a probe indicates the presence of several homologous sequences in the rice genome, suggesting that this large intron and also the transit peptide coding region may be acquired from another portion of the genome by duplication and insertion of the sequence into the gene.

1,4-alpha-Glucan Branching Enzyme↗
PubMed1993

Effect of chronic treatment with enalapril on glucose tolerance and serum insulin in non-insulin-resistant Japanese patients with essential hypertension.

The effect of enalapril, an angiotensin converting enzyme inhibitor, on glucose tolerance and serum insulin response to a glucose load has been evaluated in 8 non-obese patients (3 women and 5 men) with untreated essential hypertension (WHO Stage I or II) and without insulin resistance. Following a 2-month run-in control period, each patient received oral enalapril 10 mg once daily for 6 months, and an intravenous glucose tolerance test (IVGTT) was performed at the end of the run-in control and active treatment periods. Treatment with enalapril significantly lowered both the systolic and diastolic blood pressures. The response of plasma glucose to the IVGTT, glucose disappearance rate (k-value) and area under the serum insulin concentration time curve were comparable between the two phases. The results suggest that long-term treatment with enalapril has no effect on glucose tolerance in non-obese, non-insulin-resistant patients with mild-to-moderate essential hypertension.

Adult↗
PubMed1993

Ultrastructural study of Cryptococcus neoformans by quick-freezing and deep-etching method.

The three-dimensional ultrastructure of Cryptococcus neoformans was studied by quick-freezing and deep-etching (QF-DE) method. C. neoformans, strain CDC551, was cultured on agar. The viable yeast cells (10(7) cells) were inoculated into each mouse from the tail vein. Three weeks after the inoculation, the brains of the mice were perfused with fixatives, quickly frozen, freeze-fractured, deeply etched and rotary shadowed with platinum and carbon. In addition, the viable cells of C. neoformans on agar were picked up and quickly frozen, and replica membranes were prepared as described above. The ultrastructure of C. neoformans was three-dimensionally demonstrated by the QF-DE method. The capsule was composed of fine meshworks of microfibrils (10-13 nm in diameter), which were directly attached to the cell walls. The capsule of the in vivo yeasts (yeast cells in the brain lesion) was thicker than that of the in vitro yeasts (yeast cells on agar culture). At the outer part of the cell wall, a particle-accumulating layer was observed. This layer in vivo was thicker than that in vitro. Occasionally, the yeast cells were ingested by phagocytes in the mouse brain. Although the cytoplasm of such yeast cells was destroyed, the capsular meshworks were well preserved. The ultrastructure of the capsule was the same both in cultured and phagocytized yeasts in the cystic lesions of the brains. This lack of morphological changes of the capsular meshworks suggests that they are resistant to the digestion by phagocytes. This stability of capsular structures may provide one of the important pathogenic factors in cystic lesions by C. neoformans.

Animals↗
PubMed1993

Role of Harderian gland on differentiation and proliferation of immunoglobulin A-bearing lymphocytes in chickens.

The mechanism of accumulation of surface immunoglobulin A (sIgA) bearing cells in chicken Harderian gland (HG) was examined. Almost no sIgA-bearing cells were identified in HG of 1.5-week-old chickens. In 3.5-week-old chickens, however, 46.4% of the HG lymphocytes were sIgA-bearing cells. When bursa of Fabricius (BF) lymphocytes were transferred into recipients, BF lymphocytes migrated into the HG but the number of these lymphocytes was extremely small. In addition, no correlation was found between the surface immunoglobulin class of BF lymphocytes that migrated into HG and the migration. HG lymphocytes were co-cultured with HG whole cells (which contained 50-60% configurationally epithelial cells) in a non-contacting manner for 80 h using an Intercell culture vessel. The results showed a significant increase in the ratio of sIgA-bearing cells in HG lymphocytes compared with the control which was cultured without placing HG whole cells in the external well. B lymphocytes that migrated into HG seemed to undergo specific differentiation in situ into sIgA-bearing cells owing to microenvironmental factors, probably humoral factors excreted from HG epithelial cells, and subsequently undergo proliferation to result in accumulation of sIgA-bearing cells in HG tissue.

Animals↗