Biomedical subjects

A Kaji

Publications and source records attributed to A Kaji.

At least 73 records · Page 4Linked to original sources

PubMed1994

M13 bacteriophage DNA inhibits duck hepatitis B virus during acute infection.

We investigated effects of various DNAs on duck hepatitis B virus replication in vivo. One-day-old ducks were infected intravenously with DHBV. Various DNAs were then injected intravenously, and duck hepatitis B virus levels were followed for up to 20 days after the inoculation. When M13 bacteriophage DNA (M13 DNA), heat-denatured Escherichia coli DNA or phi X 174 phage DNA was injected intravenously at a dose of 2.45 mg/kg body wt daily for 10 days, a significant decrease of serum duck hepatitis B virus DNA was detected within 10 days. The efficacy was twice that reported with antisense DNA on a weight basis and far more than that reported on a molar basis. M13 DNA was superior, on the basis of effective dose, to acyclovir as an anti-duck hepatitis B virus agent. On treatment with M13 DNA, serum 2-5 A synthetase level was increased five to six times, suggesting that the antiviral effect of M13 DNA is at least partly due to induction of endogenous interferon, which in turn induces 2-5 A synthetase. No significant inhibitory effect on replication of duck hepatitis B virus was demonstrated by DNAs obtained from herring testes, herring sperm, salmon testes, human placenta or calf thymus. On discontinuation of M13 DNA injection on day 10, duck hepatitis B virus reappeared in the serum at later time points. Digestion of M13 DNA with S1 nuclease resulted in marked reduction of antiviral activity. These results show that M13 DNA, not its digested product, has potent antiviral activity.

2',5'-Oligoadenylate Synthetase↗

PubMed1993

Cloning of CRP2, a novel member of the cysteine-rich protein family with two repeats of an unusual LIM/double zinc-finger motif.

The cDNA coding for a novel member of the cysteine-rich protein family was isolated from a rat brain cDNA library. It encodes a protein, denoted cysteine-rich protein 2 (CRP2), of 208 amino acid residues containing two tandem repeats of an unusual LIM/double zinc-finger-like motif. The ubiquitous tissue distribution and high level of expression of CRP2 mRNA suggest an important role for CRP2 in cell functions.

Amino Acid Sequence↗

PubMed1993

In vitro cytostatic effect of TNF (tumor necrosis factor) entrapped in immunoliposomes on cells normally insensitive to TNF.

The cytostatic activity of TNF entrapped in novel immunoliposomes with a specific antibody against target cells is described. A two step conjugation method was used for the preparation of these targeted immunoliposomes. In the first step, liposomes containing N-4-(p-maleimidophenyl)butyryl phosphatidylethanolamine (MPB-PE) were conjugated with a goat anti-mouse IgG Fab' fragment which recognizes the Fc portion of a mouse antibody against the target cell markers. In the second step, the mouse antibody against human tumor cells was conjugated to the liposomes. Using these targeted immunoliposomes, we demonstrated that cells usually insensitive to TNF such as Daudi cells, MT-2 cells and T-24 cells could become sensitive to TNF in vitro. The cytostatic activity of these immunoliposomes was blocked by the addition of a lysosomotropic agent such as NH4Cl or chloroquine. Significant uptake of 125I-TNF into T-24 cells was observed when these immunoliposomes were used, and this uptake of TNF was inhibited by cytochalasin B or chloroquine. Free 125I-TNF was not taken up by these cells.

Animals↗

PubMed1993

Effect of barbiturate therapy on phenytoin pharmacokinetics.

OBJECTIVE: To evaluate the effect of high-dose pentobarbital therapy on phenytoin pharmacokinetics. DESIGN: A prospective, clinical study. SETTING: The intensive care unit of a university hospital. PATIENTS: Ten adult patients with cerebral lesions requiring anticonvulsants and control of intracranial pressure. INTERVENTIONS: Each patient received phenytoin sufficient to maintain a plasma concentration at 15 micrograms/mL (60 mumol/L) both before and after barbiturate therapy. Plasma concentrations of total phenytoin, unbound phenytoin, and the major metabolite of phenytoin, 5-(p-hydroxyphenyl)-5-phenylhydantoin, were measured, and pharmacokinetic variables obtained before and after barbiturate therapy were compared. MEASUREMENTS AND MAIN RESULTS: Plasma concentrations of total phenytoin remained within the therapeutic range during the 12-hr period preceding barbiturate therapy. After barbiturate therapy, plasma concentrations of both total and unbound phenytoin were significantly less than those concentrations before barbiturate therapy. For total phenytoin, maximum metabolic velocity was increased by 62% (1.09 +/- 0.62 to 1.77 +/- 0.52 mg/L/hr, 1.20 +/- 0.68 to 1.95 +/- 0.57 nmol/L/sec, p < .05), and area under the plasma concentration-time curve (0 to infinity) and mean residence time were each decreased by 73% (32.5 +/- 20.0 to 8.7 +/- 3.1 min.mg/mL, 2.14 +/- 1.25 to 0.57 +/- 0.19 sec.mmol/L, p < .01, and 135,000 +/- 69,000 to 37,000 +/- 11,000 secs, p < .005, respectively) after barbiturate therapy. The plasma concentration of the principal metabolite of phenytoin, 5-(p-hydroxyphenyl)-5-phenylhydantoin, was significantly increased after barbiturate therapy. CONCLUSIONS: Phenytoin metabolism is increased by barbiturate therapy, and supplemental doses of phenytoin and frequent drug monitoring may be required after barbiturate therapy.

Adult↗

PubMed1992

Studies on antisense inhibition of translation in vitro. Anomalies and re-evaluation.

Experiments were carried out to better characterize antisense control of translation. Results in an E. coli system confirmed specific inhibition of poly(U) translation. At low concentrations, certain homopolymers (including poly(rA)) stimulated translation. Oligo(dA(n)) was inhibitory at n less than or equal to 8. Translation of globin mRNA in reticulocyte lysates indicated that ssDNA 15-mers targeted at beta-globin mRNA inhibited both alpha- and beta-globin production. Sequences targeted immediately downstream of the AUG were the least effective in inhibition. These and other anomalies are discussed here in relation to those of others, emphasizing caution in performing antisense experiments.

Animals↗

PubMed1992

Nucleotide sequence of a cDNA encoding human tumor necrosis factor beta from B lymphoblastoid cell RPMI 1788.

Published sequences of cDNA for human tumor necrosis factor beta (TNF-beta) have a discrepancy within the coding region as well as exon 1. To resolve these discrepancies we have re-isolated TNF-beta cDNA from the human B cell lymphoblastoid cell line, RPMI 1788, and determined its DNA sequence. Results indicate that amino acid 26 is threonine (Thr) instead of asparagine (Asn). In contrast to published sequences, the sequence of the exon 1 region corresponded to the genomic sequence of TNF-beta. From our studies we conclude that the TNF-beta gene of the human B cell lymphoblastoid cell line, RPMI 1788, is homologous with respect to the TNF-beta gene.

B-Lymphocytes↗

PubMed1992

HIV-1 reverse transcriptase inhibitor from Phyllanthus niruri.

An aqueous extract of Phyllanthus niruri (Euphorbiaceae) inhibited human immunodeficiency virus type-1 reverse transcriptase (HIV-1-RT). The inhibitor against HIV-1-RT in this plant was purified by combination of three column chromatographies, Sephadex LH-20, cellulose, and reverse-phase high-performance liquid chromatography. The inhibitor was then identified by nuclear magnetic resonance (NMR) spectra as repandusinic acid A monosodium salt (RA) which was originally isolated from Mallotus repandus. The 50% inhibitory doses (ID50) of RA on HIV-1-RT and DNA polymerase alpha (from HeLa cells) were 0.05 microM and 0.6 microM, respectively, representing approximately a 10-fold more sensitivity of HIV-1-RT compared with DNA polymerase alpha. RA was shown to be a competitive inhibitor with respect to the template-primer while it was a noncompetitive inhibitor with respect to the substrate. RA as low as 10.1 microM inhibited HIV-1-induced cytopathogenicity in MT-4 cells. In addition, 4.5 microM of RA inhibited HIV-1-induced giant cell formation of SUP-T1 approximately 50%. RA (2.5 microM) inhibited up to 90% of HIV-1 specific p24 antigen production in a Clone H9 cell system.

Antigens, Viral↗

PubMed1992

Infection of terminally differentiated myotubes with Rous sarcoma virus (RSV): lack of DNA integration but presence of RSV mRNA.

We re-examined the generally accepted concept that replication of Rous sarcoma virus (RSV) requires host DNA synthesis. We used terminally differentiated chicken myotubes as the host because chromosomal DNA replication is completely abolished by the natural differentiation process. Southern blot analysis detected unintegrated viral DNA in both the nucleus and cytoplasm of infected myotubes. This indicated that reverse transcription of the infecting viral RNA and transport of the newly synthesized viral DNA into the nucleus proceeded normally in myotubes. However, restriction enzyme digestion of high M(r) DNA prepared from infected myotubes produced none of the fragments specific for RSV, indicating that the viral DNA had failed to integrate into the myotube chromosomal DNA. In these infected myotubes, viral RNA was detected by in situ hybridization. Northern blot analysis showed the presence of all three RSV mRNAs (38S, 28S and 21S). The amount of these viral RNAs in infected myotubes was comparable with that found in infected fibroblasts. We conclude that host DNA synthesis is required for RSV integration, but, in contrast to the generally accepted concept, viral DNA integration is not an absolute requirement for transcription of the RSV genome.

Animals↗

PubMed1992

Infection of terminally differentiated myotubes with Rous sarcoma virus: reduced synthesis of env and v-src proteins.

We studied Rous sarcoma virus (RSV) protein synthesis in RSV-infected, terminally differentiated chicken myotubes ('late-infected' myotubes), in which no viral DNA integration takes place but all three viral mRNAs (38S, 28S and 21S) are transcribed normally. With the use of specific anti-RSV protein antisera, we found that only the viral gag and pol proteins were synthesized at levels similar to those synthesized in RSV-transformed fibroblasts; the synthesis of env and v-src proteins was significantly reduced in these infected myotubes. We concluded that the viral RNA transcribed from the unintegrated RSV DNA was functional but that genes at the 3' end of the RSV genome were translated at a lower level. By contrast, when mononucleated replicating chicken myoblasts were infected with a mutant (tsNY68) carrying a temperature-sensitive v-src gene and maintained at the non-permissive temperature for this gene, they developed into myotubes with viral DNA integrated in their chromosomal DNA. These 'early-infected' myotubes expressed all four viral genes (gag, pol, env and v-src) at a level similar to that in infected fibroblasts. This result ruled out the possible presence of specific factor(s) in myotubes that preferentially inhibit the 3' genes of RSV, and suggested other translational control(s) of viral gene expression in late-infected myotubes.

Animals↗

PubMed1991

Use of single-stranded DNA oligonucleotides in programming ribosomes for translation.

Single-stranded DNA (ssDNA) oligomers were compared to synthetic RNA oligomers in their ability to program E. coli ribosomes in vitro. AUG and dATG-containing oligomers promoted the non-enzymatic binding of fmet-tRNA to ribosomes, with similar dependence on time and magnesium concentration; only at 10 mM Mg++ or at low oligomer concentration was RNA slightly preferred in complex formation. These initiation complexes were biologically active in that fmet-tRNA, bound in response to ssDNA or RNA, was fully reactive with puromycin. While dAUG could not function as an initiation codon, p-dAUG functioned as well as AUG or dATG. However, dUAA and p-dUAA could not replace UAA in directing release-factor (RF) activity, and dTAA functioned only to a slight extent. Release factors had specificity for termination complexes containing dATGTAA, dATGTAG, or dATGTGA. At Mg++ concentrations of 15 mM or higher, these hexamers directed peptidyl transferase-dependent fmet-tRNA hydrolysis in the absence of RF. We suggest this RF-independent activation of peptidyl transferase as a unique system for studying the mechanism of termination. Overall, these results indicate that ssDNA can be used in place of RNA for certain studies of protein synthesis.

Codon↗

PubMed1991

Novel muteins of human tumor necrosis factor alpha.

For chemical synthesis of a gene coding for human tumor necrosis factor alpha (TNF-alpha), DNA sequence predicted by the amino acid sequence of human TNF molecule was prepared. Codons were chosen according to the codon usage in Escherichia coli (E. coli). The 490 bp gene was assembled by enzymic ligation of 42 oligonucleotides and was cloned into a vector (pKK223-3) for high expression of active TNF-alpha in E. coli. With use of site-directed mutagenesis on this DNA, five different muteins of TNF-alpha were synthesized. TNF-M1 and TNF-M4 have deletions of His-73 and Gln-102, respectively. These deletions didn't cause loss of the cytotoxic activity against L929 cells. TNF-M5, which has a substitution of Asp-10 to Arg, had the similar cytotoxic activity to that of TNF-alpha. The cytotoxic spectra against several tumor cells were not changed by this substitution. TNF-M3 has an amino acid substitution of Glu-116 to His which occupies this position in human TNF-beta. This substitution didn't change the cytotoxicity. In addition, evidence was presented that the change of the carboxyl terminal residue doesn't always influence the cytotoxic activity of TNF-alpha. Many different muteins were also isolated by random mutagenesis with hydroxylamine-HCl. One of the muteins, which carries a mutation of His-15 to Tyr, lost the cytotoxic activity almost completely.

Amino Acid Sequence↗

PubMed1991

Expression of chimeric ras protein with OmpF signal peptide in Escherichia coli: localization of OmpF fusion protein in the inner membrane.

The ras gene was fused with the DNA sequence of OmpF signal peptide or with the DNA sequence of OmpF signal peptide plus the amino terminal portion of the OmpF gene. They were placed in plasmids together with the bacteriophage lambda PL promoter. These plasmids were introduced into Escherichia coli strain K-12 and the OmpF signal peptide fusion proteins were expressed. These fusion proteins were identified as 29.0 and 30.0 kDa proteins. However, processed products of these proteins were not found in the extract. The fusion proteins were localized mostly in the cytoplasm and the inner membrane, but none of them was secreted into the periplasmic space. On the other hand, the ras protein alone was found in the cytoplasm and not in the inner membrane. Viable counts of E. coli harbouring these plasmids decreased when these fused proteins were induced. Induction of the ras protein alone did not harm cells. These observations suggest that insertion of the heterologous proteins into the inner membrane may cause the bactericidal effect.

Amino Acid Sequence↗

PubMed1991

Parasympathetic innervation of cutaneous blood vessels examined by retrograde tracing in the rat lower lip.

The origin of vasoactive intestinal polypeptide (VIP)-immunoreactive and acetylcholinesterase (AChE)-positive perivascular nerve fibers in the lower lip of rats was investigated using the retrograde tracer, wheat germ agglutinin conjugated to enzymatically inactive horseradish peroxidase gold complex (WGAapoHRP-Au), in combination with immunohistochemistry and enzyme histochemistry, by comparing the cells of origin of projection to the parotid gland. After the application of the tracer to the lip, small- to medium-sized nerve cells were labelled exclusively in the ipsilateral otic ganglion. Most of them showed moderate VIP-immunoreactivity and AChE activity. In contrast, injection into the parotid gland resulted in labelling of mostly large-sized cells of the otic ganglion which showed intense VIP-immunoreactivity and AChE activity. These results confirmed that the parasympathetic innervation of the rat lip originates from the otic ganglion. It was further suggested that there are at least two subpopulations in the otic ganglion cells, different from each other in size and in VIP-immunoreactivity, which separately innervate the salivary gland and the blood vessels.

Acetylcholinesterase↗

PubMed1991

Identification of the promoter region of the ribosome-releasing factor cistron (frr).

Previous studies of the structure and expression of the ribosome-releasing factor (RRF) cistron (frr) have suggested that an efficient promoter region is located in the RRF cistron. We report here on the nucleotide sequence and in vivo function of the RRF promoter. The transcriptional start site was determined by primer extension to be 58 bp upstream of the translational initiation codon of frr. The location of the RRF promoter region was confirmed by means of (i) deletion analysis of the 5' proximal sequences of frr fused to the chloramphenicol acetyltransferase reporter gene, (ii) analysis of RRF produced in vivo from the deletion derivatives of frr cloned into pUC19, and (iii) gel retardation analysis with Escherichia coli RNA polymerase. The -35 and -10 regions were TTacCc and TATAcT, respectively. The strength of the RRF promoter was similar to that of the lac promoter, as determined by in vivo expression of chloramphenicol acetyltransferase activity. However, the RRF promoter was not affected by the intracellular cyclic AMP level despite the presence of a cyclic AMP receptor protein binding site downstream of the RRF promoter.

Amino Acid Sequence↗

PubMed1991

Three short fragments of Rts1 DNA are responsible for the temperature-sensitive growth phenotype (Tsg) of host bacteria.

Rts1 is a multiphenotype drug resistance factor, and one of its phenotypes is temperature-sensitive growth (Tsg) of host bacteria. A 3.65-kb fragment from Rts1 DNA was shown to cause the Tsg phenotype in host cells. This tsg fragment was split by a restriction enzyme, HincII, into four fragments. Two of these fragments were called HincII-S (short) and HincII-L (long), respectively. Each of these two fragments conferred the Tsg phenotype, indicating that, in fact, these two independent regions were responsible for the Tsg phenotype. The HincII-S 783-bp and HincII-L 1,479-bp fragments were sequenced. The region in the HincII-S fragment to which the Tsg phenotype was attributed was narrowed to a 146-bp (nucleotides 1 to 146) fragment by various restriction enzyme digestions. Further digestion of the 146-bp fragment with Bal 31 suggested that the 116-bp (nucleotides 9 to 124) fragment is the minimum sequence required for Tsg. On the other hand, in the HincII-L fragment, a fragment of 249 bp (nucleotides 1210 to 1458) and a fragment of 321 bp (nucleotides 1942 to 2262) contained separate temperature-sensitive growth activity. None of three tsg fragments contained open reading frames. The 249-bp fragment had very weak Tsg activity, while the 321-bp fragment had no Tsg activity. On the other hand, when these two fragments were together in the pUC19 vector, they exhibited very strong Tsg activity equivalent to that of the original 1,479-bp fragment. In addition, two of the 249-bp fragments gave similar, strong Tsg activity. The HincII-L 1,479-bp fragment contained an open reading frame for kanamycin resistance which was found between nucleotides 1423 and 2238. This kanamycin resistance gene sequence was different from that of the reported kanamycin resistance gene of Tn903 at 12 positions which were deduced to change seven amino acids.

Base Sequence↗

PubMed1990

Enzymic synthesis of oligonucleotides containing methylphosphonate internucleotide linkages.

Thymidine 5'-O-(pyrophosphoryl methylphosphonate) (dTTP alpha CH3) has been chemically synthesized by condensation of thymidine 5'-O-(methylphosphonate) with pyrophosphate. This novel nucleotide, which contained an alpha-phosphorus atom as methylphosphonate, was used as a substrate of terminal deoxynucleotidyltransferase (TDTase) in the presence of oligonucleotide (5'-GCTGTATCGTCAAGGCACTC-3') as an initiator. The reaction products were separated into two components by reverse-phase high-performance liquid chromatography (RP-HPLC). These products were, after purification, digested with nuclease P1 and alkaline phosphatase followed by separation of digested products by RP-HPLC. The result showed the presence of one of the isomers of 2'-deoxycytidyl-3'-methylphosphonyl-5'-thymidine (dCpCH3T) and 2'-deoxycytidyl-3'-methylphosphonyl-5'-thymidyl-3'-methyl phosphonyl-5'-thymidin e (dCpCH3TpCH3T), respectively. Fast atom bombardment mass spectrometry of these products further supported identification of the dinucleotide and the trinucleotide. These results indicated that dTTP alpha CH3 was used as a substrate of TDTase, resulting in methylphosphonate linkages. Produced oligomers were resistant to hydrolysis by snake venom phosphodiesterase I.

Base Sequence↗

PubMed1989

Molecular cloning and expression of ribosome releasing factor.

Ribosome releasing factor (RRF) is responsible for the release of ribosomes from messenger RNA at the termination of Escherichia coli protein biosynthesis (Hirashima, A., and Kaji, A. (1972) Biochemistry 11, 4037-4044). RRF has been partially analyzed by Edman degradation to obtain its amino acid sequence. Based on this analysis, a 47-nucleotide probe was synthesized and used to screen clones from the Clarke and Carbon Gene Bank which carry sequences within the 0-10 min region of the E. coli gene map. The entire RRF cistron was detected on the plasmid pLC6-32. The DNA sequence of RRF was determined, and it was deduced that RRF consists of 185 amino acids with a calculated molecular weight of 20,639. A rho-independent transcriptional termination sequence was found immediately downstream of the RRF cistron. The RRF gene was subcloned into the vector pUC19, and the resulting plasmid was named pRR1. E. coli harboring this plasmid expressed much more RRF than cells containing pUC19, and it was biologically active.

Amino Acid Sequence↗

PubMed1989

Effect of 2',5'-oligoadenylate on herpes simplex virus-infected cells and preventive action of 2',5'-oligoadenylate on the lethal effect of HSV-2.

Antiviral effects of ppp(A2'p)nA, (2-5A) on herpes simplex virus type 1 or type 2 (HSV-1 or HSV-2)-infected baby hamster kidney fibroblasts (BHK cells) and HSV-2-infected female guinea pigs were examined. 2-5A was introduced into BHK cells in the form of a calcium phosphate precipitate and as an ointment of polyethylene glycol (PEG) into guinea pig vagina. Under optimum conditions, 2-5A trimer and other 2-5A derivatives inhibited over 90% of HSV-1 syncytia formation and over 50% of HSV-2 plaque formation. The growth of uninfected cells was only slightly and transiently inhibited under these conditions. Northern analysis of viral immediate early mRNAs and cellular mRNAs showed that all transcripts determined were reduced in amount by the 2-5A trimer treatment. The reduction in level of viral mRNAs (ICP4, ICP22, and ICP47) by 2-5A trimer was significantly more than that of cellular mRNAs (represented by beta-actin). HSV-2 (strain 186) inoculation into the vagina of female guinea pigs causes severe symptoms in the genital area and high mortality. Topically applied 2-5A trimer almost completely prevented the lethal effect of HSV-2. These data are discussed from the viewpoint of mechanism of interferon action.

Adenine Nucleotides↗