Effects of endothelin (ET) family peptides and their derivatives on cellular contraction and calcium-ion level were examined by using cultured human vascular smooth muscle cells (VSM). Contraction of cultured human VSM, isolated from human fetal aortic segments, was induced within 1 min after the treatment with ET-1 (100 nM) as seen in the changes of cytosolic calcium-ion localization. In parallel with the cell contraction, cytosolic calcium-ion level in the human VSM increased very rapidly and then dropped with some oscillation as determined by Anchorage Cell Analyzing System. It was noted that transient calcium-ion mobilization rather than sustained calcium-ion influx was significant in the contraction of cultured human VSM. Vasoactive intestinal contractor (VIC), three amino acids different from ET-1, had less activity in increase of intracellular calcium-ion level and in percent of response cells than ET-1, ET-2, and VIC-S4L6 (one amino acid different from ET-1). EC50 of ET-1, VIC-S4L6, ET-2, and VIC were 0.5 nM, 0.6 nM, 2.0 nM, and 20 nM, respectively. VIC-like peptide (VIC-LP), 16 amino acids fragment of VIC precursor protein, had no effect with a single administration of up to 10 microM. However, the increase in calcium-ion level by VIC was suppressed with a prior treatment of cells with high concentration (10 microM) of VIC-LP. The establishment of cultured human VSM for the simultaneous examination of the contraction and calcium-ion level will provide a new system to study signal transduction of vasocontractor peptides.

Amino Acid Sequence↗

PubMedDate not supplied

Alteration in cellular RNAs during the in vitro lifespan of cultured human diploid fibroblasts. II. Synthesis and processing of RNA.

Incorporation of tritiated uridine into cellular RNA is decreased in senescent human fibroblast cultures when measured per cellular RNA content. Since early and late passage cells demonstrate similar kinetics of RNA precursor pool labeling and saturation, this decreased tritiated uridine incorporation reflects decreased RNA synthesis. However, cellular RNA contents are markedly elevated in senescent cell cultures. When data are measured per cell number or DNA content, net RNA synthesis is slightly increased in senescent cultures. Separation of labeled RNAs by polyacrylamide gel electrophoresis revealed the same diminished specific activities (CPM/RNA) and increased net synthesis (CPM/cell number) of major ribosomal and transfer RNAs. However, this slight increase in RNA synthesis is probably not of sufficient magnitude to completely explain the observed increase in cellular RNA content in senescent cells.

Aging↗

PubMedDate not supplied

Relationship between cell replication and volume in senescent human diploid fibroblasts.

Since alterations in cell replication rate and cell volume distribution are two of the earliest changes seen in the culture of human diploid cells, it was decided to examine the relationship between these parameters. After standardization of the conditions for cell volume measurements (enzyme treatment, temperature and stage of cell growth), a close correlation was observed between cell population doubling time and cell volume in WI-38 cells at various levels of in vitro passage. Cell populations which replicate more slowly (trisomic-21 fibroblasts, fetal skin fibroblasts) also demonstrated a shift to larger cell volumes when compared with control rapidly replicating cell populations at the same level of in vitro passage. Similar shifts to larger cell volumes were produced by reducing serum concentration, decreasing incubation temperature and inhibiting DNA synthesis. Separation of senescent WI-38 cells on the basis of cell volume revealed that the cell fractions with the largest modal cell volume contained the highest percentage of slow or nonreplicating cells. Therefore, an inverse relationship appears to exist between growth rate and cell volume in cultured human diploid fibroblasts.

Cell Count↗

PubMedDate not supplied

Changes in DNA polymerases alpha, beta and gamma in mouse liver as a function of age.

The activities of DNA polymerases alpha, beta and gamma were determined in mouse liver as a function of age by a combination of glycerol density gradient centrifugation with polymerase specific assays. Although alpha polymerase was preserved throughout the life span, the activity dropped sharply from a high level at the fetal and neonatal stages to a level one order lower after maturation through adjustment of the amount of protein administered. beta polymerase showed similar but less drastic changes than alpha. DNA polymerase gamma activity increased about two-fold in going from newborn to adult stages and remained constant after maturation. According to the amount of DNA, DNA polymerase alpha decreased after birth, but the change was less drastic compared to that through adjustment of the amount of protein. DNA polymerase beta increased the activity 2-3-fold within a period of 3 months following birth. gamma polymerase underwent more than a 10-fold increase in activity through adjustment of the amount of DNA within the same period.

Aging↗

PubMedDate not supplied

Induction of host DNA synthesis in senescent human diploid fibroblasts by infection with human cytomegalovirus.

A human diploid fibroblast strain, TIG -1, ceased to proliferate at about 60-62 population doubling level. The percentage of nuclei incorporating [3H]thymidine during 24-h culture in fresh medium containing 10% fetal bovine serum was less than 2% in the senescent cells used in this study. Infection of these cells with human cytomegalovirus (HCMV), strain AD-169, increased the percentage of [3H]thymidine-labeled cells by about ten-fold. Equilibrium density gradient centrifugation analysis of purified DNA from infected cells showed that cellular DNA synthesis was stimulated preceded by the viral DNA synthesis. Ultraviolet irradiation of HCMV reduced the ability to induce DNA synthesis. Equilibrium density gradient centrifugation analysis of DNA which was labeled with 5-bromodeoxyuridine indicated semiconservative replication rather than repair synthesis. These results suggested that in a considerable fraction of human senescent cells host DNA replication could be reinitiated by infection with HCMV, but not by the addition of fetal bovine serum.

Autoradiography↗

PubMedDate not supplied

Effects of multiple dosing of phenacetin in the micronucleus test.

As a part of the international cooperative study to identify the most sensitive regimen in the micronucleus test, phenacetin was given i.p. to male CD-1 mice at doses of 37.5, 75, 150, 300, 400, and 600 mg/kg once, twice, thrice or four times and the bone marrow cells were harvested 24 h after the final dosing. Positive responses were seen at 600 mg/kg after single and triple dosing and at 400 and 600 mg/kg after double dosing. No dose level gave a positive response after quadruple dosing. A repeated-dosing effect was detected at double and triple dosing. Although triple dosing gave the highest magnitude of micronuclei at 600 mg/kg, double dosing showed a sufficient sensitivity and was more convenient from the viewpoint of selecting a suitable test dose and carrying out the micronucleus test.

Animals↗

PubMedDate not supplied

Presynaptic effects of botulinum toxin type A on the neuronally evoked response of albino and pigmented rabbit iris sphincter and dilator muscles.

PURPOSE: To investigate the effects of botulinum toxin type A (botulinum A toxin) on the autonomic and other nonadrenergic, noncholinergic nerve terminals. METHODS: The effects of botulinum A toxin on twitch contractions evoked by electrical field stimulation (EFS) were studied in isolated albino and pigmented rabbit iris sphincter and dilator muscles using the isometric tension recording method. RESULTS: Botulinum A toxin inhibited the fast cholinergic and slow substance P-ergic component of the contraction evoked by EFS in the rabbit iris sphincter muscle without affecting the response to carbachol and substance P. These inhibitory effects were more marked in the albino rabbit than in the pigmented rabbit. Botulinum A toxin (150 nmol/L) did not affect the twitch contraction evoked by EFS in the rabbit iris dilator muscle. CONCLUSIONS: These data indicated that botulinum A toxin may inhibit not only the acetylcholine release in the cholinergic nerve terminals, but also substance P release from the trigeminal nerve terminals of the rabbit iris sphincter muscle. However, the neurotoxin has little effect on the adrenergic nerve terminals of the rabbit iris dilator muscle. Furthermore, the botulinum A toxin binding to the pigment melanin appears to influence the response quantitatively in the two types of irides.

Albinism↗

PubMedDate not supplied

Inflammatory cytokines following diethylcarbamazine (DEC) treatment of different clinical groups in lymphatic filariasis.

In an earlier study in Indonesia we reported on adverse reactions to diethylcarbamazine (DEC) in brugian filariasis patients identified as microfilaraemics (n = 26), endemic normals (n = 11) and elephantiasis patients (n = 17). To assess the link between adverse reactions and cytokines we have now analysed an array of inflammatory mediators in plasma samples collected during the same study. Pre-treatment levels of interleukin (IL)-6 and soluble tumour necrosis factor receptor 75 (sTNF-R75) were higher in elephantiasis patients compared to microfilaraemics and endemic normals, indicating the presence of an ongoing inflammation in patients with chronic disease. After initiation of treatment, the levels of IL-6 and LPS-binding protein (LBP) were consistently and significantly higher in microfilaraemics who suffered most from adverse reactions compared with endemic normals and elephantiasis patients. In microfilaraemics the levels of sTNF-R75 increased after treatment to reach levels recorded in elephantiasis patients. IL-6 increased early, concurrent with the development of adverse reactions and peaked by 24 h post treatment. The levels of LBP and sTNF-R75 in microfilaraemics also increased to peak, later than IL-6, at 32 h post DEC therapy. Although changes were recorded in IL-8 and IL-10 levels in some individuals, no significant differences were found between the 3 clinical groups. These results demonstrate that intake of DEC leads to an increase in a selected number of inflammatory mediators in the group of filarial patients who suffer most from adverse systemic reactions.

Acute-Phase Proteins↗

PubMedDate not supplied

Adverse reactions following diethylcarbamazine (DEC) intake in 'endemic normals', microfilaraemics and elephantiasis patients.

This paper reports on adverse reactions following a 12-day course of 6 mg/kg diethylcarbamazine (DEC) therapy in brugian filariasis patients in Indonesia. Microfilaria-positive individuals (n = 26), 'endemic normals' (n = 12) and elephantiasis patients (n = 17) were included in the study. Fever, headache and body aches started between 2 and 24 h after DEC intake. Adverse reactions were categorized into 'no or mild', 'moderate' or 'severe' depending on the total reaction score. Four microfilaraemic individuals (15.4%) suffered from severe adverse reactions and their pre-treatment microfilarial levels (geometric mean, GM = 3060 mf/10 mL) were significantly higher than in the 5 microfilaraemic individuals (19.2%) suffering from moderate reactions (GM = 1268 mf/10 mL) and in the 17 microfilaraemic patients (65.4%) who experienced no or mild reactions (GM = 6 mf/10 mL)(P < 0.001 and P < 0.001, respectively). Endemic normals showed no or mild adverse reactions. No or mild adverse reactions were also recorded in all but 2 elephantiasis patients after DEC intake. Two elephantiasis patients with moderate reactions had high levels of circulating microfilariae at pre-treatment (2097 and 7375 mf/10 mL). Concentrations of DEC were measured in plasma, but could not explain the differences in the severity of adverse reactions.

Adolescent↗

PubMedDate not supplied

Expression of endothelin, fibronectin, and mortalin as aging and mortality markers.

Studies on fibronectin, endothelin-1, and mortalin from our laboratory are reviewed here. Fibronectin expression has been analyzed as upregulated during in vitro serial passaging of human fetal lung and neonatal foreskin fibroblasts as well as umbilical vein endothelial cells. In vivo aging of skin fibroblasts, as well as aortic endothelial cells, are also accompanied by upregulation of fibronectin expression. Fibronectin promoter binding proteins from young and old cell nuclear extracts were further explored by gel retardation assay. Preliminary analyses have detected age-related differential binding activities with respect to AP-1, CRES, TFIID, CTF, and AP-2 regions, whereas Sp1 binding proteins remain unaltered. Endothelin-1 expression is also seen as upregulated during in vitro and in vivo aging of endothelial cells. This can contribute to the hypertension commonly observed in elderly patients. Mortalin, a novel member of hsp 70 family of proteins, was initially identified by virtue of its association with a cellular mortal phenotype. Subsequently, normal cells and the ones with an immortal phenotype have been found to have differential subcellular localization of this protein. Antiproliferative activity of this protein in normal cells and the deregulation of expression in transformed cells is observed which suggests the association of mortalin in pathways that determine cellular divisional phenotype.

Aging↗

PubMedDate not supplied

An N-terminal region of mot-2 binds to p53 in vitro.

The mouse mot-2 protein was earlier shown to bind to the tumor suppressor protein, p53. The mot-2 binding site of p53 was mapped to C-terminal amino acid residues 312-352, which includes the cytoplasmic sequestration domain. In the present study, we have found that both mot-1 and mot-2 bind to p53 in vitro. By using His-tagged deletion mutant proteins, the p53-binding domain of mot-2 was mapped to its N-terminal amino acid residues 253-282, which are identical in mot-1 and mot-2 proteins. Some peptides containing the p53-binding region of mot-2 were able to compete with the full-length protein for p53 binding. The data provided rationale for in vitro binding of mot-1 and mot-2 proteins to p53 and supported the conclusion that inability of mot-1 protein to bind p53 in vivo depends on secondary structure or its binding to other cellular factors. Most interestingly, the p53-binding region of mot-2 was common to its MKT-077, a cationic dye that exhibits antitumor activity, binding region. Therefore it is most likely that MKT-077-induced nuclear translocation and restoration of wild-type p53 function in transformed cells takes place by a competitional mechanism.

Amino Acids↗

PubMedDate not supplied

CT appearance of hepatic parenchymal changes after percutaneous microwave coagulation therapy for hepatocellular carcinoma.

PURPOSE: The purpose of this study was to evaluate the changes in the CT appearance of the hepatic parenchyma surrounding the necrotic area in the early period after percutaneous microwave coagulation therapy (PMCT) for hepatocellular carcinoma (HCC). METHOD: We reviewed enhanced CT scans obtained before and within 2 weeks, at 1 month, and at 3 months after PMCT of 61 lesions in 47 patients with HCC. RESULTS: On dynamic CT, early enhancement of the hepatic parenchyma around the treated area was a frequent finding within 1 (87%) or 2 (68%) weeks after PMCT, but such enhancement disappeared on follow-up. Arterioportal shunts were also demonstrated by enhanced CT after treatment (21% at < or =2 weeks), and these shunts tended to persist for >1 month. CONCLUSION: We should evaluate the effect of PMCT by performing dynamic enhanced CT not only within 2 weeks to determine the end-point of treatment but also at 1 month or more after finishing treatment for definite assessment of tumor necrosis.

Adult↗