Human beings, nature and medicine: perspectives of internal medicine.
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Aged↗
Biomedical subjects
T Kamada
Publications and source records attributed to T Kamada.
Laser scanning cytometry allows detection of cell death with morphological features of apoptosis in cells stained with PI.
Laser scanning cytometry (LSC), a newly developed technology, allowed simple detection of dead cells with morphological features of apoptosis for cells stained with only propidium iodide (PI). HeLa cells were treated with Adriamycin (ADM, 0.5 or 1.0 microgram/ml). A PI fluorescence value (representing DNA content) versus PI fluorescence peak (representing chromatin condensation) cytogram of LSC made it possible to segregate cells with high PI fluorescence peak from others in a cell population and concomitantly to analyze the relationship between the cells and the cell cycle. A fraction of the cells manifesting hypercondensation of chromatin was exclusively present in a cell population treated with ADM. Visual inspection of the cells defined in the cytogram revealed morphological features of apoptosis. LSC analysis facilitates monitoring effects of anticancer drugs on a cell population, because nuclear DNA staining with PI is simple and rapid.
Antibiotics, Antineoplastic↗
[Human, nature and medicine--future internal medicine].
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Aging↗
Group II phospholipase A2 as an autocrine growth factor mediating interleukin-1 action on mesangial cells.
The proliferation of mesangial cells plays a central role in the progression of glomerulonephritis. We studied the role of group II phospholipase A2 in interleukin-1-stimulated proliferation of mesangial cells. Cultured rat mesangial cells secreted 5.3 units group II phospholipase A2/24 h per 10(5) cells in response to stimulation of 200 U/ml of interleukin-1. Northern hybridization analysis showed that mRNA for group II phospholipase A2 was induced by exogenously added group II phospholipase A2 (15 U/ml) as well as interleukin-1. The pretreatment of quiescent mesangial cells with interleukin-1 augmented [3H]thymidine incorporation caused by platelet derived growth factor. Exogenous group II phospholipase A2 (5-36 U/ml) purified homogeneously from rat spleen also increased [3H]thymidine incorporation by platelet derived growth factor-stimulated mesangial cells in a dose dependent manner (36 U/ml phospholipase A2; 1.9-fold). The stimulatory effect of interleukin-1 on DNA synthesis of mesangial cells was specifically blunted by immunoglobulin raised against group II phospholipase A2. Group II phospholipase A2 (16 U/ml) amplified a platelet derived growth factor-stimulated increase in the mesangial cell number by 1.5-fold. Among the products of the phospholipase A2-catalyzed reaction, lysophospholipids including lysophosphatidylcholine, lysophosphatidylethanolamine and lysophosphatidic acid, but not fatty acids, mimicked the stimulatory effect of interleukin-1 and phospholipase A2. These results suggest that group II phospholipase A2 acts as a signaling molecule that mediates interleukin-1-induced growth of rat mesangial cells through yielding lysophospholipids.
Animals↗
Sample preparation from paraffin-embedded tissue specimens for laser scanning cytometric DNA analysis.
We have developed a simple, rapid method for isolating cells from a block of formalin-fixed, paraffin-embedded tissue specimen for laser scanning cytometric (LSC) DNA analysis by using a grater. The scraping-like tissue samples were obtained by grating a paraffin-embedded tissue block. The grated samples were collected, put into a small plastic tube, and deparaffinized with xylene. Subsequently, the samples were immersed in 100% ethanol to remove the xylene. After using a syringe with a 26-gauge needle and filtering through 40-microm nylon mesh, the cells suspended in ethanol were dropped directly onto a glass slide. As a result, isolated cells adhered tightly to the glass slide. The slides mounted with isolated cells were treated with 0.1% pepsin in 0.1 N HCl for 1 h at 37 degrees C and then 0.1% RNase for 10 min at room temperature. The slides were dipped in propidium iodide (25 microg/ml) to stain DNA and sealed with nail varnish. The coefficients of variation for histograms were small enough to detect an aneuploid peak close to the diploid peak.
Aneuploidy↗
Sensitive, selective gas chromatographic-mass spectrometric analysis with trifluoroacetyl derivatives and a stable isotope for studying tissue sorbitol-producing activity.
One of the major mechanisms involved in diabetic microangiopathy is considered to be an altered polyol pathway. However, clarifying the pathophysiology is difficult due to the lack of a sensitive method for measuring the reduction of glucose to sorbitol in tissue. Here we report a sensitive and selective method for polyol measurement using trifluoroacetyl (TFA) derivatives of polyols and stable isotope-labeled D-sorbitol (U-[13C]sorbitol, 13C6H14O6, 98.7%) as an internal standard. Gas chromatography-mass spectrometry (GC-MS) using an SE-30 capillary column gave elution of TFA derivatives of sugars, polyols and U-[13C]sorbitol within 8 min, with clear separation of sorbitol. In the calibration study, the coefficients of correlation between the amount of sorbitol added and that determined in standard solutions containing 0.1-8.0 nmol sorbitol, erythrocyte mixture and liver cytosol mixture were r = 0.999, r = 0.997 and r = 0.997, respectively. The precision of the GC-MS measurement of standard solution was C.V. = 4.3%. Because glucose is used as a substrate, the method can clarify the polyol pathway under physiological conditions. With this method, Km and Vmax values of the reductase in erythrocytes were 115 +/- 19 mmol/l and 4.42 +/- nmol/min/g of hemoglobin. In human liver, on the other hand, they were 755 +/- 132 mmol/l and 0.773 +/- 0.090 nmol/min/mg of protein, respectively. This difference of Km values suggested that aldehyde reductase rather than aldose reductase is mainly responsible for reducing glucose to sorbitol in the liver. In conclusion, this newly developed method offers a highly sensitive and selective procedure for measuring low concentrations of sorbitol in various tissues and cells and should enable clarification of the kinetics of glucose reduction to sorbitol, which in turn can be used to evaluate the role of an altered polyol pathway in the pathophysiology of diabetic microangiopathy.
Adolescent↗
Glycation-dependent, reactive oxygen species-mediated suppression of the insulin gene promoter activity in HIT cells.
Prolonged poor glycemic control in non-insulin-dependent diabetes mellitus patients often leads to a decline in insulin secretion from pancreatic beta cells, accompanied by a decrease in the insulin content of the cells. As a step toward elucidating the pathophysiological background of the so-called glucose toxicity to pancreatic beta cells, we induced glycation in HIT-T15 cells using a sugar with strong deoxidizing activity, D-ribose, and examined the effects on insulin gene transcription. The results of reporter gene analyses revealed that the insulin gene promoter is more sensitive to glycation than the control beta-actin gene promoter; approximately 50 and 80% of the insulin gene promoter activity was lost when the cells were kept for 3 d in the presence of 40 and 60 mM D-ribose, respectively. In agreement with this, decrease in the insulin mRNA and insulin content was observed in the glycation-induced cells. Also, gel mobility shift analyses using specific antiserum revealed decrease in the DNA-binding activity of an insulin gene transcription factor, PDX-1/IPF1/STF-1. These effects of D-ribose seemed almost irreversible but could be prevented by addition of 1 mM aminoguanidine or 10 mM N-acetylcysteine, thus suggesting that glycation and reactive oxygen species, generated through the glycation reaction, serve as mediators of the phenomena. These observations suggest that protein glycation in pancreatic beta cells, which occurs in vivo under chronic hyperglycemia, suppresses insulin gene transcription and thus can explain part of the beta cell glucose toxicity.
Acetylcysteine↗
B7/BB-1 expression and hepatitis activity in liver tissues of patients with chronic hepatitis C.
Cytotoxic T lymphocytes (CTL) are closely related to the mechanism of liver injury in chronic viral hepatitis. Recently, it has been suggested that antigen-specific T cell activation requires both presentation of antigen by major histocompatibility complex (MHC) molecules and the delivery of costimulatory signals. Such signals are provided by B7/BB-1, one of the most important accessory molecules, sufficient for causing antigen-specific MHC-restricted T cell activation. To evaluate the role of B7/BB-1 in chronic hepatitis C, we immunohistochemically studied its expression in liver tissues obtained from 61 patients with hepatitis C virus (HCV) infection and compared them based on hepatitis activity. In HCV-infected liver, B7/BB-1 was strongly expressed in the cytoplasm of hepatocytes. B7/BB-1-positive cells accompanied liver-infiltrating lymphocytes and were mainly detected in the periportal region. B7/BB-1 expression was closely correlated with the activity of viral hepatitis as evaluated from scores of periportal or intralobular inflammation and necrosis, or serum alanine transferase (ALT) levels. Further study by immunostaining with anti-HCV core and anti-human leukocyte antigen (HLA) class I antibody showed B7/BB-1 positive cells near HCV core antigen- and HLA class I-positive cells, with B7/BB-1-positive cells mostly included among HLA class I-positive cells. These findings suggested that B7/BB-1 expression by hepatocytes may be induced by HCV infection and may trigger generation and activation of CTL, which may cause damage to HCV-infected HLA class I-expressing hepatocytes.
Adult↗
Binding cells of 125I-iodoamphetamine in rat liver.
We recently reported that transrectal or intestinal portal scintigraphy with 123I-iodoamphetamine (IMP) could be a useful method for the non-invasive and quantitative evaluation of the portosystemic shunt in portal hypertension, but what cells in the liver trap IMP has not been clarified. This study was aimed at elucidating whether IMP was extracted by parenchymal cells, sinusoidal endothelial cells, Kupffer cells or fat storing cells. Each type of liver cell was isolated from rats and cultured. The cells were incubated with 125I-IMP and the radioactivity of the lysate was determined. Nonspecific binding was assessed in the presence of an excess of unlabeled IMP, and specific binding was determined by subtracting the nonspecific from total binding. Specific binding observed in parenchymal cells, endothelial cells and Kupffer cells was 70.2 +/- 0.4, 4.2 +/- 1.4 and 2.3 +/- 0.8 pmol/well, respectively, but no specific binding was observed in fat storing cells. The binding in parenchymal cells was much higher than that in endothelial cells or Kupffer cells (p < 0.005). In addition, the binding to parenchymal cells reached equilibrium within 20 min and was not saturable over the concentration range tested (0.5-10 microM). These findings indicate that IMP is mostly extracted by parenchymal cells in the liver.
Amphetamines↗
A temperature-sensitive mutation of Coprinus cinereus, hyt1-1, that causes swelling of hyphal tips.
The TU25 mutant strain of the basidiomycete Coprinus cinereus grows well at 28 degrees C but not at 37 degrees C. Microscopic examination revealed that TU25 exhibited swelling at hyphal apices after a shift-up from 28 degrees C to 37 degrees C. The temperature sensitivity and hyphal swelling co-segregated through meiosis as expected for a single Mendelian factor, designated hyt1 (hyphal tip). Both defects could be suppressed by the presence of osmotic stabilizers in the medium, suggesting that the hyt1 gene product is required for proper cell-wall function. Chemical analysis of the cell walls, however, failed to detect any clear difference in the composition of the cell-wall polysaccharides between the wild-type and TU25. A DNA fragment which complements the hyt1-1 mutation was cloned and sequenced. The predicted protein in the ORF essential for complementation of hyt1-1 is a novel protein.
Amino Acid Sequence↗
Disease stage of chronic hepatitis C assessed by both peritoneoscopic and histologic findings and its relationship with response to interferon therapy.
BACKGROUND: Disease stage in patients with chronic hepatitis C was assessed by both peritoneoscopy and histology and correlated with responses to interferon therapy. METHODS: The subjects were 105 patients with chronic hepatitis C treated with interferon who were classified into 28 sustained responders, 34 transient responders, and 43 nonresponders according to alanine aminotransferase normalization. The influence of various patient's characteristics on responses to interferon therapy was investigated by multivariate analysis. RESULTS: Patients were categorized into 21 patients who exhibited a "smooth liver" on peritoneoscopy and did not demonstrate histologic bridging fibrosis (group I) and 84 patients who exhibited a "granular" or "nodular liver" on peritoneoscopy and/or had histologic bridging fibrosis (group II). Multivariate analysis showed that genotype 2a/2b (p = .0002), low viremia (p = .0048), and early disease stage (group I) (p = .0290) were significant independent factors contributing to sustained response, and that early disease stage (group I) (p = .0010) and genotype 2a/2b (p = .0085) were those contributing to sustained or transient response. Neither peritoneoscopic nor histologic findings alone were a significant factor influencing responses to interferon therapy. CONCLUSION: Disease stage assessed by both peritoneoscopic and histologic findings may serve as a reliable marker for predicting responses to interferon therapy in chronic hepatitis C.
Adult↗
Circulating matrix metalloproteinase-2 and tissue inhibitor of metalloproteinase-1 as serum markers of fibrosis in patients with chronic hepatitis C. Relationship to interferon response.
BACKGROUND/AIMS/METHODS: The imbalance between matrix metalloproteinases (MMPs) and tissue inhibitors of matrix metalloproteinases (TIMPs) is considered to be an important determinant of extracellular matrix deposition and breakdown. We measured serum MMP-1, MMP-2, TIMP-1 and TIMP-2 levels using the respective one-step sandwich enzyme immunoassays in 98 patients with chronic hepatitis C treated with interferon beta to examine their clinical significance for assessment of liver histology and to determine whether they can be useful as predictors of the interferon response. RESULTS: Serum TIMP-1 levels showed a positive correlation with the degree of fibrosis (r(s)=0.30, p= 0.004). Serum MMP-2 levels revealed positive relationships with the degree of periportal necrosis (r(s)= 0.32, p=0.002), the degree of fibrosis (r(s)=0.26, p= 0.01) and total score of histological activity index (r(s)=0.24, p=0.02). Serum MMP-2 levels were significantly higher in patients with no response than in those with sustained and transient response (p<0.01 and p<0.05, respectively), while serum MMP-1 levels did not differ among the three groups. Compared with the levels in sustained responders, the total amounts of serum TIMP-1 were significantly lower in transient responders and non-responders (p<0.01 and p<0.001, respectively). As for serum TIMP-2 levels, a significant decrease was found in transient responders and non-responders (p<0.01). The ratios of serum MMP-2 to TIMP-1 levels were significantly higher in transient responders and non-responders than in sustained responders (p<0.001, respectively) even when HCV RNA levels were low in patients with HCV genome subtype 1b or when the HCV genome subtype was 2a or 2b. Sustained response was never found in type 1b patients with ratios of serum MMP-2 to TIMP-1 levels of over 6.0. In logistic multivariate regression analysis, the ratios of serum MMP-2 to TIMP-1 level (p=0.0001), HCV genome subtype (p=0.005) and serum TIMP-2 level (p=0.03) were the independent predictors for sustained response, while serum MMP-2 level (p=0.0006) was the only predictor for no response. CONCLUSIONS: Serum MMP-2 and TIMP-1 levels might be useful for estimating the degree of liver fibrosis. The ratio of serum MMP-2 to TIMP-1 levels may serve as a new predictor of interferon response in patients with chronic hepatitis C.
Alanine Transaminase↗
Prediction of the changes in cardiac output in association with preload reduction therapy in patients with hypertensive heart failure.
In treating patients with acute hypertensive heart failure we often try preload-reduction therapy using diuretics or vasodilators, but this may frequently produce a considerable decrease in cardiac output, particularly in hypertensive patients with diastolic dysfunction. Therefore, it is important from the clinical standpoint to predict the change in cardiac output following preload-reduction therapy. The objective of this study was to assess whether the change in cardiac output in association with preload-reduction therapy is predictable before treatment by analysis of mitral flow velocity patterns in patients with hypertensive heart failure. Changes in left ventricular volumes and cardiac output in association with preload-reduction therapy and Doppler echocardiographic parameters of the mitral flow velocity pattern before treatment were studied in 18 patients with hypertensive heart failure. Cardiac output increased in six patients with systolic dysfunction (fractional shortening < 25%). In the other 12 patients (those with normal systolic function), the changes in cardiac output were homogenous among the patients. In this subset there was a greater decrease in cardiac output in patients with a shorter deceleration time. Thus, analysis of deceleration time of the early diastolic filling wave before treatment may be useful in estimating the change in cardiac output following preload reduction in patients with hypertensive heart failure due to diastolic dysfunction.
Adult↗
Plasma and gastric mucosal endothelin-1 concentrations in patients with peptic ulcer.
Peptic ulcer in the human stomach causes localized destruction of the gastric wall, which may be associated with focal vascular insufficiency. Endothelin-1, an extremely potent vasoconstrictor peptide, modulates regional blood flow in the vasculature of stomach, suggesting a role for endothelin-1 in peptic ulcer. We examined the relationship among endogenous plasma and mucosal endothelin-1 concentrations and the severity and area of ulcer in 19 patients with gastric ulcers and eight healthy adults. Endothelin-1 concentrations were measured by enzyme immunoassay in plasma and gastric mucosal specimens from ulcer margins, corpus, and antrum. The severity and area of ulcer were assessed endoscopically. Plasma endothelin-1 concentrations in active (P < 0.01 compared with normal) and healing (P < 0.05) stages of ulcer were significantly greater than those in normal subjects. Plasma endothelin-1 concentrations, but not mucosal endothelin-1 concentrations in the ulcer margin, were significantly associated with the severity of the ulcer. There was a significant positive correlation between plasma endothelin-1 concentrations and area of ulcer (r = 0.70, P < 0.01). In conclusion, locally increased endothelin-1 may be an important mediator contributing to the pathogenesis of peptic ulcer.
Adult↗
Activation of ecto-5'-nucleotidase by protein kinase C and its role in ischaemic tolerance in the canine heart.
1. Ischaemic preconditioning (IP) protects the myocardium against irreversible ischaemic injury by activating protein kinase C (PKC). The mechanism by which PKC protects the myocardium is unknown. We have shown that PKC increases the activity of ecto-5'-nucleotidase (ecto-5'-N) and thereby the production of adenosine in cardiomyocytes which may protect the myocardium against ischaemia-reperfusion injury in vivo. 2. The objective of this study was to elucidate the possible role of PKC-induced activation of ecto-5'-N in the cardioprotection associated with IP in the canine heart. 3. IP increased the activities of both ecto-5'-N and PKC, and minimized ischaemic damage (infarct size: 7.5 +/- 1.8 vs. 42.3 +/- 2.8%, P < 0.01 vs. the control group). Treatment with the PKC activator (4 beta-phorbol 12-myristate-13-acetate) also reduced infarct size (13.5 +/- 2.9%, P < 0.01 vs. the control group). 8-Sulfophenyltheophylline (an antagonist of adenosine receptors) or alpha,beta-methyleneadenosine 5'-diphosphate (an inhibitor of ecto-5'-N) eliminated the cardioprotective effect of the PKC activator (infarct size: 36.6 +/- 3.9 and 34.7 +/- 4.2%, respectively), suggesting that PMA limits infarct size by increasing the activity of ecto-5'-N and the adenosine level. 4. The PMA-induced cardioprotection was blunted by GF109203X (an inhibitor of PKC, infarct size: 36.2 +/- 3.1%), but not by pretreatment with dexamethasone (infarct size, 14.2 +/- 2.6%). 5. We conclude that the PMA- and IP-induced cardioprotection is attributable to phosphorylation and activation of ecto-5'-N.
5'-Nucleotidase↗
Relation of disease activity during chronic hepatitis C infection to complexity of hypervariable region 1 quasispecies.
We studied the heterogeneity in the E2/NS1 hypervariable region 1 of the hepatitis C virus (HCV) genome in relation to the natural course after infection. The subjects were composed of 38 chronic hepatitis C carriers who had been followed for 9 to 218 months after the onset of non-A, non-B (type C) hepatitis, being tested monthly for serum alanine aminotransferase levels. The complexity of the sequence heterogeneity was assessed by single-strand conformation polymorphism analysis. The quasispecies complexity had no relation to the route of infection, the time from infection and the duration of aminotransferase elevation after the onset. However, it had a significant relationship with the degree of aminotransferase elevation in the course of the disease. The quasispecies complexity was directly correlated with the first peak of serum aminotransferase at the onset (r = .48, P < .01) and the mean aminotransferase levels during the period of persistent aminotransferase elevation (r = .58, P < .01). Twenty-three of the 38 patients were further followed for 24 months with biweekly alanine transaminase (ALT) tests. Their aminotransferase levels remained within the normal range during follow-up, and no significant change was seen in the quasispecies complexity after this asymptomatic period. However among the 23 patients, the quasispecies complexity increased in six cases (26%) and decreased in five (22%). A significant direct relation was seen between changes in the quasispecies complexity and the mean aminotransferase levels during the asymptomatic period (r = .55, P = .01). These findings suggest that the development of the HCV quasispecies nature may be related to the severity of the hepatitis in the course of infection.
Adult↗
Reduced incidence of Helicobacter pylori infection in young Japanese persons between the 1970s and the 1990s.
Although the incidence of gastric carcinoma in Japan has declined in the past several decades, there is no information about trends in helicobacter pylori infection. The purpose of the study was to clarify trends in H. pylori infection and in histologic gastritis in young Japanese subjects evaluated between 1975 and 1978 (the 1970s) versus 1991 and 1994 (the 1990s). The prevalence of H. pylori infection and the grade of gastritis in biopsy specimens were evaluated in 173 young Japanese without localized lesions in the upper gastrointestinal tract. Findings in 75 specimens obtained from subjects in the 1970s were compared with those of 98 specimens obtained from subjects in the 1990s. The prevalence of H. pylori infection in the 1970s (54.7%) was significantly higher than that in the 1990s (28.6%) (p < 0.01). The grade of inflammation and the prevalence of both mucosal atrophy and intestinal metaplasia also were significantly higher in the 1970s than in the 1990s (p < 0.01 and p < 0.05). Our data thus show a trend toward a reduced prevalence of H. pylori infection and of histologically apparent gastritis (especially atrophic gastritis). This decrease may be linked to the reported decline of gastric carcinoma in Japan.
Adolescent↗
Relationship between interleukin-8 levels and myeloperoxidase activity in human gastric mucosa.
Although interleukin (IL)-8 is well known as a chemotactic agent for neutrophil migration in vitro, the relationship between IL-8 activity and the degree of neutrophil infiltration in gastric mucosa is still unclear. In the present study, we investigated IL-8 and myeloperoxidase activity, a marker of neutrophil infiltration, in gastric antral mucosa using biopsy samples in 23 patients with no gastric lesions. The results indicate that there is a good correlation between IL-8 and myeloperoxidase activity (y = 0.173x + 13.9; r = 0.49, P < 0.01). Furthermore, IL-8 and myeloperoxidase activity are significantly higher in Helicobacter pylori-positive patients than in H. pylori-negative patients. In conclusion, an increase of IL-8 activity in the gastric mucosa causes increased neutrophil infiltration in human gastric mucosa and H. pylori infection accelerates these reactions in the mucosa.
Aged↗