Yoshikazu Mimura

Telomerase activity and telomere length in benign and malignant human thyroid tissues

Several studies have demonstrated that telomerase is activated and telomere length is altered in various types of tumors. In this study, we investigated telomerase activities and telomere length in 21 thyroid tumors. Telomerase activity was detected in 11 of 12 thyroid cancers and three of nine follicular adenomas. The mean telomere lengths in the cancers tissue and follicular adenomas were lower than in the respective background tissues, the differences being significant (P=0.0055 and P<0.006), respectively. Our findings suggest that change in telomerase activity and telomere length may be important for development of thyroid tumors.

SIMULTANEOUS ONSET OF ACUTE INFLAMMATORY RESPONSE, SEPSIS-LIKE SYMPTOMS AND INTESTINAL MUCOSAL INJURY AFTER CANCER CHEMOTHERAPY

Chemotherapy is 1 method for the treatment of cancer, but serious side effects can sometimes limit the dosage given. Mild fever and diarrhea are common side effects of cancer chemotherapy. Gastrointestinal injury induced by chemotherapeutic agents may result in bacterial/endotoxin translocation from the gut into the systemic circulation. An experimental study was therefore conducted to clarify the effect of systemic chemotherapeutic agents on gastrointestinal barrier function. Male Wistar rats were divided into a 5‐fluorouracil (5‐FU) group (100 mg/kg/day for 4 days; n = 27) and a control group (n = 5). All rats were fasted and central venous catheterization was performed for total parenteral nutrition and blood sampling. Intestinal tissue was also sampled for pathological examination. Plasma levels of interleukin‐6 (IL‐6) and tumor necrosis factor α (TNFα) were determined by ELISA, bacterial translocation was quantified by lymph node culture and plasma endotoxin content of portal blood was measured by the Limulus‐amebocyte‐lysate test. In the 5‐FU group on day 4, a proportion of rats exhibited severe watery diarrhea (73.9%) and occasional vomiting (86.2%). The levels of plasma TNFα and IL‐6 were seen to increase, peaking at day 6 (IL‐6, 350.0 ± 67.8 pg/ml; TNFα, 26.1 ± 3.2 pg/ml). The pathological findings also changed on day 4. On day 6, 90% of the rats in the 5‐FU group showed dramatic sepsis‐like manifestations, whereas the control group did not. Within the 5‐FU group, only at day 6 was bacterial translocation in the rat mesenteric lymph nodes or significantly elevated levels of endotoxin evident. These results suggest that bacterial/endotoxin translocation might cause sepsis‐like manifestations after systemic chemotherapy.

Prolonged Toll-like receptor stimulation leads to down-regulation of IRAK-4 protein

Interleukin‐1 receptor‐associated kinase (IRAK)‐4 is a key mediator in the Toll‐like receptor (TLR) signaling. We found that stimulation of TLR2, TLR4, or TLR9, but not TLR3, caused a decrease in IRAK‐4 protein without affecting its mRNA level in a mouse macrophage cell line, RAW 264. The decrease in IRAK‐4 was accompanied by the appearance of a smaller molecular weight protein (32 kD), which was recognized by an anti‐IRAK‐4 antibody raised against the C‐terminal region. The decrease in IRAK‐4 and the appearance of the 32‐kD protein occurred with slower kinetics than the activation of IRAK‐1 and were suppressed by inhibitors of the proteasome, inducible inhibitor of κBα phosphorylation or protein synthesis, but not by caspase inhibitors. These results indicate that prolonged stimulation of TLR2, TLR4, or TLR9 causes a down‐regulation of IRAK‐4 protein, which may be mediated through cleavage of IRAK‐4 by a protease induced by the activation of nuclear factor‐κB.

Antioxidant Effect of Zinc on Acute Renal Failure Induced by Ischemia-Reperfusion Injury in Rats

Zinc may have an antioxidant effect mediated by induction of metallothionein. Based on the assumption that metallothionein can scavenge oxygen free radicals, we examined whether zinc administration prior to renal ischemia would improve renal dysfunction caused by ischemia-reperfusion injury in rats. Wistar rats weighing 265 g were treated with an intraperitoneal injection of 20 mg/kg zinc 24 h prior to the renal ischemia-reperfusion procedure, which was achieved by a 30-min clamping of the bilateral renal vessels and subsequent 90-min reperfusion. Thirty-minute renal clearance tests were performed before and after renal ischemia in zinc- (n = 11) and saline-treated (n = 8) rats. Thiobarbituric acid reactive substance, conjugated diene, and metallothionein levels in the renal tissues were also determined. Sham-operated rats (n = 5 in each treatment) served as control for the ischemia-reperfusion rats. Ischemia-reperfusion resulted in significantly lower glomerular filtration rate values and marked increases in tissue concentrations of thiobarbituric acid reactive substance and conjugated diene compared with sham-operation. Zinc administration improved the reduced glomerular filtration rate values seen after the ischemia-reperfusion procedure, but not to the extent of pre-ischemic levels. Zinc pretreatment significantly reduced the increased levels of thiobarbituric acid reactive substance and conjugated diene during ischemia-reperfusion and increased metallothionein levels compared with saline injection. These findings suggest that zinc has an antioxidant effect mediated through the induction of metallothionein, but appears only to have a minor protective effect on renal function induced by renal ischemia-reperfusion injury.

Ischaemic preconditioning ameliorates functional disturbance and impaired renal perfusion in rat ischaemia-reperfused kidneys.

1. The effects of ischaemic preconditioning (IP) on renal function, haemodynamics and lipid peroxidation in the rat ischaemia–reperfused kidney model were examined.

Caspase-3 is activated and rapidly released from human umbilical vein endothelial cells in response to lipopolysaccharide

Endothelial cell injury/dysfunction is considered to play a critical role in the pathogenesis of severe sepsis and septic shock. Although it is considered that endothelial cell apoptosis is involved in endothelial injury/dysfunction, physiological involvement remains ambiguous since the induction of apoptosis requires the inhibition of endogenous apoptosis inhibitors. Here we show that caspase-3 activation, a biological indicator of apoptosis, is observed in response to lipopolysaccharide (LPS) stimulation even under the influence of endogenous apoptosis inhibitors, and that activated caspase-3 is rapidly released from human umbilical vein endothelial cells (HUVEC). In the presence of cycloheximide (CHX), an increase in intracellular caspase-3/7 activity in response to LPS was not detected in HUVEC up to 24 h following stimulation even in the presence of LPS-binding protein (LBP), soluble CD14 and soluble MD-2, whereas the decrease in cell viability and increase in release of the cellular enzyme lactate dehydrogenase (LDH) were observed in a soluble CD14/LBP-dependent manner. On the other hand, even in the absence of CHX, a significant increase in caspase-3/7 activity and a cleaved caspase-3 fragment with a slight increase in LDH release was observed in culture supernatants in response to LPS. This increase in caspase-3/7 activity was observed even when LDH release was undetected. These results indicate that caspase-3 is activated by LPS under physiological conditions and suggest that HUVEC escape from cell death by rapidly releasing activated caspase-3 into extracellular space. Failure of this escape mechanism may result in endothelial injury/dysfunction.

Telomere shortening with aging in human thyroid and parathyroid tissue.

Progressive telomere shortening with aging was studied using normal thyroid tissue specimens from 46 human subjects aged between 0 and 98 yr and normal parathyroid tissue specimens from 21 human subjects aged between 0 and 83 yrs. There has hitherto been no information documented about telomere length in such thyroid and parathyroid tissues. Age-related shortening at rates of 91 and 92 base pairs (bp) per year, respectively, were observed. Telomere lengths of normal thyroid tissues were 16.53 +/- 1.10 (mean +/- SE), 14.31 +/- 0.80, 11.27 +/- 0.68 and 8.73 +/- 1.08 kbp for age groups less than 2, 20-50, 51-80 and more than 80 yr. Telomere lengths of normal parathyroid tissues were 15.80 +/- 1.46 (mean +/- SE), 15.36 +/- 0.86 and 10.93 +/- 0.78 kbp for age groups less than 4, 20-50 and 51-80 yr. Telomere shortening occurred after 50 yr of age in thyroid and parathyroid tissues. Human thyroid and parathyroid tissues do not seem to show the rapid reduction in telomere length early in life that was reported for some human cell types, suggesting that the rate of telomere shortening has tissue-specific characteristics.

Consistent Decrease in Telomere Length in Parathyroid Tumors but Alteration in Telomerase Activity Limited to Malignancies: Preliminary Report

AbstractTelomerase is known to be activated and telomere length altered in various types of malignant and benign tumors, but whether this is also the case for parathyroid lesions has hitherto been unclear. We therefore investigated telomerase activity and telomere length in 3 parathyroid metastatic cancers, 6 adenomas, 2 cases of parathyroid hyperplasia, and 16 samples of normal parathyroid tissue. Telomerase activity, assayed by the telomeric repeat amplification protocol, was detected in all of the parathyroid cancers (100%), in none of the 8 parathyroid benign lesions, and in only 1 of the 16 normal parathyroid samples (8.3%). Telomere length, determined by the terminal restriction fragment assay, was reduced in the tumor tissues with a mean telomere length of 8.23 ± 0.86 kbp compared with the 12.61 ± 0.81 kbp for the 16 age-matched subjects (p = 0.002). The results indicate that telomerase activity and telomere length may reflect the biologic behavior of individual parathyroid lesions.

Effect of glutamine on acute lung injury in rats with endotoxemia

We administered endotoxin to rats to produce an acute lung injury model, and assessed the effect on respiratory function when glutamine was added to the solution for total parenteral nutrition. Rats given total parenteral nutrition with glutamine as 33% of the amino acid content were compared with rats given no glutamine. Endotoxin (500 microg/kg) was administered intravenously after 72 h of total parenteral nutrition. The survival rate and the nitrogen balance were significantly improved (P < 0.05) at 48 h after endotoxin administration as a result of glutamine treatment. In addition, the arterial oxygen partial pressure was significantly increased (P < 0.01) and the wet: dry lung weight ratio was decreased (P < 0.05) by glutamine. These results suggested that glutamine improved endotoxin-induced acute lung injury in rats.

Review of 41 patients operated on for primary hyperparathyroidism

We reviewed 41 cases of operation for primary hyperparathyroidism (PHPT) in our institution between 1987 and 1999. The objective of this study was 1) to evaluate the sensitivity and positive predictive value of several localization studies for an enlarged parathyroid gland; 2) to determine whether a selective. unilateral-exploration operation is safe; and 3) to investigate rates of coexisting malignancies of other organs. A total of 61 enlarged parathyroid glands (701 +/- 131 mg wt) were removed, and the lesions consisted of 32 adenomas, two cancers, and seven hyperplasias. MIBI scintigraphy had both a high sensitivity (88.9%) and positive predictive value (88.9%) for localization of abnormal parathyroid glands and yielded better performance than the other techniques, including ultrasonography, CT scanning, and Tl-Tc scintigraphy. However, all of the localization techniques failed to detect enlarged glands (18/32 glands = 62.5%) in patients with multi-glandular parathyroid lesions. Initial operations with selective unilateral exploration of the neck were successful in 23 of 24 patients (95.8%). Operative failure was due to missing the second adenoma of a double adenoma. Malignant tumors were found in 11 patients (26.8%) previously treated or concurrently managed at the time of parathyroidectomy. There was a significant increase in serum-intact PTH level in patients with concurrent malignant tumors compared to patients who had no association of malignancies. In conclusion, 1) at least two preoperative localization tests, an MIBI scan and ultrasonography, are helpful in accurately localizing an abnormal parathyroid gland; 2) selective unilateral exploration is safe and desirable if the second ipsilateral gland is normal macroscopically; and 3) systematic examination for malignant tumors is necessary in PHPT patients before and after parathyroidectomy.