Anatomy-based surgery to remove thyroglossal duct cyst: two anomalous cases.

OBJECTIVE: To review previous reports and to discuss the management of branching polycystic and giant thyroglossal duct cysts. CASE REPORT: We present two cases of thyroglossal duct cyst: one a branching, polycystic thyroglossal duct cyst in an 11-year-old boy, and the other a giant thyroglossal cyst in a 41-year-old man. Such cysts are rare. Both patients were operated upon according to the methods of Sistrunk and Horisawa, and both had a satisfactory post-operative course. DISCUSSION: We discuss the most important aspects of such cyst removal procedures. CONCLUSION: Our experience suggests that surgery to remove an anomalous thyroglossal duct cyst should be performed using a technique based on the anatomy of the hyoid bone region.

Structure-based design of specific cathepsin inhibitors and their application to protection of bone metastases of cancer cells.

We report the antihypercalcemic and antimetastatic effects of CLIK-148 in vivo, which is a specific inhibitor of cathepsin L. The decalcification during bone absorption is followed by the degradation of type-1 collagen by osteoclastic cathepsins. Tumor-bearing osteoclasts or TNF-alpha-activated osteoclasts secrete large amounts of cysteine proteases, especially procathepsin L, which powerfully degrade type-1 collagen leading to tumor-associated bone absorption and release of bone calcium. The bone pit formations in vitro, which are caused by osteoclasts derived from human bone marrow cells activated by RANKL and M-CSF and also by mice osteoclasts activated by TNF-alpha, are significantly prevented by CLIK-148 treatment. We evaluated the in vivo inhibitory effect of malignant hypercalcemia induced by LJC-1 human mandibular cancer inoculation by CLIK-148 treatment, and the CLIK-148 treatment significantly protected against the tumor-induced hypercalcemia. On the protection of bone metastasis of colon 26 PMF-15 implanted to mouse calvaria, CLIK-148 treatment significantly inhibited calvaria bone absorption (direct metastasis). The CLIK-148 treatment also reduced distant bone metastasis to the femur and tibia of melanoma A375 tumors implanted into the left ventricle of the heart.

Assays of methylenetetrahydrofolate reductase and methionine synthase activities by monitoring 5-methyltetrahydrofolate and tetrahydrofolate using high-performance liquid chromatography with fluorescence detection.

We developed a method for assays of methylenetetrahydrofolate reductase and methionine synthase activities by monitoring their products of 5-methyltetrahydrofolate (5-CH(3)-H(4)folate) and tetrahydrofolate (H(4)folate) directly, using high-performance liquid chromatography with fluorescence detection. Folate derivatives and enzymes were stable in the assay process. No reagents in the assay mixture were found to disturb the separation and detection of both H(4)folate and 5-CH(3)-H(4)folate in our assay system. The detection limit of this method was less than 20 nM H(4)folate or 5-CH(3)-H(4)folate in the enzyme assay system. This analytical method, therefore, has a sensitivity high enough to obtain accurate parameters of Michaelis-Menten kinetics and for assays of crude extracts from various biological samples. In addition, the analytical procedure is very simple and economical; it may be a useful tool for studying methylenetetrahydrofolate reductase and methionine synthase activities.

Effect of vitamin B6 deficiency on the synthesis and accumulation of S-adenosylhomocysteine and S-adenosylmethionine in rat tissues.

The effect of vitamin B6-deficiency on the B6-vitamer concentrations, level of S-adenosylhomocysteine (SAH) and S-adenosylmethionine (SAM) were studied in rat tissues. The plasma pyridoxal 5'-phosphate (PLP) and, pyridoxal (PL) levels were lower in the B6-deficient group compared to the control group. After 5 weeks of feeding the experimental diets, tissue PLP, pyridoxamine 5'-phosphate (PMP) and PL concentrations were significantly lower in the B6-deficient group compared to the control and the pair-fed control groups. Thymus PLP and PL levels were lower in the B6-deficient group. The concentration of SAM in the B6-deficient group decreased to approximately 50% and 25% in liver and thymus, respectively. However SAH concentration was 3.5 and 2 fold higher compared to the control and the pair-fed control groups. Thus, the ratio of SAM/SAH was significantly decreased in the B6-deficient group compared to the control or the pair fed-control group. In addition, the S-adenosylhomocysteine hydrolase (EC 3.3.1.1) activity increased by 45% and 15% in liver and thymus, respectively, in the B6-deficient group compared to the pair-fed control and the control groups. However, the activity of L-methionine S-adenosyltransferase (EC 2.5.1.6) was also unaffected. Concentrations of SAH and SAM, SAM/SAH ratio and activities of S-adenosylhomocysteine hydrolase and L-methionine S-adenosyltrasferase in rat brain were not affected by the B6-deficiency. We infer that the alteration of B6 metabolism, especially the reduction of PLP contents in liver and thymus, caused by the B6 deficiency, resulted in accumulation of SAH as well as reduction of SAM and the SAM/SAH ratio. The reduction of the SAM/SAH ratio was due to a block in the catabolism of methionine via the trans-sulfuration pathway. These may lead to inhibition of transmethylation reaction of DNA, RNA and protein, the synthesis and function of thymic lymphocyte and result in damage to tissues.

Vitamin B-6-supplemented diets compared with a low vitamin B-6 diet suppress azoxymethane-induced colon tumorigenesis in mice by reducing cell proliferation.

Male ICR mice were examined for the effect of vitamin B-6 [pyridoxine (PN) HCl] on azoxymethane-induced colon tumorigenesis. Mice were fed the diets containing 1, 7, 14 or 35 mg PN HCl/kg for 22 wk, and given a weekly injection of azoxymethane (5 mg/kg body) for the initial 10 wk. Compared with the 1 mg PN HCl/kg diet, 7, 14 and 35 mg PN HCl/kg diets significantly suppressed the incidence and number of colon tumors, colon cell proliferation and expressions of c-myc and c-fos proteins. For some variables, 14 and 35 mg PN HCl/kg diets were more effective than the 7 mg/kg diet. Supplemental vitamin B-6 had no influence on the number of colon apoptotic cells. The results suggest that elevating dietary vitamin B-6 suppresses colon tumorigenesis by reducing cell proliferation.

Involvement of angiotensin II in progression of renal injury in rats with genetic non-insulin-dependent diabetes mellitus (Wistar fatty rats).

Wistar fatty (WF) rats have a genetic predisposition to hyperglycemia, polyuria, hyperinsulinemia, hyperlipidemia, obesity and nephropathy. These phenotypic characteristics are similar to those observed in obese patients with non-insulin-dependent diabetes mellitus (NIDDM) nephropathy. In this study, the effects of two types of renin-angiotensin system inhibitors, an angiotensin II type 1-receptor antagonist (AT1A) and an angiotensin I-converting enzyme inhibitor (ACEI), on renal injury in WF rats were studied during the progressive phase of diabetic nephropathy. An AT1A, candesartan cilexetil (1 mg/kg), and an ACEI, enalapril (10 mg/kg), were administered orally once a day for 12 weeks, beginning when the rats were 27-week-old and already showed diabetic nephropathy and obesity. Both drugs prevented an increase in proteinuria during the experimental period. Furthermore, after 4-week intervention, the levels of proteinuria were markedly lower in drug-treated rats. At the end of the experiment, both drugs prevented the development of glomerular lesions without affecting glucose metabolism and obesity. In conclusion, the inhibition of angiotensin II activity ameliorated both existing proteinuria and the progression of proteinuria, resulting in preservation of glomerular structure. Thus angiotensin II plays important roles in the development and the progression of nephropathy in genetically obese diabetic WF rats.

Distribution and properties of rat intestinal alkaline phosphatase isoenzymes.

The ALP activities and properties of rat intestine cut into 20 segments were examined, and we were able to demonstrate that the ALP activity of upper intestine is high compared to that of lower intestine. This result coincided with those of other reports. However, we newly clarified that there is an ALP isoenzyme found in the lower intestine which can be inhibited by L-homoarginine. The molecular weight of the ALP isoenzyme was 136 kDa. In addition, it was clarified that there are several isoenzymes from upper to lower intestine. This study demonstrates that there exist isoenzymes, which are inhibited by L-HArg, in the intestine which are similar to the isoenzymes in the liver, bone and kidney.

[Distribution of intraglandular metastatic foci in the contralateral lobe of papillary thyroid carcinoma].

The distribution of metastatic foci in the opposite lobe has not been studied in detail despite of several reports on the high incidence of contralateral metastasis. Whether foci spread to the upper one-third of the contralateral lobe influences the choice of total or subtotal thyroidectomy. Metastasis was studied in 66 patients 11 men and 55 women aged 24-73 years (mean; 51.3), undergoing primary total thyroidectomy from 1988 to 1996. Serial blocks of the opposite lobe, approximately 5 mm thick were sliced and stained by hematoxylin-eosin. Metastases were found in 44 patients (67%). Based on the size of the primary focus, these patients were divided into group A (smaller than one-third of the lobe) and group B (greater than that of group A). The average size of the primary focus was 21 mm in group A and 36 mm in group B. The contralateral metastatic rate was 64% (14/22) in group A and 68% (30/44) in group B. The distribution of metastatic foci in the opposite lobe was studied in 44 positive patients. The spread to the upper one third occurred in 61% (27/44); 29% (4/14) in group A and 76% (23/30) in group B a significant difference (p < .003). We thus concluded that the larger the primary focus, the wider the spread of metastatic foci to the opposite lobe.

Vacuolar hepatocyte degeneration induced by infusion of 20% glucose solution with insulin after hepatopancreatectomy in rats.

To investigate the causes of hepatic dysfunction after extensive resection of the liver together with pancreatectomy, rats were subjected to sham operation, to 68% hepatectomy alone, to 90% pancreatectomy alone, or to 68% hepatectomy combined with 90% pancreatectomy (hepatopancreatectomy). Solutions of 5% or 20% glucose were infused post-operatively for 48 h at a constant rate (250 ml/kg body weight/day) under fasting conditions. To improve the survival rates of pancreatectomized and hepatopancreatectomized rats given 20% glucose, it was necessary to use insulin. In hepatopancreatectomized rats, infusion of 20% glucose with insulin (1 U/5 g glucose) induced prominent hepatocyte vacuolar degeneration and mitochondrial swelling, associated with reduced hepatic protein content. The severity of histological changes was proportional to the insulin dose and the activity of hepatic glucokinase, a key glycolytic enzyme. were observed in These histological changes pancreatectomized rats albeit in a milder form, but not in sham-operated or hepatectomized rats given 20% glucose nor in any rats given 5% glucose. Our results suggest that hepatopancreatectomy followed post-operatively by a high glucose load and exogenously administered insulin enhances the development of hepatocyte swelling.

Effects of mixed administration of glucose and fat emulsion on the liver of rats undergoing hepatopancreatectomy.

To prevent the development of liver failure after simultaneous massive resection of the liver and pancreas, we examined whether fat emulsion could be used as an energy source during postoperative transfusion. Using rats, the following four groups were prepared: the simple laparotomy group (S group), 68% hepatectomy group (H group), 90% pancreatectomy group (P group) and 68% hepatectomy + 90% pancreatectomy group (HP group). A constant total calorie level and transfusion dose were used. Three kinds of transfusions with different glucose-to-fat emulsion caloric ratios, that is: 75% fat emulsion (of the total calorie level) + 25% glucose (75L), 50% fat emulsion + 50% glucose (50L) and 25% fat emulsion + 75% glucose + 1 unit/5 g glucose of insulin (25L), were continuously administered for 48 hours, and the effects of these transfusions on the survival rate and liver were examined. After 75L administration, survival rates in the H group and HP group were significantly lower than those in the S group and P group. After administration of 50L, survival rates did not significantly differ among the four groups. After 25L administration, the survival rate in the HP group was significantly lower than that in other groups. In the HP group, the survival rate after 50L administration was the highest. However, necrosis was observed in a portion of the hepatic lobule, while the blood aspartate aminotransferase (AST) level was increased. After 25L administration, hepatocellular mitochondrial swelling and degeneration were noted in the HP group; furthermore, the blood total protein level was decreased. These results showed that the post-operative administration of fat emulsion at a percentage of 50% of the total calorie level caused partial hepatocellular necrosis in the HP group, but improved the survival rate.

Effects of vitamin B-6 on (n-3) polyunsaturated fatty acid metabolism.

To investigate interactions between vitamin B-6 and fatty acid metabolism, male Wistar rats were fed a vitamin B-6 (B-6)-deficient diet consisting of 70% vitamin-free casein and 10% perilla oil [approximately 63% alpha-linolenic acid, (n-3)] for 5 wk. The amounts of linoleic acid (n-6) and arachidonic acid (n-6) in the B-6-deficient group changed only slightly compared with those in a pair-fed control group. The amount of linoleic acid increased and arachidonic acid decreased in the plasma total lipid fraction, and the ratios of both eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in the B-6-deficient group were significantly lower than for the controls. The ratios of alpha-linolenic acid and EPA were higher, and DHA lower, in the B-6-deficient group than in the pair-fed control group in the total lipid as well as phospholipid fractions in liver microsomes. The activity of delta6-desaturase was significantly lower in the B-6-deficient group than in the pair-fed control group (approximately 64%), and acyl-CoA oxidase activity, an initial enzyme of the peroxisomal beta-oxidation pathway, was reduced by approximately 80% in the B-6-deficient group. These data suggest that B-6 deficiencies impair the metabolism of (n-3) PUFA from alpha-linolenic acid to EPA and DHA with the most pronounced reduction in the production of DHA.

Inhibition mechanism of cathepsin L-specific inhibitors based on the crystal structure of papain-CLIK148 complex.

Papain was used as an experimental model structure to understand the inhibition mechanism of newly developed specific inhibitors of cathepsin L, the papain superfamily. Recently, we developed a series of cathepsin L-specific inhibitors which are called the CLIK series [(1999) FEBS Lett. 458, 6-10]. Here, we report the complex structure of papain with CLIK148, which is a representative inhibitor from the CLIK series. The inhibitor complex structure was solved at 1.7 A resolution with conventional R 0.177. Unlike other epoxisuccinate inhibitors (E64, CA030, and CA074), CLIK148 uses both prime and nonprime sites, which are important for the specific inhibitory effect on cathepsin L. Also, the specificity for cathepsin L could be explained by the existence of Phe in the P2 site and hydrophobic interaction of N-terminal pyridine ring.

Metabolism of theanine, gamma-glutamylethylamide, in rats.

The metabolism of theanine, one of the major amino acid components in tea (Camellia sinensis), was studied in rats. High-performance liquid chromatography (HPLC) with fluorometric detection was used to evaluate the nature of theanine's metabolites in plasma, urine, and tissues. In the urine samples collected after administration of 100, 200, and 400 mg each of theanine, intact theanine, L-glutamic acid, and ethylamine, these compounds were detected in a dose-dependent manner. When 200 mg of theanine was orally administered to rats, the plasma concentrations of theanine and ethylamine reached their highest levels about 0.5 and 2 h after administration, respectively. It seems most likely that the enzymatic hydrolysis of theanine to glutamic acid and ethylamine was accomplished in the kidney. These results indicate that orally administered theanine is absorbed through the intestinal tract and hydrolyzed to glutamic acid and ethylamine in the rat kidney.

Structure based development of novel specific inhibitors for cathepsin L and cathepsin S in vitro and in vivo.

Specific inhibitors for cathepsin L and cathepsin S have been developed with the help of computer-graphic modeling based on the stereo-structure. The common fragment, N-(L-trans-carbamoyloxyrane-2-carbonyl)-phenylalanine-dimethyla mide, is required for specific inhibition of cathepsin L. Seven novel inhibitors of the cathepsin L inhibitor Katunuma (CLIK) specifically inhibited cathepsin L at a concentration of 10(-7) M in vitro, while almost no inhibition of cathepsins B, C, S and K was observed. Four of the CLIKs are stable, and showed highly selective inhibition for hepatic cathepsin L in vivo. One of the CLIK inhibitors contains an aldehyde group, and specifically inhibits cathepsin S at 10(-7) M in vitro.

Crystallization and preliminary X-ray studies of the Ia component of Clostridium perfringens iota toxin complexed with NADPH.

A recombinant Ia component of Clostridium perfringens iota toxin, which ADP-ribosylates actin, was crystallized by the hanging drop vapor diffusion method using PEG4000 as a precipitating agent. The crystals were obtained in the presence of NADPH, which is similar to a real substrate, NADH, and belongs to the space group P1 (a = 47.9 A, b = 54.5 A, c = 103.1 A, alpha = 99.0 degrees, beta = 93.3 degrees, and gamma = 107.2 degrees ). The Matthews coefficient of native crystal was 2.7, assuming 2 mol/asymmetric unit. Native data were collected at 2.4-A resolution. The results from a heavy-atom search showed that lanthanide ions (samarium, holmium) altered the molecular packing, judging from the unit-cell difference. The crystals also belonged to the space group P1 (a = 47.7 A, b = 53.9 A, c = 54.6 A, alpha = 68.9 degrees, beta = 78.3 degrees, and gamma = 73.7 degrees ), which is consistent with only one molecule per asymmetric unit.

Konjac mannan improves the vitamin B-6 status of rats fed a vitamin B-6-deficient diet.

To investigate how dietary fiber in the diet affects vitamin B-6 nutriture of rats which have been deprived of vitamin B-6, rats were made vitamin B-6-deficient by feeding a vitamin B-6-deficient 70% casein diet. They were fed 2% cellulose powder-based vitamin B-6-deficient diets supplemented with 3% of additional dietary fiber sources (agar, konjac mannan, pectin and cellulose powder) for subsequent 18 days. Vitamin B-6 status was evaluated according to several biological criteria (weight gain, urinary excretion of xanthurenic acid after tryptophan loading, plasma pyridoxal 5'-phosphate, apparent pyridoxal 5'-phosphate-saturation of liver kynureninase, urinary excretion of 4-pyridoxic acid and fecal output of vitamin B-6). Vitamin B-6 status evaluated by these criteria was considerably improved in the konjac mannan-fed group, when compared with the respective data of the vitamin B-6 supplemented group. The relative mean effect of the konjac mannan diet was about 40% of the vitamin B-6 supplemented diet. In conclusion, konjac mannan was effective for improving the vitamin B-6 nutritional state in vitamin B-6-deprived rats.

Hyperammonemia induced by administration of glucose and insulin after hepatopancreatectomy in rats.

Massive resection of both the liver and pancreas is performed as a radical procedure in some cases of advanced biliary cancer, but it has been reported that this disease is frequently complicated by hyperbilirubinemia or hepatic insufficiency postoperatively, which is a serious hindrance to performing such extended surgery (Nimura et al., 1991; Nakamura et al., 1992). To investigate the pathogenesis of hepatic dysfunction after hepatopancreatectomy, we performed 4 surgical procedures consisting of 68% hepatectomy, 90% pancreatectomy, 68% hepatectomy plus 90% pancreatectomy (hepatopancreatectomy) and sham-surgery in rats. Then, rats were continuously infused with 5% or 20% glucose solution at a constant speed (50 mL/day) for 24 hours in the fasting state, thus creating a total of 8 groups. During infusion of 20% glucose solution into rats with pancreatectomy or hepatopancreatectomy, insulin (1 U/5 g glucose) was added to the solution to adjust the blood glucose. In rats infused with 20% glucose solution with added insulin after hepatopancreatectomy, the blood glucose level did not differ, but adenosine 5'-triphosphate (ATP) and energy charge levels in the liver tissue were significantly lower, while the blood ammonia level was significantly higher than those in the other 7 groups. These results demonstrate that continuous infusion of high concentrations of glucose solution with added insulin after hepatopancreatectomy in rats reduces hepatic mitochondrial function, resulting in hyperammonemia due to reduced urea synthesis.

Effects of vitamin B6 deficiency on cytokine levels and lymphocytes in mice.

The effects of vitamin B6 (B6) deficiency on cytokine levels and proportions of lymphocyte subsets in BALB/c mice were investigated. The proportion of lymphocytes from the thymus and spleen of mice given no B6, that were CD4+ CD8- T cells, was larger than in mice given B6, and the ratio of CD8+ to CD4+ T cells in the thymus of mice given no B6 was lower. The concentrations of interleukin-5 and -10 in spleen cells stimulated in vitro with concanavalin A were significantly higher in the mice with B6 deficiency, as was their plasma corticosterone concentrations. These results suggested that B6 is necessary to maintain cytokine levels and lymphoid function in the thymus and spleen of mice.