Predictive performance of a genetic risk score using 11 susceptibility alleles for the incidence of Type 2 diabetes in a general Japanese population: a nested case-control study.

AIMS: To assess the predictive ability of a genetic risk score for the incidence of Type 2 diabetes in a general Japanese population. METHODS: This prospective case-control study, nested within a Japan Public Health Centre-based prospective study, included 466 participants with incident Type 2 diabetes over a 5-year period (cases) and 1361 control participants, as well as 1463 participants with existing diabetes and 1463 control participants. Eleven susceptibility single nucleotide polymorphisms, identified through genome-wide association studies and replicated in Japanese populations, were analysed. RESULTS: Most single nucleotide polymorphism loci showed directionally consistent associations with diabetes. From the combined samples, one single nucleotide polymorphism (rs2206734 at CDKAL1) reached a genome-wide significance level (odds ratio 1.28, 95% CI 1.18-1.40; P = 1.8 × 10-8 ). Three single nucleotide polymorphisms (rs2206734 in CDKAL1, rs2383208 in CDKN2A/B, and rs2237892 in KCNQ1) were nominally significantly associated with incident diabetes. Compared with the lowest quintile of the total number of risk alleles, the highest quintile had a higher odds of incident diabetes (odds ratio 2.34, 95% CI 1.59-3.46) after adjusting for conventional risk factors such as age, sex and BMI. The addition to the conventional risk factor-based model of a genetic risk score using the 11 single nucleotide polymorphisms significantly improved predictive performance; the c-statistic increased by 0.021, net reclassification improved by 6.2%, and integrated discrimination improved by 0.003. CONCLUSIONS: Our prospective findings suggest that the addition of a genetic risk score may provide modest but significant incremental predictive performance beyond that of the conventional risk factor-based model without biochemical markers.

Calreticulin mutant mice develop essential thrombocythemia that is ameliorated by the JAK inhibitor ruxolitinib.

Mutations of calreticulin (CALR) are detected in 25-30% of patients with essential thrombocythemia (ET) or primary myelofibrosis and cause frameshifts that result in proteins with a novel C-terminal. We demonstrate that CALR mutations activated signal transducer and activator of transcription 5 (STAT5) in 293T cells in the presence of thrombopoietin receptor (MPL). Human megakaryocytic CMK11-5 cells and erythroleukemic F-36P-MPL cells with knocked-in CALR mutations showed increased growth and acquisition of cytokine-independent growth, respectively, accompanied by STAT5 phosphorylation. Transgenic mice expressing a human CALR mutation with a 52 bp deletion (CALRdel52-transgenic mice (TG)) developed ET, with an increase in platelet count, but not hemoglobin level or white blood cell count, in association with an increase in bone marrow (BM) mature megakaryocytes. CALRdel52 BM cells did not drive away wild-type (WT) BM cells in in vivo competitive serial transplantation assays, suggesting that the self-renewal capacity of CALRdel52 hematopoietic stem cells (HSCs) was comparable to that of WT HSCs. Therapy with the Janus kinase (JAK) inhibitor ruxolitinib ameliorated the thrombocytosis in TG mice and attenuated the increase in number of BM megakaryocytes and HSCs. Taken together, our study provides a model showing that the C-terminal of mutant CALR activated JAK-STAT signaling specifically downstream of MPL and may have a central role in CALR-induced myeloproliferative neoplasms.

Plasma 25-hydroxy vitamin D and subsequent prostate cancer risk in a nested Case-Control study in Japan: The JPHC study.

BACKGROUND/OBJECTIVES: Although vitamin D has been experimentally reported to inhibit tumorigenesis, cell growth and prostate cancer invasion, epidemiologic data regarding prostate cancer risk are inconsistent, and some studies have suggested positive but nonsignificant associations. Further, the impact of vitamin D on prostate cancer between Western and Japanese populations may differ due to different plasma vitamin D levels. SUBJECTS/METHODS: We performed a nested case-control study within the Japan Public Health Center-based Prospective (JPHC) Study in 14,203 men (40-69 years) who answered a self-administered questionnaire at baseline (1990-1994) and gave blood samples, and were followed until 2005. We identified 201 prostate cancers which are newly diagnosed during follow-up (mean 12.8 years). We selected two matched controls for each case from the cohort. We used a conditional logistic regression model to estimate the odds ratios (ORs) and 95% confidence intervals (CIs) for prostate cancer with respect to levels of 25-hydroxy vitamin D (25(OH)D) in plasma. RESULTS: We did not observe statistically significant association between 25(OH)D level and total prostate cancer (multivariate OR=1.13 (95%CI=0.66-1.94, Ptrend=0.94) for the highest versus lowest tertile) However, 25(OH) levels were slightly positively associated with advanced cancer. The results remained substantially unchanged after stratification by intake of fish or calcium intake. CONCLUSIONS: 25(OH)D level showed no association with overall prostate cancer among Japanese men in this large cohort.

Association of plasma concentrations of branched-chain amino acids with risk of colorectal adenoma in a large Japanese population.

Background: Available evidence from animal studies suggests that branched-chain amino acids (BCAAs) may have a protective effect against colorectal carcinogenesis. However, a possible effect of BCAAs against colorectal neoplasia has not been evaluated in humans. Here, we aimed to evaluate whether plasma concentrations of BCAA are associated with the risk of colorectal adenoma (CRA), a precursor lesion of colorectal cancer. Patients and methods: CRA cases and controls were identified from examinees who underwent total colonoscopy as part of a cancer screening program between 2004 and 2005 and responded to self-administered dietary and lifestyle questionnaires. We measured plasma concentrations of leucine, isoleucine and valine in 629 patients with adenoma and 584 controls. Unconditional logistic regression models were used to estimate odds ratio (OR) and 95% confidence interval (CI) for the association between BCAA and CRA risk after adjustment for potential confounders. Results: High plasma concentrations of leucine, valine and total BCAA were inversely associated with CRA risk after adjustment of potential confounders. The multivariate-adjusted ORs for the highest versus lowest quartiles were 0.60 (95% CI 0.42-0.87, Ptrend = 0.006) for leucine, 0.68 (95% CI 0.48-0.97, Ptrend = 0.09) for valine and 0.68 (95% CI 0.48-0.98, Ptrend = 0.10) for total BCAA. Further analysis by gender revealed that this inverse association was clearly evident in men, but not in women: the corresponding OR for leucine, valine and total BCAA was 0.50 (95% CI 0.32-0.80, Ptrend = 0.003), 0.60 (95% CI 0.38-0.95, Ptrend = 0.01) and 0.58 (95% CI 0.37-0.93, Ptrend = 0.04), respectively, in men and 0.78 (95% CI 0.42-1.45, Ptrend = 0.44), 0.77 (95% CI 0.41-1.43, Ptrend = 0.85) and 0.84 (95% CI 0.45-1.57, Ptrend = 0.81), respectively, in women. Conclusion: Our finding suggests that BCAAs may have a beneficial influence against the process of colorectal carcinogenesis, at least in the early stage. The mechanisms underlying this potential association between BCAA and colorectal carcinogenesis warrant further investigation.

Coping strategies and risk of cardiovascular disease incidence and mortality: the Japan Public Health Center-based prospective Study.

AIMS: Coping strategies may be significantly associated with health outcomes. This is the first study to investigate the association between baseline coping strategies and cardiovascular disease (CVD) incidence and mortality in a general population cohort. METHODS AND RESULTS: The Japan Public Health Center-based prospective Study asked questions on coping in its third follow-up survey (2000-04). Analyses on CVD incidence and mortality included 57 017 subjects aged 50-79 without a history of CVD and who provided complete answers on approach- and avoidance-oriented coping behaviours and strategies. Cox regression models, adjusted for confounders, were used to determine hazard ratios (HRs) according to coping style. Mean follow-up time was 7.9 years for incidence and 8.0 years for mortality.The premorbid use of an approach-oriented coping strategy was inversely associated with incidence of stroke (HR = 0.85; 95% CI, 0.73-1.00) and CVD mortality (HR = 0.74; 95% CI, 0.55-0.99). Stroke subtype analyses revealed an inverse association between the approach-oriented coping strategy and incidence of ischaemic stroke (HR = 0.79; 95% CI, 0.64-0.98) and a positive association between the combined coping strategy and incidence of intra-parenchymal haemorrhage (HR = 2.03; 95% CI, 1.01-4.10). Utilizing an avoidance coping strategy was associated with increased mortality from ischaemic heart disease (IHD) only in hypertensive individuals (HR = 3.46; 95% CI, 1.07-11.18). The coping behaviours fantasizing and positive reappraisal were associated with increased risk of CVD incidence (HR = 1.24; 95% CI, 1.03-1.50) and reduced risk of IHD mortality (HR = 0.63; 95% CI, 0.40-0.99), respectively. CONCLUSION: An approach-oriented coping strategy, i.e. proactively dealing with sources of stress, may be associated with significantly reduced stroke incidence and CVD mortality in a Japanese population-based cohort.

Soy food and isoflavone intake and endometrial cancer risk: the Japan Public Health Center-based prospective study.

OBJECTIVE: Compared with western populations, the consumption of soy foods among Japanese is very high and the incidence of endometrial cancer very low. We evaluated the association of soy food and isoflavone intake with endometrial cancer risk in Japanese women. DESIGN: Prospective cohort study. SETTING: Ten public health centre areas in Japan. POPULATION: Forty nine thousand one hundred and twenty-one women of age 45-74 years who responded to a 5-year follow-up survey questionnaire. METHODS: Intakes of soy foods as well as other covariates were assessed in 1995-1998 by a self-administered food frequency questionnaire. Cox proportional hazards regression models were used to estimate hazard ratios (HR) and 95% confidence intervals (CI). MAIN OUTCOME MEASURE: Incidence of endometrial cancer. RESULTS: During an average of 12.1 years of follow up, 112 newly diagnosed endometrial cancer cases were identified. Energy-adjusted intakes of soy food and isoflavone were not associated with the risk of endometrial cancer. The multivariate-adjusted HR per 25 g/day increase in the intake of soy food was 1.02 (95% CI 0.94-1.10), and the corresponding value for isoflavone intake per 15 mg/day was 1.01 (95% CI 0.84-1.22). CONCLUSION: In this population-based prospective cohort study of Japanese women, we observed no evidence of a protective association between soy food or isoflavone intake and endometrial cancer risk.

Rice, bread, noodle and cereal intake and colorectal cancer in Japanese men and women: the Japan Public Health Center-based prospective Study (JPHC Study).

BACKGROUND: Colorectal cancer (CRC) incidence rate increased rapidly in Japan between the 1950s and 1990s. We examined the association between rice intake and CRC risk in comparison with bread, noodles and cereal among Japanese adults enrolled in the Japan Public Health Center-based prospective Study. METHODS: A total of 73,501 Japanese men and women were followed-up from 1995 to 1999 until the end of 2008 for an average of 11 years. During 801,937 person-years of follow-up, we identified 1276 incident cases of CRC. Hazard ratios (HRs) and 95% confidence intervals (95% CIs) of CRC for rice, noodle, bread and cereal intake were calculated by Cox proportional hazards model. RESULTS: Overall, no significant association was observed for the highest quartile of rice intake compared with the lowest and the risk of CRC and its subsites in men (HR, 0.77; 95% CI, 0.56-1.07) and women (HR, 1.10; 95% CI, 0.71-1.68). However, a non-significant inverse trend was observed between rice intake and rectal cancer in men. No clear patterns of association were observed in bread, noodle and cereal intake. CONCLUSION: Our findings suggest that the consumption of rice does not have a substantial impact on the risk of CRC in the Japanese population.

TET2 is essential for survival and hematopoietic stem cell homeostasis.

Ten-Eleven-Translocation 2 (TET2) is an enzyme that catalyzes the conversion of 5-methylcytosine into 5-hydroxymethylcytosine (5-hmC) and thereby alters the epigenetic state of DNA; somatic loss-of-function mutations of TET2 are frequently observed in patients with diverse myeloid malignancies. To study the function of TET2 in vivo, we analyzed Ayu17-449 (TET2(trap)) mice, in which a gene trap insertion in intron 2 of TET2 reduces TET2 mRNA levels to about 20% of that found in wild-type (WT) mice. TET2(trap/trap) mice were born at Mendelian frequency but died at a high rate by postnatal day 3, indicating the essential role of TET2 for survival. Loss of TET2 results in an increase in the number of hematopoietic stem cells (HSCs)/progenitors in the fetal liver, and TET2(trap/trap) HSCs exhibit an increased self-renewal ability in vivo. In competitive transplantation assays, TET2(trap/trap) HSCs possess a competitive growth advantage over WT HSCs. These data indicate that TET2 has a critical role in survival and HSC homeostasis.

Gender difference in the association of insulin and the insulin-like growth factor axis with colorectal neoplasia.

OBJECTIVE: Accumulating evidence has implicated insulin and the insulin-like growth factor (IGF) axis in colorectal carcinogenesis. Of interest, adiposity is likely to impose a greater risk on men than on women, which indicates that the association of insulin and the IGF axis with colorectal neoplasia may differ by gender. However, epidemiological evidence for this possible gender difference is limited to date. METHODS: We measured plasma concentrations of C-peptide, IGF-I and IGF-binding proteins (IGFBPs) 1 and 3 in 1520 healthy volunteer examinees who underwent total colonoscopy between February 2004 and February 2005, and cross-sectionally investigated the association of these biomarkers with colorectal adenoma by gender. An unconditional logistic regression model was used to estimate odds ratios (ORs) and their 95% confidence intervals (CIs) for colorectal adenoma after adjustment for potential confounders. RESULTS: We observed a positive association of C-peptide and IGF-I (P (trend)<0.001 and 0.02, respectively) and an inverse association of IGFBP-1 (P (trend)=0.002) with colorectal adenoma in men. Adjusted ORs of colorectal adenoma for the highest compared with the lowest quartile were also statistically significant for C-peptide (OR: 2.62, 95% CI: 1.71-4.01), IGF-I (OR: 1.63, 95% CI: 1.08-2.46) and IGFBP-1 (OR: 0.49, 95% CI: 0.32-0.75). In contrast, no measurable association was seen in women. Corresponding ORs for C-peptide, IGF-I and IGFBP-1 were 0.98 (95% CI: 0.56-1.71), 0.79 (95% CI: 0.44-1.43) and 1.05 (95% CI: 0.60-1.86), respectively. The gender difference was statistically significant for C-peptide (P (interaction)=0.03) and marginally significant for IGF-I and IGFBP-1 (P (interaction)=0.14 and 0.12, respectively). CONCLUSION: Our observations suggest that insulin and the IGF axis act differently by gender in colorectal carcinogenesis, at least in its early stage. The findings of this study further our understanding of the complexities of the gender difference in the association between adiposity and colorectal neoplasia.

Dietary isoflavone and the risk of colorectal adenoma: a case-control study in Japan.

We conducted a case-control study in a Japanese population to investigate the association between dietary isoflavone intake and the risk of colorectal adenoma. Participants who underwent magnifying colonoscopy with dye spreading as part of a cancer screening programme responded to a self-administered questionnaire, which included lifestyle information and intake of 145 food items, before the colonoscopy. A total of 721 case and 697 control subjects were enrolled. Odds ratios (ORs) and 95% confidence intervals (CIs) were estimated using unconditional logistic regression models. We found a significant inverse association between dietary isoflavone intake and the risk of colorectal adenoma in men and women combined. However, the inverse association was not linear; rather, all quartiles above the first showed a similar decrease in risk, with multivariable-adjusted ORs and 95% CIs compared with the lowest quartile of 0.77 (0.57-1.04), 0.76 (0.56-1.02) and 0.70 (0.51-0.96) in the second, third and highest quartiles, respectively (P for trend=0.03). Of interest, the observed association was more prominent in women than in men. The observed ceiling effect associated with higher isoflavone intake suggests that a lower intake of dietary isoflavone might be associated with an increased risk of colorectal adenoma in Japanese populations.

Glucocorticoid receptor activation is involved in producing abnormal phenotypes of single-prolonged stress rats: a putative post-traumatic stress disorder model.

Post-traumatic stress disorder (PTSD) is a stress-related mental disorder caused by traumatic experience, and presents with characteristic symptoms, such as intrusive memories, a state of hyperarousal, and avoidance, that endure for years. Single-prolonged stress (SPS) is one of the animal models proposed for PTSD. Rats exposed to SPS showed enhanced inhibition of the hypothalamo-pituitary-adrenal (HPA) axis, which has been reliably reproduced in patients with PTSD, and increased expression of glucocorticoid receptor (GR) in the hippocampus. In this study, we characterized further neuroendocrinologic, behavioral and electrophysiological alterations in SPS rats. Plasma corticosterone recovered from an initial increase within a week, and gross histological changes and neuronal cell death were not observed in the hippocampus of the SPS rats. Behavioral analyses revealed that the SPS rats presented enhanced acoustic startle and impaired spatial memory that paralleled the deficits in hippocampal long-term potentiation (LTP) and depression. Contextual fear memory was enhanced in the rats 1 week after SPS exposure, whereas LTP in the amygdala was blunted. Interestingly, blockade of GR activation by administering 17-beta-hydroxy-11-beta-/4-/[methyl]-[1-methylethyl]aminophenyl/-17-alpha-[prop-1-ynyl]estra-4-9-diene-3-one (RU40555), a GR antagonist, prior to SPS exposure prevented potentiation of fear conditioning and impairment of LTP in the CA1 region. Altogether, SPS caused a number of behavioral changes similar to those described in PTSD, which marks SPS as a putative PTSD model. The preventive effects of a GR antagonist suggested that GR activation might play a critical role in producing the altered behavior and neuronal function of SPS rats.

Influence of location of acceleration sensor on physiological tremor of upper limb.

The physiological tremor of the upper limb in three positions of pronation, neutrality, and supination due to the movement of forearm was measured on four locations at the tip of the finger, the root of the finger in the hand, the wrist, and the elbow with use of an accelerated sensor. The evaluation of the total power, which was the summation of the power spectrum in the frequency range from 1 to 50 Hz, showed no significant difference in any of the positions. The maintenance of the upper limb at the horizontal level showed the coordination of the central nervous system due to the body parts of the upper arm, forearm, hand, and finger connected by the joint. The coherence spectra showed clear activation of the joint of the wrist in the main peak frequency of around 2.5 and 12.5 Hz in their respective positions. The value of the correlation coefficient in the location between the hand and finger was the largest at over 0.8, and those of the locations which connected the joint of the wrist between the forearm and hand and between the forearm and finger were significantly large with a value from 0.6 to 0.8. The mean time (i.e., arrival time) of the transmission from the proximal side (i.e., upper arm and forearm) to the distal side (i.e., hand and finger) in the upper limb was evaluated quantitatively to be 20 ms for pronation and supination, but the value was small for neutrality.

Possibility of clinical application of tremor in the upper limb.

A procedure to evaluate the function of the glenohumeral joint (i.e., shoulder joint) for persons with shoulder disorders was proposed by analyzing tremor. In order to compare subjects with shoulder disorders to healthy subjects without disorders, characteristics of tremor for the healthy persons under various angles of glenohumeral joint and under the dominant and no-dominant sides of the upper limb were obtained by peak frequency and power spectrum as basic data. As for the persons with rotator cuff injury, the ratio of total powers for the affected side and the healthy side was obtained as the affective side ratio. The ratio showed the characteristic of the persons with the disorder, and discriminated the total power spectra of persons who were healthy and those with disorders.

Coffee consumption and glucose tolerance status in middle-aged Japanese men.

AIMS/HYPOTHESIS: Several studies have reported that coffee has a protective effect against the development of type 2 diabetes. However, few of these studies used the standard glucose tolerance test to diagnose type 2 diabetes. The aim of this study was to investigate the relationship between coffee and green tea consumption and glucose tolerance status as determined using a 75-g OGTT. METHODS: We performed a cross-sectional study of 3224 male officials of the self-defence forces. Glucose tolerance status was determined in accordance with the 1998 World Health Organization criteria, and average intakes of coffee and green tea over the previous year were assessed by a self-administered questionnaire. The figures obtained were adjusted for BMI, physical activity and other factors. RESULTS: A total of 1130 men were identified as having glucose intolerance (IFG, IGT or type 2 diabetes). Compared with those who did not consume coffee on a daily basis, fasting and 2-h post-load plasma glucose levels were 1.5% and 4.3% lower in those who drank 5 cups of coffee or more per day respectively. The adjusted odds ratios of glucose intolerance for categories of <1, 1-2, 3-4 and >/=5 cups of coffee per day were 1.0 (referent), 0.8 (95% CI 0.6-1.0), 0.7 (95% CI 0.6-0.9) and 0.7 (95% CI 0.5-0.9) respectively (p=0.0001 for trend). No clear association was observed between green tea drinking and glucose tolerance status. CONCLUSIONS/INTERPRETATION: Coffee consumption may inhibit postprandial hyperglycaemia and thereby protect against the development of type 2 diabetes mellitus.

Hepatitis B virus genomes of chronic hepatitis patients do not contain specific mutations related to acute exacerbation.

To determine the specific viral variants associated with acute exacerbation of chronic hepatitis from hepatitis B virus (HBV) infection, we analyzed the complete nucleotide sequences of the HBV genome in serial serum samples from two chronic active hepatitis patients who seroconverted from HBeAg to anti-HBe. HBV DNA was amplified by polymerase chain reaction (PCR) and sequenced. A 1896 precore stop codon mutant (G to A at nt 1896) coexisting with the wild sequence was found in both patients prior to seroconversion from HBeAg to anti-HBe. Core promoter mutations at nucleotide positions 1762 (A to T) and 1764 (G to A) were found in both patients throughout the observation period. Mutations were observed in the HBV genome of the two patients at different time points, and there was no correlation between the mutations and liver disease or DNA polymerase levels. The nucleotide divergence rate and the composition of quasispecies in the HBV sequence at the time of acute exacerbation were almost the same as were found at other time points. These results suggest that acute exacerbation does not appear to be caused by a characteristic HBV species. The multiple factors that cause generalized HBV replication activation may contribute to acute exacerbation.

Apoptosis of CTLL-2 cells induced by an immunosuppressant, ISP-I, is caspase-3-like protease-independent.

In our previous study, the sphingosine-like immunosuppressant ISP-1 was shown to induce apoptosis in the mouse cytotoxic T cell line CTLL-2. In this study, we characterized the ISP-1-induced apoptotic pathway. Although caspase-3-like protease activity increases concomitantly with ISP-1-induced apoptosis in CTLL-2 cells, the apoptosis is not inhibited by caspase-3-like protease inhibitors, i.e. DEVD-cho and z-DEVD-fmk. In contrast, sphingosine-induced apoptosis in CTLL-2 cells is caspase-3-like protease-dependent. A caspase inhibitor with broad specificity, z-VAD-fmk, protects cells from apoptosis induced by ISP-1, indicating that ISP-1-induced apoptosis is dependent on caspase(s) other than caspase-3. Overexpression of Bcl-2 or Bcl-xL suppresses the apoptosis induced by ISP-1, although sphingosine-induced apoptosis is not efficiently inhibited by Bcl-2. Finally, ISP-1-induced mitochondrial depolarization, which is thought to be a checkpoint dividing the apoptotic pathway into upstream and downstream stages, is not inhibited by DEVD-cho, but is inhibited by z-VAD-fmk. These data suggest that a pathway dependent on caspase(s) other than caspase-3 is involved upstream of mitochondrial depolarization in ISP-1-induced apoptosis.

The effect of micromovement on callus formation.

Micromovement at fracture sites is known to promote callus formation and bridging of the bony fragments. The present study was conducted to identify the suitable amount of micromovement, and to analyze the location and timing of callus proliferation. A standardized transverse osteotomy, in the right metatarsus of 32 sheep, was used as a fracture model. The osteotomy was externally fixed with a special ring fixator, which allowed axial micromovements of defined sizes. The animals were divided into four groups, with gaps of 2 mm and 6 mm, and micromovements of 0.3 mm and 0.7 mm, respectively. The labeling of new bone formation was performed by the intravenous injection of calcein green in the fourth week and tetracycline in the eighth week. Nine weeks postoperatively the sheep were killed. The explanted metatarsals were radiographed for the measurement of the periosteal callus area and were nondestructively loaded in a three-point bending test to determine their flexural rigidity. Histological analysis of undecalcified bone was performed in bone slices in the sagittal plane. Fluorescent green (callus formed in the fourth week) and yellow areas (callus formed in the eighth week) and the area of connective tissue were determined, using fluorescence microscopy. Bone formation was larger in the eighth week than that in the fourth week in all groups. In the fourth week, large micromovements in the small gap resulted in increased bone formation, whereas, for large gaps, the large micromovements diminished new bone formation. With large micromovement, the amount of newly formed bone within the gap decreased with increasing gap size, suggesting a delay of bone healing. Stimulation of new bone formation by micromovement was mainly effective in the early healing phase (4 weeks postoperatively). Large gaps showed the least new bone formation at the fracture site and the lowest flexural rigidity. From the histological analysis, it was found that the flexural rigidity correlated with the new bone area in the periosteal region.

Type V collagen is increased during rabbit medial collateral ligament healing.

To understand the reparative process of medial collateral ligament (MCL), fibrillar collagen and their relative ratios in healing MCL with anterior cruciate ligament (ACL) reconstruction were analyzed. Skeletally mature New Zealand white rabbits were subjected to a mop-end tear of MCL without repair with ACL reconstruction. Rabbits were killed 6 and 52 weeks after injury. Ligamentous tissues from the injury site and sham controls were soaked in 0.5 M acetic acid for 24 h, minced, and treated with pepsin to solubilize collagen. Pepsin solubilized about 80% of the total collagen as determined by hydroxyproline analysis of the pepsin residues. Sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis of the solubilized collagen revealed presence of fibrillar collagen types I, III, and V. Densitometric scanning of the protein bands corresponding to types I, III, and V collagen indicated that in sham controls types III and V collagen represented about 8% and 12%, respectively, of the type I collagen whereas the healed MCL ligaments at 6 weeks showed significant increase in type III and V collagen to about 19% and 24%, respectively. By 52 weeks type III collagen in the healed MCL had returned to that of sham controls while type V collagen remained elevated at approximately 18%. These data suggest that presence of type V collagen in high concentration in healing ligaments may have an influence on collagen fibril diameters seen in healed ligament and should be included in the analysis when evaluating ligament healing.

Malignant pheochromocytoma lacking clinical features of catecholamine excess until the late stage.

A malignant pheochromocytoma is described in a 71-year-old man. Osseous metastases became manifest 12 years after successful removal of the primary tumor which originated in paraganglionic tissue near the right adrenal gland. Although the patient had no symptoms of catecholamine excess initially, hypertension, tachycardia and excessive sweating appeared several months before his death, concomitantly with a sharp increase in noradrenaline secretion due to an accelerated growth of metastatic tumors. Since there is no histologic criterion of malignancy in this neoplasm, it would be prudent to consider every case of pheochromocytoma as potentially malignant and to follow-up carefully for a long time after removal of the primary tumor.