Functional Regulatory Mechanisms Underlying Bone Marrow Mesenchymal Stem Cell Senescence During Cell Passages.

Mesenchymal stem cell (MSC) transplantation is an effective periodontal regenerative therapy. MSCs are multipotent, have self-renewal ability, and can differentiate into periodontal cells. However, senescence is inevitable for MSCs. In vitro, cell senescence can be induced by long-term culture with/without cell passage. However, the regulatory mechanism of MSC senescence remains unclear. Undifferentiated MSC-specific transcription factors can regulate MSC function. Herein, we identified the regulatory transcription factors involved in MSC senescence and elucidated their mechanisms of action. We cultured human MSCs (hMSCs) with repetitive cell passages to induce cell senescence and evaluated the mRNA and protein expression of cell senescence-related genes. Additionally, we silenced the cell senescence-induced transcription factors, GATA binding protein 6 (GATA6) and SRY-box 11 (SOX11), and investigated senescence-related signaling pathways. With repeated passages, the number of senescent cells increased, while the cell proliferation capacity decreased; GATA6 mRNA expression was upregulated and that of SOX11 was downregulated. Repetitive cell passages decreased Wnt and bone morphogenetic protein (BMP) signaling pathway-related gene expression. Silencing of GATA6 and SOX11 regulated Wnt and BMP signaling pathway-related genes and affected cell senescence-related genes; moreover, SOX11 silencing regulated GATA6 expression. Hence, we identified them as pair of regulatory transcription factors for cell senescence in hMSCs via the Wnt and BMP signaling pathways.

Anterior insular volume decrease is associated with dysfunction of the reward system in patients with chronic pain.

BACKGROUND: Chronification of pain is associated with both anatomical and functional alterations of the brain. Alteration in regional grey matter volume might potentially be associated with modified activity of specific brain networks. In this cross-sectional, observational study, we sought to identify brain regions with grey matter volume changes in patients with chronic pain and to reveal its significance by analysing alteration in functional connectivity from those regions. We further explored relevance of such alterations with psychometrics of chronic pain. METHODS: We recruited 23 patients with chronic pain and 17 age-, gender-matched healthy control subjects. After completing multiple psychophysical questionnaires, each subject underwent resting-state functional magnetic resonance imaging and three-dimensional anatomical imaging on a 3 Tesla magnetic resonance imaging scanner. RESULTS: Patients with chronic pain showed significant volume decrease at the right anterior insular cortex (p < 0.001) and the left middle cingulate cortex (p < 0.001) compared with healthy controls. They also showed decreased connectivity between the right anterior insular cortex and the left nucleus accumbens in negative association with the Pain Catastrophizing Scale (R2  = 0.20, p = 0.046) and the Beck's Depression Inventory scores (R2  = 0.24, p = 0.017). CONCLUSIONS: Decreased grey matter volumes of those core regions for affective processing of pain might be a common cerebral feature shared by, at least some of, different aetiologies of chronic pain. Dysfunctional network between the anterior insular cortex and the nucleus accumbens might reflect affective and motivational disability involved in chronic pain. Such anatomical and functional profiles could potentially be part of a cerebral signature for chronification of pain. SIGNIFICANCE: This article illustrates decreased network activity of the reward system in association with insular cortical volume decrease in patients with chronic pain, and its close relationships with affective and cognitive morbidity of pain. Attenuation of brain's reward system involving cortical plastic changes might have a key role in chronification of pain.

Olfactory schwannoma.

We report a case of olfactory schwannoma with calcification. Intraoperative findings indicated that the tumour originated from the olfactory groove. Intraoperative findings of previous studies have not indicated a clear relationship between subfrontal schwannoma and the olfactory nerve, which seems strange, given the association between tumours and cranial nerves at other sites. We suggest this observation has not been reported because the growing olfactory schwannoma changes the local morphology, affecting the appearance of the olfactory nerve.

Precocious decrease of chymotrypsinogen by thyroxine in the pancreas of suckling mice.

Chymotrypsinogen activity in mouse pancreas gradually decreased from birth, reaching the adult level on day 20 after birth. In suckling mice the enzyme activity was decreased to 1/9 of the control value by injection of thyroxine. The activity was not affected by insulin, and was slightly increased, rather than decreased, by daily injection of hydrocortisone. The effect of thyroxine seemed to be direct, not due to modification of adrenal function.

Induction of osteoblastic cell differentiation by forskolin. Stimulation of cyclic AMP production and alkaline phosphatase activity.

The effects of forskolin on differentiation of osteoblastic cells (clone MC3T3-E1) cultured in alpha-minimum essential medium containing 0.1% bovine serum albumin were investigated by assays of intracellular cyclic AMP level and alkaline phosphatase activity in the cells. Forskolin increased cyclic AMP production in the cells in a dose-related manner, the maximum increase being 250-fold above that of the controls. Alkaline phosphatase activity in the cells was also elevated as early as 24 h and rose to nearly its maximum at 48 h. The elevation was dose-dependent, with a maximum increase at 5 X 10(-6) M forskolin. Forskolin and prostaglandin E2 showed a supraadditive effect on cyclic AMP production in the cells and had an additive effect on alkaline phosphatase activity, whereas forskolin and dibutyryl cyclic AMP had little additive effect on either cyclic AMP production or enzyme activity. These results suggest that cyclic AMP is closely linked to the differentiation of osteoblastic cells in vivo.

Precocious differentiation of chick embryo pancreas in vitro. Roles of prednisolone, insulin and L-thyroxine.

The hormonal requirement for functional differentiation of chick embryo pancreas were investigated by using organ cultures in chemically defined medium. The hormones tested were prednisolone, insulin and thyroxine, and the parameters examined were alpha-amylase (EC 3.2.1.1) and chymotrypsinogen (EC 3.4.4.5) activities, and the ultrastructure of the tissues. Addition of prednisolone alone to explants from 14-day-old chicken embryo pancreas for 3 days increased the activities of amylase and chymotrypsinogen in the tissues by 3.4- and 6.6-fold, respectively, those of tissues before cultivation. Neither thyroxine or insulin alone, nor both hormones together affected pancreatic exocrine differentiation. Thyroxine enhanced the effect of prednisolone on both enzymes, but insulin did not. When the explants were cultured in the medium containing all three hormones, maximum enzyme activities were observed; amylase or chymotrypsinogen activity being 7- or 18-fold, respectively, that of tissues before cultivation. But these three hormones were not simultaneously necessary. Morphological differentiation was also observed in explants cultuvated in medium containing these three hormones. These results suggest that glucocorticoids are essential for normal differentiation of chick pancreas during the late fetal period, possibly with insulin and thyroxine, and also support the idea that pancreatic enzymes are controlled separately.

Two distinct mechanisms operate in the reactivation of heat-denatured proteins by the mitochondrial Hsp70/Mdj1p/Yge1p chaperone system.

The yeast mitochondrial Hsp70, Ssc1p, functions as a molecular chaperone with its partner proteins, Mdj1p (DnaJ homologue) and Yge1p (GrpE homologue). We have purified a mature form of Ssc1p from yeast mitochondria and those of Mdj1p and Yge1p from Escherichia coli overexpresser cells. With these purified components of the mitochondrial Hsp70 chaperone system, we have succeeded in reconstituting their chaperone functions in the protection of firefly luciferase against thermal damage in vitro. Heat-denatured luciferase is prevented from irreversible aggregation and is maintained in a refolding-competent state by Ssc1p and/or Mdj1p at 42 degreesC. Luciferase denatured at 42 degreesC is actively reactivated by Ssc1p, Mdj1p and/or Yge1p after lowering the temperature to 25 degreesC. The reactivation process of heat-denatured luciferase shows two-phase kinetics. The slow refolding process requires either Ssc1p or Mdj1p at 42 degreesC but the presence of Ssc1p, Mdj1p and Yge1p, and ATP hydrolysis, is essential at 25 degreesC. The slow refolding of luciferase involves multiple rounds of formation and dissociation of the complex between luciferase and Mdj1p/Ssc1p. On the other hand, the fast refolding process is most enhanced when luciferase is incubated with Ssc1p alone at 42 degreesC, and it requires neither the assistance of Mdj1p and Yge1p nor ATP hydrolysis. We have observed a similar two-pathway reactivation of heat-denatured luciferase by the bacterial Hsp70 and the yeast cytosolic Hsp70 systems.

Venepuncture is preferable to heel lance for blood sampling in term neonates.

BACKGROUND: The analgesic effect of oral sucrose in newborn infants undergoing painful procedures is generally accepted. For blood sampling, some studies have shown that venepuncture (VP) is less painful than heel lance (HL). OBJECTIVE: To determine the least painful and most effective method among blood sampling by VP or HL with or without sucrose. DESIGN: Randomised, double blind, placebo controlled trial. SUBJECTS: A total of 100 healthy, full term newborn infants being screened for inborn errors of metabolism were randomly allocated to one of four experimental groups (25 infants in each). Intervention and OUTCOME MEASURE: Seven specially trained nurses took turns to carry out blood sampling two minutes after administration of oral sucrose or water. Neonatal pain was assessed by the neonatal facial coding system (NFCS), as well as by crying. RESULTS: Without sucrose, the NFCS score was higher in the HL group than the VP group during blood sampling (median 58 v 23, p<0.001). Oral sucrose significantly reduced the score of the HL group (58 v 47, p<0.01) and also tended to reduce the score of the VP group (23 v 2, p<0.1). However, the HL with sucrose group still had a higher score than the VP without sucrose group (47 v 23, p<0.01). Crying and the total procedure time showed the same trends as the NFCS score. CONCLUSIONS: VP is less painful and more effective than HL for blood sampling in newborn infants. Although oral sucrose may have an additive analgesic effect, it is not necessarily required if VP is used for blood sampling.

Receptor-type protein tyrosine phosphatase κ directly dephosphorylates CD133 and regulates downstream AKT activation.

Although CD133 has been considered to be a molecular marker for cancer stem cells, its functional roles in tumorigenesis remain unclear. We here examined the molecular basis behind CD133-mediated signaling. Knockdown of CD133 resulted in the retardation of xenograft tumor growth of colon cancer-derived HT-29 and LoVo cells accompanied by hypophosphorylation of AKT, which diminished ß-catenin/T-cell factor-mediated CD44 expression. As tyrosine residues of CD133 at positions 828 and 852 were phosphorylated in HT-29 and SW480 cells, we further addressed the significance of this phosphorylation in the tumorigenesis of SW480 cells expressing mutant CD133, with substitution of these tyrosine residues by glutamate (CD133-EE) or phenylalanine (CD133-FF). Forced expression of CD133-EE promoted much more aggressive xenograft tumor growth relative to wild-type CD133-expressing cells accompanied by hyperphosphorylation of AKT; however, CD133-FF expression had negligible effects on AKT phosphorylation and xenograft tumor formation. Intriguingly, the tyrosine phosphorylation status of CD133 was closely linked to the growth of SW480-derived spheroids. Using yeast two-hybrid screening, we finally identified receptor-type protein tyrosine phosphatase κ (PTPRK) as a binding partner of CD133. In vitro studies demonstrated that PTPRK associates with the carboxyl-terminal region of CD133 through its intracellular phosphatase domains and also catalyzes dephosphorylation of CD133 at tyrosine-828/tyrosine-852. Silencing of PTPRK elevated the tyrosine phosphorylation of CD133, whereas forced expression of PTPRK reduced its phosphorylation level markedly and abrogated CD133-mediated AKT phosphorylation. Endogenous CD133 expression was also closely associated with higher AKT phosphorylation in primary colon cancer cells, and ectopic expression of CD133 enhanced AKT phosphorylation. Furthermore, lower PTPRK expression significantly correlated with the poor prognosis of colon cancer patients with high expression of CD133. Thus, our present findings strongly indicate that the tyrosine phosphorylation of CD133, which is dephosphorylated by PTPRK, regulates AKT signaling and has a critical role in colon cancer progression.

Prostaglandin E1 and F2 alpha stimulate differentiation and proliferation, respectively, of clonal osteoblastic MC3T3-E1 cells by different second messengers in vitro.

The effect of several prostaglandins (PGs) on osteoblastic cells was investigated using clone MC3T3-E1 under serum-free conditions. PGA1, A2, B1, and B2 had little effect on intracellular cAMP, alkaline phosphatase (ALP) activity, and DNA synthesis in the cells. At 4-2000 ng/ml, PGE1 among PG analogs tested had a dose-dependent stimulatory effect on ALP activity in the cells, and this effect was amplified by isobutyl methylxanthine. Also, PGE1 strongly augmented the amount of intracellular cAMP over the same concentration range. However, PGE1 had little effect on ornithine decarboxylase activity and DNA synthesis, and at high doses it rather depressed DNA synthesis. Furthermore, PGE1 did not affect the intracellular cGMP level. The effect of PGE1 on the cells closely mimics that of forskolin, suggesting that the PG stimulates the differentiation of the osteoblastic cells predominantly via the stimulation of adenylate cyclase. In contrast with PGE1, PGF2 alpha strongly increased ornithine decarboxylase activity and DNA synthesis in the cells in a dose-related fashion at low concentrations (4-100 ng/ml), at which concentrations it had little effect on the intracellular cAMP or cGMP level and depressed ALP activity. Moreover, PGF2 alpha depressed the stimulatory effect of PGE1 on ALP activity but did not affect the elevation of cAMP level by PGE1. The accumulation of inositol phosphates was greatly increased by PGF2 alpha in the concentration range effective in stimulating DNA synthesis, but was increased little by PGE1, suggesting that PGF2 alpha is a potent stimulator of phosphatidyl inositol turnover in the cells. In addition, A23187, a Ca ionophore, alone did not influence the DNA synthesis, but the effects of tetradecanoyl phorbol acetate, a direct activator of protein kinase C, were very similar to those of PGF2 alpha. Moreover, the stimulation of DNA synthesis or the inhibition of ALP activity by PGF2 alpha was partially counteracted by H-7, a strong inhibitor of protein kinase C. These results suggest that PGF2 alpha stimulates the proliferation of osteoblastic cells predominantly through the phosphatidyl inositol turnover system following in part the activation of protein kinase C. Our data presented here indicate that PGE1 and PGF2 alpha are closely involved in the differentiation and proliferation, respectively, of osteoblasts in vitro and that their action may be mediated by second messengers which differ from each other.

YGE1 is a yeast homologue of Escherichia coli grpE and is required for maintenance of mitochondrial functions.

The grpE gene is a heat shock gene of Escherichia coli whose product functions as a chaperone to (re)fold proteins. We found a yeast homologue of grpE and designated it YGE1. YGE1 can replace grpE in E. coli, indicating that YGE1 is a functional homologue of grpE. Deletion of YGE1 is lethal. During depletion of the Yge1 product, mitochondria are sequestered in mother cells thereby accumulating cells without mitochondria, suggesting that Yge1 protein plays a pivotal role in maintaining mitochondrial functions.

Age-related changes in the cerebral distribution of 99mTc-ECD from infancy to adulthood.

UNLABELLED: Although cerebral blood flow in infants differs from that in older individuals, the distribution of 99mTc-ethyl cysteinate dimer (ECD) in infants has not been well studied. This study compared 99mTc-ECD distribution in infants and children with that in young adults. METHODS: 99mTc-ECD SPECT was performed on 37 patients suspected of having epilepsy, ranging in age from 3 mo to 26 y. The patients were divided into two age-matched groups, a drug-free group (n = 19) and a drug-taking group (n = 18), according to their anticonvulsant medication status at the time of examination. 99mTc-ECD (100-740 MBq) was injected interictally, and SPECT data were acquired using a triple-head gamma camera. Mean whole-brain counts were obtained from 10 sequential SPECT images. Regions of interest were set bilaterally on five areas of the cerebral cortex and on the basal ganglia, thalamus and cerebellum. The brain perfusion index (BPI) was obtained as a ratio of the mean counts in each region of interest to the mean whole-brain counts. The relationship between BPI and age in each region in the drug-free and drug-taking groups was analyzed separately and together using linear regression. The relationship between five patient age groups (<1 y, n = 4; 1-4 y, n = 9; 5-9 y, n = 8; 10-15 y, n = 7; >15 y, n = 9) and BPI in each region was also examined using multiple comparison analyses. RESULTS: Significant positive correlations between BPI and age in the frontal cortex and cerebellum were confirmed in the drug-free group. Anticonvulsant drugs did not affect the regression lines of BPI in the frontal cortex and cerebellum. Significant differences in BPI between age groups were seen in the parietal cortex, frontal cortex, occipital cortex, basal ganglia, thalamus and cerebellum in all patients. CONCLUSION: Age-related changes in cerebral 99mTc-ECD distribution were confirmed and found to be unaffected by the administration of anticonvulsant drugs. 99mTc-ECD uptake in children and infants is different from cerebral blood flow glucose metabolism as previously reported, especially in the cerebellum.

Angiotensin II-stimulated vascular endothelial growth factor expression in bovine retinal pericytes.

PURPOSE: Angiotensin II (AII) has been shown to play a role in many vascular diseases. In the study described, the effect of AII on vascular endothelial growth factor (VEGF) expression and related intracellular signaling mechanism was investigated in bovine retinal microcapillary pericytes. METHODS: Cultured bovine retinal microvascular endothelial cells and pericytes were prepared. VEGF expression was determined by Northern blot analysis and immunoprecipitation assay. Cell proliferation was assessed by DNA content growth assay. Reporter gene studies were performed to identify the AII responsible transcription-activating region of VEGF gene. RESULTS: Angiotensin II induced a significant increase in VEGF mRNA in a time- and dose-dependent manner. Angiotensin II type I receptor antagonist inhibited this effect. Angiotensin II activates the transcription of VEGF gene without changing the mRNA half-life, and the AII responsible region was found in the 5'-flanking region of the VEGF gene. Angiotensin II also increased the expression of c-fos and c-jun mRNA, and antisense oligonucleotides against c-Fos blocked the AII-induced VEGF mRNA expression. The conditioned media of AII-stimulated pericyte cultures had a growth-promoting effect on endothelial cells, and this effect was inhibited almost completely by VEGF neutralizing antibody. CONCLUSIONS: These findings suggest that AII might induce angiogenic activity through a paracrine function of VEGF in retinal microvascular cells.

Coexpression of VEGF receptors VEGF-R2 and neuropilin-1 in proliferative diabetic retinopathy.

PURPOSE: To elucidate vascular endothelial growth factor (VEGF)-mediated pathogenesis of fibrovascular proliferation in diabetic retinopathy. METHODS: Fibrovascular tissues were obtained at vitrectomy from 22 cases with proliferative diabetic retinopathy. The half-divided tissues were processed for reverse transcription-polymerase chain reaction (RT-PCR) analysis to examine the expression of VEGF isoforms and their receptors. Paraffin sections of the other half were used for immunohistochemistry for CD34, glial fibrillary acidic protein and VEGF, and in situ hybridization for VEGF. RESULTS: RT-PCR analysis demonstrated the expression of VEGF receptors VEGF-R1, VEGF-R2, and neuropilin-1 in 12, 14, and 14 of 22 cases, respectively. Notably, VEGF-R2 and neuropilin-1 were simultaneously expressed in the identical 14 tissues. The isoform VEGF121 was constitutively expressed in all the tissues examined, whereas the expression of VEGF165 was confined to the 7 tissues that also expressed VEGF-R2 and neuropilin-1. The vascular density of fibrovascular tissues evaluated by immunohistochemistry for CD34 was significantly higher in the cases with the expression of VEGF-R2 and neuropilin-1 than in those without their expression (P < 0.01), whereas VEGF-R1 expression had no such relationship with the vascular density. The fibrovascular tissues that expressed VEGF165 together with VEGF-R2 and neuropilin-1 were found in significantly younger patients (P < 0.01). In situ hybridization and immunohistochemical studies demonstrated that glial cells in the fibrovascular tissues express and produce VEGF. CONCLUSIONS: Coexpression of VEGF-R2 and neuropilin-1 is suggested to facilitate fibrovascular proliferation in diabetic retinopathy.

Expression of Leu M1 antigen on a monoclonal B cell line established from a patient with rheumatoid arthritis.

The purpose of this study is to show that anti-Leu M1 antibody (anti-CD15), which has different staining characteristics in lymphoid and non-lymphoid cells, reacted against the surface antigen of a defined monoclonal B cell line. This antibody recognizes the sugar moiety, lacto-N-fucopentaose (LNF-III), which is linked to the cell membrane protein in several kinds of cells, but not in B cells. However, a human monoclonal B-cell line (TKS-1) which was established from the peripheral blood of a patient with rheumatoid arthritis, expressed the Leu M1 antigen spontaneously. The analysis of surface markers using a fluorescence-activated cell sorter (FACS) has revealed that the surface markers of TKS-1 were anti-mu, delta, kappa, HLA-DR, DQ, Leu 12 (CD19) and Leu M1 (CD15). TKS-1 cells were not reactive with any of the following antibodies: anti-OK M1 (CD11b), Leu M2, Leu M3 (CD14), Leu M4, Leu 1 (CD5), Leu 2 (CD8), Leu 3 (CD4), Leu 4 (CD3), Leu 7 and Leu 11 (CD16). In addition, TKS-1 was positive to Epstein-Barr nuclear antigen, weakly positive to non-specific esterase without staining inhibition by NaF, and negative to peroxidase. TKS-1 cells produced IgM in the culture supernatant and have kappa-light chain rearrangement in its DNA. As shown in other studies, distribution of Leu M1 is very wide. This antigen is not a specific immunodiagnostic marker to distinguish the cell type. We conclude that it is possible to express Leu M1 antigen on the membrane of a B-cell lineage cell.

Permissive role of L-thyroxine in induction of pancreatic amylase by cortisol in neonatal rats.

The effect of L-thyroxine (T4) on amylase activity in the developing rat pancreas has been investigated. Administration of T4 (0.2 microgram/g body wt) alone to intact rats on days 5-10 after birth did not induce pancreatic amylase but the enzyme was induced significantly by daily injection of cortisol (10 microgram/g body wt) alone into intact rats over the same period. In thyroidectomized, adrenalectomized rats pancreatic amylase was not induced by the injection of cortisol alone but it was induced by the administration of cortisol plus T4. Increase in enzyme activity was much less in thyroidectomized animals than in intact animals. These results suggested that T4 does not have a direct effect in increasing pancreatic amylase activity but plays a permissive role in increasing enzyme activity.

L-Thyroxine, cortisol and diet affect the level of amylase in the parotid gland of developing rats.

The effects of cortisol (10 microgram/g body weight) and L-thyroxine (T4; 0.2 microgram/g body weight) on the activity of parotid gland amylase in young rats were investigated. Administration of cortisol or T4 for 5 consecutive days from day 5 after birth caused the precocious appearance of amylase, T4 having almost twice the effect of cortisol. Cortisol and T4 did not have synergistic effects. In thyroidectomized-adrenalectomized rats, T4 increased amylase activity but cortisol did not. The increase in enzyme activity after day 20 was much less in rats thyroidectomized on day 10 than in rats adrenalectomized on day 10. These results suggest that T4 has a direct effect on the early increase of amylase activity (days 15-25) and that the action of glucocorticoid requires the presence of endogenous thyroid hormones. The hormone-induced level of amylase in intact rats was less than that of normal adult rats. Forced weaning of intact rats resulted in a further increase in amylase activity, suggesting that further amylase accumulation (after day 25) may be due to dietary factors.

Inhibitory effect of phenothiazine derivatives on bone in vivo and osteoblastic cells in vitro.

We tested the inhibitory effects of phenothiazine derivatives on bone in vivo and osteoblastic cells in vitro. Chlorpromazine (CPZ) and trifluoperazine (TFPZ) dose-dependently decreased alkaline phosphatase activity in calvariae of rats: half-maximal inhibitory effects of CPZ and TFPZ were at 2.0 and 4.0 mg/kg, respectively. These effects were more specific for calvaria and ileum than for liver and duodenum. CPZ inhibited the proliferation of osteoblastic clone MC3T3-E1 cells to a greater extent than that of liver epithelial clone RLC-18(4) cells in vitro. CPZ, TFPZ and perphenazine (PNZ) also affected rather specifically alkaline phosphatase activity and collagen synthesis and were not cytotoxic. These in vivo and in vitro findings suggest inhibitory effects on osteoblastic cell function(s). However, promethazine (PMZ) had little effect in vivo and in vitro. In addition, increases in serum calcium and phosphate induced by CPZ indicate its possible involvement in bone resorption.

Immunohistochemical detection of p21waf1/cip1/sdi1 and p53 proteins in formalin-fixed, paraffin-embedded tissue sections of colorectal carcinoma.

Wild-type p53 protein is a critical participant in G1 cell cycle arrest through the induction of waf1/cip1/sdi1 gene product p21, but mutant p53 proteins have lost transcriptional activity. To know whether the view obtained from studies using cultured cells is adaptable to human solid tumors, the authors immunohistochemically stained p21 and p53 in formalin-fixed, paraffin-embedded tissue sections from 67 cases of colorectal carcinoma. Of these, 54 cancers showed positive staining of p53, whereas p21-positive cells were identified in 46 tumors. However, the proportion of cells positive for these proteins varied considerably among cases, and moreover wide variation in p21 immunoreactivity was noted within the same tumor. Although there was an inverse relationship between p21 and p53 staining in tumors, namely, p21-positive cells were p53 negative and vice versa, an intermingling of tumor cells expressing concomitantly both genes was noted in 34 (51%) of 67 tumors. In these tumors, both proteins were usually moderately stained, but tumor cells intensely coexpressing both were found in one sample. This study supports the notion that although p21 expression is regulated by p53 under physiological conditions, it can be regulated by an additional pathway(s) in solid malignant tumors.

A patient with diabetes mellitus and severe arterial embolism.

An 89-year-old man with diabetes mellitus was admitted to the hospital because of a low-grade fever and a disturbance in consciousness. He had been diagnosed as having diabetes mellitus at the age of 22 years and had been taking oral hypoglycemic drugs for 16 years at least. A few days before admission, a loss of appetite was noticed by his family; he developed a stupor on the day of admission. On physical examination, his lower extremities were pale and his skin temperature was low. Laboratory tests showed an increase in his white blood cell count and his blood culture was positive for Staphylococcus aureus. An MRI showed that the abdominal aorta was totally occluded beneath the renal arteries, and no significant collateral circulation was observed. He was given antibiotics and anticoagulants, but his general condition continued to worsen. Laboratory tests showed renal failure and liver dysfunction, indicating multi-organ failure. On the 24th day of admission, he died of respiratory and heart failure. An autopsy showed the aorta to be totally occluded beneath the renal arteries by an embolism; atherosclerotic changes were rather mild. Acute plaque change on the surface of the aorta may have induced the sudden development of emboli in the aorta.