Pancreatic MRI associated with pancreatic fibrosis and postoperative fistula: comparison between pancreatic cancer and non-pancreatic cancer tissue.

AIM: To evaluate the potential value of magnetic resonance imaging (MRI) for predicting postoperative pancreatic fistula (POPF) in patients with pancreatic cancer (PC) and non-pancreatic cancer (non-PC). MATERIAL AND METHODS: This retrospective study was approved by the institutional review board and written informed consent was waived. Forty patients underwent pancreatoduodenectomy due to PC (n=31) and non-PC (n=9). The pancreas-to-muscle signal intensity ratio (SIR) on three-dimensional (3D)- fast field echo (FFE) T1-, in- and opposed-phase T1-, and T2-weighted images, as well as the apparent diffusion coefficient (ADC) value of the pancreas were measured. The frequency of POPF and MRI measurements were compared between patients with PC and non-PC. The MRI measurements were also compared with the grade of pancreatic fibrosis on pathological findings, fat deposition, and interstitial oedema. RESULTS: The frequency of POPF was significantly higher in patients with non-PC than in those with PC (p=0.0067), with an odds ratio of 10.4. The SIR on 3D-FFE T1-weighted images was significantly higher in patients with non-PC (p=0.0001) and those with POPF (p=0.017) than in those with PC and those without POPF, respectively. Multiple regression analysis demonstrated that the SIR on 3D-FFE T1-weighted image was independently associated with the grade of pancreatic fibrosis (p<0.0001). CONCLUSION: The frequency of POPF was significantly higher in patients with non-PC than in those with PC was inversely related to the grade of pancreatic fibrosis. The SIR on 3D-FFE T1-weighted image might be a potential imaging biomarker for predicting POPF.

Ultraconserved region-containing Transformer 2ß4 controls senescence of colon cancer cells.

Ultraconserved regions (UCRs) are >200 bp genomic segments with perfect human-to-rodent sequence identity. Transcribed UCRs constitute a new category of noncoding RNAs whose functions remain poorly understood. The human transformer 2ß (TRA2B) gene contains a 419-bp UCR spanning the 276-bp exon 2 and its neighboring introns. TRA2B exon 2 has premature stop codons, whereas an exon 2-containing splice variant (TRA2ß4) was expressed preferentially in the nuclei of human colon cancer cells. TRA2ß4 knockdown p53-independently stimulated CDKN1A transcription and increased p21, resulting in the appearance of senescent cells. Biotin pull-down and RNA immunoprecipitation assays revealed that TRA2ß4 interacted with Sp1 through a Sp1-binding sequence (485-GGGG-488) in a stem-loop structure of exon 2. Mutation of this sequence (485-AAGG-488) disrupted the stem-loop structure, blocked the interaction with Sp1 and increased CDKN1A transcription. Overexpression of TRA2ß4 significantly decreased CDKN1A mRNA levels and accelerated cell growth, but the introduction of the mutation in the Sp1-binding sequence completely canceled these effects. Taken together, TRA2ß4 may sequester Sp1 from occupying promoters of target genes including CDKN1A, promoting cell growth by interrupting the senescence-related gene expression program. This novel function of TRA2ß4 may uncover an oncogenic function of transcribed UCRs.

Transformer 2ß and miR-204 regulate apoptosis through competitive binding to 3' UTR of BCL2 mRNA.

RNA-binding proteins and microRNAs are potent post-transcriptional regulators of gene expression. Human transformer 2ß (Tra2ß) is a serine/arginine-rich-like protein splicing factor and is now implicated to have wide-ranging roles in gene expression as an RNA-binding protein. RNA immunoprecipitation (RIP) with an anti-Tra2ß antibody and microarray analysis identified a subset of Tra2ß-associated mRNAs in HCT116 human colon cancer cells, many of which encoded cell death-related proteins including Bcl-2 (B-cell CLL/lymphoma 2). Tra2ß knockdown in HCT116 cells decreased Bcl-2 expression and induced apoptosis. Tra2ß knockdown accelerated the decay of BCL2α mRNA that encodes Bcl-2 and full-length 3' UTR, while it did not affect the stability of BCL2ß mRNA having a short, alternatively spliced 3' UTR different from BCL2α 3' UTR. RIP assays with anti-Tra2ß and anti-Argonaute 2 antibodies, respectively, showed that Tra2ß bound to BCL2α 3' UTR, and that Tra2ß knockdown facilitated association of miR-204 with BCL2α 3' UTR. The consensus sequence (GAA) for Tra2ß-binding lies within the miR-204-binding site of BCL2 3' UTR. Mutation of the consensus sequence canceled the binding of Tra2ß to BCL2 3' UTR without disrupting miR-204-binding to BCL2 3' UTR. Transfection of an anti-miR-204 or introduction of three-point mutations into the miR-204-binding site increased BCL2 mRNA and Bcl-2 protein levels. Inversely, transfection of precursor miR-204 reduced their levels. Experiments with Tra2ß-silenced or overexpressed cells revealed that Tra2ß antagonized the effects of miR-204 and upregulated Bcl-2 expression. Furthermore, TRA2ß mRNA expression was significantly upregulated in 22 colon cancer tissues compared with paired normal tissues and positively correlated with BCL2 mRNA expression. Tra2ß knockdown in human lung adenocarcinoma cells (A549) increased their sensitivity to anticancer drugs. Taken together, our findings suggest that Tra2ß regulates apoptosis by modulating Bcl-2 expression through its competition with miR-204. This novel function may have a crucial role in tumor growth.

Homeodomain-interacting protein kinase 2 regulates DNA damage response through interacting with heterochromatin protein 1γ.

Homeodomain-interacting protein kinase 2 (HIPK2) is a potential tumor suppressor that has a crucial role in the DNA damage response (DDR) by regulating cell-cycle checkpoint activation and apoptosis. However, it is unclear whether HIPK2 exerts distinct roles in DNA damage repair. The aim of this study was to identify novel target molecule(s) of HIPK2, which mediates HIPK2-dependent DNA damage repair. HIPK2-knockdown human colon cancer cells (HCT116) or hipk1/hipk2 double-deficient mouse embryonic fibroblasts could not remove histone H2A.X phosphorylated at Ser139 (γH2A.X) after irradiation with a sublethal dose (10 J/m(2)) of ultraviolet (UV)-C, resulting in apoptosis. Knockdown of HIPK2 in p53-null HCT116 cells similarly promoted the UV-C-induced γH2A.X accumulation and apoptosis. Proteomic analysis of HIPK2-associated proteins using liquid chromatography-tandem mass spectrometry identified heterochromatin protein 1γ (HP1γ) as a novel target for HIPK2. Immunoprecipitation experiments with HCT116 cells expressing FLAG-tagged HIPK2 and one of the HA-tagged HP1 family members demonstrated that HIPK2 specifically associated with HP1γ, but not with HP1α or HP1ß, through its chromo-shadow domain. Mutation of the HP1box motif (883-PTVSV-887) within HIPK2 abolished the association. HP1γ knockdown also enhanced accumulation of γH2A.X and apoptosis after sublethal UV-C irradiation. In vitro kinase assay demonstrated an HP1γ-phosphorylating activity of HIPK2. Sublethal UV-C irradiation phosphorylated HP1γ. This phosphorylation was absent in endogenous HIPK2-silenced cells with HIPK2 3'UTR siRNA. Overexpression of FLAG-HIPK2, but not the HP1box-mutated or kinase-dead HIPK2 mutant, in the HIPK2-silenced cells increased HP1γ binding to trimethylated (Lys9) histone H3 (H3K9me3), rescued the UV-C-induced phosphorylation of HP1γ, triggered release of HP1γ from histone H3K9me3 and suppressed γH2A.X accumulation. Our results suggest that HIPK2-dependent phosphorylation of HP1γ may participate in the regulation of dynamic interaction between HP1γ and histone H3K9me3 to promote DNA damage repair. This HIPK2/HP1γ pathway may uncover a new functional aspect of HIPK2 as a tumor suppressor.

Downregulation of serine/arginine-rich splicing factor 3 induces G1 cell cycle arrest and apoptosis in colon cancer cells.

Serine/arginine-rich splicing factor 3 (SRSF3) likely has wide-ranging roles in gene expression and facilitation of tumor cell growth. SRSF3 knockdown induced G1 arrest and apoptosis in colon cancer cells (HCT116) in association with altered expression of 833 genes. Pathway analysis revealed 'G1/S Checkpoint Regulation' as the most highly enriched category in the affected genes. SRSF3 knockdown did not induce p53 or stimulate phosphorylation of p53 or histone H2A.X in wild-type HCT116 cells. Furthermore, the knockdown induced G1 arrest in p53-null HCT116 cells, suggesting that p53-dependent DNA damage responses did not mediate the G1 arrest. Real-time reverse transcription-polymerase chain reaction and western blotting confirmed that SRSF3 knockdown reduced mRNA and protein levels of cyclins (D1, D3 and E1), E2F1 and E2F7. The decreased expression of cyclin D and E2F1 likely impaired the G1-to-S-phase progression. Consequently, retinoblastoma protein remained hypophosphorylated in SRSF3 knockdown cells. The knockdown also induced apoptosis in association with reduction of BCL2 protein levels. We also found that SRSF3 knockdown facilitated skipping of 81 5'-nucleotides (27 amino acids) from exon 8 of homeodomain-interacting protein kinase-2 (HIPK2) and produced a HIPK2 Δe8 isoform. Full-length HIPK2 (HIPK2 FL) is constantly degraded through association with an E3 ubiquitin ligase (Siah-1), whereas HIPK2 Δe8, lacking the 27 amino acids, lost Siah-1-binding ability and became resistant to proteasome digestion. Interestingly, selective knockdown of HIPK2 FL induced apoptosis in various colon cancer cells expressing wild-type or mutated p53. Thus, these findings disclose an important role of SRSF3 in the regulation of the G1-to-S-phase progression and alternative splicing of HIPK2 in tumor growth.

Periodontitis-associated up-regulation of systemic inflammatory mediator level may increase the risk of coronary heart disease.

BACKGROUND AND OBJECTIVE: Although an elevation in the concentration of high-sensitivity C-reactive protein (hs-CRP) as a result of periodontal infection may account for an increased risk of developing coronary heart disease (CHD), the effect of periodontal infection on the level of hs-CRP in an otherwise healthy Japanese population has not yet been reported. The aim of the present study was to confirm, on a larger scale, our previous pilot study findings that both chronic periodontitis and subsequent periodontal treatment alter the serum levels of C-reactive protein (CRP), interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha). MATERIAL AND METHODS: The concentrations of serum hs-CRP, IL-6 and TNF-alpha were measured in 78 periodontitis patients at baseline and at re-assessment, and in 40 periodontally healthy subjects at the time of examination. RESULTS: The concentrations of hs-CRP and IL-6 in the sera of periodontitis patients were significantly higher than those in control subjects. By contrast, the concentration of TNF-alpha was significantly lower in periodontitis patients than in control subjects. Whereas periodontal treatment decreased the levels of serum hs-CRP and IL-6, no such effect was observed for TNF-alpha. When the patients were subdivided into four groups according to their initial concentration of hs-CRP, only the CRP and IL-6 concentrations of the highest quartile group showed a significant reduction following periodontal treatment. No significant difference in the initial clinical parameters was observed in any quartile. CONCLUSION: Although periodontal infection does affect the concentration of hs-CRP and IL-6 in serum, a subgroup of patients exist who are highly susceptible to an increased risk of CHD associated with periodontitis, suggesting that there may be subjects who have an elevated risk of CHD independent of susceptibility to periodontal tissue destruction per se.

Hypoglycaemia induced by Trichinella infection is due to the increase of glucose uptake in infected muscle cells.

The present study investigates how Trichinella infection induces host hypoglycaemia and explores a potential relationship between infection and the insulin signalling pathway. The results showed that mice infected with Trichinella spiralis or Trichinella pseudospiralis exhibited a temporary decrease in blood glucose level between 8 and 28 days p.i. and the kinetics of the glucose levels corresponded to the process of muscle larval growth and development. Histochemical results showed that glycogen accumulation increased in infected muscle cells during the period of hypoglycaemia. Analysis of gene expression profiles with quantitative PCR demonstrated that insulin signalling pathway-related genes, such as insulin receptor (IR), insulin receptor substance 1 (IRS-1), IRS-2, phosphatidylinositol 3-kinase (PI3-K) and V-akt murine thymoma viral oncogene homologue 2 (Akt2) were up-regulated in infected muscle cells during infection and these expression changes correlated with the kinetics of blood glucose level, glycogen accumulation and the process of larval growth and development in infected muscle cells. Western blot analysis clarified that the expression of IR and Akt2 proteins increased in muscle tissues infected with both species of Trichinella. This study suggests that hypoglycaemia induced by Trichinella infection is the result of an increase in glucose uptake by infected muscle cells via up-regulation of insulin signalling pathway factors.

Quantitative messenger RNA expression of Toll-like receptors and interferon-alpha1 in gingivitis and periodontitis.

INTRODUCTION: In addition to bacteria, viruses have been reportedly implicated in periodontitis. However, the available data are confined to Toll-like receptor 2 (TLR2) and TLR4, which recognize bacterial products in periodontitis. In the present study, we investigated the expression levels of TLR5, -7, and -9 messenger RNAs (mRNAs) in addition to those of TLR2 and -4, and compared gingivitis and periodontitis. Interferon-alpha1 (IFN-alpha1), which is important for the antiviral response, was also compared. METHODS: Gene expression was analyzed using quantitative real-time polymerase chain reaction for 59 periodontitis and 27 gingivitis tissue samples together with viral serology in some patients. The presence of plasmacytoid dendritic cells (pDCs), a robust producer of IFN-alpha, was immunohistochemically analyzed in an additional seven periodontitis and two gingivitis specimens. RESULTS: The expression levels of TLR2, -4, -7, and -9 were significantly higher in periodontitis lesions than gingivitis lesions. The expression level of TLR5 was comparable to levels of TLR2 and -4; however, no significant difference was found between gingivitis and periodontitis. Although the expression of IFN-alpha1 mRNA was higher in periodontitis lesions compared with gingivitis lesions, the level was quite low. Only a few pDCs were found in some periodontitis specimens. No difference was found for antibody-positivity between gingivitis and periodontitis. CONCLUSION: This is the first study to show that a variety of TLRs are up-regulated in periodontitis lesions compared with gingivitis lesions, suggesting that diverse microbial and possibly viral antigens are involved in the pathogenic mechanisms for periodontal diseases. However, the ligands recognized by the various TLRs in periodontal lesions remain to be determined.

Relationship of periodontal infection to serum antibody levels to periodontopathic bacteria and inflammatory markers in periodontitis patients with coronary heart disease.

Several reports have demonstrated a possible association of periodontal infections with coronary heart disease (CHD) by elevated antibody titre to periodontopathic bacteria in CHD patients compared with non-diseased controls. Although each periodontopathic bacterium may vary in virulence for periodontitis and atherosclerosis, antibody response to multiple bacteria in CHD patients has not been understood fully. Therefore, serum levels of antibody to 12 periodontopathic bacteria together with other atherosclerotic risk markers were compared among 51 patients with CHD, 55 patients with moderate to severe chronic periodontitis and 37 healthy individuals. The antibody response was the most prevalent for Porphyromonas gingivalis, a major causative organism, in CHD as well as periodontitis patients. However, antibody positivity was different between CHD and periodontitis if the response was analysed for two different strains of P. gingivalis, namely FDC381 and Su63. While periodontitis patients were positive for both P. gingivalis FDC381 and Su63, a high frequency of antibody positivity for P. gingivalis Su63 but not for FDC381 was observed in CHD patients. The results indicate that the presence of particular periodontopathic bacteria with high virulence may affect atherogenesis. Identifying the virulence factors of P. gingivalis Su63 may gain insight into the new therapeutic modality for infection-induced deterioration of atherosclerosis.

Balance of inflammatory response in stable gingivitis and progressive periodontitis lesions.

The balance between inflammatory mediators and their counter-regulatory molecules may be crucial for determining the outcome of immune pathology of periodontal diseases. Based on clinical and immunological findings, the immune response in stable gingivitis lesion is supposed to be in balance, whereas the response is skewed towards the predominance of proinflammatory reactivity in progressive periodontitis lesion. However, this hypothesis has not been verified. Therefore, the aim of this study was to compare the gene expression profile of inflammatory mediators including proinflammatory cytokines and other inflammatory molecules, and anti-inflammatory cytokines by using quantitative real-time polymerase chain reaction in gingivitis and periodontitis lesions showing distinct clinical entities. For inflammatory mediators, interleukin (IL)-1beta, interferon (IFN)-gamma and receptor activator of nuclear factor (NF)-kappaB ligand tended to be higher in periodontitis, whereas tumour necrosis factor (TNF)-alpha and IL-12 p40 showed no difference. Heat-shock protein 60 (HSP60) expression was up-regulated significantly in periodontitis. For anti-inflammatory cytokines, transforming growth factor (TGF)-beta1 expression tended to be higher in periodontitis compared with gingivitis, whereas no difference was observed for IL-10 and IL-4. These findings support further our previous finding that autoimmune response to HSP60 may exert in periodontitis lesion, and suggest that perhaps subtle differences in the balance of cytokines may result in different disease expression.

Effect of tumor necrosis factor-alpha on insulin signal transduction in rat adipocytes: relation to PKCbeta and zeta translocation.

Although much evidence has been accumulated suggesting that tumor necrosis factor-alpha (TNF-alpha) is an important mediator of insulin resistance, the precise mechanism involved is still unclear. Recently, it has been reported that insulin-induced glucose uptake is mediated by activation of second messengers such as insulin receptor substrate 1 (IRS-1), phosphatidylinositol 3-kinase (PI3K), and diacylglycerol (DG)-protein kinase C (PKC). We have examined the effect of TNF-alpha on insulin-induced glucose uptake and activations of tyrosine kinase, IRS-1, PI3K and PKC in rat adipocytes. Pretreatment with 0.1-100 nM TNF-alpha for 60 min resulted in a significant decrease in 10 nM insulin- or 1 microM 12-O-tetradecanoyl phorbol-13-acetate (TPA)-induced [3H]2-deoxyglucose uptake without affecting basal glucose uptake. 10 nM insulin-stimulated activation of tyrosine kinase, IRS-1 and PI3K was suppressed by preincubation with 0.1-10 nM TNF-alpha for 60 min. 10 nM TNF-alpha pretreatment also suppressed 10 nM insulin- and 1 microM TPA-induced increases in membrane-associated PKCbeta and PKCzeta. Furthermore, 10 nM TNF-alpha, by itself, altered PKCbeta translocation from the membrane to cytosol. These results suggest that TNF-alpha inhibits insulin-stimulated activation of both the tyrosine kinase-IRS-1-PI3K-PKCzeta pathway and DG-PKC pathway. Finally, TNF-alpha contributes to insulin resistance in rat adipocytes.

Inhibitory effect of glyburide on thrombin-induced platelet aggregation and phosphoinositide metabolism in normal human platelets.

We previously reported the effects of diet, sulphonylureas or insulin on thrombin-induced platelet aggregation, phosphoinositide metabolism and protein phosphorylation in non-insulin-dependent diabetes mellitus (NIDDM) patients. To clarify the mechanism of glyburide and insulin on platelet function, here we studied the in vitro effects of glyburide and insulin on thrombin-induced metabolic changes using normal human platelets. Platelet aggregation stimulated with <0.5 U/ml thrombin, 0.75-3 microM adenosine diphosphate (ADP) or 1 microg/ml collagen was significantly lower in glyburide-treated platelets, but not in insulin-treated platelets, than in untreated ones (control). Thrombin-induced incorporation of 32P radioactivity into phosphatidic acid (PA) in glyburide-treated platelets was lower than that in control but not in insulin-treated platelets. Phosphorylated proteins of platelets induced by thrombin and 12- O -tetradecanoylphorbol 13-acetate (TPA) in glyburide-treated platelets were suppressed, but not in insulin-treated platelets, compared with control. These results suggest that glyburide induces suppression of thrombin-induced activation of phospholipase C, which mediates hydrolysis of PIP and PIP(2) and production of PA, and subsequently inhibits platelet aggregation.

Glucose- and phorbol ester-induced insulin secretion in human insulinoma cells--association with protein kinase C activation.

This study examined the effect of glucose and 12-O-tetradecanoylphorbol-13-acetate (TPA) on insulin secretion in isolated human insulinoma cells. In addition, we analyzed conventional PKC alpha and beta activation in the membrane fractions, respectively. Treatment with 5 mM and 20 mM glucose for 5 min and 20 min resulted in 6-7-fold increases in insulin secretion, and treatment with 1 microM TPA for 5 min also resulted in 3-fold increases in insulin secretion from the basal level. Immunoblot analysis of membrane fractions showed increases in PKC alpha and beta immunoreactivities after treatment with 5 mM, 20 mM glucose and 1 microM TPA. Translocations of PKC alpha after treatment with glucose and TPA were greater than those of PKC beta in membrane fractions. These results suggest that TPA independently provokes insulin secretion via PKC activation and that PKC alpha and beta activation may be involved in insulin secretion in human insulinoma cells.

Differential effect of the antidiabetic thiazolidinediones troglitazone and pioglitazone on human platelet aggregation mechanism.

Troglitazone and pioglitazone, antidiabetic thiazolidinediones, are known to improve insulin resistance. However, the effect of these drugs on platelet aggregation remains unclear. The chemical structure of troglitazone contains vitamin E. Accordingly, we studied the effect of troglitazone, pioglitazone, and vitamin E on thrombin-induced platelet aggregation, metabolism of phosphoinositide, protein phosphorylation, protein kinase C (PKC)-alpha and -beta, and phosphatidylinositol (PI) 3-kinase activation in vitro in human platelets. Maximum platelet aggregation by ADP, collagen, and thrombin decreased in the presence of 0.1-1 micromol/l troglitazone and 500 nmol/l vitamin E for 60 min compared with controls. However, pioglitazone did not inhibit ADP-, collagen-, or thrombin-induced platelet aggregation. Pretreatment with troglitazone and vitamin E, but not with pioglitazone, resulted in decreases in thrombin-induced phosphatidic acid production, hydrolysis of phosphatidylinositol 4,5-bisphosphate by phospholipase C, and 47-kDa protein phosphorylation. Thrombin-induced PKC-alpha and -beta activation in membrane fraction was suppressed by pretreatment with troglitazone and vitamin E, but not with pioglitazone. Separately, troglitazone and pioglitazone stimulated PI 3-kinase activity, but thrombin-induced PI 3-kinase activation was suppressed by pretreatment with troglitazone and pioglitazone for 60 min. These results suggest that troglitazone and vitamin E, but not pioglitazone, have a potent inhibitory effect on platelet aggregation via suppression of the thrombin-induced activation of phosphoinositide signaling in human platelets. Finally, the chemical structure of vitamin E may contribute to the inhibitory effect of troglitazone on platelet aggregation in human platelets.

ACTH-induced cortisol secretion is mediated by cAMP and PKC in various adrenocortical adenomas.

We examined the role of PKC in cortisol secretion from adrenocortical adenomas. Isolated cells were prepared from aldosterone producing adenoma (APA, n=5), APA complicated with pre-clinical Cushing's syndrome (APA+PC, n=1), PC (n=2), and cortisol producing adenoma (CPA, n=5). They were stimulated with 100 nM ACTH, 1 microM forskolin (FS), 1 microM tetradecanoyl phorbol 13-acetate (TPA), and 100 nM angiotensin II (AngII). ACTH was most potent to secret cortisol. FS also stimulated cortisol secretion, but did less-potently. TPA and AngII also stimulated cortisol secretion significantly in cells from CPA. Furthermore, ACTH- and TPA-induced PKCalpha and beta translocations from cytosol to membrane were observed in adenoma cells from APA+PC, PC, and CPA. In conclusion, it was suggested that ACTH-induced cortisol secretion may be mediated by both PKC and protein kinase A in adrenocortical adenomas, and that PKC-mediated signal transduction may be involved in ACTH-induced cortisol secretion in CPA.

Insulin regulated PKC isoform mRNA in rat adipocytes.

Insulin and 12-O-tetradecanoyl phorbol-13-acetate (TPA) induce both glucose uptake and translocation of protein kinase C (PKC) from cytosol to membrane in insulin-sensitive tissues as previously reported by several investigators. We examined insulin-mediated PKC beta I, beta II, and epsilon translocation from cytosol to cytoskeleton, and expression of PKC alpha, beta I, beta II, gamma, and epsilon isoforms using the reverse transcription polymerase chain reaction (RT-PCR) method during treatment with insulin for 240 min in rat adipocytes. Insulin-induced increases in PKC beta I, beta II, and epsilon were greater in the cytoskeleton fraction than those in the membrane fraction. Insulin induced time-dependent increases in PKC alpha, gamma, epsilon and zeta mRNA levels for up to 240 min (555%, 117%, 236% and 138% increase, respectively). TPA also induced time-dependent increases in PKC alpha and gamma (34% and 500% increase, respectively) but not in PKC zeta. However, PKC beta I mRNA was decreased for up to 60 min and then maintained at under the basal level during stimulation with insulin and TPA. On the other hand, PKC beta II mRNA was markedly increased for up to 240 min. These results suggest that insulin-regulated PKC alpha, gamma and epsilon mRNA levels and PKC beta mRNA alternative splicing may occur in rat adipocytes.

Successful percutaneous transluminal angioplasty of the intracranial vertebral artery 1 month after total occlusion--case report.

A 51-year-old male suffering from recurrent cerebral ischemia due to total occlusion of the bilateral intracranial vertebral arteries more than 1 month old was successfully treated by percutaneous transluminal angioplasty (PTA). The totally occluded portion from the right intracranial vertebral artery to the basilar artery was adequately dilated. Follow-up angiography approximately 3 months after angioplasty demonstrated no evidence of restenosis. His symptoms have not recurred. PTA is potentially a much less invasive and safer reconstruction than bypass surgery for total occlusions of the intracranial vertebral arteries less than 3 months old.

ACTH and phorbol ester stimulated redistribution of protein kinase C in human cortisol-producing adrenal adenoma.

We studied the steroidogenetic action and concomitant subcellular redistribution of protein kinase C (PKC) in cortisol hypersecreting adrenal adenoma cells obtained from two patients with Cushing's syndrome. Isolated adenoma cells were treated with 10(-9) M and 10(-6) M 12-O-tetradecanoyl phorbol-13-acetate (TPA) or 10(-6) M ACTH. Treatment of the isolated adenoma cells with TPA resulted in cortisol secretion equivalent to that with ACTH-treatment. Immunoblot analysis of PKC during treatment with ACTH or TPA showed that PKC beta translocated from cytosol to membrane. A small amount of PKC alpha, but not membrane-associated PKC alpha, was detectable in the cytosolic fraction. It appeared that TPA-induced cortisol secretion mimicked ACTH-induced cortisol secretion, and that PKC beta translocated from cytosol to membrane on stimulation by both ACTH and TPA. We suggested that ACTH-induced cortisol secretion in human cortisol hypersecreting adrenal adenoma is mediated by PKC beta activation.

[A study of anti-pituitary-antibodies in patients with hypopituitarism and their hereditary background].

Recently several types of anti-pituitary-antibodies (APA) have been found in patients with pituitary disorders including hypopituitarism and diabetes insipidus, and in postpartum women. However, the pathophysiological role(s) of APA still remains unknown. In order to elucidate the clinical significance of APA, longitudinal follow-up and family study of APA in patients with hypopituitarism were performed. APA in serum was examined in a total of 11 patients with various types of hypopituitarism (7 of isolated ACTH deficiency, 1 of partial hypopituitarism, 3 of Sheehan's syndrome, 6 males and 5 females). Chronic thyroiditis was associated in 3 out of 7 patients with isolated ACTH deficiency, and empty sella was found in each one patient with isolated ACTH deficiency and partial hypopituitarism, and in 3 patients with Sheehan's syndrome. APA was examined on 2 or 3 occasions at more than a 6 month interval (longitudinal study). In 5 patients, their 16 family members were examined for the presence of APA, and pituitary functions were evaluated in 3 out of 7 family members with positive APA (family study). For pituitary function tests, arginine infusion test, TRH, LH-RH or CRH test and insulin tolerance test were performed. APA reacting to rat pituitary cytoplasmic antigens (pituitary cell antibodies: PCA) and APA reacting to rat GH3 cells and/or mouse AtT20 cells surface antigens (pituitary cell surface antibodies: PCSA) were assayed with indirect immunofluorescence method. At the initial examination, 6 out of 11 patients (55%) showed positive APA. The patients were divided into 3 subgroups according to the longitudinal study: the group with disappearance of initially positive APA (3 patients), the group with altered titers or types of initially positive APA (3 patients), and the group with sustained initially negative APA (4 patients). No effects of replacement therapy on the alterations of APA were observed. In 16 family members of 5 patients (each 1 with partial hypopituitarism and isolated ACTH deficiency syndrome, and 3 with Sheehan's syndrome), APA in their sera were investigated. Seven out of 16 members (44%) showed positive APA. Among 6 first-degree relatives of 16 family members, both or either one of APA and PCSA were positive in 4 (67%). Out of 10 of their second- or third-degree relatives, 3 (30%) were positive for PCA or PCSA. All of 3 relatives with positive APA studied showed mild pituitary hypofunction without any clinical manifestations.(ABSTRACT TRUNCATED AT 400 WORDS)

Anti-pituitary antibodies in patients with hypopituitarism and their families: longitudinal observation.

In an attempt to investigate the clinical significance of anti-pituitary antibodies in patients with hypopituitarism, anti-pituitary antibody in plasma was examined in 10 such patients (7 cases of isolated ACTH deficiency, 1 of partial hypopituitarism, and 2 of Sheehan's syndrome), on two or three occasions with an interval of more than 6 months (longitudinal study). In a total of 16 relatives of these 4 patients (2 cases of Sheehan's syndrome, one in each of partial hypopituitarism and isolated ACTH deficiency) and one patient not involved in the longitudinal study, anti-pituitary antibodies were also examined (family study). Anti-pituitary antibodies reacting with rat pituitary cytoplasmic antigens (pituitary cell antibodies: PCA) and pituitary cell surface antibodies (PCSA) reacting with GH3 cells and/or AtT-20 cells were measured with indirect immunofluorescence. The longitudinal study revealed the disappearance of antibodies in 3 patients, 2 PCA positive and one both PCA and PCSA positive. In 3 patients, altered antibody titers or a newly appearing antibody were found during the follow-up period. In 4 patients, the pituitary antibodies were negative during the follow-up periods. Of 16 family members studied, positive PCA was found in 3 members (2 in the families of patients with PCA positive Sheehan's syndrome, and 1 in the family of the patients with PCA positive partial hypopituitarism). Positive PCSA was found in 4 members (one in each of families of patients with partial hypopituitarism and isolated ACTH deficiency and of two cases of Sheehan's syndrome), and weakly positive PCSA was found in one family member of a patients with PCA positive Sheehan's syndrome.(ABSTRACT TRUNCATED AT 250 WORDS)