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In order to elucidate the influence of dietary constituents on colonic intracellular signal transduction, the effect of different fats on rat colonic epithelial protein kinase C (PKC) alpha (classical), delta (novel) and lambda-zeta (atypical) expression was determined in carcinogen-treated animals. Sprague-Dawley rats were provided with one of two fats (corn oil and fish oil); plus or minus the carcinogen azoxymethane (AOM) and killed at two time points (15 and 37 weeks) in a 2x2x2 factorial design. At 5 and 6 weeks of age, animals were injected s.c. with either AOM at a dose of 15 mg/kg body weight or saline once a week for 2 weeks and continued on the same diet until termination of the study. At 15 and 37 weeks after the second injection, 10 rats from each treatment group were killed. Colonic PKC alpha, delta and lambda-zeta steady-state protein and mRNA levels were determined using immunoblotting and relative quantitative polymerase chain reaction, respectively. Colonic mucosa from rats injected with AOM had significantly suppressed membrane and cytosolic PKC alpha and cytosolic lambda-zeta protein levels (P < 0.05) as compared to saline-injected control animals at both time points. In contrast, rats fed fish oil diets had significantly higher (P < 0.05) cytosolic PKC delta and lambda-zeta protein levels relative to animals fed corn oil diets. However, the effect of diet and AOM on the steady-state expression of PKC alpha, delta and zeta mRNA was not consistent with changes in the respective isozyme protein levels, suggesting regulation at the post-transcriptional level. These data demonstrate that dietary fish oil blocks the carcinogen-induced decrease in the steady-state levels of colonic mucosal PKC delta and lambda-zeta, which may in part explain why this fat source protects against colon cancer development.

In conclusion, dietary n-3 polyunsaturated fatty acids found in fish oil are capable of suppressing carcinogen-induced ras activation in the colon prior to overt neoplasia. This in turn blocks the oncogene driven increase in colonic diacylglycerol mass, preventing the persistent activation and chronic down-regulation of PKC isozymes, thereby maintaining tissue PKC levels. Since the maintenance of crypt PKC levels may sustain the homeostatic balance between cell proliferation, differentiation and apoptosis, the ability of dietary n-3 polyunsaturated fatty acids to block the carcinogen-induced decrease in steady-state levels of colonic mucosal PKC may in part explain why these fatty acids protect against colon tumorigenesis. Additional studies are required in order to elucidate the mechanisms by which select dietary lipids reduce colonic tumor incidence. This research focus is absolutely essential, because if we do not know why a dietary component is protective or promotive of cancer, then we have no right to attempt to modify eating behaviors.

Angelman syndrome (AS) is associated with maternal deletions of human chromosome 15q11-q13 and with paternal uniparental disomy for this region indicating that deficiency of an imprinted, maternally expressed gene within the critical interval is the likely cause of the syndrome. Although the gene for E6-AP ubiquitin-protein ligase (UBE3A) was mapped to the critical region for AS, evidence of expression from both parental alleles initially suggested that it was an unlikely candidate gene for this disorder. Because attempts to identify any novel maternally expressed transcripts were unsuccessful and because the UBE3A gene remained within a narrowed AS critical region, we searched for mutations in UBE3A in 11 AS patients without known molecular defects (large deletion, uniparental disomy, or imprinting mutation). This analysis tested the possibility that deficiency of an undefined, maternally expressed transcript or isoform of the UBE3A gene could cause AS. Four mutations were identified including a de novo frameshift mutation and a de novo nonsense mutation in exon 3 and two missense mutations of less certain significance. The de novo truncating mutations indicate that UBE3A is the AS gene and suggest the possibility of a maternally expressed gene product in addition to the biallelically expressed transcript. Intragenic mutation of UBE3A in AS is the first example of a genetic disorder of the ubiquitin-dependent proteolytic pathway in mammals. It may represent an example of a human genetic disorder associated with a locus producing functionally distinct imprinted and biallelically expressed gene products.

Elucidation of the mechanism(s) by which dietary fish oil, enriched in eicosapentaenoic acid (EPA, 20:5(n-3)] and docosahexaenoic acid [DHA, 22:6(n-3)], suppresses the inflammatory process is essential in maximizing this potentially therapeutic effect. Murine T-lymphocyte function and signal transduction were examined in response to a low fat, short term diet enriched in highly purified EPA or DHA ethyl esters. For 10 d, mice were fed comparable diets containing either 3% safflower oil ethyl esters (SAF), 2% SAF + 1% arachidonic acid triglyceride (AA), 2% SAF + 1% EPA, or 2% SAF + 1% DHA. Concanavalin A-induced T-lymphocyte proliferation in splenocyte cultures was significantly suppressed by dietary EPA and DHA while AA had no effect relative to the SAF control. The suppressed proliferative response in EPA- and DHA-fed mice was preceded temporally by a significant reduction in IL-2 secretion. Kinetics of mitogen-induced diacyl-sn-glycerol (DAG) and ceramide production did not differ significantly between SAF and AA diet groups. In contrast, DAG production was significantly suppressed in EP- and DHA-fed mice relative to the SAF and AA groups. The reduced DAG mass was paralleled by reduced ceramide mass following EPA and DHA feeding compared to the SAF and AA groups. Thus, low dose, short term dietary exposure to highly purified EPA or DHA appears to suppress mitogen-induced T-lymphocyte proliferation by inhibiting IL-2 secretion, and these events are accompanied by reductions in the production of essential lipid second messengers, DAG and ceramide.

There is great interest in utilizing butyrate as a chemotherapeutic agent. To elucidate its mechanism of action, the effect of butyrate on cAMP receptor protein kinase (PKA) activity in young adult mouse colon (YAMC) cells isolated from transgenic mice bearing a temperature sensitive mutation of the SV40 large T antigen gene was investigated. Conditionally immortalized cultures were plated at the permissive temperature (33 degrees C) or growth arrested by incubation at the nonpermissive temperature (39 degrees C). In addition, cells were incubated at 33 degrees C with or without 1 mmol/L butyrate for 24 h. Butyrate treatment reduced cell proliferation by 28% and enhanced apoptosis by 350% compared with cultures not exposed to butyrate. The PKA type I/II isozyme activity ratio was lower (P < 0.05) in cells incubated with butyrate. The relative level of PKA I isozyme was higher in proliferating cells at 33 degrees C (63% of total PKA), while the relative level of PKA II was higher in nonproliferating cells undergoing apoptosis at 39 degrees C (59% of total PKA). Neither incubation conditions (33 vs. 39 degrees C) nor butyrate treatment altered total PKA activity. When YAMC cells were incubated with 8-CI-cAMP, an activator of PKA II, growth was markedly inhibited in cells at both temperatures. Consistent with in vitro data, increased PKA I isozyme levels were associated with dysregulated growth in vivo. Specifically, the relative level of PKA I isozyme was three- to fivefold higher in rat colonic tumors compared with normal nontransformed colonic mucosa. These data indicate that the biological effects of butyrate on colonocyte proliferation and apoptosis are associated with changes in PKA isozyme-dependent signal transduction, and the YAMC cell line is a relevant model to examine the molecular mechanisms by which dietary-derived factors affect relative cancer risk.

The effect of fat, fiber and carcinogen on colonic epithelial intracellular second messengers 1,2-diacyl-sn-glycerol (DAG), ceramide, and the steady-state level of phospholipase C (PLC-gamma1) was determined in 160 male Sprague-Dawley rats (10 rats per group). The study was a 2 x 2 x 2 x 2 factorial design with two types of fat (corn oil or fish oil), two types of fiber (cellulose or pectin), two injected subgroups (with or without azoxymethane (AOM), and two time points (15 and 37 weeks). At the final time point (37 weeks) there were an additional 20 rats per diet in each of the carcinogen-treated groups for tumor analyses only (n = 80), for a total of 240 animals in the entire study. At each time point (15 and 37 weeks), 80 rats were killed and colonic mucosa obtained for DAG, ceramide and PLC-gamma1 assays. At the first time point (15 weeks), there was no microscopic evidence of tumors. At the final time point (37 weeks), fish oil resulted in a lower proportion of animals with adenocarcinomas relative to corn oil feeding (56.1 % versus 69.6 %, P < 0.05). There was no significant main effect of fiber on the percentage of animals with tumors. At 15 weeks post-injection, AOM injected animals fed corn oil-containing diets had a significantly (P < 0.001) higher DAG mass and steady-state levels of PLC-gamma1 compared with AOM-injected animals fed fish oil and saline injected rats on all diets. Animals fed corn oil diets also had a significantly (P < 0.01) elevated mucosal ceramide mass compared with fish oil fed animals. Moreover, rats injected with AOM had a significantly (P < 0.02) elevated colonic mucosal DAG/ceramide ratio versus saline injected animals. In contrast, dietary fiber had no effect on any of the parameters measured at 15 weeks. However, at 37 weeks post-injection, dietary fiber significantly altered DAG (P < 0.02), and PLC-gamma1 expression (P < 0.05) in the absence of an effect on tumor incidence. These data demonstrate that the ability of dietary fish oil to reduce experimental colon carcinogenesis may be mediated by changes in colonic intracellular mediators during the initial stages of tumorigenesis.

Reverse transcriptase (RT)-PCR is widely used to study gene transcription in many biological systems. Despite the development of a variety of procedures, quantification of RT-PCR products remains difficult, particularly when processing a large number of samples. Therefore, we developed a novel alternative PCR technique that we term "rapid competitive PCR" (RC-PCR), designed to study the relative expression of specific genes in a large number of small tissue biopsies. RC-PCR is characterized by measuring relative gene expression at the mRNA level of two or more samples with a nonradioactive assay based on competitive PCR amplification between identical sequences of internal standard and target cDNA. Only a single reaction tube per sample is used in this technique, and it was validated by comparing RC-PCR of protein kinase C zeta and alpha expression in rat colonic mucosa samples with competitive RT-PCR analysis (requiring 6-8 reaction tubes per sample). We conclude that RC-PCR is a simple, rapid, highly sensitive technique that is capable of detecting less than twofold differences in mRNA expression.

Skeletal muscle uses voltage sensors in the transverse tubular membrane that are linked by protein-protein interactions to intracellular ryanodine receptors, which gate the release of calcium from the sarcoplasmic reticulum. Here we show, by using voltage-clamped single fibres and confocal imaging, that stochastic calcium-release events, visualized as Ca2+ sparks, occur in skeletal muscle and originate at the triad. Unitary triadic Ca(2+)-release events are initiated by the voltage sensor in a steeply voltage-dependent manner, or occur spontaneously by a mechanism independent of the voltage sensor. Large-amplitude events also occur during depolarization and consist of two or more unitary events. We propose a 'dual-control' model for discrete Ca2+ release events from the sacroplasmic reticulum that unifies diverse observations about Ca(2+)-signalling in frog skeletal muscle, and that may be applicable to other excitable cells.

We have demonstrated previously the presence of classical (alpha), novel (delta and epsilon), and atypical (zeta) protein kinase C (PKC) isozymes in human and rat colonic mucosa (L. A. Davidson et al., Arch. Biochem. Biophys., 312:547-553, 1994). To gain insight into the functions of individual PKC isozymes in colonic epithelium in situ, we determined the localization of the major PKC isozymes expressed in normal rat colonic epithelial cells using in situ reverse transcription (RT)-PCR and immunohistochemistry (IH). Cytokeratin, a positive biological control known to be expressed in epithelial cells, was shown by in situ RT-PCR and IH to be expressed only in epithelial cells within the colonic crypt. PKC gamma, a negative control for the colon since it is expressed only in the central nervous system, was not detectable in colon sections by either methodology. In situ RT-PCR analysis revealed that PKC alpha, delta, epsilon, and zeta mRNAs are expressed in epithelial cells along the entire colonic crypt. In addition, PKC delta and zeta mRNA are expressed in the stromal layer. All four PKC isozymes in the colonic epithelial cells were also detected by IH. However, in general, isozyme protein expression was greater at the top of the crypt axis, associated primarily with cells having acquired a differentiated phenotype. These results suggest that PKC isozyme protein expression may be localized to mature differentiated cells at the top of the colonic crypt. Therefore, PKC isozyme-dependent signal transduction may play a role in colonic epithelial cell ontogeny along the colonic crypt axis.

Deaths from colon cancer number over 60,000 each year in the United States. Because early detection results in a high cure rate, development of noninvasive techniques for detection of colon cancer has received much interest. The ability to detect early changes in colonocyte genes and gene expression would provide valuable information. We have shown previously that alterations in protein kinase C (PKC) isoform expression are associated with changes in colonic cell proliferation, a key intermediate marker for the prediction of tumorigenesis. Here, we describe a method for the quantitative detection of mRNAs for select PKC isoforms isolated from rat feces containing exfoliated colonocytes. After total RNA extraction from fresh fecal material, polyadenylated RNA was selectively purified and quantitated with slot blotting and hybridization to oligodeoxythymidylic acid. Fecal polyadenylated RNA was used for semiquantitative (mimic) RT-PCR to quantitate PKC isoform mRNA expression. We detected mRNA for PKC-alpha, PKC-delta, PKC-epsilon, and PKC-sigma, but not for PKC-beta or PKC-gamma, which is consistent with the profile of isoforms detected previously in scraped colonic mucosa using immunoblot analysis. This noninvasive method, utilizing feces containing exfoliated colonocytes, is a sensitive noninvasive technique for quantitating luminal mRNAs. This provides a means to monitor gene expression of colonic epithelial cells, which may have predictive value in monitoring the neoplastic process.

To better understand the biochemical mechanisms by which select fats and fibers modulate colonic cell proliferation, we determined the profile of protein kinase C (PKC) isozymes and cell proliferation in rat proximal and distal colonic mucosa following diet manipulation, because enhanced cell proliferation has been correlated with colon cancer incidence. Rats were assigned to one of four diets (each with 15 g fat + 6 g fiber/100 g diet) for 3 wk: fiber-free fish oil (FF), fiber-free corn oil (FC), cellulose + corn oil (CC), or pectin + corn oil (PC). Stead-state levels of colonic mucosal cytosolic and membrane PKC isozymes were determined. In vivo cell proliferation was determined by bromodeoxyuridine incorporation into DNA. In addition, viable exfoliated colonic epithelial cells were isolated from feces using Percoll-bovine serum albumin gradients. We found that 1) proximal and distal colonic mucosa possessed different steady-state levels and relative proportions of PKC isozymes; 2) PKC alpha and delta expression were significantly greater in distal membrane of the PC-fed group compared with the other dietary groups; 3) the number of exfoliated cells per 4-h fecal collection generally was proportional to the diet-induced changes in cell proliferation (PC > FC > CC > FF). These data demonstrate that dietary treatment altered colonic PKC isozyme expression, with animals fed the fiber-containing diets generally expressing higher steady-state levels of PKC alpha and delta.

Labial salivary gland biopsy (LSG-B) along with Schirmer test and kerato-conjunctival staining with fluorescein dye was performed in 196 patients with suspected Sjogren's syndrome (SS). Of the 196 patients, 117 were found to have 2 or more lymphocytic focus scores (LFS/4mm2) on their LSG-B specimens. 92 of them having keratoconjunctivitis sicca (KCS) as well met the diagnostic criteria of SS. Among the cases (25) without KCS, 16 with another CTD were diagnosed as secondary SS. 9 cases were classified as probable primary SS in this study because they had clinical manifestations of SS. Of the 11 cases with KCS only, 6 with another CTD were diagnosed as secondary SS; 5 befitted primary SS for abnormal findings on sialography. We also found that lymphocytic infiltration in LSG-B was much more severe in primary SS than in secondary SS; this could be attributed to the use of immunotherapy for the accompanying CTD in secondary SS. There was good association in our study between the severity of lymphocytic infiltration and systemic involvement.

The protein kinase C (PKC) family of enzymes plays a key role in the regulation of cellular events, including cell proliferation and differentiation. Work from our laboratory has shown that the effects of dietary fat and fiber on colonic cell proliferation were positively correlated with membrane/cytosol PKC activity ratios (Chapkin et al., 1993, J. Nutr. 123, 649-655). The presence and subcellular distribution of specific PKC isoforms in rat and human colon were therefore determined in cytosolic and membrane extracts. Tissue extracts were probed with antibodies to individual PKC isoforms. PKC alpha, beta, delta, epsilon, and zeta were detected in both rat and human colonic mucosa, while PKC eta was detected in human colonic mucosa only. PKC alpha, beta, and zeta were predominantly localized in the cytosolic fraction, whereas the majority of PKC delta, epsilon, and eta were found in the membrane-associated fraction. Presence of mRNA for individual PKC isoforms was determined by reverse transcriptase PCR (RT-PCR). Using rat colonic mucosa, mRNA for PKC alpha, beta, delta, epsilon, eta, and zeta were detected by RT-PCR with identity confirmed by sequencing. The relative steady-state levels of PKC isoforms in human colon adenocarcinoma as compared with normal colonic mucosa were determined, with adenocarcinomas having higher amounts of cytosolic PKC beta, delta, epsilon, eta, and zeta. PKC isoforms were also detected in viable, exfoliated colonic cells isolated from human feces, demonstrating that this noninvasive method can be utilized to examine PKC expression in colonic cells. These results demonstrate that colonic mucosa expresses both calcium-dependent (classical) and calcium-independent (novel and atypical) PKC isoforms with distinct subcellular distributions for each. The dynamics of these PKC isoforms may have implications in the development of colon carcinogenesis.

Due to the numerous health benefits associated with consumption of antioxidants such as alpha-tocopherol and beta-carotene, an experiment was conducted to determine dietary levels that would significantly enhance their concentration in chicken egg yolks. In the experiment, 127 Single Comb White Leghorn laying hens were divided into treatment groups (n = 40 per treatment group) and fed diets containing 0, 50, 100, 200, or 400 mg/kg beta-carotene, dl-alpha-tocopheryl acetate, or their combination. Yolk alpha-tocopherol increased (P < .05) from the control level of 144 micrograms/g of yolk to 477 micrograms/g of yolk when 400 mg dl-alpha-tocopheryl acetate/kg of diet was supplemented. Yolk retinol levels increased (P < .05) from 11.6 micrograms/g of yolk in controls to 13.9 micrograms/g of yolk at 200 mg beta-carotene/kg of diet. beta-Carotene content in the yolk also increased (P < .05) from .14 micrograms/g of yolk in controls to 5.19 micrograms/g of yolk at 200 mg beta-carotene/kg of diet. Supplemental beta-carotene markedly decreased the yolk deposition of alpha-tocopherol when the two compounds were fed together. Egg production, egg weight, and egg yield were not affected by dietary supplementations. Although the data indicated that it is possible to significantly increase the concentration of all three compounds in chicken eggs, because of the relative expense involved it may not be commercially viable to increase egg yolk concentrations of beta-carotene or retinol by supplementing beta-carotene in the diet.

Hysteroscopic resection of submucous myomas was done in 27 patients with the Karl Storz equipment. Among them 20 were peduncular myomas, 5 of which dropped out of the external of cervix, 7 were sessile myomas. Under ultrasonography the largest sessile myoma was 5cm in diameter. The depth of the uterine cavity was less than 10cm in all cases. For the sessile submucous myomas, the surface vessels were electro-coagulated first, then the myomas were electro-resected chip-by-chip. Peduncular for the myomas, if the pedicle was larger than 1cm, it was electro-resected, if it was less than 1cm, the myoma was removed by grasping and twisting the pedicle. Hysteroscopic resection of submucous myomas under video camera monitor is safe, less traumatic and requires, shorter stay in hospital. There was no significant postoperative complications. Follow up for one to twenty-nine months showed satisfactory results.

This paper showed that in 110 cases of Sjogren's syndrome(SS),the caries incidence was high and was related to the salivary gland lesions.In comparison with 90 matched no SS group,the incidence of caries in SS was 88.18%,and in no SS group 73.33%(P<0.01).The average of caries in cases with more than ten inflammatory foci were significantly increased than in those less than ten,no significant difference in the average of caries was observed between primary and secondary SS.The amounts of salivary excretion measured were significantly decreased in 30 cases with SS than in no SS group cases,and the more the inflammatory foci,the less the salivary excretion.

This article is to present the experience in 1,000 cases given intrathecal Iohexol injection during 1985-1988, including conventional myelography in 343 cases, conventional and CT myelography in 572, only CT myelography in 60 and CT cisternography in 25. No convulsions were observed. The frequency of headache was 11.6% and the total uncomfortable subjective reaction was 19.6% after intrathecal injection, but no serious complications were found. Because of very low frequency of side effects after this injection, most of the examinations can be made in the outpatient departments. In our clinical experience, Iohexol appears to be a myelographic contrast medium with diagnostic capabilities and less morbidity compared to Metrizamide. Thus Iohexol seems to be well suited for intrathecal injection and will replace metrizamide in this respect.

The effects of sodium ascorbate (vitamin C) and 2-methyl-1,4-naphthoquinone (vitamin K3) administered separately or in combination on the in vitro cultured human neoplastic cell lines MCF-7 (breast carcinoma), KB (oral epidermoid carcinoma), and AN3-CA (endometrial adenocarcinoma) have been examined. When given separately, vitamin C or K3 had a growth inhibiting action only at high concentrations (5.10(3) mumol/1 and 10(5) nmol/l, respectively). Combined administration of both vitamins demonstrated a synergistic inhibition of cell growth at 10 to 50 times lower concentrations. At this level separately given vitamins are not toxic. The sensitivity to this treatment was somewhat different in the three cell lines, being slightly higher for KB line. This tumor cell growth inhibitory effect was completely suppressed by the addition of catalase to the culture medium containing vitamins C and K3, suggesting an excessive production of hydrogen peroxide as being implied in mechanisms responsible for the above-mentioned effects.