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Placental Na(+)/H(+) exchanger (NHE-1), which plays an important role in maintaining fetal and maternal Na+ and H+ homeostasis, is uniquely regulated. However, the role of this protein in type-2 diabetic placentas, and the molecular basis for its unique regulation in normal placenta remain poorly understood. To address these issues, a C-terminus regulatory domain of NHE-1 was cloned and sequenced from normal human placentas, and was used to prepare a GST-fusion protein for raising polyclonal antibodies. For this study, age-matched type 2 diabetic (n=8) and normal (n=8) pregnant women were recruited to investigate the effects of controlled hyperglycemia on the expression of placental NHE-1 at term delivery. The C-terminal sequence in the normal human placental isoform was identical to that reported for other tissues. The antibodies reacted selectively with a 110 kD protein. The level of NHE-1 protein was decreased significantly ( p<0.05) in diabetic placentas, whereas beta-actin, an internal control, remained unaltered. Yield of placental crude microsomes was significantly higher from diabetic placentas. Interestingly, the levels of NHE-1 mRNA and beta-actin mRNA did not change in diabetic pregnancies. Blood pressure values of the mothers in both groups were also normal. The placental mass and weight of babies were slightly increased, whereas the gestational age was lower in diabetic pregnancies. These results suggest that the unique regulation of placental NHE-1 is not due to differences in its C-terminus structure. Lack of a significant correlation between the suppression of NHE-1 and gestational age, placental mass or birth weight in the diabetic pregnancies suggests that suppression is independent of these parameters, and is regulated post-transcriptionally. This change in NHE-1 may contribute to an adequate provision of electrolytes and nutrients to the fetus.

Background: Na+/H+ exchanger (NHE) is responsible for a net uptake of sodium chloride and water from the gastrointestinal tract and maintains electrolyte and water homeostasis. However, its status in human inflammatory bowel disease such as ulcerative colitis (UC) and Crohn's disease (CD) remains poorly understood., Objectives: To investigate the role of NHE-1 isoform in human CD and UC., Methods: Expression of NHE-1 protein and messenger ribonucleic acid and sodium pump activity were examined in the colonic biopsy samples taken from UC (n=11) and CD (n=13) patients using enhanced chemiluminescence-Western blot analysis, reverse transcription polymerase chain reaction and spectrophotometry. Subjects presenting with abdominal pain and endoscopically normal colon served as normal controls (n=11). Myeloperoxidase (MPO) activity and histology were performed to confirm tissue inflammation., Results: MPO activity increased significantly (P<0.05) in both UC and CD patients compared with the normal controls. Parallel to MPO activity profile, there was also a significantly higher infiltration of inflammatory cells in both cases. P-nitrophenylphosphatase activity, a marker of the sodium pump, remained unchanged in CD but increased significantly (P<0.05) in UC compared with the normal controls. On the contrary, the level of NHE-1 protein and messenger ribonucleic acid was significantly decreased (P<0.05) in both cases, whereas the internal control, a-actin remained unaltered., Conclusions: These findings demonstrate a transcriptionally regulated suppression of NHE-1 in both UC and CD. This NHE-1 suppression may reduce an uptake of sodium chloride and water from the inflamed colonic lumen and thus contribute to diarrhea and neuromuscular alterations in these conditions.

Background: The effects of cyclooxygenase-2 (cox-2) inhibition by a cox-2 selective antisense phosphorothioated oligonucleotide (AS) and meloxicam were examined in experimental colitis., Methods: Colitis was induced by trinitrobenzenesulphonic acid (TNBS) and acetic acid (Hac) separately in male Sprague-Dawley rats. Both groups of animals were treated daily intraperitoneally with AS and a mismatched control oligo (CO) (3 mg/kg), and orally with meloxicam (7.5 mg/kg) 1 h before induction of colitis. The animals were killed on day 4 (Hac) and on day 5 (TNBS). Tissue samples from colon, ileum, liver, kidney and spleen were collected for mRNA, myeloperoxidase activity (MPO), prostaglandin E2 (PGE2) estimation and for histology, and blood samples for PGE2, thromboxane B2 (TxB2) and TNF-alpha., Results: Both TNBS and Hac increased colonic MPO activity, PGE2 concentrations and infiltration of colonic wall by inflammatory cells. Serum levels of TNF-alpha were increased in both models, whereas PGE2 was increased only in TNBS colitis. Only meloxicam suppressed the level of PGE2 significantly below the basal level. The animals in both models also showed splenomegaly. The colitis-induced changes were significantly suppressed by the treatment of the test compounds but not by the CO. Cox-2 mRNA but not cox-1 was decreased by the AS, but not by meloxicam or in CO-treated colitic animals., Conclusion: The findings demonstrate comparable beneficial effects of the cox-2 selective antisense oligonucleotide and meloxicam, which seem to be mediated by a combined inhibition of both PGE2 and TNF-alpha in the present models of colitis.

Inhibition of cyclooxygenase-2 (cox-2) is considered to be anti-inflammatory, whereas inhibition of the constitutive isozyme cox-1 causes renal and gastrointestinal toxicity. Therefore, to achieve an optimal anti-inflammatory effect, an inhibitor should be cox-2 selective without inhibiting cox-1. For this purpose, 10 different cox-2-selective phosphorothioated oligonucleotides (S-oligos) were tested to inhibit the cox-2 enzyme selectively in vivo. An aqueous solution of these S-oligos (3 mg/kg body weight) was injected intraperitoneally (i.p.) into male Sprague-Dawley rats with colitis induced by trinitrobenzene sulfonic acid (TNBS). The colonic levels of cox-2 protein, mRNA, myeloperoxidase (MPO), and prostaglandin E2 (PGE2) were increased significantly on day 1 and remained significantly elevated until day 7 post-TNBS administration, whereas cox-1 remained unaltered. Two S-oligos were found to be effective in reducing the level of cox-2 protein selectively without any effect on the cox-1. The effective S-oligo, but not the mismatched control oligo, reduced the tissue levels of PGE2 and MPO activity significantly. The effective S-oligo reduced the level of cox-2 but not the cox-1 mRNA significantly, whereas a mismatched or a sense control oligo did not affect the levels of these isoforms. M-fold analysis demonstrated extensive secondary structure formation in the cox-2 mRNA. These findings demonstrate that only a few selected sites in the cox-2 target mRNA are accessible in vivo, probably because of the presence of secondary structures. Suppression of cox-2 protein, PGE2, and MPO activity by the S-oligo might prove to be an anti-inflammatory property.

Studies on camels' milk, whether with respect to concentration or bioavailability of trace elements from this milk, are limited and warrant further investigation. The object of this study was to analyse the concentration and distribution of zinc, copper, selenium, manganese and iron in camel milk compared to those in human milk, cows' milk and infant formula under similar experimental conditions. Camels' milk and cows' milk were collected from local farms, human milk samples were obtained from healthy donors in Kuwait and infant formula was purchased locally. Milk fractionation was performed by ultra-centrifugation and gelcolumn chromatography. The concentration of trace elements was analysed by atomic absorption spectrometry and that of protein was determined spectrophotometrically. The concentration of manganese and iron in camels' milk was remarkably higher (7-20-fold and 4-10-fold, respectively) than in human milk, cows' milk and infant formula. The zinc content of camels' milk was higher than that of human milk but slightly lower than in cows' milk and infant formula. The concentration of copper in camels' milk was similar to that of cows' milk but lower than in human milk and infant formula. The selenium content of camels' milk was comparable to those of other types of milk, Approximately 50-80% of zinc, copper and manganese in camels' milk were associated with the casein fraction, similar to that of cows' milk, The majority of selenium and iron in camels' milk was in association with the low molecular weight fraction, It is recommended that camels' milk be considered as a potential source of manganese, selenium and iron, perhaps not only for infants, but also for other groups suspected of mild deficiency of these elements. Further investigations are required to confirm this proposal.

Products of certain species of Cordia are reported to have antiinflammatory properties. In the present study we examined the effects of Cordia myxa fruit on experimentally induced colitis in rats. Colitis was induced by intrarectal administration of 4% acetic acid. Colitic, normal, and corresponding control animals were included. Body weight was recorded daily. All the animals were sacrificed 4 days after the fruit treatment. Colitis was monitored histologically and by activity of myeloperoxidase. Glutathione peroxidase, superoxide dismutase, as well as total antioxidant status and concentrations of zinc, copper, manganese, selenium, and iron were assayed in plasma, liver, and colon using standard methods. Histology of the colon of colitic rats showed acute colitis that was confirmed by a significant increase in the myeloperoxidase activity. Colitis was associated with significant decreases in the tissue activities of glutathione peroxidase and superoxide dismutase and lower concentrations of trace elements. Histologic examination and myeloperoxidase activity showed that the fruit treatment reversed the above findings in the inflamed colon, and in liver and plasma of colitic rats. The present results suggest that the observed antiinflammatory effect of the Cordia myxa may be attributed partly to its antioxidant property and to restoration of the levels of trace elements in the inflamed colon, liver, and plasma.

Colitis reduces the blood and tissue levels of adenosine deaminase and adenylate deaminase. Whether this has any effect on blood purines remains to be determined. The aim of this study was to measure the adenylate pool, substrates of the above enzymes, and energy status in blood from rats with colitis. Colitis was induced by intrarectal administration of acetic acid and followed over a period of seven days. The levels of ATP, ADP, AMP, adenosine, inosine, and uric acid were analyzed by HPLC, and energy status was estimated. Myeloperoxidase was used as a marker of colitis. Concentrations of ATP, ADP, AMP and adenosine decreased during days 1-5, whereas energy status decreased on day 2. The concentrations of inosine, uric acid, and hemoglobin remained unaltered, whereas colonic myeloperoxidase activity increased. These, findings demonstrate colitis-induced reduction of the circulating purines, which may be due to their enhanced usage for the repair of the inflamed colon.

The distribution of breast, colon, gastric, thyroid, oral, rectal, pancreatic and renal cancers were determined in 71 Kuwaitis, 45 other Arabs, and 26 Indians. Plasma levels of micronutrient antioxidants, retinol, alpha-tocopherol, lycopene, and beta-carotene were measured in the groups and in 90 matched controls for comparison. Cholesterol was measured to determine its association with the micronutrient antioxidants. Pancreatic cancer occurred exclusively in Kuwaitis, while breast and colon cancers were disproportionately higher in Kuwaitis than in the other groups. Micronutrient antioxidant levels were similar in the groups, except for higher lycopene levels in Kuwaitis. In most instances, the micronutrient antioxidants, except beta-carotene, decreased significantly in levels in patients than in controls. Low levels of retinol, lycopene, and beta-carotene were strongly associated with pancreatic cancer. Compared to controls, significantly increased levels of beta-carotene occurred in breast, colon, thyroid, and renal cancers; increased lycopene occurred in oral cancer, and increased alpha-tocopherol occurred in pancreatic cancer. Alpha-tocopherol strongly correlated with cholesterol. Generally, changes in alpha-tocopherol/ cholesterol ratios mimicked those of alpha-tocopherol levels. Micronutrient antioxidant levels were significantly lower in male patients than female patients. Age showed a negative but statistically insignificant relationship with micronutrient antioxidants. Lycopene strongly correlated with alpha-carotene and alpha-tocopherol with retinol. Among the patients, all micronutrient antioxidants except retinol decreased significantly in levels in smokers than nonsmokers, suggesting susceptibility to cigarette smoke oxidative stress. We conclude that micronutrient antioxidant depletions and altered associations may imply tumor utilization or antioxidant burden in oxidative stress or both. Furthermore, the incidence of pancreatic, colon and breast cancers among Kuwaitis warrants further study.

There is a wide variation in the reported data on the concentrations of trace elements in human milk from different countries, but such data are not available for Kuwait. The objective of this study was to analyze the concentration of zinc, copper, manganese, and iron in milk and plasma of Kuwaiti and non-Kuwaiti mothers during prolonged lactation. Milk samples (from 34 donors) were collected early in the morning before feeding the infant. Trace elements were analyzed using atomic absorption spectrophotometry. Protein content and activity of superoxide dismutase were assayed spectrophotometrically. Concentration of zinc, copper, iron, and total protein and activity of superoxide dismutase in milk and of only zinc in plasma of Kuwaiti mothers were significantly higher than those of non-Kuwaitis. Concentration of zinc, copper, manganese, and total protein in milk of both groups decreased as lactation continued but that of milk iron and plasma trace elements remained unchanged. The data of Kuwaiti mothers are consistent with those of previous reports on hyperuricemia, and the prevalence of obesity was found to be higher in the Kuwaiti population than in other countries. High protein content in association with high concentration of trace elements in milk of Kuwaiti versus non-Kuwaiti mothers may indicate that protein content in milk is an important determining factor for the concentration and bioavailability of these elements.

Background: Vitamin A is an important nutritional factor that regulates normal growth and functions of epithelial cells of the gastrointestinal tract., Objective: The objective of this study was to examine the role of vitamin A on the histological and biochemical changes in the colon of mice., Methods: To address this issue, vitamin A deficiency (VAD) was developed in mice by placing them on a VAD diet from weaning up to 120-170 days. Infiltration of inflammatory cells in the colon was determined histologically. Activities of adenosine deaminase, adenylate deaminase, purine nucleoside phosphorylase and myeloperoxidase were determined., Results: VAD in mice induced a significant increase in the number of mast cells per 100 crypts. There was also an abundance of other connective tissue cells such as plasma cells, lymphocytes and neutrophils around the crypts in the lamina propria. The colonic activity of adenosine deaminase and adenylate deaminase was increased due to VAD, whereas purine nucleoside phosphorylase activity remained unchanged. Immunohistochemical analysis showed an increased expression of adenosine deaminase in VAD mice colon. The increase in myeloperoxidase activity was not statistically significant., Conclusions: VAD causes upregulation of purine enzyme, which together with an increased number of inflammatory cells might exacerbate colonic injuries in VAD condition., (Copyright 2000 S. Karger AG, Basel.)

Although the role of adenosine deaminase (ADA), adenylate deaminase (AMP-DA), purine nucleoside phosphorylase (PNP) is well documented in gastric and intestinal carcinoma, their role in inflammatory bowel diseases remains unknown. In the present study, we investigated the profile of these enzymes in blood and intestinal tissues during colitis. Colitis induced in Wistar rats by acetic acid was monitored by a marker enzyme myeloperoxidase (MPO). The tissue levels of MPO increased on 1, 2, 5 and 6 days post-administration (PA) of acetic acid and declined to the control levels by day 7 PA. In parallel the blood levels of ADA and AMP-DA decreased on days 1, 2 and 5 without any significant change on days 6 and 7 PA. Similar observations were recorded for these enzymes in the cytosolic extracts of colonic tissue specimens. In contrast, PNP remained unaltered in both blood and tissue samples. These findings suggest an inverse-relationship between inflammation and purine deaminases in both blood and tissues.

The sodium hydrogen exchanger (NHE) plays an important role in the absorption of NaCl, the regulation of intracellular pH and cell growth. These functions are compromised in the inflammatory bowel diseases. The objective of this study was to examine the expression of the NHE-1 isoform during colitis induced by acetic acid or trinitrobenzenesulfonic acid in Sprague-Dawley male rats. We also examined the effect of dexamethasone on the expression of NHE-1. Levels of mRNA were estimated using the reverse transcription-polymerase chain reaction and slot blot analysis, and levels of protein were estimated by enhanced chemiluminescence light Western blot analysis. The levels of the NHE-1 mRNA and protein in colonic mucosa increased as assessed at 1, 2, 5 and 7 days post-acetic acid administration and 7 days post-trinitrobenzenesulphonic acid administration in the rats. The levels of mRNA were not suppressed by dexamethasone treatment in either case. These findings demonstrate that colitis-induced expression of the NHE-1 mRNA and protein is independent of the way colitis is induced. Although factor(s) responsible for the induction remain to be identified, our findings showing similar changes in the NHE-2 and NHE-3 mRNA isoforms, together with the lack of their suppression by dexamethasone, suggest that cytokines and intracellular pH are secondary factors.

Trace elements constitute important prosthetic groups in a number of antioxidant enzymes which neutralize free radicals generated during inflammatory conditions such as colitis. However, the status of trace elements in colitis remains to be found. In the present study the concentrations of zinc, copper, manganese and selenium in the colon, liver and serum of rats with acetic acid (HAc)- or trinitrobenzenesulfonic acid (TNBS)-induced colitis were measured using atomic absorption spectrophotometer. Myeloperoxidase and glutathione peroxidase activities were measured spectrophotometrically. Our results show that the selenium concentration was significantly decreased by 33 and 37.5% in the colon and 69 and 78% in liver by HAc and TNBS treatment, respectively. Similarly the zinc concentration in the colon was decreased by 21 and 28% by HAc- and TNBS-induced colitis as compared to the controls, but manganese and copper, remained unaltered. The serum concentrations of copper, zinc and selenium also remained unaltered during colitis. The weight of HAc-treated rats did not decrease while there was a significant weight loss in the TNBS-treated rats. Myeloperoxidase activity was increased, whereas glutathione peroxidase activity was significantly decreased in the colon inflamed by HAc or TNBS as compared to the controls. These findings suggest that colitis induces a reduction in the tissue levels of trace elements which is independent of the way colitis is induced. Our findings of a reduction in Se and glutathione peroxidase activity together suggest that the reduction in the trace element concentrations is not due to dietary factors or malabsorption. The decrease may severely affect the antioxidant potential of the colon and therefore is a putative factor for the progression of disease.

Background: Inflammatory bowel diseases are accompanied by alterations in the contractile function of colonic muscle. Whether these changes are also accompanied by the production of interleukin-1 beta (IL-1 beta) mRNA remains unknown., Aims: To investigate the profile of IL-1 beta mRNA in the colonic muscle of rats with acetic acid induced colitis., Methods: Colitis was induced by intrarectal administration of 1 ml of 4% acetic acid 8 cm from anal margin. Myeloperoxidase activity was used as a marker of inflammation in the colon. RNA was extracted from colonic muscle and was used to amplify the level of IL-1 beta mRNA with a reverse transcription coupled polymerase chain reaction (RT-PCR) method developed in this study., Results: Colonic muscle exhibited constitutive expression of IL-1 beta mRNA. The level was enhanced significantly, one, two, and five days after acetic acid administration. On day 7 after treatment, the level of IL-1 beta mRNA had returned baseline values. This profile of IL-1 beta mRNA was consistent with myeloperoxidase activity., Conclusions: IL-1 beta is a putative mediator of the changes in muscle function in this model of colitis.

The energy status of HeLa cells arrested in the G2 phase of the cell cycle by the antineoplastic agent cis-acid was compared with that of normal G2-phase-synchronized cells. The results showed that treatment with the drug has significantly decreased the adenylate pool, adenylate energy charge and phosphorylation potential.

1. Activity of glutamate dehydrogenase and adenylate deaminase were measured in the livers of carnivores (animals characterised by intake of a high dietary protein). 2. Animals studied (ferret, cat, dog, hedgehog, rat, hamster, mouse, cow, pig and rabbit) were kept on their natural diet. 3. Glutamate dehydrogenase activity showed no variation between carnivores and non-carnivores. 4. Adenylate deaminase activity was significantly higher in carnivores than in non-carnivores. 5. In carnivores, adenylate deaminase might be the rate limiting enzyme in the terminal deamination of L-amino acids. 6. Elevation of adenylate deaminase might be due to the acidogenic effect of the diet.

The efficacy of a hypoglycaemic plant extract, in common use by Kuwaiti diabetic individuals, was evaluated using both streptozotocin-induced diabetic and normal rats. A significant decrease in blood glucose concentration was demonstrated on glucose tolerance tests, as compared to untreated animals. The sum of the fasting, 1 and 2 h blood glucose values, decreased from 18.5 +/- 0.72 to 13.6 +/- 0.62 mmol/l (p less than 0.001) and from 58.6 +/- 2.83 to 44.5 +/- 3.12 mmol/l (p less than 0.005) in normal and diabetic animals treated for 1 week, respectively. Treatment with the extract was not found to significantly alter insulin levels or intestinal glucose absorption. The mode of action of the hypoglycaemic preparation remains to be elucidated.

In streptozotocin induced diabetes in rats, excretion of urinary protein fractions were studied in relation to structural changes in the renal glomeruli, using light and transmission electron microscopy. After six weeks of induced diabetes only beta 1 and beta 2 plasma globulins were significantly elevated. The amount of excreted proteins and degree of glomerular changes were not proportional. In the initial stages (1-2 weeks) glomerular structural changes were very mild and were accompanied by significantly elevated proteinuria. This progressed (4-8 weeks) to moderate to prominent structural changes with intermittent proteinuria except for the fractions beta 1 & beta 2 which were elevated throughout the duration of the experiment. The amount of proteinuria was not proportional to changes in the plasma protein levels. The following conclusions may be made: 1) The mild early glomerular abnormalities seem to be mainly due to acute metabolic disturbances. 2) An early indication of diabetic nephropathy is provided not only by albuminuria, but may also be an elevated excretion of beta-globulin fractions. 3) Decrease of albuminuria in the later stages of diabetes may be related to the deposition of albumin as a basement membrane-like material in the mesangium.

A blood glucose lowering extract of a mixture of five plants in use by Kuwaiti diabetics was studied for the identification of its active component(s). Only the extracts of myrrh and aloe gums effectively increased glucose tolerance in both normal and diabetic rats. The remaining components, gum olibanum, Nigella sativa seeds and gum assafoetida were without effect.