Effect of diet upon intestinal disaccharidases and disaccharide absorption.

The administration of a carbohydrate-containing diet for 24 hours to rats previously fasted for 3 days led to a twofold increase in total intestinal sucrase and sucrase specific activity. The specific activity of maltase was similarly increased, but lactase activity was unaffected. The sucrose-containing diet led to a greater increase in sucrase than maltase activity, whereas the converse was true of the maltose-containing diet. A carbohydrate-free isocaloric diet led to a slight increase in the total intestinal sucrase, but sucrase specific activity was unchanged. Assay of sucrase activity of mixed homogenates from casein-fed and sucrose-fed rats or fasted and sucrose-fed animals yielded activities that were additive. The Michaelis constant (Km) of the enzyme hydrolyzing sucrose was similar in the fasted, casein-fed, and sucrose-fed rats. The maximal velocity (Vmax) was twice greater in sucrose-fed as compared to casein-fed or fasted rats, suggesting an increased quantity of enzyme subsequent to sucrose feeding. Adrenalectomized rats maintained on 1.0% salt intake had sucrase and maltase levels comparable to those of controls. Steroid administration did not significantly increase their activities. The response to sucrose feeding was similar in both control and adrenalectomized rats, indicative of the absence of steroidal control on sucrase and maltase activity in the adult animal. Studies using intestinal ring preparations indicated that sucrose hydrolysis by the intact cells proceeded more rapidly when animals were fed sucrose. Additional corroboration of the physiologic significance of the increased enzyme levels in homogenates was afforded by intestinal perfusion studies. Sucrose hydrolysis increased twofold and fructose absorption fourfold in animals fed sucrose when compared to either fasted or casein-fed rats.

Vitamin B12 uptake by intestinal microorganisms: mechanism and relevance to syndromes of intestinal bacterial overgrowth.

The mechanism of bacterial uptake of vitamin B(12), the spectrum of microorganisms capable of such uptake, and the factors involved were the subject of this study. Bacterial uptake of vitamin B(12) was found to be at least a two stage process. A primary uptake phase which was rapid (1 min or less), pH dependent, nontemperature dependent, did not require viable organisms and was insensitive to either the metabolic inhibitor dinitrophenol or to the sulfhydryl inhibitor N-ethyl-maleimide. Protein denaturation (formalin treatment or autoclaving) abolished all B(12) uptake. This primary uptake phase is thought to represent adsorption to binding or "receptor" sites on the cell wall. Second stage uptake was slower, pH and temperature dependent, required living bacteria, and was abolished by either dinitrophenol or N-ethyl-maleimide. This phase is dependent upon metabolic processes and may reflect transfer of B(12) from surface "receptor" sites into the bacterial cell. Although differences among organisms were observed in total 1 hr uptake, number of surface "receptor" sites, and relative avidities for B(12), all organisms except Streptococcus fecalis shared the two stage mechanism. Two Gram-positive organisms. Bacillus subtilis and Group A streptococcus, demonstrated the highest 1 hr vitamin B(12) uptake values; Gram-negative bacteria required 2,000-10,000 the number of organisms for comparable uptake. Binding constants (K(m)) varied from 5.05 +/-1.67 x 10(-10)M for B. subtilis to 6.18 +/-3.08 x 10(-9)M for Klebsiella pneumoniae which approximate the Km for human intrinsic factor (0.38 x 10(-10)M). Competition between bacteria and intrinsic factor for vitamin B(12) may be inferred from the similarity of these constants. These observations suggest that a variety of enteric and nonenteric organisms, not requiring exogenous B(12), may play a role in the pathogenesis of the vitamin B(12) malabsorption found in the intestinal bacterial overgrowth syndromes.

Demonstration and immunochemical characterization of carcinoembryonic antigen in human pancreatic juice.

Pancreatic juice collected from 10 patients without evidence of malignant disease of the pancreas or other organs was pooled, extracted, and fractionated by Sepharose 6-B and Sephadex G-200 gel filtration. The carcinoembryonic antigen (CEA) activity in the material was demonstrated and studied by: a) radioimmunoassay, b) competitive binding to antibodies against CEA, c) precipitin inhibition, and d) Ouchterlony analysis. The immunochemical identity of the active material to CEA purified from liver metastases of colon cancer was demonstrated.

Cholesterol excretion and colon cancer.

Populations consuming diets high in fat and cholesterol exhibit a greater incidence of colon cancer than those consuming less fat and cholesterol. Lowering elevated serum cholesterol levels experimentally or clinically is associated with increased large-bowel tumorigenesis. Thus, cholesterol lost to the gut, either dietary or endogenously synthesized, appears to have a role in large-bowel cancer. Whether the effect(s) is mediated by increases in fecal bile acid excretion or some other mechanism is not clear.

Lipids and immune function.

There is in vitro and in vivo evidence to suggest that dietary lipids play a role in modulating immune function. A review of the current literature on the interrelationships among dietary lipids, blood cholesterol levels, immunosuppression, and tumorigenesis makes for a very strong argument that (a) immunosuppression may be causally related to lymphoproliferative disorders, as well as to tumorigenesis and (b) diets high in polyunsaturated fat, relative to diets high in saturated fat, are more immunosuppressive and are better promotors of tumorigenesis. The effects of dietary fat on immune function seem to be mediated though its component parts, the unsaturated fatty acids, specially linoleic, linolenic, and arachidonic. It is not clear how these components affect immune function. Several studies suggest that one effect is mediated by altering the lipid component of the cell membrane and thus its fluidity; the more fluid the membrane, the less responsive it is. Thus, fluidity of both immune cells and those to be destroyed or protected may be affected. The effects of saturated as well as unsaturated fatty acids may be mediated by modulating serum lipoprotein levels, prostaglandin metabolism, and cholesterol concentrations and metabolism.

Evidence for deficiency of low density lipoprotein receptor on human colonic carcinoma cell lines.

Cells from six human colonic adenocarcinoma lines (CaCo-2, HT29, LS174T, SW480, SW403, and SW1417) and a normal skin fibroblast cell line (AG1519) were assayed in vitro for their ability to use low density lipoprotein (LDL). All tumor cell lines grew well in lipoprotein-deficient serum, implying that LDL in culture medium was not critical for cell growth. When cell growth was inhibited with mevinolin, a cholesterol synthesis inhibitor, the addition of LDL to the medium had no effect on the growth of cells from five of six tumor cell lines. CaCo-2 cells showed a moderate reversal while the fibroblast control showed total reversal of inhibition. A monoclonal antibody to bovine/human LDL receptor, used in an enzyme-linked immunosorbent assay, indicated that only CaCo-2 cells and human skin fibroblast cells consistently demonstrated the presence of LDL receptors. Thus, five of six colon tumor cell lines were unable to overcome a mevinolin block in cholesterol metabolism indicating that these cells were deficient in LDL receptors.

Postpromotional effects of dietary marine or safflower oils on large bowel or pulmonary implants of CT-26 in mice.

Marine oils containing n-3 fatty acids exhibit variable antineoplastic effects. Diets containing low (11.6% of kcal) or high (46.5% of kcal) levels of marine oils as the exclusive fat source were compared to diets containing identical amounts of safflower oil (n-6) in weanling, male BALB/c ByJ mice. All diets provided approximately 90 kcal/100 g body weight/day, and contained identical quantities of vitamins, minerals, protein, and fiber. The growth of transplantable colon carcinoma, CT-26, (10(6) cells/animal) implanted, subserosally, into the descending colon via laparotomy, was observed weekly over 28 days by necropsy in all dietary groups. At each time period animals fed safflower oil had larger tumors than those fed marine oil. Tumor volumes at 21 days postimplantation were as follows: low fat marine, 55 mm3 (5-196 mm3) [median (range)]; high fat marine, 70 (26-194); low fat safflower, 216 (32-800); high fat safflower, 247 (70-1352). Marine oil tumors were smaller than safflower oil tumors (P less than 0.005 by analysis of variance; P less than 0.01 by Scheffe test). Metastatic potential was assessed by pulmonary colonization. CT-26 was injected i.v. in tail veins (10(5) cells/animal). Mice were sacrificed and colonies were counted after 21 days. Mice fed low fat marine, high fat marine, and low fat safflower oil diets, 10-14 colonies; high fat safflower, 55 colonies (P less than 0.001 by analysis of variance). Hence, dietary marine oil significantly suppressed growth of this colon carcinoma at all intake levels studied and inhibited pulmonary colonization at higher intakes relative to safflower oil.

Receptor-mediated endocytosis of carcinoembryonic antigen by rat liver Kupffer cells.

In vivo, carcinoembryonic antigen (CEA) is removed from the circulation by the liver Kupffer cells. Immunologically identifiable CEA is transferred from these macrophages to the hepatocytes, where degradation is completed. Circulatory clearance of CEA is specific, rapid [t1/2 = 3.7 +/- 0.9 (S.D.) min], and saturable. In vitro, Kupffer cells take up CEA by a saturable process which is time/temperature dependent and colchicine sensitive. Isolated Kupffer cells endocytose CEA with an apparent Km of 6 X 10(-8) M. There are approximately 16,000 CEA binding sites per cell. Nonspecific cross-reacting antigen (NCA), a glycoprotein structurally similar to CEA, is recognized with lower affinity by the same receptor. Endocytosis is independent of the nonreducing terminal sugars on the molecule: CEA modified by Smith degradation inhibits Kupffer cell recognition of native CEA. Since performic acid oxidized CEA also inhibits endocytosis, receptor binding is similarly independent of intact protein conformation. Isolated Kupffer cells have mannose and/or N-acetyl glucosamine receptor activity but do not internalize CEA by that mechanism. Galactose-terminated glycoproteins impede CEA and NCA clearance in vivo but not Kupffer cell endocytosis in vitro. Radiolabeled CEA released from isolated Kupffer cells following endocytosis shows no apparent molecular weight change. However, the released CEA contains species with higher isoelectric points, suggesting that perhaps the removal of sialic acid and the resulting exposure of galactose residues mediate the subsequent transfer to the hepatocyte.

Beneficial effects of post-transfusional hepatitis in acute myelogenous leukemia may be mediated by lipopolysaccharides, tumor necrosis factor alpha and interferon gamma.

Post-transfusional hepatitis is often a complication in patients with acute myelogenous leukemia (AML) in whom survival is paradoxically prolonged. The etiology is unknown. In previous studies, we showed that impaired hepatic endotoxin (lipopolysaccharide, LPS) clearance in patients with acute viral hepatitis A, B, or C versus controls results in endotoxemia and tumor necrosis factor alpha (TNF-alpha) release. TNF-alpha mediates anti-proliferative and differentiating effects in AML cell lines. Interferon-gamma (IFN-gamma) released in acute viral hepatitis, acts in synergy with TNF-alpha. HL60, KG1, and U937 AML cells treated 3, 6, and 9 days with physiologically attainable TNF-alpha (10 U/ml), IFN-gamma (100 U/ml) and LPS (10 ng/ml) levels, have significantly diminished viability and cell growth versus controls. Treatment of HL60 AML cells with LPS/TNF-alpha/IFN-gamma also resulted in significantly increased monocytic pathway differentiation not seen with KG1 or U937 AML cells. HL60 AML cells treated with TNF-alpha/IFN-gamma for 6 days released endogenous TNF-alpha (1.57 U/10(6) cells) upon LPS stimulation compared to less than 0.01 U/10(6) cells in non-LPS-stimulated TNF-alpha/IFN-gamma-treated cells or untreated cells (p less than 0.0001). Untreated HL60 AML cells co-cultured with HL60 cells pretreated for 6 days with TNF-alpha/IFN-gamma and then subjected to LPS stimulation had significantly diminished cell growth compared to controls (p less than 0.0001). This effect could be reversed with anti-TNF-alpha antibody, supporting the concept that endogenous TNF-alpha release by LPS/TNF-alpha/IFN-gamma treated HL60 AML cells may act by paracrine means to suppress growth of other AML cells. The beneficial effects of post-transfusional hepatitis in AML patients may be mediated via LPS/TNF-alpha/IFN-gamma-induced AML cell growth suppression and/or terminal differentiation in which AML cells participate by releasing TNF-alpha after being acted upon by LPS/TNF-alpha/IFN-gamma. Endogenously released TNF-alpha might then act by autocrine/paracrine means to mediate further suppression and terminal differentiation.

Growth restraint and differentiation by LPS/TNF-alpha/IFN-gamma reorganization of the microtubule network in human leukemia cell lines.

The microtubule (MT) network of the cytoskeleton has been implicated as a mediator of cellular signal transduction; disorganization of this network may allow for mitogenesis. In previous work, loss of MT network organization in human MOLT4 and HUT78 T-cell leukemias was demonstrated in contrast to an organized "spoke-wheel-like arrangement" in normal human T-lymphocytes. In this study, loss of MT network organization was shown in several representative acute myeloid leukemia (AML) cell lines: KG1 myeloblastic, HL60 promyelocytic, and U937 myelomonocytic cells. Re-organization of the MT network was observed in HL60 and U937 AML cells treated with combined lipopolysaccharide (LPS), tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma). This re-organization paralleled earlier work which showed this combination was effective in inducing monocytic pathway differentiation and growth restraint in HL60 cells, and growth restraint in U937 cells. In contrast, KG1 cells exhibited growth restraint, but did not re-organize with LPS/TNF-alpha/IFN-gamma treatment. These results are consistent with a role for the MT network in mitogenesis. Loss of MT network organization appeared to parallel the neoplastic phenotype in three AML cell lines, whereas MT network re-organization accompanied recovery of growth control in 2 of 3 AML cell lines.

Pathophysiological aspects of coxsackievirus B intestinal infection.

Our findings reveal that intestinal infection with coxsackie B5 results in decreased intestinal epithelial cell division in association with an increase in carbohydrate (glucose) and amino acid (leucine) absorption in the small intestine. These findings are contrasted with those occurring during Salmonella infection, which results in increased intestinal cell division rate but decreased carbohydrate (glucose) absorption. The changes in intestinal function and physiology that have been described occurred during an asymptomatic viral infection characterized by normal intestinal histology. A reasonable hypothesis is that these pathophysiological changes may be due not only to a variety of local factors but also to hormonal effects induced by systemic spread of coxsackievirus B.

CD14 mediated endogenous TNF-alpha release in HL60 AML cells: a potential model for CD14 mediated endogenous cytokine release in the treatment of AML.

In previous studies, HL60 AML cells treated with tumor necrosis factor-alpha (TNF), interferon-gamma (IFN), and lipopolysaccharides (LPS) displayed decreased growth and viability, enhanced monocytic pathway differentiation and endogenous TNF release. Endogenous TNF release by LPS/TNF/IFN treated HL60 cells was postulated to play a role with the above findings. In these studies, HL60 cells expressed CD14 when treated with TNF, IFN, and LPS. CD14 mediates TNF release in monocytes/macrophages in response to binding of LPS with LPS binding protein (LBP). CD14 was not expressed in either untreated or LPS only treated HL60 cells. CD14 expression was present and greater with HL60 cells cultured with LPS/TNF/IFN vs TNF/IFN (47.47% vs 9.07% positive, respectively) suggesting synergism for LPS in CD14 induction. CD14 expression was associated with endogenous TNF release, and with significantly higher levels by HL60 cells treated with LPS/TNF/IFN vs TNF/IFN (p < 0.001). Addition of anti-CD14 antibody significantly reduced release of TNF in TNF/IFN (p < 0.001) and LPS/TNF/IFN (p = 0.0013) treated cells. KG1 and U937 AML cells treated with LPS, TNF, and IFN did not express CD14, nor release TNF. A model for inducing release of endogenous growth inhibitory cytokines by CD14 bearing AML cells is proposed as an approach to AML therapy.

Effect of serotonergic and catecholaminergic antagonists on mild-stress-induced excessive grooming in the rat.

Excessive grooming was induced in male rats by two ip injections of physiological saline. This mild stressful procedure did not modify open-field locomotion in 5-min trials. Methysergide (15 mg/kg) and pizotifene (5 mg/kg), serotonergic blockers, selectively prevented the grooming response to saline without affecting locomotion. Haloperidol (.4 mg/kg) also prevented the excessive grooming. However, this dopaminergic blocker impaired locomotion. The alpha- or beta-adrenoceptor antagonists phentolamine (20 mg/kg) and l-propranolol (20 mg/kg) did not prevent the excessive grooming in response to saline and did not affect locomotion. The results suggest that some serotonergic pathways in the brain are involved in the grooming response to a mild stress and support previous findings on the role of dopaminergic systems on this activity.

Effects of a histamine synthesis inhibitor and antihistamines on the sexual behavior of female rats.

Intraventricular administration of alpha-hydrazinohistidine, a histamine synthesis inhibitor, at different doses and times before testing produced a significant decrease of lordotic responses and sexual receptivity in ovariectomized estrogen plus progesterone-primed female rats. The H1-antihistamines pyrilamine and chlorfeniramine and the H2-antihistamine metiamide, injected in the lateral ventricle, significantly decreased the lordosis quotient but did not modify receptivity; antihistamine-injected rats showed no soliciting behavior. Exploratory activity was decreased by both alpha-hydrazinohistidine and metiamide but not by the H1-antihistamines. It is concluded that treatments which either deplete histamine or block their receptors can alter female copulatory responsiveness. The mechanism of this antihistamine effect appears to be unrelated to that of other side effects, such as motor impairment, sedation, or local anesthesia.

Effect of prenatal and postnatal exposure to therapeutic doses of chlorimipramine on emotionality in the rat.

Prenatal administration of high doses of tricyclic antidepressants have been reported to produce teratogenic and behavioral effects in rat offspring. In the present work, behavioral abnormalities are described in offspring of rats treated with therapeutic doses of chlorimipramine (CIM) during pregnancy (CIM-P), lactation (CIM-L) and during the whole pregnancy-lactation period (CIM-PL). CIM-P treatment did not produce teratogenic effects, did not affect number or body weight of pups at birth and did not induce neonatal mortality. At 2 months of age, the CIM-P males showed a significant increase in digging and grooming (familiar environment test), a decrease in "exploration" (novel environment test) and a decrease in active social interactions (social behavior test). Females were more resistant than males to the prenatal CIM treatment. The results suggest increased emotionality in CIM-P pups. Some behavioral abnormalities were also observed in the tests performed at 4 months of age. CIM-L treatment had minor effects on litter behavior. CIM-PL treatment potentiated the effects of the CIM-P treatment. In the CIM-PL males, impairment of exploration of a novel environment still remained in the tests performed at 4 months of age. It is speculated that when prenatal brain development is altered by CIM, further postnatal treatment may impair compensatory processes occurring in early postnatal life.

E. coli peritonitis and bacteremia cause increased blood-brain barrier permeability.

Impaired mental status is a poorly understood manifestation of sepsis and may be associated with altered permeability of the blood-brain barrier. To examine the possibility that sepsis affects permeability of the blood-brain barrier, rats were infected with a peritoneal implant consisting of sterilized feces, barium sulfate, and 10(8) colony forming units (CFU) of Escherichia coli. Using this model, reproducible episodes of peritonitis with bacteremia resulted. Rats were sacrificed hourly after 5 min circulation of 100 mg horseradish peroxidase. Animals were perfused-fixed and the brains removed. Representative coronal sections were stained for peroxidase reaction product and cerebral blood vessels were examined microscopically for evidence of HRP staining and extravasation. The number of stained cerebral vessels from infected rats was increased at all times compared to uninfected control rats. Extravasation of horseradish peroxide within neuropil was significantly higher in hours 1, 4 and 5 as compared to controls. The lack of significant increase in hours 2 and 3 may suggest transient closing or repair of the tight junctions. We conclude that peritonitis and bacteremia are associated with increased permeability of the blood-brain barrier.

R-(+)-perillyl alcohol-induced cell cycle changes, altered actin cytoskeleton, and decreased ras and p34(cdc2) expression in colonic adenocarcinoma SW480 cells.

Monoterpenes as S-(-)-perillyl alcohol (PA) have been shown to inhibit the isoprenylation of such growth regulatory proteins as ras. In this study, we investigated the effects of the R-(+) enantiomer of PA on cell cycle, signaling, and cytoskeletal control in the colonic adenocarcinoma cell line SW480, which carries a K-ras mutation. Cell cycle analysis by flow cytometry of SW480 cells treated with 1 mM PA for 24 hours demonstrated an increase in the number of cells in G0/G1 with a decrease in S phase, compared with untreated control cells. These cell cycle changes correlated with an inhibition of protein isoprenylation from (14)C-mevalonate and decreased expression of the cell cycle regulatory kinase p34(cdc2). Additionally, PA-treated cells acquired a flattened morphology with a condensation of cytoskeletal actin spikes to the periphery. This was in contrast to treatment with 15 microM mevinolin (MVN), a direct mevalonate synthesis inhibitor, which imparted to SW480 cells a more rounded and spindly morphology, associated with the depolymerization of actin microfilaments. Together, these data suggest that fluctuations in mevalonate and isoprenoid pools may involve different morphologic phenomenon. Because ras mediated signaling is related to the organization of the actin cytoskeleton, we investigated the effects of PA on the isoprenylation of ras. Although MVN treatment inhibited ras farnesylation, PA treatment decreased the expression of total ras protein. In summary, R-(+)-PA-induced cell signaling events correlated with alterations in the organization of cytoskeletal actin and decreased protein expression of growth regulatory proteins, such as ras and cdc2 kinase. These effects may contribute to the growth inhibitory activity of R-(+)-PA.

Preferential nesting in lemon-scented environment in rats reared on lemon-scented bedding from birth to weaning.

Newborn rats (males and females) were reared on lemon scented shavings from birth to weaning. The experienced rats (111 animals) and their controls (135 animals) were tested for lemon odor preference at 21, 51, 81, 111 and 141 days of age. The test box consisted of two preference chambers, containing wood shavings, interconnected by a starting compartment. Shavings of one of the preference chambers were sprinkled with natural lemon juice. The preference ratio was obtained on the basis of the chamber selected for nesting. The results clearly show that (1) sex differences occur in both experienced and control groups of rats and (2) rats reared on lemon scented bedding from birth to weaning acquire a permanent preference for nesting in the lemon scented chamber. The results are consistent with the hypothesis that an imprinting-like process takes place.

Effect of the chronic ingestion of chlorimipramine and desipramine on the hole board response to acute stresses in male rats.

The effect of the chronic ingestion of chlorimipramine (CI) or desipramine (DS) on the alterations of hole board behavior caused by a model stress (2 IP injections of physiological saline) and by a short restraint stress (5 min) is analyzed in this study. The experimental groups ingested about 3 mg/kg/24 hr CI or DS for 15 days. Then some experimental and control rats were assigned to control of drug effects on baseline activity. The remaining rats were submitted to saline stress (Experiment I) or restraint stress (Experiment II). The baseline scores of hole board locomotion, head dipping, grooming and defecation were not affected by DS treatment but locomotion slightly increased in the CI treated group. Saline stress impaired significantly head dipping and caused excessive grooming in control rats. The CI treatment induced almost full protection against these behavioral effects of saline stress but DS treatment was ineffective. Restraint stress was found to cause a pronounced inhibition of head dipping as well as a great increase of the scores of grooming in the control group. The CI treatment clearly attenuated these effects of restraint but DS treatment was not effective. The results suggest that male rats treated chronically with CI tolerated both acute stresses better than untreated rats, and that a similar treatment with DS did not provide protection against the effect of such stresses on hole board responding. Inasmuch as CI and DS have different relative potency at noradrenergic and serotonergic systems, it is speculated that this might be in part responsible for their differences as stress protectors.

Behavioral responses of high and low active male rats to the chronic ingestion of desipramine.

Male rats arbitrarily selected for high and low motor activity (HA and LA-rats) were submitted to the chronic ingestion (30 days) of desipramine (DSP) in doses of about 1.5, 3 and 6 mg/kg/24 hr. Their motor activity was assessed in an animal activity monitor providing a measure of total horizontal movements and vertical movements and in a hole-board providing a measure of locomotion, head-dipping and grooming. There were significant differences between HA and LA-rats in their behavioral response to DSP treatment. At the doses used DSP did not affect horizontal and vertical movements and hole-board locomotion or exploration in HA-rats (Experiment 1). In LA-rats, however (Experiment 2), these motor activities were significantly stimulated by DSP. Such effect was dose dependent; 1.5 mg/kg/24 hr was ineffective while 6 mg/kg/24 hr produced a clear cut reversion of hypoactivity. It is speculated that DSP treatment increased resistance of LA-rats to the mild stress caused by testing.