Probiotics - do they have a role in the pig industry?

The delivery of certain living microorganisms in food has long been suggested as having positive health effects in humans. This practice has extended into food animal production, with a variety of microorganisms being used; lactic acid bacteria, various Bacillus species and the yeast Saccharomyces cerevisiae have been particularly used in the pig industry. The increased interest in probiotics is essentially due to the problem of microbial resistance to antibiotics and following the ban of the use of antibiotics in animal production, probiotics being considered an alternative means to reduce pathogen infection and improve animal health especially around the time of weaning. However, there is still a need to clarify the probiotic effectiveness in pigs, and the underlying mechanisms. When assessing the efficacy of probiotics one must consider the particular strain of organism being used and the production stage of the pigs being treated. The reproducible delivery of probiotics in industrial pig production is problematic as maintenance of viability is key to their beneficial activity, but difficult to achieve with commonly used feed processing technologies. One specific context where probiotics organisms may be reliably delivered is in systems utilising fermented liquid feeds. Liquid feed may be fermented by the activity of wild lactic acid bacteria or may be stimulated using specific isolates as 'starters'; the latter system has advantages in terms of reproducibility and speed of fermentation. The farm context in which the organism is used is likely to be critical; the use of probiotics is more likely to result in measurable economic gains in animals living in sub-optimal conditions rather than in those reared in the highest welfare and husbandry conditions. The establishment of a beneficial lactic acid bacteria population at birth may lead to healthier animals, this may be most effectively achieved by treating sows, which provide an amplification step and flood the neonatal pigs' environment with desirable bacterial strains. In contrast, it may be sufficient to provide a supportive, protective microbiota around the time of weaning as this is a time of major crisis with instability and loss of certain bacterial populations.

Isolation and characterization of circulating tissue transglutaminase-specific T cells in coeliac disease.

Tissue transglutaminase (TG2) was identified as the humoral autoantigen in coeliac disease, but whether it can also serve as T cell autoantigen is still unknown. We aimed, therefore, to firstly explore the presence of TG2-specific T cells in peripheral blood of ten adult patients (four active, i.e. carrying both serological and histological features of the disease; four treated, i.e. with proven mucosal recovery and disappearance of specific antibodies after an adequate period of gluten free diet; and two potential coeliacs, i.e. carrying the serological stigmata of the disease, but not the intestinal lesions), and four healthy controls (two carrying the HLA-DQ2 haplotype of susceptibility to the disease), and secondly to carry out a detailed in vitro characterization of the isolated antigen-specific T cells. T cell lines were first established by means of weekly stimulation with human recombinant TG2 followed by generation of T cell clones through distribution of T cells on plates at one cell/well limiting dilution and further rounds of stimulation. Antigen specificity and HLA-DQ2 restriction were both assessed by evaluating the proliferative response to TG2 in the absence and presence of human sera blocking HLA-DQ2 molecules, after exclusion of impurities in the antigen preparation. Immune phenotyping of T cell clones was performed by flow cytometry, and the expression of IL-1â, IL-4, IL-6, IL-10, IL-12, TGF-beta, IFN-gamma and TNF-alpha was determined by ELISA assay on the supernatants of these clones. A total of 91 T cell clones were isolated from the three HLA-DQ2-positive, active patients, but none from the other patients and controls. The immune phenotyping showed that the majority of them (85.7 percent) were CD3/CD4+ and only a small percentage (14.3 percent) were CD3/CD8+, all carried the TCR alphabeta, and had a memory phenotype. The cytokine profile showed high levels of IFN-gamma and IL-6 that, together with the absence of IL-4, placed these T cell clones in the T helper type 1-like category. Further in vitro analysis was carried out on 32/91 CD4+ clones and showed a specific and dose-dependent proliferative response towards TG2 and an HLA-DQ2 restriction. Finally, when incubating duodenal mucosal specimens of treated patients with the supernatant of TG2-specific T cell clones, characteristic disease lesions were found, indicating a role for TG2-specific cellular immune response in the pathogenesis of coeliac disease.

Effect of different plant extracts and natural substances (PENS) against membrane damage induced by enterotoxigenic Escherichia coli K88 in pig intestinal cells.

Pig weaning period is frequently associated with infectious disease, mainly caused by enterotoxigenic Escherichia coli (ETEC) K88. Plant extracts exert different beneficial effects and may represent antibiotic alternatives to reduce piglet infection. In this study, plant extracts and other natural substances (PENS) have been evaluated on the pig intestinal IPEC-1 cells, for potential protection against ETEC K88 induced membrane damage. Several PENS have been considered: yeast extract, yeast nucleotides, unsaturated oligo-mannuronic acid, ulvan, bromelain and three fractions of bovine colostrums, as anti-inflammatory and immunomodulatory compounds; daidzein and Chlorella vulgaris extract, as anti-oxidant compounds; allicin, cinnamaldehyde and carvacrol, as anti-bacterial compounds. First, possible toxic effect of PENS on cell membrane permeability was verified by assessing the transepithelial electrical resistance (TEER) and paracellular flux of the extracellular marker phenol red. The highest non-toxic PENS concentration was added to ETEC infected cells to test the protection against membrane damage. The results showed that yeast extract, daidzein, bovine colostrum, bromelain and allicin protected the cells against the increased membrane permeability caused by ETEC, whereas the other PENS did not show this ability. Allicin protection was not due to its anti-bacterial activity, since ETEC growth was unaffected by the presence of allicin.

Immune response in relation to zinc status, sex and antioxidant defence in Italian elderly population: the ZENITH study.

OBJECTIVE: Evaluation of some immune markers in Italian elderly population in relation to zinc status, gender and antioxidant defence. DESIGN: Observational study. SETTING: Italian population. SUBJECTS: Apparently healthy, free-living subjects, 56 men and 52 women, aged 70-85 y, enrolled in Italy. METHODS: Lymphocytes were unstimulated or stimulated with the mitogen phytohemoagglutinin (PHA). The proliferative capacity was measured as incorporation of [3H]-thymidine and reported as stimulation index (SI). Cytokine secretion by lymphocytes was determined by ELISA. The antioxidant enzyme activities were measured using commercial kits. RESULTS: Dietary zinc intake, as well as zinc in serum, red blood cells and urine were on the normal range of values and did not show any difference between men and women. The proliferative response showed a high variability without significant differences between men and women. The amount of secreted pro- and anti-inflammatory cytokines was similar in men and women. No differences were found in the activity of antioxidant enzymes in lymphocytes, namely superoxide dismutase, glutathione peroxidase and catalase, between men and women. An association between SI and serum zinc level in men was found. SI resulted negatively correlated with interleukin (IL)-1beta (R2 = 0.036 and P = 0.012) and IL-10 (R2 = 0.34 and P = 0.040) only in men. IL-10 of PHA-stimulated lymphocytes was negatively correlated with red blood zinc in men (R2 = 0.41 and P = 0.008), while IL-10 of unstimulated and PHA-stimulated lymphocytes were negatively correlated with serum zinc in women (R2 = 0.38 and P = 0.020; R2 = 0.31 and P = 0.040, respectively). No correlation was observed between immune markers and antioxidant enzyme activities. CONCLUSIONS: Only weak differences on immune response between men and women were observed. However, zinc status appears to have more influence on the ability of lymphocytes to proliferate in men than in women.

Spray-dried plasma improves growth performance and reduces inflammatory status of weaned pigs challenged with enterotoxigenic Escherichia coli K88.

We investigated whether spray-dried plasma (SDP) improved growth and health of piglets challenged with enterotoxigenic Escherichia coli K88 (ETEC). Forty-eight pigs weaned at 21 d (BW = 4.88 +/- 0.43 kg) received one of four diets containing 6% SDP or fish proteins (as-fed basis) either nonmedicated (SDP-NM and FP-NM diets) or medicated with 0 or 250 mg/kg of colistine + 500 mg/kg of amoxycycline (SDP-M and FP-M diets), for 15 d. On d 4, pigs were orally challenged with ETEC. On d 15, eight pigs per dietary group were killed, blood and saliva were collected for analysis of K88 fimbriae-specific immunoglobulin (Ig)-A, and jejunum was removed for villi preparation, histological analysis, and cytokine expression. The presence or absence of K88 receptors (K88+ and K88- pigs respectively) was determined by villous adhesion assay. Effects of protein source on ADG (P = 0.04) and ADFI (P < 0.01), as well of medication on ADFI (P < 0.02), of all pigs were observed. In sacrified pigs, there was an effect of protein source on ADG (P = 0.03) and ADFI (P < 0.001), as well an interaction between medication and presence of K88 receptor (P = 0.02) for feed:gain ratio. Plasma K88 specific IgA were low in all K88 pigs and higher in K88+ pigs fed FP-NM compared with all the other groups (P < 0.05), except SDP-M. An interaction was found among protein source, medication, and presence of K88 receptors (P = 0.04). Saliva IgA concentrations were high in all pigs fed FP-NM and low in all other pigs. Jejunum of pigs fed FP-NM showed some ulcerations, edema, and mild inflammatory cell infiltration (ICI). In pigs fed FP-M, edema was reduced. Conversely, only a mild ICI was observed in pigs fed SDP-NM and SDP-M. Crypt depth was increased in K88+ pigs fed SDP-NM and an interaction between protein source and presence of K88 receptors was observed (P < 0.05). Expressions of tumor necrosis factor-alpha and interleukin (IL)-8 were lower in pigs fed SDP-NM and SDP-M than in those fed FP-NM and FP-M, either K88- or K88+ (P < 0.01). In pigs fed FP diets, expression of IL-8 tended to increase (P = 0.08) in K88+ compared with K88- subjects. Expression of interferon-gamma increased in K88 and K88+ pigs fed FP-M as compared with other pigs (P < 0.01). These results indicate that feeding with SDP improved growth performance and protected against E. coli-induced inflammatory status, and suggest that use of SDP-NM can be considered a valid antibiotic alternative.

Isolation, expression and characterization of carp retinol-binding protein.

Vitamin A alcohol and its precursors carotenoids are introduced in the organism with the diet, transported to the liver and from there as retinol to target tissues by a specific carrier, the retinol-binding protein (RBP). RBP, isolated and characterized in many vertebrates, shows very high homology among the species investigated; however, very little is known in fish. In the present work RBP cDNA isolated from a carp liver library was transcribed and translated in vitro and the corresponding protein characterized. Carp RBP amino acid sequence and tertiary structure are highly conserved, but the protein shows two peculiar and unique characteristics: the signal sequence is not processed by the ER signal peptidase and two N-glycosylations are present at the N-terminus portion of the protein. It was also demonstrated that RBP glycosylation is not a feature common to all teleosts. Transfection experiments show that the green fluorescent protein (GFP) can be directed into the secretory pathway by the carp RBP N-terminal region, both in fish and in mammal cells, demonstrating that the sequence, although not processed, is recognized as a secretory signal in different species. Results obtained from different investigators indicated that in fish plasma RBP circulates without interacting with transthyretin (TTR) or other proteins, suggesting that the complex with TTR, whose postulated function is to hamper easy kidney filtration of circulating RBP, has evolved later in the evolutionary scale. This hypothesis is reinforced by the finding that carp RBP, as well as trout and other lower vertebrates in which circulating complex has never been demonstrated, lacks a short C-terminal sequence that seems to be involved in RBP-TTR interaction. In carp, carbohydrates could be involved in the control of protein filtration through the kidney glomeruli. Moreover, experiments of carp RBP expression in Cos-1 cells and in the yeast Saccharomyces cerevisiae show that glycosylation is necessary for protein secretion; in particular, additional in vitro experiments have shown it is involved in protein translocation through ER membranes.

Unique biochemical nature of carp retinol-binding protein. N-linked glycosylation and uncleavable NH2-terminal signal peptide.

Retinol transport and metabolism have been well characterized in mammals; however, very little is known in fish. To study the mechanism by which fish retinol-binding protein (RBP) is able to remain in plasma besides its small molecular size, we isolated RBP cDNA from a carp liver cDNA library. Comparison of the deduced amino acid sequence with that of known vertebrate RBPs showed that carp RBP has high homology to the other cloned vertebrate RBPs, but it lacks the COOH-terminal tetrapeptide, RNL(S)L, which is most likely involved in the interaction with transthyretin in mammalian RBPs. In addition, the primary structure of carp RBP contains two consensus N-linked glycosylation sites that represent a unique feature. We have obtained experimental evidence, by in vitro and in vivo expression experiments, that both sites are indeed glycosylated. We have also characterized the protein as a complex type N-linked glycoprotein by lectin binding assay, neuraminidase and endoglycosidase H and F digestion. Inhibition of glycosylation by tunicamycin treatment of transfected cells caused a great reduction of RBP secretion. Since kidney filtration of anionic proteins is less than half that of neutral protein of the same size, this finding strongly suggests that the amount of carp RBP filtration through kidney glomeruli may be reduced by a glycosylation-dependent increase in the molecular size and negative charge of the protein. A second unique feature of carp RBP as secretory protein is the presence of a nonconserved NH(2)-terminal hydrophobic domain, which functions as an insertion signal but is not cleaved cotranslationally and remains in the secreted RBP.

Reduction of oxidative stress and cytokine-induced neutrophil chemoattractant (CINC) expression by red wine polyphenols in zinc deficiency induced intestinal damage of rat.

Consumption of red wine has been associated with health promotion and disease prevention. We have previously found that the intestine of zinc-deficient (ZD) rats develop oxidative damage associated with inflammation. Here we have used this model to investigate whether red wine polyphenols could protect against intestinal injury and, if so, whether this protection was achieved through antioxidant and anti-inflammatory activity. The intestinal alterations induced by zinc deficiency such as morphological damage, increased TBA-RS level and CuZn-superoxide dismutase activity, and decreased glutathione peroxidase activity, did not develop with the administration to ZD rats of a suspension of dealcoholated red wine (RWS). The same treatment induced in control rats a decrease of TBA-RS level but also of glutathione peroxidase and catalase activity. Treatment with RWS to ZD rats prevented a marked mucosal macrophage and neutrophil infiltration. The expression of pro-inflammatory cytokines, such as tumor necrosis factor alpha and cytokine-induced neutrophil chemoattractant (CINC), was induced by zinc deficiency, whereas that of the anti-inflammatory interleukin-10 was suppressed. Treatment with RWS reduced CINC expression. These results report a novel activity of red wine polyphenols in downregulation of intestinal CINC expression, which likely protects cells against inflammatory processes.

Bifidobacterium animalis protects intestine from damage induced by zinc deficiency in rats.

We investigated the potential beneficial effects of Bifidobacterium animalis on intestinal damage using zinc-deficient (ZD) rats as a model for intestinal alterations. The ZD rats were fed diets containing 1 mg Zn/kg for 20 (ZD(20)) or 40 (ZD(40)) d to induce damage that differed in severity. Subgroups of these rats, the ZD(20) + B and ZD(40) + B groups, received a suspension of B. animalis (3.5 x 10(8) colony forming units) daily for the last 10 d. Another subgroup, the ZD(40) + B + 7 d group, was fed the ZD diet for 7 d after the B. animalis treatment period. Zinc deficiency induced ulcerations, edema, inflammatory cell infiltration and dilatation of blood vessels in duodenum, jejunum and ileum, with increasing severity between 20 and 40 d of zinc deficiency. The mucosa of the ZD(20) + B group was well preserved, and most of the morphologic alterations induced by zinc deficiency were normalized in the ZD(40) + B group. The high fecal concentrations of B. animalis in the ZD(40) + B and ZD(40) + B + 7 d groups indicate that these bifidobacteria survived passage through the gastrointestinal tract and proliferated. Electron microscopy confirmed the elevated numbers of bifidobacteria in cecum. Treatment with B. animalis resulted in greater epithelial cell proliferation and disaccharidase activities in the ZD(40) + B group compared with the ZD(40) group. These findings indicate that B. animalis can protect the intestine from alterations induced by zinc deficiency, suggesting that this bacterium may play a role in intestinal mucosal defense.

Intestinal damage induced by zinc deficiency is associated with enhanced CuZn superoxide dismutase activity in rats: effect of dexamethasone or thyroxine treatment.

Zinc has a wide spectrum of biological activities and its deficiency has been related to various tissue dysfunctions and alterations of normal cell metabolism. Zinc also plays an important role in the antioxidant cellular defenses being a structural element of the non-mitochondrial form of the enzyme superoxide dismutase (CuZnSOD). We have already reported that Zn deficiency induces severe alterations in the rat intestine, that are reverted by treatment with dexamethasone (Dex) or thyroxine (T4). Here we report a paradoxical increase of CuZnSOD activity in rat intestine after 20 and 40 days of zinc deficiency. The increase of CuZnSOD activity is not due to an upregulation of gene expression because both Northern and Western blot analysis indicate that CuZnSOD mRNA and protein levels are not affected by zinc deficiency. A significant increase of lipid peroxidation was also observed in duodenum and jejunum associated with zinc deficiency. Treatment with either Dex or T4 to zinc-deficient rats protects against intestinal oxidative damage and results in SOD activity similar to control rats. Because glutathione peroxidase and catalase activities decreased in zinc deficiency, we speculate that the increase in SOD activity may be associated with an accumulation of hydrogen peroxide that may activate inflammatory molecules, further worsening tissue damage.

Involvement of interleukin-1beta in zinc deficiency-induced intestinal damage and beneficial effect of cyclosporine A.

In a previous study we have shown that zinc deficiency caused several alterations in intestine of rats. Here we report that interleukin-1beta (IL-1beta) is involved in the zinc deficiency-induced mucosal damage and that cyclosporine A (CsA) protects the intestine against both structural and functional alterations by different mechanisms. The zinc deficient (ZD) rats were maintained on a zinc deficient diet for 40 days. They received a daily injection of CsA (12 mg/kg) for the last 10 days. The histological analysis of small intestine revealed that the dramatic alterations induced by zinc deficiency (ulcerations, inflammation, edema, vasodilatation), were not present after CsA treatment. The IL-1beta gene expression, analyzed by PCR, was increased in the three intestinal regions of ZD rats, as compared to C rats. There was a relation between increasing IL-1beta expression and increased severity of damage, and the highest cytokine elevation was in the most damaged region, i.e. the jejunum. After CsA administration the IL-1beta mRNA was similar to control rats. The intestinal cell proliferation, measured as crypt cell production rate and labelling index, as well the cell renewal, measured as cell migration rate and turnover time, were affected by zinc deficiency. After CsA treatment, all these variables were similar to control rats, suggesting that CsA induces a stimulation of intestinal cell proliferation in zinc deficiency. Finally, the decrease in the disaccharidase activities induced by zinc deficiency was abrogated by CsA treatment.

Treatment of rats with dexamethasone or thyroxine reverses zinc deficiency-induced intestinal damage.

Structural and functional damage to the intestine and the potential beneficial effects of dexamethasone (Dex) and thyroxine (T4) were examined in zinc-deficient rats. Rats were assigned to zinc deficient (ZD), control (C) or pair-fed (PF ) groups and fed for 40 d a zinc deficient (1 mg/kg) diet (ZD rats) or a similar diet supplemented with 50 mg Zn/kg (C and PF rats). Some rats of the ZD group were treated for the last 10 d with low (250 mg/kg) or high (5 mg/kg) doses of Dex or with T4 (100 mg/kg). Serum corticosterone of T4-treated ZD rats did not differ from untreated ZD rats. Serum T4 of T4-treated ZD rats did not differ from C rats. ZD rats developed ulcerations, inflammation and edema in the small intestine, particularly in the jejunum. PF rats did not show mucosal changes relative to C rats. ZD rats showed significantly lower crypt cell production rate (CCPR) and labeling index (LI) in the three intestinal regions, and lower cell migration rate and higher turnover time in the duodenum relative to C rats. Sucrase and maltase activities of ZD rats were significantly lower than C rats in the three mucosal regions. Treatment with the low dose of Dex resulted in fewer ulcerations compared with ZD rats. In rats administered the high dose of Dex or T4, all morphological alterations disappeared; the CCPR, LI, cell migration rate, cell turnover time and disaccharidase activities did not differ from C rats. In conclusion, Dex and T4 exert beneficial effects on zinc deficiency-induced intestinal alterations in rats.

Cytokine gene expression in intestine of rat during the postnatal developmental period: increased IL-1 expression at weaning.

In the present study we have investigate whether cytokines are constitutively and differently expressed in intestine during the differentiative processes that take place at weaning. We have analyzed the expression of IL-1 beta, IL-2, IL-4 and IFN gamma by polymerase chain reaction in Peyer's patches (PP) and in intestine deprived of PP (I-PP) of rats from 16 to 30 days of age. The results showed a constitutive and marked expression of the cytokines already before weaning, with the exception of IL-2 in PP and IFN gamma in I-PP. IL-beta was the only cytokine to show a different expression at various ages with an initial increase at 19 days and a further elevation at 21 days when intestinal epithelium passes through major differentiative stages, suggesting an involvement of this cytokine in intestinal development. We have also tested whether treatment of rats with the immunosuppressor cyclosporin A (CsA) could affect intestinal differentiation. The results showed that only some markers of differentiation were affected (proliferation of staminal crypt cells and length of crypts). This was probably due to a direct effect rather than an immunomediated effect of CsA, since treatment of three intestinal cell lines (Caco-2, HT-29, FRIC) with CsA indicated that this drug can exert a cytostatic activity on intestinal cells.

Metallothionein gene is expressed in developing rat intestine and is induced by zinc but not by corticosteroids.

The expression of metallothionein (MT) mRNA during perinatal development of rat intestine and its induction by zinc and corticosteroids were studied. Pregnant rats from d 17 to 22 of gestation and rats at 2, 4, 13 and 21 d of postnatal life were injected with saline solution (control) or with zinc (10 mg/kg body wt) or corticosteroids (1 mg/kg body wt). After 6 h, tissues were removed for analysis. Northern hybridization of polyA + RNA to 32P-MT-cDNA revealed that MT was expressed already at d 17 of fetal life, increased afterwards (reaching the maximal expression around birth) and decreased soon after until weaning. Metallothionein mRNA was markedly induced by zinc at d 18 of fetal life to a level that remained constant throughout postnatal life. Corticosteroids were ineffective in inducing MT gene expression during prenatal and postnatal development. In 21-d-old adrenalectomized rats the level of MT mRNA was similar to that of control rats of the same age and was not changed by hormone treatment. The results indicate that MT gene expression can be induced by zinc during fetal life and that its expression without exogenous inducers cannot be ascribed to circulating corticosteroids.

Protein starvation impairs the ability of activated lymphocytes to produce interferon-gamma.

The impairment of lymphocyte function in nutritionally deprived rats was studied. Lymphocytes from rats fed a diet lacking protein showed a severe decrease in their ability to proliferate when stimulated with mitogens. This was accompanied by a dramatic inhibition of interferon-gamma (IFN-gamma) production and a lesser decrease in the release of interleukin-2 (IL-2). Analysis of RNA levels by Northern blot hybridization confirmed that lymphocytes from protein-starved rats had lower levels of mRNAs for IFN-gamma, IL-2, and IL-2 receptor than those from control animals. Protein deprivation therefore leads to a modification of lymphocyte function such that IFN-gamma production in particular is severely impaired.

Vitamin A intake and in vivo expression of the genes involved in retinol transport.

Two different metabolic alterations in vitamin A status are known to cause changes in the amount of circulating retinol-binding protein (RBP) and cellular retinol-binding protein (CRBP) in experimental animals; namely vitamin A deficiency, characterized by depleted retinol-liver stores and hypervitaminosis A, characterized by hepatic accumulation of retinyl esters. We have induced vitamin A deficiency and hypervitaminosis A in two groups of rats with the aim of determining whether the expression of the genes coding for these two proteins might be directly regulated by retinol. Using human RBP and CRBP cDNAs as probes, we measured the rate of transcription of the two genes in liver nuclei from control and treated rats by run-on transcription assays, and the steady-state level of the mRNAs by Northern blot analysis of total liver RNA. The distribution profile of RBP and CRBP mRNAs on fractionated liver polysomes was also examined. We have found a threefold decrease in the hepatic level of CRBP mRNA in vitamin-A-deficient animals, while the RBP mRNA is not affected by this nutritional deprivation. The decreases does not correspond to a lower transcription rate of the gene and therefore it is likely to result from lower stability of the CRBP mRNA. In hypervitaminosis A, we do not observe any differences in both the steady-state level of the mRNAs and in the rate of transcription of the two genes. The results are discussed in terms of retinol-dependent stabilization of the mRNA coding for CRBP.

Evidence that polyunsaturated lecithin induces a reduction in plasma cholesterol level and favorable changes in lipoprotein composition in hypercholesterolemic rats.

For 30 d adult rats were fed a hypercholesterolemic (H) diet (25% saturated fat, 1% cholesterol and 0.5% cholic acid) containing different amounts of saponins (1% or 0.2%) and/or purified polyunsaturated lecithin (2.5% or 0.7%). Lecithin induced a striking reduction in the plasma levels of very low density lipoprotein (VLDL), intermediate density lipoprotein (IDL) and low density lipoprotein (LDL) cholesterol as well as an increase in the level of high density lipoprotein (HDL) cholesterol. Saponins had only a very slight effect in lowering the level of VLDL cholesterol. Apoprotein A-I was unexpectedly present in VLDL, IDL and LDL after feeding rats the H diet and disappeared only after lecithin feeding. The activity of plasma lecithin-cholesterol acyltransferase was higher when the two lecithin diets were fed than when the other diets were fed. Fecal excretion of neutral sterols was unmodified by the various diets whereas acid steroid excretion increased after lecithin feeding. Saponins, when added with lecithin to the diet, reduced the beneficial effect of lecithin. The results indicate that polyunsaturated lecithin induced a reduction in plasma cholesterol, possibly through an increased formation of HDL particles.

Expression of liver-specific genes coding for plasma proteins in protein deficiency.

Protein deficiency leads to a decreased concentration of plasma proteins, although it is not clear whether this response is caused by alterations in gene transcription or in post-transcriptional events. The aim of this study was to investigate the expression of some liver-specific genes coding for plasma proteins in rats kept on a protein-free diet for 30 days. Cloned cDNA probes for the albumin, transthyretin, retinol-binding protein and prothrombin genes were used in Northern hybridizations to total liver RNA to compare their transcript levels in protein-deficient and control animals. Liver polysomes were also isolated and fractionated from the two groups of animals to examine the possible effects of protein deficiency on translation of the mRNAs. The results indicate that the albumin and transthyretin mRNAs are present in lower amounts in protein deficiency. The distribution profile along sucrose gradients shows that all mRNAs are undergoing translation, but in protein-deficient animals a small but consistent fraction of each mRNA is also present in the non-polysomal, low molecular weight fractions.

Modifications in plasma cholesterol and apolipoproteins of hypercholesterolaemic rats induced by ethanol-soluble factors of Vicia faba.

High-fat-high-cholesterol diets containing casein or a Vicia faba bean (faba bean) protein concentrate as the protein source were given to rats for 5 weeks. When the faba bean protein concentrate or its ethanol extract was present in the diet, a marked decrease was found in the level of circulating cholesterol associated with the lower-density lipoproteins (very-low-, intermediate- and low-density lipoproteins) compared with the level found on the diets containing casein or the faba bean protein concentrate deprived of ethanol-soluble factors. Alterations in apoprotein pattern were detected after the different dietary treatments. In particular, apoA-I appeared in an unusual form with electrophoretic mobility faster than normal in all lipoprotein fractions after feeding the diets that did not lower plasma cholesterol. When the diets contained the faba bean protein concentrate or its ethanol extract, the apoA-I disappeared from the lower-density lipoproteins but its normal form and the unusual one were apparent in the high-density lipoproteins. A moderate increase in faecal excretion of acidic steroids was found after feeding the diets containing the ethanol-soluble factors, irrespective of the protein source. The results are discussed in relation to the presence of saponin and polyunsaturated lecithin in the ethanol extract of the faba bean protein concentrate.

Thymidine kinase genes and the induction of anti-viral responses by interferon.

A mouse fibroblast cell-line deficient in thymidine kinase (Ltk(-) aprt(-)) fails to show an anti-viral response when treated with interferon. After introduction of a viral tk gene into these cells the resultant clones showed normal responses to interferon. However, one such tk-containing clone (C6) spontaneously lost its ability to respond to interferon by inducing an antiviral state although it retained its ability to induce the enzyme oligo(2'-5' A)-synthetase. This sub-clone (6A) still expressed thymidine kinase activity but restriction endonuclease analysis indicated an alteration in the sequences flanking the exogenous viral tk gene. Our results suggest that a modification in the exogenous viral DNA sequences led to a loss of interferon sensitivity.