SOS3 (salt overly sensitive 3) from Arabidopsis thaliana: expression, purification, crystallization and preliminary X-ray analysis.

The salt-tolerance gene SOS3 (salt overly sensitive 3) of Arabidopsis thaliana encodes a calcium-binding protein that is able to sense the cytosolic calcium signal elicited by salt stress. SOS3 activates the SOS2 protein kinase, which activates various ion transporters. SOS3 was cloned into a plasmid and expressed in Escherichia coli, allowing purification of the protein to homogeneity. Two crystals with different additive contents were grown. Both diffract to 3.2 A resolution and belong to space group I4(1), with unit-cell parameters a = 93.65, c = 80.08 A and a = 91.79, c = 85.78 A, respectively. A promising molecular-replacement solution has been found using neuronal calcium-sensor 1 as the search model. Interestingly, no solution was found using AtCBL2 (A. thaliana calcineurin B-like protein) structure as a search model, although this protein belongs to the same family and displays 50% sequence identity.

Structure of bacteriocin AS-48: from soluble state to membrane bound state.

The bacteriocin AS-48 is a membrane-interacting peptide, which displays a broad anti-microbial spectrum against Gram-positive and Gram-negative bacteria. The NMR structure of AS-48 at pH 3 has been solved. The analysis of this structure suggests that the mechanism of AS-48 anti-bacterial activity involves the accumulation of positively charged molecules at the membrane surface leading to a disruption of the membrane potential. Here, we report the high-resolution crystal structure of AS-48 and sedimentation equilibrium experiments showing that this bacteriocin is able to adopt different oligomeric structures according to the physicochemical environment. The analysis of these structures suggests a mechanism for molecular function of AS-48 involving a transition from a water-soluble form to a membrane-bound state upon membrane binding.

Effect of growth hormone, epidermal growth factor, and insulin on bacterial translocation in experimental short bowel syndrome.

BACKGROUND/PURPOSE: An adaptive process starts in the remaining intestine after massive resection, and several trophic factors including growth hormone (GH), epidermal growth factor (EGF), and insulin (INS) have been shown to have a positive effect on it. Bacterial translocation (BT) is frequent after extensive small bowel resection, but the effects of GH, EGF, or INS have not been investigated in experimental short bowel syndrome (SBS). This study tests the hypothesis that GH, EGF, or INS decrease BT in SBS in rats with parenteral nutrition (PN). METHODS: Thirty-eight adult Wistar rats underwent central venous cannulation and were assigned randomly to 1 of 4 groups receiving for 10 days 4 treatment regimes: (1) PN group (n = 10): fasting, all-in-one PN solution (300 mL/kg/24 h, 280 kcal/kg/24 h), 80% gut resection including ileo-cecal valve; (2) GH group (n = 9): fasting, same PN regime and resection, GH (1 mg/kg/d, subcutaneously); (3) EGF group (n = 9): fasting, PN, resection, EGF (150 microg/24 h intravenously); (4) INS group (n = 9): fasting, PN, resection, INS (1 UI/100 g/24 h subcutaneously). At the end of the experiment they were killed, and mesenteric lymph nodes (MLN) and peripheral and portal blood samples were recovered and cultured. Several fragments of intestine were taken to determine cell proliferation (PCNA index) and morphometric parameters (villous height, crypt depth). RESULTS: GH, EGF, and INS groups showed a 28%, 29%, and 30% increase in gut mucosal thickness, and PCNA index rose 21%, 20%, and 25%, respectively in comparison with PN controls. Bacterial translocation to peripheral blood was detected in 0% of PN animals and in 44%, 40%, and 28% of GH, EGF, or INS rats, respectively (P < .05). No differences were found in BT in MLN or portal blood among groups. CONCLUSION: Administration of GH, EGF, or INS improves gut mucosal structure in rats with SBS under PN, but, surprisingly, the incidence of BT detected in peripheral blood was increased rather than decreased in animals receiving these treatments.

Bacterial translocation is favored by the preservation of the ileocecal valve in experimental short bowel with total parenteral nutrition.

Sepsis in short-bowel syndrome (SBS) is in part due to bacterial translocation (BT). Parenteral nutrition (PN) is often necessary in SBS and promotes BT. The aim of this study was to asses the effect of the presence or absence of ileocecal valve (ICV) on BT in parenterally-fed rats with massive intestinal resection. Sixty-five adult Wistar rats underwent central venous cannulations and were randomly assigned to one of five groups receiving for ten days five treatment regimes: Sham (n = 17) standard rat chow + i.v. saline. PN (n = 17) fasting + PN. Res-Sham (n = 10) standard rat chow + i.v. saline + 80% gut resection. Res-PN (n = 11) fasting, PN + 80% gut resection. Res-ICV-PN (n = 10) fasting, PN + 80% gut resection including ICV. At the end of the experiment they were euthanized and mesenteric lymph nodes (MLN), spleen and peripheral and portal blood specimens were recovered and cultured. BT was found in 47% of PN animals, 91% of Res-PN rats, 100% of Res-Sham group and 60% of Res-ICV-PN animals, but not in Sham ones. 97% of BT+ animals had positive cultures in MLN and/or portal blood, whereas germs beyond liver were detected in 30% of Res-Sham, 37% of PN, 50% of Res-PN and 0% of Res-ICV-PN rats. The present study confirms that both massive intestinal resection and PN promote BT. In addition, it shows that animals deprived of ICV have lower incidence of BT in this setting than those with it and that the germs do not reach in them peripheral blood in the same proportions as in ICV-intact animals. These results suggest that the presence of an intact ICV favor BT in parenterally-fed rats with massive intestinal resection.

Effect of growth hormone on bacterial translocation in experimental short-bowel syndrome.

Despite the adaptive process triggered in the remaining intestine by massive bowel resection, bacterial translocation (BT) is frequent under these conditions. Several trophic factors, including growth hormone (GH), are involved in the process of adaptation in short-bowel syndrome (SBS). However, the effect of GH on BT has not been investigated experimentally. The aim of this study was to test the hypothesis that GH administration prevents BT in rats with SBS receiving only parental nutrition (PN). Nineteen adult Wistar rats underwent central venous cannulation and were randomly assigned to one of two groups receiving for 10 days two treatment regimes: PN group (n = 10): fasting, all-in-one PN solution (300 ml. kg. 24 h, 280 kcal/kg. 24 h), 80% gut section including ileocecal valve; GH group (n = 9): fasting, same PN regime and resection plus GH 1 mg/kg s.c). At the end of the experiment, the rats were killed and mesenteric lymph nodes (MLN) and samples of systemic and portal blood were obtained and cultured. Several samples of full-thickness jejunal wall were taken for determining cell proliferation index (PCNA) and mucosal thickness. Jejunal mucosal thickness increased by 30% and PCNA index by 35% in GH-treated rats in comparison with those treated with PN alone. However, contrary to our expectations, BT expressed by positive culture of intestinal flora in portal blood, MLN, or systemic blood was found in 60% of PN and 87% of GH animals (P = 0.1). Translocation to the general circulation expressed by the presence of organisms in systemic blood was detected in 0% of PN and 44% of GH rats (P < 0.05). Although exogenous GH improves gut mucosal structure in rats with SBS treated with PN, it seems to increase rather than decrease mucosal permeability to intestinal bacteria.

Bacterial translocation and T-lymphocyte populations in experimental short-bowel syndrome.

Bacterial translocation (BT) accounts in part for sepsis in short-bowel syndrome in which total parenteral nutrition (TPN) is routinely necessary. TPN "per se" facilitates BT and it has been suggested that decreased T-lymphocyte populations (TLP) in newborn rabbits and nude mice promote BT as well. We have tested the hypothesis that BT and modifications in TLP are to be expected in rats subjected to TPN and gut resection. Forty-five adult Wistar rats underwent central venous cannulations and were randomly assigned to one of three groups receiving for ten days three treatment regimes: - Group Sham (n = 17) oral intake of rat chow + saline (300 ml/kg/24 h) through a jugular vein catheter. - Group TPN (n = 17) fasting + infusion of all-in-one TPN solution (300 ml/kg/24 h). - Group RES (n = 11) fasting, same TPN regime + 80% gut resection. At the end of the experiment they were sacrified and specimens (peripheral and portal blood, spleen and mesenteric lymph nodes) were recovered, cultured and/or assessed for CD4+ and CD8+. Bacterial translocation was found in 47% of TPN animals, 92% of RES rats, but not in SHAM ones. Lymphocyte populations were not different in BT+ (n = 8) or BT- (n = 9) rats in the TPN group. TPN and resected animals showed a rise in CD4+ and a drop in CD8+ (then a better CD4+/CD8 ratio) when comparing with SHAM group rats. From this data we may conclude that: 1) BT is frequent if TPN is administered, and constant in resected animals. 2) No apparent relationship between the proportions of CD4+ and CD8+ lymphocytes and BT could be shown in TPN group. 3) High CD4+/CD8+ ratio in TPN and RES groups demonstrate that BT is possible even having good TLP.

Immune dysfunction in Down's syndrome: primary immune deficiency or early senescence of the immune system?

Multiple immunologic disturbances are commonly observed in individuals with Down's syndrome including abnormal proportions of peripheral blood lymphoid subsets, cellular dysfunction, and autoimmune phenomena. However, a majority of the individuals with this syndrome do not show clear features of immunological disease. Many of these immunological alterations are age-related changes and can be enclosed in the spectrum of multiple signs of early senescence characteristic of Down's syndrome.

Lymphocyte subpopulations after extensive small bowel resection in the rat.

The increased risk of infection after massive intestinal resection (MIR) may be attributable to impaired nutrition, loss of intestinal lymphoid tissue, or both. This study examines whether MIR itself changes the immune cell populations in laboratory animals when nutritional status is preserved. The authors studied cellular immunity (lymphocyte subsets T4 and T8 and the T4:T8 ratio) and humoral immunity (IgG, IgM, IgA, and B lymphocytes) in the blood, spleen, and mesenteric lymph nodes of unresected Wistar rats (control group, n = 6) and of animals that underwent 80% bowel resection followed by 7 days of either oral feeding (resection-oral group, n = 6) or parenteral nutrition (resection-TPN group, n = 6). The increase in body weight was similar among all groups, and the levels of total protein, albumin, prealbumin, and immunoglobulin remained unchanged. All resected animals, irrespective of their feeding route, had significantly lower proportions of T4 and B lymphocytes and T4:T8 ratio in blood, T4 and T8 in mesenteric lymph nodes, and T4 and T4:T8 ratio in the spleen. The author's results suggest that removal of large amounts of lymphoid tissue along with the bowel during MIR might lead to inadequate immune response even when the nutritional status is preserved.

Quantification of chicken alpha-fetoprotein: a useful tool in studies of embryo development and pathology.

Chicken alpha-fetoprotein (AFP) from the plasma of 12-day-old chick embryos was purified by electroelution from SDS/PAGE gels, and used to produce polyclonal and monoclonal antibodies. Both reagents were then used to design a sandwich-type enzyme-immunoassay for the quantification of AFP in biological fluids. The assay was used to quantify AFP in the serum and amniotic fluid of chick embryos with abnormalities of the neural tube. Serum AFP was significantly greater in these embryos than in normal ones of similar age. Moreover, substantial amounts of AFP were demonstrated in the amniotic fluid, whereas this protein was undetectable in the amniotic fluid of normal embryos. This method of assay may provide a reliable tool for studies of chick embryogenesis and abnormalities of embryonal differentiation.

Imbalance of the CD4+ subpopulations expressing CD45RA and CD29 antigens in the peripheral blood of adults and children with Down syndrome.

Peripheral blood lymphoid subsets expressing either CD45RA or CD29 antigens, were quantified in 30 children and 59 adults with Down Syndrome and appropriate age-matched controls, by dual immunofluorescence and flow cytometry. Down's patients, both adults and children, displayed a significant decrease of CD4+CD45RA+ cells in comparison with the observed in their age-matched controls, but no difference was found in the CD4+CD29+ subset. These results show clearly the imbalance of these subpopulations in the peripheral blood of individuals with Down syndrome that result in the inversion of the CD45RA/CD29 ratio, due to a major reduction of the CD45RA subset. No obvious difference was found in the CD45RA/CD29 ratio within the CD4 negative cells. Abnormalities of these subpopulations could be indicative of early senescence of the immune system, since age-related changes in Down's persons were in parallel with those observed in normal individuals and the proportion of both subpopulations were roughly similar in Down's children and normal adults.

[The defense against infection in the short bowel syndrome]. / La defensa contra la infección en el intestino corto.

The high risk of infection in the short-bowel syndrome (SBS) may be due to malnutrition, lost of lymphoid bowel structures or both. Total parenteral nutrition (TPN) may alleviate the malnutrition, but we do not know what will happen with immune response in SBS with good nutritional state. We have studied the cellular immunity (lymphocytic subsets T4 and T8 and T4/T8 ratio) and the humoral one (IgG, IgM, IgA and B lymphocytes) in blood, spleen and mesenteric lymph nodes, in 12 wistar rats with 80% bowel resection, 6 of them with oral feeding and 6 with TPN, and 6 control rats, during 7 days. The weight increased and the total protein, albumin and prealbumin levels were the same in all groups. There was not difference between the resected groups. No difference was observed in the rate of immunoglobulins and the resected groups showed significatively lower figures than the control group in T4, B lymphocytes and T4/T8 ratio in blood, T4 and T8 in mesenteric nodes and in T4 and T4/T8 ratio in the spleen. These results suggest that the resection of large amounts of bowel could produce a fall in the immune response even when adequate nutritional state is preserved.

[Amniotic and serum alphafetoprotein in the chick embryo with neural tube defect]. / Alfafetoproteína sérica y amniótica en el embrión de pollo con defecto del tubo neural.

Although alpha-feto-protein (AFP) is a widely used marker for human neural tube defects (NTD) little is known about the mechanisms for its increase in the amniotic fluid in this condition. For investigating this issue we developed a chick embryo AFP assay and tested it in a NTD experimental model. AFP obtained by electroelution on PAGE/SDS gels from the plasma of 12-day-old embryos was used to produce rabbit polyclonal and mouse monoclonal antibodies. A specific sandwich-type enzyme-immune-assay was developed using both reagents. Sterile aspiration of 5 ml. of albumen from 602 fertile hen eggs on the 27th hour of incubation (Hamburger stages 8 to 11) led to the appearance of NTD in 36 out of the 270 survivors (13%). Amniotic and seric AFP levels were measured on the 15th day of incubation in NTD chicks (n = 11) and in control ones (n = 9) and the results were compared by non-parametric tests. Serum AFP was five times higher in NTD chicks than in controls (119.2 +/- 32.6 vs 523.3 +/- 173.62 micrograms/ml., p < 0.001) and amniotic AFP was absent in control and very increased in NTD animals (0.15 +/- 0.02 vs 87.14 +/- 84 micrograms/ml., p < 0.001). It is concluded that: 1) serum AFP is intriguingly increased in the chick with NTD; 2) since urine is not diversed into the amniotic sac in the avian embryo, the only source of AFP in its fluid is exudation through an open defect. This conclusion is further supported by the absence of amniotic AFP in a chick with a large closed NTD.

Differential expression of lymphocyte function-associated antigen (LFA-1) on peripheral blood leucocytes from individuals with Down's syndrome.

We analysed the expression of lymphocyte function-associated antigen LFA-1 on the cell surface of peripheral blood lymphocytes, monocytes and granulocytes from 20 children with Down's syndrome. No differences in LFA-1 expression was found within monocytes or granulocytes from either normal or Down's syndrome children; however, a clear-cut difference was observed on lymphoid cells. Both normal and Down's syndrome lymphocytes displayed a bimodal pattern of LFA-1 staining by flow cytometry, with a predominance of cells with low expression in normal population, and an increased proportion of lymphocytes with high level of LFA-1 expression in Down's syndrome children. This difference correlates well with the abnormal proportion of T cell subsets and inversion of CD4/CD8 observed in a majority of our cases, and therefore, it could merely reflect the increase of certain T cell subsets normally expressing higher number of LFA-1 molecules. Taken together, our results do not support an abnormally increased expression of leucocytes integrins in trisomy 21 cells, and raise some doubt about the suggested role of the abnormal cellular expression of LFA-1 in the pathogensis of secondary immunodeficiency associated to Down's syndrome.