Effects of exogenous ammonia or free amino acids on proteolytic activity and protein breakdown products in Streptococcus bovis,Prevotella albensis,and Butyrivibrio fibrisolvens

We studied in details the ammonia or free amino acids (AA) effects on proteolytic activity of three ruminal bacteria: enzymatic activities and protein breakdown products were measured at the end of the exponential growth phase. In Streptococcus bovis the simultaneous uptake of ammonia and probably small peptides induced a decline in total proteolytic activity as a result of changes in endopeptidasic activities. With free AA, the tendency for the endopeptidasic activities to specialise was more evident and the total proteolytic activity decreased too. In Prevotella albensis, the inhibition of proteolysis with free AA was linked to the disappearance of free endopeptidases, to the specialization of cell-associated endopeptidases and to the decrease in exopeptidases. The decrease of proteolysis in P. albensis when ammonia was added was more difficult to interpret. With ammonia or AA Butyrivibro fibrisolvens developed a distinct behavior of those expressed by the other species: the increase of the total proteolytic activity could be explained by a better balance of the endopeptidases expressed. It then clearly appeared that the expression of the proteolytic activities are linked to the nature and/or to the quantity of the nitrogen source. This leads each species to adopt its own nutritional strategy in order to adapt to the environmental conditions of the ruminal ecosystem. Received: 2 July 2001 / Accepted: 28 September 2001     

Nitric oxide and inducible nitric oxide synthase expression are downregulated in acute cholestasis in the rat accompanied by liver ischemia

Hepatic blood flow decreases under cholestasis and there is evidence that NO regulates liver microvascular perfusion. Thus, the aim of the present study was to evaluate NO synthesis in cholestasis. Cholestasis was induced by bile-duct ligation (BDL) in male Wistar rats. Bilirubins and enzyme activities were measured in serum. Lipid peroxidation, GSH, GSSG and glycogen were determined in liver. Histopathological analysis was performed. Serum NO2- + NO3- concentration was measured by the Gries reaction. iNOS immunoblot analysis was carried out using an iNOS polyclonal antibody. After 7 days of BDL lipid peroxidation increased while GSH/GSSG ratio decreased. Serum NO2- + NO3- and liver iNOS protein were reduced, accompanied by ischemia as revealed by the histopathological analysis. GSH upregulates NO synthesis by increasing iNOS mRNA levels and iNOS activity, thus the reduction of GSH/GSSG ratio may be responsible for the downregulation of iNOS protein and NO synthesis, which in turn may explain the observed ischemia and the decreased hepatic blood perfusion in cholestasis reported by others.     

Unravelling the complex genetics of cleft lip in the mouse model

Nonsyndromic cleft lip in ``A' strain mice and humans is genetically complex and is distinct from isolated cleft palate. Cleft lip embryos recovered in 2.4% of 1485 first backcross (BC₁) segregants from a cross of A/WySnJ (24% cleft lip) and C57BL/6J (no cleft lip) in A/WySnJ mothers, and in testcrosses of 10 recombinant inbred (RI) strains (AXB/Pgn or BXA/Pgn), were used for gene mapping and for inference of genetic architecture. The A/WySnJ maternal genotype increased cleft lip risk in reciprocal crosses; the relevant genetic difference between AXB-6/Pgn (8%) and A/WySnJ (24%) is entirely maternal. A combination of new mapping panels (325 meioses), new markers, and a recombinant cleft lip embryo redefined the location of a recessive factor essential to cleft lip risk, clf1, and candidate genes Itgb3 and Crhr, to between D11Mit146/360 and D11Mit166/147. A screen of 54 YACs for 46 genes and SSLP loci located Wnt15, Wnt3, Crhr, Mtapt, Itgb3, Dlx3, and Dlx7 within the clf1 candidate region. The clf2 locus was newly mapped to Chromosome (Chr) 13 by a genome screen of BC₁ segregants, and further defined to a 4-cM region between D13Mit13/54 and D13Mit231 by strain distribution patterns of cleft lip liability and markers in testcrossed RI strains. Specific combinations of marker genotypes associated with cleft lip risk indicated that high risk in A/WySnJ mice is caused by epistatic interaction between clf1 and clf2 in the context of a genetic maternal effect. Human homologs of clf1 and clf2 are expected to be on 17q and 5q/9q. Received: 17 May 2000 / Accepted: 30 November 2000     

Nitric Oxide Production in Striatum and Pallidum of Cirrhotic Rats

Ammonium and manganese are neurotoxic agents related to brain metabolic disturbances observed after prolonged liver damage. The aim of this study was to assess the production of nitric oxide (NO) in the brain of cirrhotic rats exposed to manganese. We induced cirrhosis by bile duct ligation for 4 weeks in rats. From brain, striatum and globus pallidus were dissected out, and NO synthase activity and the content of nitrites plus nitrates (NOx) were determined. In pallidum we found a diminished constitutive NO synthase activity from cirrhotic rats, independently of manganese exposure. This result was confirmed by low levels of NOx in the same brain area (P<0.05, two-way ANOVA). This finding was not related to protein expression of NO synthase since no differences were observed in immunoblot signals between cirrhotic and sham-operated animals. Results from present study suggest that the production of NO is reduced in basal ganglia during cirrhosis.     

Severe bacterial neonatal infections in Madagascar,Senegal, and Cambodia: A multicentric community-based cohort study

BackgroundSevere bacterial infections (SBIs) are a leading cause of neonatal deaths in low- and middle-income countries (LMICs). However, most data came from hospitals, which do not include neonates who did not seek care or were treated outside the hospital. Studies from the community are scarce, and few among those available were conducted with high-quality microbiological techniques. The burden of SBI at the community level is therefore largely unknown. We aimed here to describe the incidence, etiology, risk factors, and antibiotic resistance profiles of community-acquired neonatal SBI in 3 LMICs.Methods and findingsThe BIRDY study is a prospective multicentric community-based mother and child cohort study and was conducted in both urban and rural areas in Madagascar (2012 to 2018), Cambodia (2014 to 2018), and Senegal (2014 to 2018). All pregnant women within a geographically defined population were identified and enrolled. Their neonates were actively followed from birth to 28 days to document all episodes of SBI. A total of 3,858 pregnant women (2,273 (58.9%) in Madagascar, 814 (21.1%) in Cambodia, and 771 (20.0%) in Senegal) were enrolled in the study, and, of these, 31.2% were primigravidae. Women enrolled in the urban sites represented 39.6% (900/2,273), 45.5% (370/814), and 61.9% (477/771), and those enrolled in the rural sites represented 60.4% (1,373/2,273), 54.5% (444/814), and 38.1% (294/771) of the total in Madagascar, Cambodia, and Senegal, respectively. Among the 3,688 recruited newborns, 49.6% were male and 8.7% were low birth weight (LBW). The incidence of possible severe bacterial infection (pSBI; clinical diagnosis based on WHO guidelines of the Integrated Management of Childhood Illness) was 196.3 [95% confidence interval (CI) 176.5 to 218.2], 110.1 [88.3 to 137.3], and 78.3 [59.5 to 103] per 1,000 live births in Madagascar, Cambodia, and Senegal, respectively. The incidence of pSBI differed between urban and rural sites in all study countries. In Madagascar, we estimated an incidence of 161.0 pSBI per 1,000 live births [133.5 to 194] in the urban site and 219.0 [192.6 to 249.1] pSBI per 1,000 live births in the rural site (p = 0.008). In Cambodia, estimated incidences were 141.1 [105.4 to 189.0] and 85.3 [61.0 to 119.4] pSBI per 1,000 live births in urban and rural sites, respectively (p = 0.025), while in Senegal, we estimated 103.6 [76.0 to 141.2] pSBI and 41.5 [23.0 to 75.0] pSBI per 1,000 live births in urban and rural sites, respectively (p = 0.006). The incidences of culture-confirmed SBI were 15.2 [10.6 to 21.8], 6.5 [2.7 to 15.6], and 10.2 [4.8 to 21.3] per 1,000 live births in Madagascar, Cambodia, and Senegal, respectively, with no difference between urban and rural sites in each country. The great majority of early-onset infections occurred during the first 3 days of life (72.7%). The 3 main pathogens isolated were Klebsiella spp. (11/45, 24.4%), Escherichia coli (10/45, 22.2%), and Staphylococcus spp. (11/45, 24.4%). Among the 13 gram-positive isolates, 5 were resistant to gentamicin, and, among the 29 gram-negative isolates, 13 were resistant to gentamicin, with only 1 E. coli out of 10 sensitive to ampicillin. Almost one-third of the isolates were resistant to both first-line drugs recommended for the management of neonatal sepsis (ampicillin and gentamicin). Overall, 38 deaths occurred among neonates with SBI (possible and culture-confirmed SBI together). LBW and foul-smelling amniotic fluid at delivery were common risk factors for early pSBI in all 3 countries. A main limitation of the study was the lack of samples from a significant proportion of infants with pBSI including 35 neonatal deaths. Without these samples, bacterial infection and resistance profiles could not be confirmed.ConclusionsIn this study, we observed a high incidence of neonatal SBI, particularly in the first 3 days of life, in the community of 3 LMICs. The current treatment for the management of neonatal infection is hindered by antimicrobial resistance. Our findings suggest that microbiological diagnosis of SBI remains a challenge in these settings and support more research on causes of neonatal death and the implementation of early interventions (e.g., follow-up of at-risk newborns during the first days of life) to decrease the burden of neonatal SBI and associated mortality and help achieve Sustainable Development Goal 3.

In a community-based, prospective cohort study, Bich-Tram Huynh and colleagues investigate the incidence and factors associated with several bacterial infections among neonates in rural and urban areas of three low-middle income countries.     

Down-regulated expression of plant-specific glycoepitopes in alfalfa

Compared with other plant expression systems used for pharmaceutical protein production, alfalfa offers the advantage of very homogeneous N -glycosylation. Therefore, this plant was selected for further attempts at glycoengineering. Two main approaches were developed in order to humanize N -glycosylation in alfalfa. The first was a knock-down of two plant-specific N -glycan maturation enzymes, β1,2-xylosyltransferase and α1,3-fucosyltransferases, using sense, antisense and RNA interference strategies. In a second approach, with the ultimate goal of rebuilding the whole human sialylation pathway, human β1,4-galactosyltransferase was expressed in alfalfa in a native form or in fusion with a targeting domain from N -acetylglucosaminyltransferase I, a glycosyltransferase located in the early Golgi apparatus in Nicotiana tabacum . Both knock-down and knock-in strategies strongly, but not completely, inhibited the biosynthesis of α1,3-fucose- and β1,2-xylose-containing glycoepitopes in transgenic alfalfa. However, recombinant human β1,4-galactosyltransferase activity in transgenic alfalfa completely prevented the accumulation of the Lewis a glycoepitope on complex N -glycans.     

Solution structure of NEMO zinc finger and impact of an anhidrotic ectodermal dysplasia with immunodeficiency-related point mutation

The regulatory NEMO (NF-κB essential modulator) protein has a crucial role in the canonical NF-κB signaling pathway notably involved in immune and inflammatory responses, apoptosis and oncogenesis. The regulatory domain is located in the C-terminal half of NEMO and contains a classical CCHC-type zinc finger (ZF). We have investigated the structural and functional effects of a cysteine to phenylalanine point mutation (C417F) in the ZF motif, identified in patients with anhidrotic ectodermal dysplasia with immunodeficiency. The solution structures of the wild type and mutant ZF were determined by NMR. Remarkably, the mutant adopts a global ββα fold similar to that of the wild type and retains thermodynamic stability, i.e., the ability to bind zinc with a native-like affinity, although the last zinc-chelating residue is missing. However, the mutation induces enhanced dynamics in the motif and leads to an important loss of stability. A detailed analysis of the wild type solution structure and experimental evidences led to the identification of two possible protein-binding surfaces that are largely destabilized in the mutant. This is sufficient to alter NEMO function, since functional complementation assays using NEMO-deficient pre-B and T lymphocytes show that full-length C417F pathogenic NEMO leads to a partial to strong defect in LPS, IL-1β and TNF-α-induced NF-κB activation, respectively, as compared to wild type NEMO. Altogether, these results shed light onto the role of NEMO ZF as a protein-binding motif and show that a precise structural integrity of the ZF should be preserved to lead to a functional protein-recognition motif triggering full NF-κB activation.     

Disruption of the mitotic kinesin Eg5 gene (Knsl1) results in early embryonic lethality

Eg5, a member of the widely conserved kinesin-5 family, is a plus-end-directed motor involved in separation of centrosomes, and in bipolar spindle formation and maintenance during mitosis in vertebrates. To investigate the requirement for Eg5 in mammalian development, we have generated Eg5 deficient mice by gene targeting. Heterozygous mice are healthy, fertile, and show no detectable phenotype, whereas Eg5^−/− embryos die during early embryogenesis, prior to the implantation stage. This result shows that Eg5 is essential during early mouse development and cannot be compensated by another molecular motor.     

Heating and microwave assisted SPPS of C-terminal acid peptides on trityl resin: the truth behind the yield

Despite correct purity of crude peptides prepared on trityl resin by Fmoc/tBu microwave assisted solid phase peptide synthesis, surprisingly, lower yields than those expected were obtained while preparing C-terminal acid peptides. This could be explained by cyclization/cleavage through diketopiperazine formation during the second amino acid deprotection and third amino acid coupling. However, we provide here evidence that this is not the case and that this yield loss was due to high temperature promoted hydrolysis of the 2-chlorotrityl ester, yielding premature cleavage of the C-terminal acid peptides.     

The immunomodulatory protein B7-H4 is overexpressed in breast and ovarian cancers and promotes epithelial cell transformation

B7-H4 protein is expressed on the surface of a variety of immune cells and functions as a negative regulator of T cell responses. We independently identified B7-H4 (DD-O110) through a genomic effort to discover genes upregulated in tumors and here we describe a new functional role for B7-H4 protein in cancer. We show that B7-H4 mRNA and protein are overexpressed in human serous ovarian cancers and breast cancers with relatively little or no expression in normal tissues. B7-H4 protein is extensively glycosylated and displayed on the surface of tumor cells and we provide the first demonstration of a direct role for B7-H4 in promoting malignant transformation of epithelial cells. Overexpression of B7-H4 in a human ovarian cancer cell line with little endogenous B7-H4 expression increased tumor formation in SCID mice. Whereas overexpression of B7-H4 protected epithelial cells from anoikis, siRNA-mediated knockdown of B7-H4 mRNA and protein expression in a breast cancer cell line increased caspase activity and apoptosis. The restricted normal tissue distribution of B7-H4, its overexpression in a majority of breast and ovarian cancers and functional activity in transformation validate this cell surface protein as a new target for therapeutic intervention. A therapeutic antibody strategy aimed at B7-H4 could offer an exciting opportunity to inhibit the growth and progression of human ovarian and breast cancers.