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We developed a protocol for the preparation of a single-cell suspension of human intestinal (ileal) biopsies, optimized for contemporary multi-omics single-cell assays including high-dimensional flow cytometry and single-cell RNA sequencing (scRNA-seq) assays. The protocol provides a rapid and efficient method of digesting human intestinal biopsies into a single-cell suspension resulting in a high viable cell yield and preservation of cell-surface markers. Notably, the protocol utilizes a Collagenase-I/Benzonase-based 37°C dissociation method, which collects already digested cells at several steps during the process to prevent over-digestion (fractioning technique). We obtained a total cell yield of ~5.8E6 +/- 1.9E6 cells, with a viability of 71% +/- 6% (n=9) (Figures 1A and 1B) from biopsy weights ranging 10-14 mg. By flow cytometry, all major immune cell lineages were detected, as well as a CD45-/EpCAM+ epithelial cell population (20%-60% of total viable singlets; Figure 1C). scRNA-seq analysis (10X Genomics; 5’/VDJ with feature barcoding) showed high-quality viable cells with low mitochondrial gene counts, indicating this protocol exerts minimal stress on the digested cells (Figure 1D).

Here we present an optimized protocol for the single-cell suspension of murine intestines (here we used ileum) optimized for contemporary multi-omics single-cell assays including high-dimensional flow cytometry and single-cell RNA-seq. This protocol provides a rapid and efficient method of digesting mouse ileum into a single-cell suspension with a short processing time, high viable cell yield, and it preserves cell-surface markers throughout the process. Notably, the protocol utilizes a Collagenase-I/Benzonase-based 37°C water bath dissociation method and collects already digested cells at several steps during the process to prevent over-digestion (fractioning technique). Tissues were normalized by length (2.5 cm) and weight (averaged 79.5 mg) and measured for cell yield and viability. We obtained an average total cell yield of ~4.4E6 +/- 1.4E6 cells, and average viability of ~56 +/- 13% (n=7) (Figure 1). By flow cytometry, ~30%-50% of cells were CD45+ immune cells, with the remainder of the cell population being CD45- non-immune cells (i.e., epithelial and stromal cells). To optimize this protocol, several other conditions were tested, including different digestive enzymes, mechanical digestion techniques, the inclusion of an intraepithelial layer (IEL) digestion, and an antioxidant cocktail addition. We summarize our main findings about the optimization of this protocol below: Collagenase-I provided the highest number of total yield and viable cells when compared to Collagenase-IV and Liberase. Enzymatic digestion in a 37°C waterbath and vortexing the cell solutions proved more successful in cell yield and viability than different combinations of the gentleMACS Tissue Dissociator (Myltenyi) intestine digestion programs, including its m_ intestine _1 and 37_m_LPDK programs and variations in their run times. We have also validated a protocol to separate the IEL before the rest of the ileum, and have found it compatible and therefore able to be adopted with the recommended protocol while maintaining a similar yield of viable cells. An IEL separation protocol has been included in a separate section at the end. By using our "fractioning technique", in which cells that are digested early on are removed before digesting the rest of the tissue separately, total cell yield and viability are increased. We tested several antioxidant combinations (Gluthatione, Vitamin C, and Vitamin E) and found higher live cell yield when excluding these antioxidants from the Collagenase-I digestion step. Based on these findings, this murine intestinal digestion protocol is rapid and efficient and can be used for multi-omics single-cell assays such as high-dimensional flow cytometry and single-cell RNA-sequencing.

The Na+-Taurocholate cotransporting polypeptide (NTCP; gene symbol SLC10A1) was cloned in 1991 and shown to function in a sodium-dependent fashion to efficiently take up all the major glycine and taurine-conjugated bile acids.1 NTCP only weakly transports unconjugated bile acids, and in subsequent years, members of the Organic Anion Transporting Polypeptide (OATP/SLCO) family of facilitative carriers such as OATP1B1 and OATP1B3 in humans and Oatp2b1 in mice were identified as the transporters responsible for hepatic sodium-independent uptake of bile acids, particularly the unconjugated species. NTCP’s properties clearly established it as the major candidate mechanism for hepatic bile acid uptake in multiple species. However definitive in vivo support was lacking until only recently when a pediatric patient was described with a loss-of-function mutation in NTCP and highly elevated serum levels of conjugated bile acids.2 Although the child currently exhibits no jaundice, pruritus, or other signs of liver disease, the finding also highlighted the gaps in our ability to predict the long-term physiologic and metabolic consequences of reduced hepatic bile acid clearance. Following closely on the heels of the first human case of an isolated NTCP defect and partially filling that void, Slijpcevic et al now report the first phenotypic characterization of an Slc10a1 knockout mouse.3 In brief, after confirming the absence of NTCP mRNA and protein in liver, the authors used freshly isolated hepatocytes in sandwich cultures to demonstrate that sodium-dependent taurocholate transport was abolished in the Slc10a1−/− mice. Given that observation, it was expected that the Slc10a1-deficient mice would exhibit markedly elevated serum bile acid levels (hypercholanemia) similar to the recently described NTCP-deficient patient. Surprisingly, serum bile acid levels were in the normal range (1 to 20 μM) for the majority (~70%) of the Slc10a1−/− mice, with the remaining subpopulation exhibiting dramatically elevated levels (> 1000 μM). Slc10a1−/− mice with intermediate levels of serum bile acids were not evident, and the heterogeneity was not explained by gender or by mouse strain genetic background. The remaining studies then proceeded to tell the tale of these two phenotypes. Overall, the Slc10a1−/− mice were grossly similar to their wild type littermates except for reduced body weights, a common observation for knockout mouse models with defects in bile acid biosynthetic enzymes or other transporters important for the enterohepatic circulation of bile acids. The reduced body weight was particularly evident for those mice with highest serum bile acid levels. There was no hepatomegaly or histological evidence of liver injury; serum markers of liver function were unremarkable in the normocholanemic mice, but elevated in hypercholanemic Slc10a1−/− mice. In the normocholanemic Slc10a1−/− mice, the clearance of intravenously administered taurocholate was clearly slowed compared to wild type mice, and the hepatic bile acid clearance defect could be unmasked by feeding a diet containing ursodeoxycholic acid. Bile acid synthesis and enterohepatic cycling appeared to be relatively unperturbed in the normocholanemic Slc10a1−/− mice, with no changes in bile flow, biliary bile acid secretion, or fecal bile acid excretion. In contrast, the hypercholanemic Slc10a1−/− mice exhibited a more substantial defect in taurocholate clearance, as well as decreased bile flow, biliary bile secretion, and fecal bile excretion, and increased urinary bile acid excretion. Overall, the results generally support the concept that NTCP/Slc10a1 is an important hepatic transporter for clearance of conjugated bile acids. However, the phenotypic heterogeneity observed for the Slc10a1−/− mice raises questions regarding alternate hepatic bile acid clearance mechanisms. The sodium-independent bile acid transporters that preferentially function in clearance of unconjugated bile acids may be sufficiently active to compensate and prevent the rise in serum conjugated bile acids in the majority of Slc10a1−/− mice. Indeed, changes were observed in hepatic mRNA and protein levels for several of the Oatps including Oatp1b2. But these changes did not strictly correlate with the presence or absence of hypercholanemia, and additional unidentified modifiers of the phenotype may be involved. Comparison of the Slc10a1−/− mice and index case for human NTCP-deficiency suggests the presence of compensatory mechanisms in mice that are not engaged in humans. This could include higher hepatocyte levels of Oatp1b2 (or another transporter such as other members of the Oatp family), or a greater proportion of the hepatocytes being involved in bile acid clearance (pericentral/zone III as well as periportal/zone I of the liver acinus). Additional studies such as analysis of knockout mice lacking both Slc10a1 and Slco1b2 (Oatp2b1) will be needed to explore those mechanisms. In addition, it is not known if the hypercholanemic phenotype described for NTCP-deficiency is fully penetrant in humans. Only a single NTCP-deficient patient with hypercholanemia has been described, even though non-functional NTCP variants have turned up in population-based sequencing studies.4 As such, the existence of NTCP-deficient normocholanemic human subjects cannot be excluded. In addition to being the bile acid uptake transporter, NTCP was identified as a cell surface receptor for the Hepatitis B virus (HBV) and Hepatitis Delta virus (HDV).5 HBV infection is initiated via low affinity binding of the HBV small surface protein to heparin-sulfate proteoglycans and attachment to the basolateral membrane of hepatocytes. In the next step high-affinity binding occurs between the HBV and the host receptor and allows for viral cellular entry. The identity of the HBV entry receptor(s) had long remained a mystery. An important breakthrough came recently when Yan et al demonstrated that binding of the HBV large surface protein pre-S1 domain myristoylated peptide (amino acids 2–48) to NTCP is essential for infection of human hepatocytes.5 Much of the work to characterize the HBV-NTCP interactions have been performed using the pre-S1 domain-derived myristoylated lipopeptide Myrcludex-B, and the Slc10a1−/− mice provided a new opportunity to further explore the in vivo role of Ntcp in HBV binding. Indeed, in vivo positron emission tomography imaging showed that after administration, radiolabeled Myrcludex B-derived peptides were enriched in liver of wild-type mice but essential absent from liver of the Slc10a1−/− mice. The results further support a key role for NTCP as the HBV entry receptor. It should be noted that mouse Ntcp binds the pre-S1 peptide but does not allow for HBV infections in vitro.6 Nor does introduction of human NTCP confer susceptibility to HBV infection for the mouse hepatoma cell lines.6 As such, the utility of mice to further dissect the role of NTCP in HBV entry is unclear. The dual functions of NCTP in bile acid transport and binding of HBV preS1 peptide raised the question of whether there is mutual interference. In vitro studies showed that binding of preS1 peptide inhibited taurocholate uptake by NTCP, whereas bile acids such as taurocholate blocked/inhibited HBV infection.6,7 In vivo findings with human liver chimeric mice further supported the concept that HBV binding to NTCP limits bile acid uptake and leads to compensatory changes in bile acid metabolism.8 The Slc10a1−/− model may also prove useful as a platform to further dissect the mechanisms responsible for the metabolic changes induced by HBV. Altogether, the first description of the Slc10a1-knockout mouse is supportive of a central role for NTCP in the hepatic uptake of conjugated bile acids and HBV preS1/Myrcludex B binding in vivo. This important work by Slijepcevic et al also provides a valuable new tool to further our understanding of the complex intersection between bile acids, HBV, and hepatic function.

Background & Aims Retinoid X Receptor α (RXRα) is the principal heterodimerization partner of class II Nuclear Receptors (NRs), and a major regulator of gene expression of numerous hepatic processes, including bile acid (BA) homeostasis through multiple partners. Specific contributions of hepatic RXRα domains in heterodimer function in response to either BA load or ductular cholestasis are not fully characterized. Methods Wild-type (WT) mice and mice expressing a hepatocyte-specific RXRα lacking the DNA-Binding-Domain (hs- Rxrα Δex4 −/− ), which retains partial ability to heterodimerize with its partners, were fed a 1% cholic acid (CA) diet for 5days, a 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) diet for 3weeks, or control diet. Results Serum ALT (6.5-fold; p p =0.06) and BA (2.8-fold; p Rxα Δex4 −/− mice compared to CA-fed WT mice, but were equally induced between genotypes by DDC-feeding. CA-feeding elevated total (4.4-fold; p =0.06) and unconjugated (2.2-fold; p Rxrα Δex4 −/− mice compared to WT mice, but not in DDC-fed hs- Rxrα Δex4 −/− mice. Increased necrosis and inflammation was observed in CA-fed, but not in DDC-fed hs- Rxrα Δex4 −/− mice. Apoptotic markers DR5 , CK8 , CK18 RNA were increased in CA- and DDC-fed hs- Rxrα Δex4 −/− mice. Cleaved caspase 3, CK18 and p-JNK protein were elevated in CA-fed but not in DDC-fed hs- Rxrα Δex4 −/− mice. Induction of Ostβ and Cyp2b10 RNA was impaired in CA-fed and DDC-fed hs- Rxrα Δex4 −/− mice. Surprisingly, DDC-fed hs- Rxrα Δex4 −/− mice showed attenuated fibrosis compared to DDC-fed WT mice. Conclusions These two models of cholestasis identify common and injury-specific roles for RXRα heterodimers and the functional relevance of an intact RXRα-DBD in the hepatocytic adaptive cholestatic response.

Two peptides, [f135–158] and [f135–162]-SH, were used to study the binding of the peptides to native ß-lactolobulin, as well as the subsequent effects on aggregation and gelation of ß-lactoglobulin. The binding of the peptide [f135–158] to ß-lactoglobulin at room temperature was confirmed by SELDI-TOF-MS. It was further illustrated by increased turbidity of mixed solutions of peptide and protein (at pH 7), indicating association of proteins and peptides in larger complexes. At pH below the isoelectric point of the protein, the presence of peptides did not lead to an increased turbidity, showing the absence of complexation. The protein–peptide complexes formed at pH 7 were found to dissociate directly upon heating. After prolonged heating, extensive aggregation was observed, whereas no aggregation was seen for the pure protein or pure peptide solutions. The presence of the free sulfhydryl group in [f135–162]-SH resulted in a 10 times increase in the amount of aggregation of ß-lactoglobulin upon heating, illustrating the additional effect of the free sulfhydryl group. Subsequent studies on the gel strength of heat-induced gels also showed a clear difference between these two peptides. The replacement of additional ß-lactoglobulin by [f135–158] resulted in a decrease in gel strength, whereas replacement by peptide [f135–162]-SH increased gel strength.

Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease in the Western world, and safe and effective therapies are needed. Bile acids (BAs) and their receptors [including the nuclear receptor for BAs, farnesoid X receptor (FXR)] play integral roles in regulating whole-body metabolism and hepatic lipid homeostasis. We hypothesized that interruption of the enterohepatic BA circulation using a luminally restricted apical sodium-dependent BA transporter (ASBT) inhibitor (ASBTi; SC-435) would modify signaling in the gut-liver axis and reduce steatohepatitis in high-fat diet (HFD)-fed mice. Administration of this ASBTi increased fecal BA excretion and messenger RNA (mRNA) expression of BA synthesis genes in liver and reduced mRNA expression of ileal BA-responsive genes, including the negative feedback regulator of BA synthesis, fibroblast growth factor 15. ASBT inhibition resulted in a marked shift in hepatic BA composition, with a reduction in hydrophilic, FXR antagonistic species and an increase in FXR agonistic BAs. ASBT inhibition restored glucose tolerance, reduced hepatic triglyceride and total cholesterol concentrations, and improved NAFLD activity score in HFD-fed mice. These changes were associated with reduced hepatic expression of lipid synthesis genes (including liver X receptor target genes) and normalized expression of the central lipogenic transcription factor, Srebp1c Accumulation of hepatic lipids and SREBP1 protein were markedly reduced in HFD-fed Asbt(-/-) mice, providing genetic evidence for a protective role mediated by interruption of the enterohepatic BA circulation. Together, these studies suggest that blocking ASBT function with a luminally restricted inhibitor can improve both hepatic and whole body aspects of NAFLD.

The plant sterol diosgenin has been shown to stimulate biliary cholesterol secretion in mice without affecting the expression of the adenosine triphosphate-binding cassette transporter heterodimer Abcg5/g8. The aim of this study was to investigate the mechanism of diosgenin-induced cholesterol hypersecretion and to identify the genes involved. Surprisingly, despite its lack of effect on Abcg5/g8 expression in wild-type mice, diosgenin did not stimulate biliary cholesterol secretion in mice deficient for Abcg8. Analysis of the kinetics of cholesterol secretion suggested that diosgenin probably activates a step before Abcg5/g8. To identify potential diosgenin targets, gene expression profiling was performed in mice fed a diosgenin-supplemented diet. Diosgenin feeding increased hepatic expression of genes involved in cholesterol synthesis as well as genes encoding for several cytochrome P450s. No significant change in expression of known cholesterol transporters was found. Comparison with published expression-profiling data for Srebp2-overexpressing mice, another mouse model in which biliary cholesterol secretion is elevated, revealed a number of genes with unknown function that were upregulated in both diosgenin-fed mice and mice overexpressing Srebp2. In conclusion, we found that although Abcg8 is essential for most diosgenin-induced biliary cholesterol hypersecretion, diosgenin probably does not interact directly with Abcg5/Abcg8, but rather increases cholesterol delivery to the heterodimer. Supplementary material for this article can be found on the HEPATOLOGY website (http://interscience.wiley.com/jpages/0270-9139/suppmat/index.html). (HEPATOLOGY 2005;41:141–150.)

In the formation of cholesterol gallstones, cholesterol hypersecretion into bile causing cholesterol supersaturation and crystallization appears to be the primary factor, with disturbed gallbladder and intestinal motility as secondary factors. Although intestinal uptake mechanisms have not yet been fully elucidated, the HDL receptor scavenger receptor B1 (SRB1) may be involved. Since HDL-cholesterol, both from the intestine and peripheral sources, is the preferred type of cholesterol for biliary secretion, increased HDL transport to the liver can also cause cholesterol hypersecretion in bile. In the hepatocyte, bile formation is regulated by several transmembrane proteins, all belonging to the ABC family. A change in the activity in one of these proteins can have a profound impact on biliary lipid secretion. The bile salt export pump (BSEP or ABCB 11) regulates the excretion of bile salts into bile and mutations cause severe cholestasis. The second ABC transporter, ABCB4 (MDR3) regulates the secretion in bile of phosphatidylcholine (PC), while ABCG5/G8 is active in the excretion of cholesterol and sterols into bile. These transporters also facilitate transport of sterols back into the intestinal lumen. Mutations in either of these genes cause sitosterolaemia with increased absorption of plant sterols and cholesterol. Until now, evidence for a genetic background of human gallstone disease is mostly indirect and based on ethnic differences. Only two single gene defects are associated with gallstones. One is an ABCB4 mutation which causes a deficiency in biliary PC secretion and the other is a CYP7A 1 mutation. the rate-limiting enzyme in the synthesis of bile salts from cholesterol in the liver. Recently, several common DNA polymorphisms in the ABCG8 gene were discovered that are associated with variations in plasma sterols, which could also influence biliary cholesterol secretion, but there is still a paucity of human studies

Background/Aims: Mutations in genes encoding the ATP-binding cassette (ABC)-transporters ABCG5 and ABCG8 underlie sitosterolemia, which is characterized by elevated plasma levels of phytosterols due to increased intestinal absorption and impaired biliary secretion of sterols. The aim of our study was to correlate the expression levels of Abcg5 and Abcg8 to biliary cholesterol secretion in various (genetically-modified) mouse models.Methods: Bile was collected from genetically-modified mice fed a chow diet, or from mice fed either a chow diet, or chow supplemented with either 1% diosgenin, 0.1% simvastatin, or a synthetic liver X receptor agonist, for determination of biliary lipids. Livers and small intestines were harvested and expression levels of Abcg5, Abcg8 and Abcb4 were determined by real-time polymerase chain reaction.Results: Intestinal expression of Abcg5 and Abcg8 did not show much variation between the various models. In contrast, a linear correlation between hepatic expression levels of Abcg5 and Abcg8 and biliary cholesterol secretion rates was found. This relation was independent of Abcb4-mediated phospholipid secretion. However, in diosgenin-fed mice showing cholesterol hypersecretion, hepatic Abcg5 and Abcg8 expression levels remained unchanged.Conclusions: Our results strongly support a role for Abcg5 and Abcg8 in regulation of biliary cholesterol secretion, but also indicate the existence of a largely independent route of cholesterol secretion. (C) 2003 European Association for the Study of the Liver. Published by Elsevier Science B.V. All rights reserved.

Processing of ovalbumin may result in proteins that differ more than 23°C in denaturation temperature while the structural fold is not significantly affected. This is achieved by 1) conversion of positive residues into negative ones (succinylation); 2) elimination of negative charges (methylation); 3) reducing the proteins hydrophobic exposure (glycosylation); 4) increasing the hydrophobic exposure (lipophilization); or by 5) processing under alkaline conditions and elevated temperature (Sovalbumin). The effect on the structural fold was investigated using a variety of biochemical and spectroscopic tools. The consequences of the modification on the thermodynamics of the protein was studied using differential scanning calorimetry and by monitoring the tryptophan fluorescence or ellipticity at 222 nm of protein samples dissolved in different concentrations of guanidine-HCl. The impact of the modification on the denaturation temperature scales for all types of modifications with a free energy change of about 1 kJ per mol ovalbumin per Kelvin (or 0.0026 kJ per mol residue per K). The nature of the covalently coupled moiety determines the impact of the modification on the protein thermodynamics. It is suggested that especially for lipophilized protein the water-binding properties are substantially lowered. Processing of globular proteins in a controlled manner offers great opportunities to control a desired functionality, for example, as texturizer in food or medical applications. © 2003 Wiley Periodicals. Chemicals/CAS: guanidine, 113-00-8, 25215-10-5, 50-01-1; ovalbumin, 77466-29-6; tryptophan, 6912-86-3, 73-22-3; Ovalbumin, 9006-59-1

Divalent calcium ions have been suggested to be involved in intermolecular protein-Ca2+-protein cross-linking, intramolecular electrostatic shielding, or ion-induced protein conformational changes as a trigger for protein aggregation at elevated temperatures. To address the first two phenomena in the case of beta-lactoglobulin, a combination of chemical protein modification, calcium-binding, and aggregation studies was used, while the structural integrity of the modified proteins was maintained. Although increasing the number of carboxylates on the protein by succinylation results in improved calcium-binding, calcium appears to be less effective in inducing protein aggregation. In fact, the larger the number of carboxylates, the higher the concentration of calcium that is required to trigger the aggregation. Lowering the number of negative charges on the protein surface via methylation of carboxylates reduces calcium-binding properties, but calcium-induced aggregation at low concentration is improved. Monovalent sodium ions cannot take over the specific role of calcium. The relation between net surface charge and number of calcium ions bound required to trigger the aggregation suggests that calcium needs to bind site specific to carboxylates with a threshold affinity. Subsequent site-specific screening of surface charges results in protein aggregation, driven by the partial unfolding of the protein at elevated temperatures, which is then facilitated by the absence of electrostatic repulsion.

Ischemia-reperfusion injury (IRI) is a common clinical consequence of hepatic surgery, cardiogenic shock, and liver transplantation. A steatotic liver is particularly vulnerable to IRI, responding with extensive hepatocellular injury. Autophagy, a lysosomal pathway balancing cell survival and cell death, is engaged in IRI, although its role in IRI of a steatotic liver is unclear. The role of autophagy was investigated in high-fat diet (HFD)-fed mice exposed to IRI in vivo and in steatotic hepatocytes exposed to hypoxic IRI (HIRI) in vitro. Two inhibitors of autophagy, 3-methyladenine and bafilomycin A1, protected the steatotic hepatocytes from HIRI. Exendin 4 (Ex4), a glucagon-like peptide 1 analog, also led to suppression of autophagy, as evidenced by decreased autophagy-associated proteins [microtubule-associated protein 1A/1B-light chain 3 (LC3) II, p62, high-mobility group protein B1, beclin-1, and autophagy-related protein 7], reduced hepatocellular damage, and improved mitochondrial structure and function in HFD-fed mice exposed to IRI. Decreased autophagy was further demonstrated by reversal of a punctate pattern of LC3 and decreased autophagic flux after IRI in HFD-fed mice. Under the same conditions, the effects of Ex4 were reversed by the competitive antagonist exendin 9-39. The present study suggests that, in IRI of hepatic steatosis, treatment of hepatocytes with Ex4 mitigates autophagy, ameliorates hepatocellular injury, and preserves mitochondrial integrity. These data suggest that therapies targeting autophagy, by Ex4 treatment in particular, may ameliorate the effects of IRI in highly prevalent steatotic liver.

In preclinical studies of fructose-induced NAFLD, endotoxin appears to play an important role. We retrospectively examined samples from three pediatric cohorts (1) to investigate whether endotoxemia is associated with the presence of hepatic steatosis; (2) to evaluate postprandial endotoxin levels in response to fructose beverage in an acute 24-hour feeding challenge, and (3) to determine the change of fasting endotoxin amounts in a 4-week randomized controlled trial comparing fructose to glucose beverages in NAFLD. We found that adolescents with hepatic steatosis had elevated endotoxin levels compared to obese controls and that the endotoxin level correlated with insulin resistance and several inflammatory cytokines. In a 24-hour feeding study, endotoxin levels in NAFLD adolescents increased after fructose beverages (consumed with meals) as compared to healthy children. Similarly, endotoxin was significantly increased after adolescents consumed fructose beverages for 2 weeks and remained high although not significantly at 4 weeks. In conclusion, these data provide support for the concept of low level endotoxemia contributing to pediatric NAFLD and the possible role of fructose in this process. Further studies are needed to determine if manipulation of the microbiome or other methods of endotoxin reduction would be useful as a therapy for pediatric NAFLD.

The antimicrobial peptide nisin contains the uncommon amino acid residues lanthionine and methyl-lanthionine, which are post-translationally formed from Ser, Thr and Cys residues. To investigate the importance of these uncommon residues for nisin activity, a mutant was designed in which Thr13 was replaced by a Cys residue, which prevents the formation of the thioether bond of ring C. Instead, Cys13 couples with Cys19 via an intramolecular disulfide bridge, a bond that is very unusual in lantibiotics. NMR analysis of this mutant showed a structure very similar to that of wild-type nisin, except for the configuration of ring C. The modification was accompanied by a dramatic reduction in antimicrobial activity to less than 1% of wild-type activity, indicating that the lanthionine of ring C is very important for this activity. The nisin Z mutants S5C and M17C were also isolated and characterized; they are the first lantibiotics known that contain an additional Cys residue that is not involved in bridge formation but is present as a free thiol. Secretion of these peptides by the lactococcal producer cells, as well as their antimicrobial activity, was found to be strongly dependent on a reducing environment. Their ability to permeabilize lipid vesicles was not thiol-dependent. Labeling of M17C nisin Z with iodoacetamide abolished the thiol-dependence of the peptide. These results show that the presence of a reactive Cys residue in nisin has a strong effect on the antimicrobial properties of the peptide, which is probably the result of interaction of these residues with thiol groups on the outside of bacterial cells.

Two-dimensional Fourier transform infrared (2D FT-IR) spectroscopy was applied to study the slower states of the membrane protein bacterioRhodopsin (bR) photocycle, with bR adsorbed on a ZnSe attenuated total reflectance (ATR) crystal. The M and the N states of the bR photocycle could be discriminated, and 2D spectra revealed an N state absorption band at 1536 cm−1 that is normally covered by the broader and bigger 1527 cm−1 M state absorption band. 2D FT-IR absorbance changes are presented in a more convenient way for analysis than in traditional 2D FT-IR spectroscopy by using 2D-phase spectra and phase/amplitude calculations.

The importance of signal-to-noise ratio (SNR) calculations for an optimum Fourier transform-infrared spectroscopy (FT-IR) detection of thin, isotropic layers on dielectric substrates is discussed; some illustrative examples are given. It is found that the SNR increases with increasing refractive index of the substrate whereas the use of p-polarized light is preferable. Optimum measurement conditions for oriented layers were found for a Ge substrate at an angle of incidence of 50–55°.

Background Triggers and exacerbants of cirrhotic cardiomyopathy (CC) are poorly understood, limiting treatment options in patients with chronic liver diseases. Liver transplantation alone reverses some features of CC, but the physiology behind this effect has never been studied. Aims We aimed to determine whether reversal of liver injury and fibrosis in mouse affects cardiac parameters. The second aim was to determine whether cardiomyopathy can be induced by specifically increasing systemic bile acid (BA) levels. Methods 6–8 week old male C57BL6J mice were fed either chow (n = 5) or 3,5-diethoxycarbonyl-1,4-dihydroxychollidine (DDC) (n = 10) for 3 weeks. At the end of 3 weeks, half the mice in the DDC fed group were randomized to chow (the reversed [REV] group). Serial ECHOs and electrocardiographic analysis was conducted weekly for 6 weeks followed by liver tissue and serum studies. Hearts were analysed for key components of function and cell signalling. Cardiac physiological and molecular parameters were similarly analysed in Abcb11−/− mice (n = 5/grp) fed 0.5% cholic acid supplemented diet for 1 week. Results Mice in the REV group showed normalization of biochemical markers of liver injury with resolution of electrocardiographic and ECHO aberrations. Catecholamine resistance seen in DDC group resolved in the REV group. Cardiac recovery was accompanied by normalization of cardiac troponin-T2 as well as resolution of cardiac stress response at RNA level. Cardiovascular physiological and molecular parameters correlated with degree of cholanemia. Cardiomyopathy was reproduced in cholanemic BA fed Abcb11−/− mice. Conclusions Cardiomyopathy resolves with resolution of liver injury, is associated with cholanaemia, and can be induced by BA feeding.

To assess the protection against cabbage root fly larvae achieved in cauliflower crops by filmcoating the seed with insecticide, six field experiments were carried out between 1990 and 1991. Various dosages of insecticides were tested at four widely separated locations in The Netherlands with high and moderate population densities of the cabbage root fly. The plants were raised in ‘loose-filled cells’ and peat ‘blocks’, or as peg plants. The efficacy of formulations of isofenphos, chlorpyrifos and fonofos, at two and three dosages of seed filmcoating, were compared with a conventional post-planting treatment. Filmcoating with fonofos was less effective in controlling cabbage root fly larvae than the post-planting treatment. Chlorpyrifos at 28.8 g a.i. kg−1 seed and isofenphos at 30 g a.i. kg−1 seed consistently gave as good control as a post-planting treatment of each plant.

A bovine β-lactoglobulin hydrolysate, obtained by the hydrolysis by the Glu specific enzyme Bacillus licheniformis protease (BLP), was fractionated at pH 7.0 into a soluble and an insoluble fraction and characterized by LC-MS. From the 26 peptides identified in the soluble fraction, five peptides (A[f97-112] = [f115-128], AB[f1-45], AB[f135-157], AB[f135-158], and AB[f138-162]) bound to β-lactoglobulin at room temperature. After heating of β-lactoglobulin in the presence of peptides, eight peptides were identified in the pellet formed, three of them belonging to the previously mentioned peptides. Principle component analysis revealed that the binding at room temperature (to β-lactoglobulin) was related to the total hydrophobicity and the total charge of the peptides. The binding to the unfolded protein could not be attributed to distinct properties of the peptides. The presence of the peptides caused a 50% decrease in denaturation enthalpy (from 148 ± 3 kJ/mol for the protein alone to 74 ± 2 kJ/mol in the presence of peptides), while no change in secondary structure or denaturation temperature was observed. At temperatures85 °C, the addition of peptides resulted in a 30-40% increase of precipitated β-lactoglobulin. At pH6, no differences in the amount of aggregated β-lactoglobulin were observed, which indicates the lack of binding of peptides to β-lactoglobulin at those pH values as was also observed by SELDI-TOF-MS. Although only a few peptides were found to participate in aggregation, suggesting specificity, principal component analysis was unable to identify specific properties responsible for this.

Cardiac dysfunction is a major cause of morbidity and mortality in patients with end-stage liver disease; yet the mechanisms remain largely unknown. We hypothesized that the complex interrelated impairments in cardiac structure and function secondary to progression of liver diseases involve alterations in signaling pathways engaged in cardiac energy metabolism and hypertrophy, augmented by direct effects of high circulating levels of bile acids. Biliary fibrosis was induced in male C57BL/6J mice by feeding a 0.1% 3,5-diethoxycarbonyl-1,4-dihydroxychollidine (DDC) supplemented diet. After 3 weeks, mice underwent live imaging (dual energy x-ray absorptiometry [DEXA] scanning, two-dimensional echocardiography [2DE], electrocardiography, cardiac magnetic resonance imaging), exercise treadmill testing, and histological and biochemical analyses of livers and hearts. Compared with chow-fed mice, DDC-fed mice fatigued earlier on the treadmill, with reduced VO(2). Marked changes were identified electrophysiologically (bradycardia and prolonged QT interval) and functionally (hyperdynamic left ventricular [LV] contractility along with increased LV thickness). Hearts of DDC-fed mice showed hypertrophic signaling (activation of v-akt murine thymoma viral oncogene/protein kinase B [AKT], inhibition of glycogen synthase kinase-3beta [GSK3beta], a 20-fold up-regulation of beta myosin heavy chain RNA and elevated G(s)alpha/G(i)alpha ratio. Genes regulating cardiac fatty acid oxidation pathways were suppressed, along with a threefold increase in myocardial glycogen content. Treatment of mouse cardiomyocytes (which express the membrane bile acid receptor TGR5) with potent natural TGR5 agonists, taurochenodeoxycholic acid and lithocholic acid, activated AKT and inhibited GSK3beta, similar to the changes seen in DDC-fed mouse hearts. This provides support for a novel mechanism whereby circulating natural bile acids can induce signaling pathways in heart associated with hypertrophy.Three weeks of DDC feeding-induced biliary fibrosis leads to multiple functional, metabolic, electrophysiological, and hypertrophic adaptations in the mouse heart, recapitulating some of the features of human cirrhotic cardiomyopathy.

The increasing use of thermoplastic carbon fiber-reinforced plastic (CFRP) materials in the aerospace industry for primary aircraft structures, such as wing leading-edge surfaces and fuselage sections, has led to rapid growth in the field of structural health monitoring (SHM). Impact, vibration, and load can all cause failure, such as delamination and matrix cracking, in composite materials. Moreover, the internal material damage can occur without being visible to the human eye, making inspection of and clear insight into structural integrity difficult using currently available evaluation methods. Here, we describe the detection of impact and its localization in materials and structures by high-speed interrogation of multiple-fiber Bragg grating (FBG) sensors mounted on a composite aircraft component.

Background and aim Chronic cholangiopathies have limited therapeutic options and represent an important indication for liver transplantation. Curcumin, the yellow pigment of the spice turmeric, has pleiotropic actions and attenuates hepatic damage in animal models of chemically-induced liver injury. Whether curcumin has beneficial effects in cholangiopathies is unknown. Methods Potential anticholestatic, anti-inflammatory and antifibrotic mechanisms of curcumin were explored in vivo in Mdr2 −/− mice as a murine model of chronic cholangiopathy; as well as in vitro in a cholangiocyte cell line (HuCCT1) and portal myofibroblasts (MFBs) isolated from Mdr2 −/− mice. Results Liver damage, cholestasis and fibrosis were reduced in Mdr2 −/− mice after curcumin feeding. Moreover, curcumin inhibited cholangiocyte proliferation and expression of activation marker vascular cell adhesion molecule-1 in Mdr2 −/− mice. Curcumin—similar to PPARγ synthetic agonist troglitazone—directly inhibited TNF-α-induced inflammatory activation of cholangiocytes in vitro, whereas these beneficial effects of curcumin were largely blocked by a PPARγ synthetic antagonist. In addition, curcumin blocked proliferation and activation of portal MFBs by inhibiting ERK1/2 phosphorylation, thus contributing to reduced fibrogenesis. Conclusions These results show that curcumin may have multiple targets in liver including activation of PPARγ in cholangiocytes and inhibition of ERK1/2 signalling in MFBs, thereby modulating several central cellular events in a mouse model of cholangiopathy. Targeting these pathways may be a promising therapeutic approach to cholangiopathies.

Contains fulltext : 81818.pdf (Publisher’s version ) (Open Access) The Mycobacterium avium complex (MAC) consists of four recognized species, Mycobacterium avium, Mycobacterium colombiense, Mycobacterium intracellulare and Mycobacterium chimaera, and a variety of other strains that may be members of undescribed taxa. We report on two isolates of a scotochromogenic, slowly growing, non-tuberculous Mycobacterium species within the M. avium complex from a lymph node and an infected wound after a dogbite of separate patients in The Netherlands. The extrapulmonary infections in immunocompetent patients suggested a high level of virulence. These isolates were characterized by a unique nucleotide sequence in the 16S rRNA gene, 99% similar to Mycobacterium colombiense, and the MAC-Q 16S-23S internal transcribed spacer (ITS) sequence. Sequence analyses of the hsp65 gene revealed 97% similarity to M. avium. The rpoB gene sequence was 98% similar to M. colombiense. Phenotypically, the scotochromogenicity, positive semi-quantitative catalase and heat-stable catalase tests, negative tellurite reductase and urease tests and susceptibility to hydroxylamine and oleic acid set these isolates apart from related species. High-performance liquid chromatography analysis of cell-wall mycolic acid content revealed a unique pattern, related to that of M. avium and M. colombiense. Together, these findings supported a separate species status within the Mycobacterium avium complex. We propose elevation of scotochromogenic M. avium complex strains sharing this 16S gene and MAC-Q ITS sequence to separate species status, for which the name Mycobacterium vulneris sp. nov. is proposed. The type strain is NLA000700772T (=DSM 45247T=CIP 109859T).

A variety of hydroxy- and methoxy-substituted 2-(acetoxymethyl)9,10-anthraquinones were reduced electrochemically and with dithionite in 50% aqueous CH 3 CN buffers over a wide pH range. Substituent effects upon the reductive cleavage process were determined by measuring rate constants for those acetates that react cleanly with dithionite at pH 8

The middle Eocene Avon Park Formation of west central Florida contains a carbonate sequence that accumulated under supratidal to shallow subtidal conditions. Vertically repetitive sedimentary sequences document transgressive regressive, open marine to shoreline cyclicity. Newly discovered cross-bedding and abundant fossils support these paleoenvironmental interpretations. The rocks are dolomitic and bioclastic packstones and grainstones. Allochems are chiefly benthic foraminifers, bivalves, echinoids, and algae. Planar cross-bedding occurs within megaripple sets. Bed thicknesses, lateral extent of bed types, and paleocurrent directions are nonuniform, indicating migrating or shifting hydrodynamic regimes. Trace fossils, including Ophiomorpha, are well preserved and suggest maximum water depths of three to four meters in a shoreline transition zone. Invertebrate, vertebrate, trace, and plant fossils represent more than 70 species. Abundant irregular echinoids (clypeasteroids, spatangoids, cassiduloids, and a holectypoid) also suggest sandy shoreline conditions. Erosional truncations of burrows indicate periodic storm action along the shoreface. Modern environmental and ecological analogues to this study area include the south Florida shelf and the Bahamian Platform. Mud-free, carbonate sands displaying megarippled cross-beds and extensive burrowing occur adjacent to sea grass beds with finer-grained sediments and greater faunal diversity/density. Shifting conditions often result in redistribution of facies.

Because the formation of protein/polysaccharide complexes is dominated by electrostatic interaction, polysaccharide charge density is expected to play a major role in the adsorption behavior of the complexes. In this study, pullulan (a non-charged polysaccharide) carboxylated to four different charge densities (fraction of carboxylated subunits: 0.1, 0.26, 0.51, and 0.56) was used to investigate the effect of charge density on the properties of mixed protein/polysaccharide adsorbed layers at air/water interfaces. With all pullulan samples, soluble complexes with β-lactoglobulin could be formed at low ionic strength, pH 4.5. It was shown that the higher was the pullulan charge density, the more the increase of surface pressure in time was retarded as compared to that for pure β-lactoglobulin. The retardation was even more pronounced for the development of the dilatational modulus. The lower dilatational modulus can be explained by the ability of the polysaccharides to prevent the formation of a compact protein layer at the air/water interface due to electrostatic repulsion. This ability of the polysaccharides to prevent "layer compactness" increases with the net negative charge of the complexes. If charge density is sufficient (≥0.26), polysaccharides may enhance the cohesion between complexes within the adsorbed layer. The charge density of polysaccharides is shown to be a dominant regulator of both the adsorption kinetics as well as the resulting surface rheological behavior of the mixed layers formed. These findings have significant value for the application of complex protein-polysaccharide systems. © 2007 American Chemical Society.

To study the influence of the carbohydrate-moiety of ovalbumin on the formation of the heat-stable conformer S-ovalbumin, ovalbumin is deglycosylated with PNGase-F under native conditions. Although the enzymatic deglycosylation procedure resulted in a complete loss of the ability to bind to Concavalin A column-material, only in about 50% the proteins lost their complete carbohydrate moiety, as demonstrated by mass spectrometry and size exclusion chromatography. Thermal stability and conformational changes were determined using circular dichroism and differential scanning calorimetry and demonstrated at ambient temperature no conformational changes due to the deglycosylation. Also the denaturation temperature of the processed proteins remained the same (77.4 ± 0.4°C). After heat treatment of the processed protein at 55°C and pH 9.9 for 72 h, the condition that converts native ovalbumin into the heat-stable conformer (S-ovalbumin), only the material with the intact carbohydrate moiety forms this heat-stable conformer. The material that effectively lost its carbohydrate moiety appeared fully denatured and aggregated due to these processing conditions. These results indicate that the PNGase-F treatment of ovalbumin prohibits the formation and stabilization of the heat-stable conformer S-ovalbumin. Since S-ovalbumin in egg protein samples is known to affect functional properties, this work illustrates a potential route to control the quality of egg protein ingredients. © 2006 Wiley Periodicals, Inc.

The main player in biliary cholesterol secretion is the heterodimeric transporter complex, ABCG5/ABCG8, the function of which is necessary for the majority of sterols secreted into bile. It is not clear whether the primary step in this process is flopping of cholesterol from the inner to the outer leaflet of the canalicular membrane, with desorption by mixed micelles, or decreasing of the activation energy required for cholesterol desorption from the outer membrane leaflet. In this study, we investigated these mechanisms by infusing Abcg8+/+, Abcg8+/−, and Abcg8−/− mice with hydrophilic and hydrophobic bile salts. In Abcg8−/− mice, this failed to substantially stimulate biliary cholesterol secretion. Infusion of the hydrophobic bile salt taurodeoxycholate also resulted in cholestasis, which was induced in Abcg8−/− mice at a much lower infusion rate compared with Abc8−/− and Abcg8+/− mice, suggesting a reduced cholesterol content in the outer leaflet of the canalicular membrane. Indeed, isolation of canalicular membranes revealed a reduction of 45% in cholesterol content under these conditions in Abcg8−/− mice. Our data support the model that ABCG5/ABCG8 primarily play a role in flopping cholesterol (and sterols) from the inner leaflet to the outer leaflet of the canalicular membrane.—Kosters, A., C. Kunne, N. Looije, S. B. Patel, R. P. J. Oude Elferink, and A. K. Groen. The mechanism of ABCG5/ABCG8 in biliary cholesterol secretion in mice.

The stability of adsorbed protein layers against deformation has in literature been attributed to the formation of a continuous gel-like network. This hypothesis is mostly based on measurements of the increase of the surface shear elasticity with time. For several proteins this increase has been attributed to the formation of intermolecular disulfide bridges between adsorbed proteins. However, according to an alternative model the shear elasticity results from the low mobility of the densely packed proteins. To contribute to this discussion, the actual role of disulfide bridges in interfacial layers is studied. Ovalbumin was thiolated with S-acetylmercaptosuccinic anhydride (S-AMSA), followed by removal of the acetylblock on the sulphur atom, resulting in respectively blocked (SX) and deblocked (SH) ovalbumin variants. This allows comparison of proteins with identical amino acid sequence and similar globular packing and charge distribution, but different chemical reactivity. The presence and reactivity of the introduced, deblocked sulfhydryl groups were confirmed using the sulfhydryl-disulfide exchange index (SEI). Despite the reactivity of the introduced sulfhydryl groups measured in solution, no increase in the surface shear elasticity could be detected with increasing reactivity. This indicates that physical rather than chemical interactions determine the surface shear behaviour. Further experiments were performed in bulk solution to study the conditions needed to induce covalent aggregate formation. From these studies it was found that mere concentration of proteins (to 200 mg/mL, equivalent to a surface concentration of around 2 mg/m2) is not sufficient to induce significant aggregation to form a continuous network. In view of these results, it was concluded that the adsorbed layer should not be considered a gelled network of aggregated material (in analogy with three-dimensional gels formed from heating protein solutions). Rather, it would appear that the adsorbed proteins form a highly packed system of proteins with net-repulsive interactions. © 2005 Elsevier B.V. All rights reserved.

The liver and (small) intestine are key organs in maintenance of cholesterol homeostasis: both organs show active de novo cholesterogenesis and are able to transport impressive amounts of newly synthesized and diet-derived cholesterol via a number of distinct pathways. Cholesterol trafficking involves the concerted action of a number of transporter proteins, some of which have been identified only recently. In particular, several ATP-binding cassette (ABC) transporters fulfil critical roles. For instance, the ABCG5/ABCG8 couple is crucial for hepatobiliary and intestinal cholesterol excretion, while ABCA1 is essential for high-density lipoprotein formation and, hence, for inter-organ trafficking of the highly water-insoluble cholesterol molecules. Very recently, the Niemann-Pick C1-like 1 protein has been identified as a key player in cholesterol absorption by the small intestine and may represent a target of the cholesterol absorption inhibitor ezetimibe. Alterations in hepatic and intestinal cholesterol transport affect circulating levels of atherogenic lipoproteins and thus the risk for cardiovascular disease. This review specifically deals with the processes of hepatobiliary cholesterol excretion and intestinal cholesterol absorption as well as the interactions between these important transport routes. During the last few years, insight into the mechanisms of hepatic and intestinal cholesterol transport has greatly increased not in the least by the identification of involved transporter proteins and the (partial) elucidation of their mode of action. In addition, information has become available on (transcription) factors regulating expression of the encoding genes. This knowledge is of great importance for the development of a tailored design of novel plasma cholesterol-lowering strategies.

A lanthanide complex, named CLaNP (caged lanthanide NMR probe) has been developed for the characterisation of proteins by par- amagnetic NMR spectroscopy. The probe consists of a lanthanide chelated by a derivative of DTPA (diethylene- triaminepentaacetic acid) with two thiol reactive functional groups. The CLaNP molecule is attached to a pro- tein by two engineered, surface-ex- posed, Cys residues in a bidentate manner. This drastically limits the dy- namics of the metal relative to the pro- tein and enables measurements of pseudocontact shifts. NMR spectrosco- py experiments on a diamagnetic con- trol and the crystal structure of the probe-protein complex demonstrate that the protein structure is not affect- ed by probe attachment. The probe is able to induce pseudocontact shifts to at least 40 a from the metal and causes residual dipolar couplings due to align- ment at a high magnetic field. The mol- ecule exists in several isomeric forms with different paramagnetic tensors; this provides a fast way to obtain long- range distance restraints.

Using a variety of spectroscopic techniques, a number of molecular functionalities have been studied in relation to the adsorption process of proteins to air-water interfaces. While ellipsometry and drop tensiometry are used to derive information on adsorbed amount and exerted surface pressure, external reflection circular dichroism, infrared, and fluorescence spectroscopy provide, next to insight in layer thickness and surface layer concentration, molecular details like structural (un)folding, local mobility, and degree of protonation of carboxylates. It is shown that the exposed hydrophobicity of the protein or chemical reactivity of solvent-exposed groups may accelerate adsorption, while increased electrostatic repulsion slows down the process. Also aggregate formation enhances the fast development of a surface pressure. A more bulky appearance of proteins lowers the collision intensity in the surface layer, and thereby the surface pressure, while it is shown to be difficult to affect protein interactions within the surface layer on basis of electrostatic interactions. This work illustrates that the adsorption properties of a protein are a combination of molecular details, rather than determined by a single one. © 2004 Wiley Periodicals, Inc.

Background Mutations in either of two genes comprising the STSL locus, ATP-binding cassette (ABC)-transporters ABCG5 (encoding sterolin-1) and ABCG8 (encoding sterolin-2), result in sitosterolemia, a rare autosomal recessive disorder of sterol trafficking characterized by increased plasma plant sterol levels. Based upon the genetics of sitosterolemia, ABCG5/sterolin-1 and ABCG8/sterolin-2 are hypothesized to function as obligate heterodimers. No phenotypic difference has yet been described in humans with complete defects in either ABCG5 or ABCG8. These proteins, based upon the defects in humans, are responsible for regulating dietary sterol entry and biliary sterol secretion. Methods In order to mimic the human disease, we created, by a targeted disruption, a mouse model of sitosterolemia resulting in Abcg8/sterolin-2 deficiency alone. Homozygous knockout mice are viable and exhibit sitosterolemia. Results Mice deficient in Abcg8 have significantly increased plasma and tissue plant sterol levels (sitosterol and campesterol) consistent with sitosterolemia. Interestingly, Abcg5/sterolin-1 was expressed in both liver and intestine in Abcg8/sterolin-2 deficient mice and continued to show an apical expression. Remarkably, Abcg8 deficient mice had an impaired ability to secrete cholesterol into bile, but still maintained the ability to secrete sitosterol. We also report an intermediate phenotype in the heterozygous Abcg8+/- mice that are not sitosterolemic, but have a decreased level of biliary sterol secretion relative to wild-type mice. Conclusion These data indicate that Abcg8/sterolin-2 is necessary for biliary sterol secretion and that loss of Abcg8/sterolin-2 has a more profound effect upon biliary cholesterol secretion than sitosterol. Since biliary sitosterol secretion is preserved, although not elevated in the sitosterolemic mice, this observation suggests that mechanisms other than by Abcg8/sterolin-2 may be responsible for its secretion into bile.

dTDP-rhamnose is an important precursor of cell wall polysaccharides and rhamnose-containing exopolysaccharides (EPS) in Lactococcus lactis . We cloned the rfbACBD operon from L. lactis MG1363, which comprises four genes involved in dTDP-rhamnose biosynthesis. When expressed in Escherichia coli , the lactococcal rfbACBD genes could sustain heterologous production of the Shigella flexneri O antigen, providing evidence of their functionality. Overproduction of the RfbAC proteins in L. lactis resulted in doubled dTDP-rhamnose levels, indicating that the endogenous RfbAC activities control the intracellular dTDP-rhamnose biosynthesis rate. However, RfbAC overproduction did not affect rhamnose-containing B40-EPS production levels. A nisin-controlled conditional RfbBD mutant was unable to grow in media lacking the inducer nisin, indicating that the rfb genes have an essential role in L. lactis . Limitation of RfbBD activities resulted in the production of altered EPS. The monomeric sugar of the altered EPS consisted of glucose, galactose, and rhamnose at a molar ratio of 1:0.3:0.2, which is clearly different from the ratio in the native sugar. Biophysical analysis revealed a fourfold-greater molecular mass and a twofold-smaller radius of gyration for the altered EPS, indicating that these EPS are more flexible polymers with changed viscosifying properties. This is the first indication that enzyme activity at the level of central carbohydrate metabolism affects EPS composition.

A number of relevant properties of Woodward's reagent K have been determined, such as the stability of the reactant and the optimal reaction conditions of the reactant with protein carboxylates. A Woodward's reagent K stock solution was stable at 4°C for prolonged time, whereas upon storage at 22°C, almost 20% of the reactive compound was lost within 1 week. The pH-dependency of the spontaneous degradation reaction of Woodward's reagent K was studied and was shown to be basemediated. A molar extinction coefficient of 3150 M-1 cm-1 at 269 nm for the enol ester resulting from the reaction between Woodward's reagent K and the protein carboxylates was established using the conditions laid out in this work. This value was validated using a variety of proteins that were modified by Woodward's reagent K. In addition, upon methylation of the carboxylates of a single protein, ovalbumin in this case, the degree of modification could be determined accurately and was confirmed by cation exchange chromatography elution profiles. Chemicals/CAS: ovalbumin, 77466-29-6; Carboxylic Acids; Esters; Isoxazoles; N-ethyl-5-phenylisoxazolium-3'-sulfonate, 4156-16-5; Ovalbumin, 9006-59-1; Proteins

One of the most powerful methods available to assign function to a gene is to inactivate or knockout the gene. Recently,we described the first target-selected knockout in zebrafish. Here,we report on the further improvements of this procedure,resulting in a highly efficient and easy method to do target-selected mutagenesis in zebrafish. A library of 4608 ENU-mutagenized F1 animals was generated and kept as a living stock. The DNA of these animals was screened for mutations in 16 genes by use of CEL-I-mediated heteroduplex cleavage (TILLING) and subsequent resequencing. In total,255 mutations were identified,of which 14 resulted in a premature stop codon,7 in a splice donor/acceptor site mutation,and 119 in an amino acid change. By this method,we potentially knocked out 13 different genes in a few months time. Furthermore,we show that TILLING can be used to detect the full spectrum of ENU-induced mutations in a vertebrate genome with the presence of many naturally occurring polymorphisms.

2D-FTIR Spectroscopy was applied to study the slower states of the membrane protein bacterioRhodopsin (bR) photocycle, with the bR adsorbed on a ZnSe ATR-crystal. 2D-FTIR absorbance changes are presented in a more convenient way for analysis than in traditional 2D-FTIR spectroscopy.

Whereas protein engineering of enzymes and structural proteins nowadays is an established research tool for studying structure-function relationships of polypeptides and for improving their properties, the engineering of posttranslationally modified peptides, such as the lantibiotics, is just coming of age. The engineering of lantibiotics is less straightforward than that of unmodified proteins, since expression systems should be developed not only for the structural genes but also for the genes encoding the biosynthetic enzymes, immunity protein and regulatory proteins. Moreover, correct posttranslational modification of specific residues could in many cases he a prerequisite for production and secretion of the active lantibiotic, which limits the number of successful mutations one can apply. This paper describes the development of expression systems for the structural lantibiotic genes for nisin A, nisin Z, gallidermin, epidermin and Pep5, and gives examples of recently produced site-directed mutants of these lantibiotics. Characterization of the mutants yielded valuable information on biosynthetic requirements for production. Moreover, regions in the lantibioties were identified that are of crucial importance for antimicrobial activity. Eventually, this knowledge will lead to the rational design of lantibiotics optimally suited for fighting specific undesirable microorganisms. The mutants are of additional value for studies directed towards the elucidation of the mode of action of lantibiotics.

Long-term trends in the concentrations of SF6, CHClF2 (CFC-22), and COF2 in the lower stratosphere have been derived from analysis of ca. 1980 and more recent infrared solar occultation spectra recorded near 32 deg N latitude at approx. 0.02/ cm resolution. Consistent sets of line parameters and spectral calibration methods have been used in the retrievals to minimize systematic error effects. Quoted error limits are 1 sigma estimated precisions. The SF6 and CHClF2 results are based on spectra recorded by balloon-borne interferometers in March 1981 and June 1988 and a comparison of these results with the Atmospheric Trace Molecule Spectroscopy (ATMOS) Experiment/Spacelab 3 measurements obtained in May 1985 near 30 deg N latitude. In the 13-18 km altitude range the mean measured SF6 mixing ratio in parts per trillion by volume (pptv) increased from 1.17 +/- 0.21 in March 1981 to 2.02 +/- 0.20 pptv in June 1988, and the CHClF2 mixing ratio below 15 km altitude increased from 51 +/- 8 pptv in March 1981 to 102 +/- 10 pptv in June 1988. The CHClF2 retrievals used new empirical CHClF2 line parameters derived from 0.03/cm resolution laboratory spectra recorded at six temperatures between 203 and 293 K; the derived mixing ratios are approx. 30% higher than obtained with earlier sets of line parameters, thereby removing a large discrepancy noted previously between IR and in situ measurements of CHClF2. Assuming an exponential growth model for fitting the trends, SF6 and CHClF2 mean increase rates of 7.4% +/- 1.9% and 9.4% +/- 1.3% /year, are obtained, respectively, which correspond to cumulative increases by factors of approx. 1.7 and -2.0 in the concentrations of these gases over the 7.2-year measurement period. Analysis of spectra recorded in October 1979 and April 1989 yields COF2 volume mixing ratios that are respectively 0.44 +/- 0.17 and 1.21 +/- 0.24 times the ATMOS/Spacelab 3 values, from which an average COF2 increase rate of 10.3 +/- 1.8%/ year over this time period has been estimated. The present results are compared with previously reported observations and trends and with one-dimensional model calculations. The model calculated trends are in reasonably good agreement with the observations.

Absorption bands of CF2Cl2, CFCl3 and CCl4 have been observed in infrared sunset solar spectra in the 800-1000 cm−1 region from 30 km altitude during a balloon flight made in September 1975. The infrared absorption bands were used to derive the distribution of these constituents in the upper troposphere and lower stratosphere. The results are compared with 1968 data and with current results of other authors.

IR solar absorption spectra at 0.002-0.0003/cm resolution in the 10-micron region obtained during two balloon flights near 32 deg N latitude are examined to determine the isotopic ratios of (O-16)(O-16)(O-18) and (O-16)(O-18)(O-16) relative to normal ozone in the stratosphere. For November 18, 1987, the results show column-averaged isotopic enhancement ratios of 1.20 + or - 0.14 and 1.40 + or - 0.18 for (O-16)(O-18)(O-16)/(O-16)(O-16)(O-16) and (O-16)(O-16)(O-18)/(O-16)(O-16)(O-16), respectively. The corresponding values for June 6, 1988, show ratios of 1.16 + or - 0.08 and 1.25 + or - 0.12. The results are compared with heavy-to-normal O3 ratios obtained using other techniques.

The Holocene Mississippi Delta System consists of six cyclic deltaic packages. Associated peat deposits are planar and eutrophic, and accumulate in areas abandoned by deltaic sedimentation. Peats that formed in large-scale interlobe basins are the subject of this paper. True peats (>75% organic matter) average 81.7% organic matter on a dry-weight basis. Controls on quality and lateral distribution of peat deposits are: botanical parent material, the balance between subsidence and accretion rates, detrital clastic influx and marine inundation. Botanical parent material, inferred from 13 C signatures and microtome sections, is always freshwater, originating either in forested swamps, or under herbaceous (floating) mats. Sphagnum spp. is essentially absent. In order for peat to accumulate, rates of accretion and subsidence must balance. Long-term subsidence rates in the study area increase from about 0.10 to 0.30 cm a -1 over a distance of 40 km from upper to lower delta plain. Subsurface accretion rates for true peats at a depth of 2-3 m, however, average about 0.05 cm a -1 . Renewed coarse detrital clastic influx terminates peat accumulation. The largest quantities of true peat are found together with relatively large amounts of organic-poor clastic sediments (mostly clays). Delta lobe abandonment and consequent coastal erosion causes salt water intrusion into freshwater environments. Resulting saline marsh sediments have maximum organic matter contents of 35%. In a transgressive setting, these may overlie older high-quality (low-ash) freshwater peats. Leaching experiments reveal the presence of water-soluble salts in both transgressed and fresh peats. Leaching reduces ash values by 1/5 to 1/3, suggesting that such losses may occur during early diagenesis. It is possible that some commercial coal seams originated in analogous interlobe environments, where leaching and possibly silica mobilization reduced the originally high ash contents to acceptable levels.

The blood of the Rock-Hopper Penguin contains only one hemoglobin component, corresponding to the Hb A of other birds. The primary structures of the alpha- and beta-chains are presented. The chains were separated by high-performance liquid chromatography and cleaved either enzymatically (alpha) or both enzymatically and chemically (beta). Both the native chains and their peptides were sequenced using liquid and gas phase sequenators. The peptides were aligned using their homology to the sequence of human hemoglobin and other bird hemoglobins. As compared to human hemoglobin, 44 amino-acid replacements are found in the alpha-chains (68% homology) and 47 in the beta-chains (67.8% homology). These exchanges involve seven alpha 1/beta 1 and one alpha 1/beta 2 contact in the alpha-chains, whereas in the beta-chains eight alpha 1/beta 1, one alpha 1/beta 2 and one hem contact are substituted. The influence of these replacements on the structure-function relationships in hemoglobin, as well as their importance for the diving ability of penguins, are discussed.

A method for visualizing molecular surfaces is described that uses a grid to store the distance to the nearest atom. Using on-the-fly three-dimensional (3D) contouring of a molecular graphics program such as FRODO, one can obtain a good impression of van der Waals surfaces and solvent-accessible surfaces. The main advantages of the method described here are its high speed and the fact that no recalculations need to be done to obtain the solvent-accessible surface visualized for a probe with another radius.