Effects of cashew nut shell extract and monensin on in vitro ruminal fermentation, methane production, and ruminal bacterial community.

The objective of this study was to evaluate the effects of cashew nut shell extract (CNSE) and monensin on ruminal in vitro fermentation, CH4 production, and ruminal bacterial community structure. Treatments were as follows: control (CON, basal diet without additives); 2.5 µM monensin (MON); 0.1 mg CNSE granule/g DM (CNSE100); and 0.2 mg CNSE granule/g DM (CNSE200). Each treatment was incubated with 52 mL of buffered ruminal content and 500 mg of total mixed ration for 24 h using serum vials. The experiment was performed as a complete randomized block design with 3 runs. Run was used as a blocking factor. Each treatment had 5 replicates, in which 2 were used to determine nutrient degradability, and 3 were used to determine pH, NH3-N, volatile fatty acids, lactate, total gas, CH4 production, and bacterial community composition. Treatment responses for all data, excluding bacterial abundance, were analyzed with the GLIMMIX procedure of SAS v9.4. Treatment responses for bacterial community structure were analyzed with a PERMANOVA test run with the R package vegan. Orthogonal contrasts were used to test the effects of (1) additive inclusion (ADD: CON vs. MON, CNSE100, and CNSE200); (2) additive type (MCN: MON vs. CNSE100 and CNSE200); and (3) CNSE dose (DOS: CNSE100 vs. CNSE200). We observed that pH, acetate, and acetate:propionate ratio in the CNSE100 treatment were lower compared with CNSE200, and propionate in the CNSE100 treatment was greater compared with CNSE200. Compared with MON, CNSE treatments tended to decrease total lactate concentration. Total gas production of CON was greater by 2.63% compared with all treatments, and total CH4 production was reduced by 10.64% in both CNSE treatments compared with MON. Also, compared with MON, in vitro dry matter degradabilities in CNSE treatments were lower. No effects were observed for NH3-N or in vitro neutral detergent fiber degradability. Finally, the relative abundances of Prevotella, Treponema, and Schwartzia were lower, whereas the relative abundances of Butyrivibrio and Succinivibrio were greater in all treatments compared with CON. Overall, the inclusion of CNSE decreased CH4 production compared with MON, making CNSE a possible CH4 mitigation additive in dairy cattle diets.

Effects of cashew nutshell extract and monensin on microbial fermentation in a dual-flow continuous culture.

The objective of this study was to compare cashew nutshell extract (CNSE) to monensin and evaluate changes in in vitro mixed ruminal microorganism fermentation, nutrient digestibility, and microbial nitrogen outflow. Treatments were randomly assigned to 8 fermenters in a replicated 4 × 4 Latin square design with 4 experimental periods of 10 d (7 d for diet adaptation and 3 d for sample collection). Basal diets contained 43.5:56.5 forage: concentrate ratio and each fermenter was fed 106 g of DM/d divided equally between 2 feeding times. Treatments were control (CON, basal diet without additives), 2.5 µM monensin (MON), 0.1 mg CNSE granule/g DM (CNSE100), and 0.2 mg CNSE granule/g DM (CNSE200). On d 8 to10, samples were collected for pH, lactate, NH3-N, volatile fatty acids (VFA), mixed protozoa counts, organic matter (OM), and neutral detergent fiber (NDF) digestibility. Data were analyzed with the GLIMMIX procedure of SAS. Orthogonal contrasts were used to test the effects of (1) ADD (CON vs. MON, CNSE100, and CNSE200); (2) MCN (MON vs. CNSE100 and CNSE200); and (3) DOSE (CNSE100 vs. CNSE200). We observed that butyrate concentration in all treatments was lower compared with CON and the concentration for MON was lower compared with CNSE treatments. Protozoal population in all treatments was lower compared with CON. No effects were observed for pH, lactate, NH3-N, total VFA, OM, or N utilization. Within the 24-h pool, protozoal generation time, tended to be lower, while NDF digestibility tended to be greater in response to all additives. Furthermore, the microbial N flow, and the efficiency of N use tended to be lower for the monensin treatment compared with CNSE treatments. Overall, our results showed that both monensin and CNSE decreased butyrate synthesis and protozoal populations, while not affecting OM digestibility and tended to increase NDF digestibility; however, such effects are greater with monensin than CNSE nutshell.

Effects of cashew nut shell extract supplementation on production, rumen fermentation, metabolism, and inflammatory biomarkers in transition dairy cows.

Cashew nut shell extract (CNSE) is a byproduct of the cashew nut industry, containing bioactive compounds that alter rumen fermentation patterns. Therefore, study objectives were to evaluate the effects of CNSE (59% anacardic acid and 18% cardol) on production, rumen fermentation variables, metabolism, and inflammation in transition dairy cows. A total of 51 multiparous Holstein cows were used in a randomized design and assigned to treatment based on their previous 305-d mature equivalent milk and parity. Cows were assigned to 1 of 2 treatments 21 d before expected calving: (1) CON (control diet; n = 17) or (2) CNSE-5.0 (control diet and 5.0 g/d CNSE granule [containing 50% CNSE]; n = 34). Following parturition, 17 cows (preselected at initial treatment assignment) from the CNSE-5.0 treatment were reallocated into a third treatment group: CNSE-2.5 (control diet and 2.5 g/d CNSE granule; n = 17), resulting in 3 total treatments postpartum: (1) CON, (2) CNSE-2.5, and (3) CNSE-5.0. Prepartum rumen pH was unaltered by treatment; however, postpartum rumen pH was increased (0.31 units) in CNSE cows relative to CON. Prepartum rumen ammonia N concentration tended to be decreased (34%) in CNSE-5.0 cows compared with CON, and there tended to be a quadratic effect on postpartum ammonia N, as it was decreased in CNSE-2.5 compared with CON and CNSE-5.0. Prepartum dry matter intake (DMI) was unaffected by treatment; however, postpartum DMI was increased (8%) in CNSE cows relative to CON. No treatment differences were observed in pre- or postpartum digestibility measurements. Milk and protein yields from cows fed CNSE tended to be increased (6% and 7%, respectively) relative to CON. No treatment differences were detected for energy-corrected milk, feed efficiency, body weight, body condition score, energy balance, milk composition, milk urea nitrogen, or somatic cell count. Prepartum fecal pH decreased (0.12 units) in CNSE-5.0 cows relative to CON cows but was similar between treatments postpartum. Supplementing CNSE did not affect prepartum glucose, nonesterified fatty acids (NEFA), ß-hydroxybutyrate (BHB), or insulin. However, prepartum circulating blood urea nitrogen tended to be decreased and glucagon was decreased in CNSE-5.0 cows compared with CON (9 and 20%, respectively). Additionally, CNSE supplementation decreased glucose and insulin concentrations postpartum relative to CON cows (6% and 20%, respectively). Quadratic effects were detected for postpartum circulating NEFA and BHB such that their levels were increased in CNSE-2.5 cows relative to CON and CNSE-5.0. Pre- and postpartum circulating serum amyloid A, lipopolysaccharide-binding protein, and haptoglobin were unaffected by treatment. Overall, CNSE influenced some key rumen fermentation variables, altered postabsorptive metabolism, and increased production parameters in transition dairy cows.

Hensin remodels the apical cytoskeleton and induces columnarization of intercalated epithelial cells: processes that resemble terminal differentiation.

Intercalated epithelial cells exist in a spectrum of phenotypes; at one extreme, beta cells secrete HCO3 by an apical Cl/HCO3 exchanger and a basolateral H+ ATPase. When an immortalized beta cell line is seeded at high density it deposits in its extracellular matrix (ECM) a new protein, hensin, which can reverse the polarity of several proteins including the Cl/HCO3 exchanger (an alternately spliced form of band 3) and the proton translocating ATPase. When seeded at low density and allowed to form monolayers these polarized epithelial cells maintain the original distribution of these two proteins. Although these cells synthesize and secrete hensin, it is not retained in the ECM, but rather, hensin is present in a large number of intracellular vesicles. The apical cytoplasm of low density cells is devoid of actin, villin, and cytokeratin19. Scanning electron microscopy shows that these cells have sparse microvilli, whereas high density cells have exuberant apical surface infolding and microvilli. The apical cytoplasm of high density cells contains high levels of actin, cytokeratin19, and villin. The cell shape of these two phenotypes is different with high density cells being tall with a small cross-sectional area, whereas low density cells are low and flat. This columnarization and the remodeling of the apical cytoplasm is hensin-dependent; it can be induced by seeding low density cells on filters conditioned by high density cells and prevented by an antibody to hensin. The changes in cell shape and apical cytoskeleton are reminiscent of the processes that occur in terminal differentiation of the intestine and other epithelia. Hensin is highly expressed in the intestine and prostate (two organs where there is a continuous process of differentiation). The expression of hensin in the less differentiated crypt cells of the intestine and the basal cells of the prostate is similar to that of low density cells; i.e., abundant intracellular vesicles but no localization in the ECM. On the other hand, as in high density cells hensin is located exclusively in the ECM of the terminally differentiated absorptive villus cells and the prostatic luminal cell. These studies suggest that hensin is a critical new molecule in the terminal differentiation of intercalated cell and perhaps other epithelial cells.

Induction of terminal differentiation in epithelial cells requires polymerization of hensin by galectin 3.

During terminal differentiation, epithelia become columnar and develop specialized apical membrane structures (microvilli) and functions (regulated endocytosis and exocytosis). Using a clonal intercalated epithelial cell line, we found that high seeding density induced these characteristics, whereas low density seeding maintained a protoepithelial state. When cells were plated at low density, but on the extracellular matrix of high density cells, they converted to the more differentiated phenotype. The extracellular matrix (ECM) protein responsible for this activity was purified and found to be a large 230-kD protein, which we termed hensin. High density seeding caused hensin to be polymerized and deposited in the extracellular matrix, and only this form of hensin was able to induce terminal differentiation. Antibodies to hensin blocked the change in phenotype. However, its purification to homogeneity resulted in loss of activity, suggesting that an additional protein might be necessary for induction of terminal differentiation. Here, we found that a 29-kD protein specifically associates with hensin in the ECM. Addition of purified p29 restored the activity of homogenously purified hensin. Mass fingerprinting identified p29 as galectin 3. Purified recombinant galectin 3 was able to bind to hensin and to polymerize it in vitro. Seeding cells at high density induced secretion of galectin 3 into the ECM where it bundled hensin. Hence, the high density state causes a secretion of a protein that acts on another ECM protein to allow the new complex to signal the cell to change its phenotype. This is a new mechanism of inside-out signaling.

Hensin, a new collecting duct protein involved in the in vitro plasticity of intercalated cell polarity.

Two forms of intercalated cells are present in kidney collecting tubules, the alpha cell has apical endocytosis, apical H+-ATPase and basolateral band 3, while beta cells have reversed polarity of these proteins and no apical endocytosis. When a beta cell line was seeded at high density, it changed into the alpha form. We previously showed that a partially purified 230 kD extracellular matrix protein of high density cells was able to retarget band 3 from apical to basolateral domains and stimulated apical endocytosis in vitro (Van Adelsberg, J., J.C. Edwards, J. Takito, B. Kiss, and Q. Al-Awqati. 1994. Cell. 76:1053-1061). We now purify this protein, which was named hensin, to near homogeneity and find that it belongs to the macrophage scavenger receptor cysteine rich (SRCR) family. An antibody, generated against a fusion protein made from a partial cDNA recognized a 230-kD protein in rabbit kidney and in the intercalated cell line. In vitro, the hensin antibody inhibited expression of apical endocytosis. Hensin was secreted in a polarized manner and bound to the basolateral membrane and extracellular matrix. Immunohistochemistry of the kidney showed that it was expressed only in collecting tubules. Double immunofluorescence with hensin and peanut lectin, H+-ATPase, or band 3 showed many patterns; most alpha-cells had hensin staining while 50% of beta-cells did not. These results suggest that hensin may also be involved in the polarity reversal of intercalated cells in vivo.

The hydrophobic region of signal peptides is involved in the interaction with membrane-bound SecA.

The positive charges of signal peptides are important for the interaction with SecA, a translocation ATPase. To examine whether or not the hydrophobic region of signal peptides also interacts with SecA, we constructed model preproteins, proOmpF-Lpps, possessing no positively charged amino acid residues at the amino-terminus and different numbers of alanine/leucine residues in the hydrophobic region of signal peptides. When the hydrophobic stretch was sufficiently long, amino-terminal positively charged residues were not required for the translocation of preproteins across the cytoplasmic membrane of Escherichia coli both in vitro and in vivo. Chemical cross-linking between SecA and preproteins possessing no positively charged residues at the amino-terminus was observed only in the presence of liposomes containing acidic phospholipids. The degree of cross-linking increased as the length of the hydrophobic stretch increased irrespective of whether positively charged residues were present or not. A preprotein possessing no positively charged residues at the amino-terminus, which is competent in the presence of liposomes, competitively inhibited the cross-linking of wild-type proOmpF-Lpp with SecA under the same conditions. It is concluded that both the amino-terminal positive charges and central hydrophobic domains are involved in the interaction with SecA in the initial stage of translocation in addition to their possible roles in transmembrane movement of preproteins.

Phosphatidylglycerol dependent protein translocation across the Escherichia coli inner membrane is inhibited by the anti-cancer drug doxorubicin. Evidence for an electrostatic interaction between the signal sequence and phosphatidylglycerol.

OmpF-Lpp, a model secretory protein, requires both a positively charged signal sequence and phosphatidylglycerol (PG) for efficient translocation across the E. coli inner membrane. Modification of the signal sequence can, however, remove both these prerequisites for translocation providing OmpF-Lpp mutants which undergo either PG and charge dependent or PG and charge independent translocation. Here we show that positively charged membrane interactive compounds (polylysine & doxorubicin) are able to inhibit PG dependent translocation of the OmpF-Lpp signal sequence mutants but not PG independent translocation. Doxorubicin is also shown to bind more efficiently to liposomes containing increased levels of anionic lipid indicating that in these assays it may be inhibiting translocation by preventing electrostatic interaction between the anionic lipid head group and the positively charged signal sequences.

Structural and functional characterization of the OmpF and OmpC porins of the Escherichia coli outer membrane: studies involving chimeric proteins.

The roles of submolecular regions of OmpF and OmpC, major outer membrane proteins of Escherichia coli, as concerns their biogenesis, structure and function were studied using a large number of chimeric genes constructed from the ompF and ompC genes through single or double homologous in vivo recombination. When recombination between the two genes took place at certain regions of their central regions, no chimeric protein was detected, irrespective of whether the amino-terminal and carboxy-terminal regions were derived from OmpF or OmpC. Biochemical studies revealed that these proteins were synthesized and exported across the cytoplasmic membrane normally, but that they were not properly assembled into the outer membrane and hence were degraded rapidly. Characterization of these chimeric proteins, in which recombination between OmpF and OmpC took place once or twice, suggested that the central region of each of these proteins plays an important role in the respective assembly, whereas the roles of the amino-terminal and carboxy-terminal regions may be marginal. Functional characterization of these chimeric proteins revealed the regions important for the receptor functions of OmpF and OmpC for phages TuIa and TuIb, respectively.

Terminal differentiation in epithelia: the Hensin pathway in intercalated cells.

The intercalated cell of the collecting tubule exists in a spectrum of types. The alpha form secretes acid by an apical H+-ATPase and a basolateral CI:HCO3 exchanger, which is an alternatively spliced form of the red cell band 3 (kAE1), and the beta form secretes HCO3 by having these transporters on the reverse membranes. In a clonal cell line of the beta form, we found that seeding density causes conversion of beta cells to the alpha form. A new protein, termed hensin, was deposited in the extracellular matrix (ECM) of high-density cells, which, on purification, reversed the polarity of the transporters. Hensin also induced the expression of the microvillar protein villin and caused the appearance of the apical terminal web proteins, cytokeratin 19 and actin; all of which led to the development of an exuberant microvillar structure. In addition, hensin caused the beta cells to assume a columnar shape. All of these studies show that the conversion of polarity in the intercalated cell, at least in vitro, represents terminal differentiation and that hensin is the first protein in a new pathway that mediates this process. Hensin, DMBT1, CRP-ductin, and ebnerin are alternately spliced products from a single gene located in human chromosome 10q25-26, a region often deleted in several cancers, especially malignant gliomas. Hensin is expressed in many epithelial cell types and it is possible that it plays a similarly important role in the differentiation of these epithelia as well.

Effects of total hydrophobicity and length of the hydrophobic domain of a signal peptide on in vitro translocation efficiency.

The hydrophobic domain of the signal peptide of OmpF-Lpp, a model secretory protein, was systematically engineered so as to be composed of different lengths of polyleucine residues or polymers with alternate leucine and alanine residues, and the effects of the length and nature of the hydrophobic stretch on the rate of in vitro translocation were studied using everted membrane vesicles of Escherichia coli. The translocation reaction exhibited high substrate specificity as to the number of hydrophobic residues. The results suggest that the hydrophobic domain is recognized specifically by a component(s) of the secretory machinery rather than nonspecifically by the hydrophobic region of the membrane. The in vitro translocation thus demonstrated required SecA and ATP and was markedly enhanced upon imposition of the proton motive force, as in the case of secretory proteins possessing a natural signal peptide. The highest translocation rate was obtained with the octamer in the case of polyleucine-containing signal peptides, whereas it was the decamer in the case of ones containing both leucine and alanine. These results suggest that the total hydrophobicity of the hydrophobic region of the signal peptides is an important determinant of the substrate specificity.

The requirement of a positive charge at the amino terminus can be compensated for by a longer central hydrophobic stretch in the functioning of signal peptides.

A variety of model presecretory proteins, proOmpF-Lpps, possessing different numbers of lysine residues (0, 2, and 4) as positively charged amino acid residues and different numbers of leucine residues (7, 8, and 9) as hydrophobic amino acid residues in their signal peptides were constructed. The effect of positive charges on the in vitro translocation efficiency markedly differed with the number of leucine residues. Positive charges were strongly required for translocation when the hydrophobic region comprised 7 or 8 leucine residues, whereas the translocation of proOmpF-Lpps possessing 9 leucine residues took place efficiently even in the absence of positive charges and the introduction of positive charges did not significantly enhance the translocation efficiency. The translocation of all the proOmpF-Lpps, including one possessing no positive charge, was ATP-, protonmotive force-, and SecA-dependent and accompanied by signal peptide cleavage, indicating that they are translocated via the usual secretory pathway. It is likely that the requirement of positive charges can be compensated for by a longer hydrophobic stretch in the functioning of the signal peptide.

Only multimeric hensin located in the extracellular matrix can induce apical endocytosis and reverse the polarity of intercalated cells.

When an intercalated epithelial cell line was seeded at low density and allowed to reach confluence, it located the anion exchanger band 3 in the apical membrane and an H+-ATPase in the basolateral membrane. The same clonal cells seeded at high density targeted these proteins to the reverse location. Furthermore, high density cells had vigorous apical endocytosis, and low density cells had none. The extracellular matrix of high density cells was capable of inducing apical endocytosis and relocation of band 3 to the basolateral membrane in low density cells. A 230-kDa extracellular matrix (ECM) protein termed hensin, when purified to near-homogeneity, was able to reverse the phenotype of the low density cells. Antibodies to hensin prevented this effect, indicating that hensin is necessary for conversion of polarity. We show here that hensin was synthesized by both low density and high density cells. Whereas both phenotypes secreted soluble hensin into their media, only high density cells localized it in their ECM. Analysis of soluble hensin by sucrose density gradients showed that low density cells secreted monomeric hensin, and high density cells secreted higher order multimers. When 35S-labeled monomeric hensin was added to high density cells, they induced its aggregation suggesting that the multimerization was catalyzed by surface events in the high density cells. Soluble monomeric or multimeric hensin did not induce apical endocytosis in low density cells, whereas the more polymerized hensin isolated from insoluble ECM readily induced it. These multimers could be disaggregated by sulfhydryl reagents and by dimethylmaleic anhydride, and treatment of high density ECM by these reagents prevented the induction of endocytosis. These results demonstrate that hensin, like several ECM proteins, needs to be precipitated in the ECM to be functional.

OmpF-Lpp signal sequence mutants with varying charge hydrophobicity ratios provide evidence for a phosphatidylglycerol-signal sequence interaction during protein translocation across the Escherichia coli inner membrane.

Using inverted Escherichia coli inner membrane vesicles we have analyzed the phosphatidylglycerol dependence of translocation of an OmpF-Lpp fusion protein carrying a signal sequence with varying positive charge at the N terminus and a hydrophobic core of varying length. It is shown that there is a direct relationship between the phosphatidylglycerol requirement of translocation and the requirement within the translocation process for positive charges on the signal sequence. This provides further evidence that the negative head group of the lipid is required for functional interaction with the positively charged N terminus of the signal sequence.

Suppression of the lethal effect of acidic-phospholipid deficiency by defective formation of the major outer membrane lipoprotein in Escherichia coli.

The Escherichia coli pgsA3 allele encoding a defective phosphatidylglycerophosphate synthase is lethal for all but certain strains. Genetic analysis of such strains has revealed that the lethal effect is fully suppressed by the lack of the major outer membrane lipoprotein that consumes phosphatidylglycerol for its maturation.

Phenotypic plasticity and terminal differentiation of the intercalated cell: the hensin pathway.

The intercalated cell of the collecting tubule exists in a spectrum of types. The alpha form secretes acid by an apical H(+) ATPase and a basolateral Cl:HCO(3) exchanger which is an alternatively spliced form of the red cell band 3 (kAE1), while the beta form secretes HCO(3) by having these transporters on the reverse membranes. In a clonal cell line of the beta form we found that seeding density causes this conversion. A new protein, termed hensin, was deposited in the extracellular matrix of high-density cells which on purification reversed the polarity of the transporters. Hensin also induced the expression of the microvillar protein, villin, and caused the appearance of the apical terminal web proteins, cytokeratin 19 and actin, all of which led to the development of an exuberant microvillar structure. In addition, hensin caused the beta cells to assume a columnar shape. All of these studies demonstrate that the conversion of polarity in the intercalated cell, at least in vitro, represents terminal differentiation and that hensin is the first protein in a new pathway that mediates this process. Hensin, DMBT1, CRP-ductin, and ebnerin are alternately spliced products from a single gene located on human chromosome 10q25-26, a region often deleted in several cancers, especially malignant gliomas. Hensin is expressed in many epithelial cell types, and it is possible that it plays a similarly important role in the differentiation of these epithelia as well.