The nonfermentable dietary fiber hydroxypropyl methylcellulose modulates intestinal microbiota.

Diet influences host metabolism and intestinal microbiota; however, detailed understanding of this tripartite interaction is limited. To determine whether the nonfermentable fiber hydroxypropyl methylcellulose (HPMC) could alter the intestinal microbiota and whether such changes correlated with metabolic improvements, C57B/L6 mice were normalized to a high-fat diet (HFD), then either maintained on HFD (control), or switched to HFD supplemented with 10% HPMC, or a low-fat diet (LFD). Compared to control treatment, both LFD and HPMC reduced weight gain (11.8 and 5.7 g, respectively), plasma cholesterol (23.1 and 19.6%), and liver triglycerides (73.1 and 44.6%), and, as revealed by 454-pyrosequencing of the microbial 16S rRNA gene, decreased microbial α-diversity and differentially altered intestinal microbiota. Both LFD and HPMC increased intestinal Erysipelotrichaceae (7.3- and 12.4-fold) and decreased Lachnospiraceae (2.0- and 2.7-fold), while only HPMC increased Peptostreptococcaceae (3.4-fold) and decreased Ruminococcaceae (2.7-fold). Specific microorganisms were directly linked with weight change and metabolic parameters in HPMC and HFD mice, but not in LFD mice, indicating that the intestinal microbiota may play differing roles during the two dietary modulations. This work indicates that HPMC is a potential prebiotic fiber that influences intestinal microbiota and improves host metabolism.

Quantification of the sulfated cholecystokinin CCK-8 in hamster plasma using immunoprecipitation liquid chromatography-mass spectrometry/mass spectrometry.

Cholecystokinin (CCK) and the different molecular forms of CCK are well established as biomarkers for satiety but accurate analysis has been limited by the multiple naturally occurring forms and extensive similarities to gastrin. Changes in levels of one form, CCK-8, a naturally occurring eight amino acid peptide of CCK, have been correlated with satiety responses. Endogenous CCK-8 has not been well characterized in Syrian Golden hamsters, an important model in the study of fat uptake and digestion. We have cloned and sequenced hamster CCK and identified and characterized endogenous CCK-8 from hamster plasma. Hamster CCK-8 is composed of eight amino acid residues which are highly conserved among other species. Following accurate identification and characterization of hamster CCK-8, we have developed a highly specific and sensitive immunoprecipitation based LC-MS/MS assay for its quantification. The present assay enables determination of active CCK-8 over a concentration range from 0.05 to 2.5 ng/mL in hamster plasma samples. This range covers both the basal and postprandial levels of CCK-8. Method performance validation samples were examined at three concentrations replicated over the course of 4 days. The assay accuracy (percent relative error, % RE) average was 11.3% with a precision (percent coefficient of variation, % CV) of 15.5% over all samples in this 4 day period. Additionally, the method was used to determine increases of endogenous plasma CCK-8 in hamsters challenged with a high-fat meal.

Supplementation of hydroxypropyl methylcellulose into yeast leavened all-whole grain barley bread potentiates cholesterol-lowering effect.

We investigated in Syrian Golden hamsters the biological impact and its underlying mechanism of single whole grain breads supplemented with 2-3% hydroxypropyl methylcellulose (HPMC), a semisynthetic viscous soluble dietary fiber (SDF) as a substitute for gluten. Hamsters were fed high-fat diets supplemented with 48-65% (w/w) differently ground, freeze-dried single grain breads including whole grain wheat, barley, barley supplemented with HPMC, debranned oat, and oat supplemented with HPMC which were compared to a diet containing microcrystalline cellulose (control). All single grain breads significantly lowered plasma LDL-cholesterol concentrations compared to the control. Enrichment with HPMC further lowered plasma and hepatic cholesterol concentrations. Despite the reduced molecular weight of naturally occurring soluble (1--->3),(1--->4)-ß-d-glucan (ß-glucan) caused by the bread-making process, whole grain barley breads downregulated hepatic expression of CYP7A1 and HMG-CoAR genes that are responsible for bile acid and cholesterol synthesis, suggesting a possible role of bioactive compounds such as short-chain fatty acids and phenolic compounds from barley bread. Barley bread enriched with HPMC downregulated expression of ABCG5 gene. Taken together, it appears that distinctive modulation of synthesis and excretion of hepatic cholesterol and bile acid contributes to the cholesterol-lowering properties of whole grain barley breads and breads enriched with HPMC. These data suggests that alternative whole grain breads supplemented with HPMC may provide consumers with a staple food that can assist in cholesterol management.

O-linked glycosylation in maize-expressed human IgA1.

O-Linked glycans vary between eukaryotic cell types and play an important role in determining a glycoprotein's properties, including stability, target recognition, and potentially immunogenicity. We describe O-linked glycan structures of a recombinant human IgA1 (hIgA1) expressed in transgenic maize. Up to six proline/hydroxyproline conversions and variable amounts of arabinosylation (Pro/Hyp + Ara) were found in the hinge region of maize-expressed hIgA1 heavy chain (HC) by using a combination of matrix-assisted laser-desorption ionization mass spectrometry (MALDI MS), chromatography, and amino acid analysis. Approximately 90% of hIgA1 was modified in this way. An average molar ratio of six Ara units per molecule of hIgA1 was revealed. Substantial sequence similarity was identified between the HC hinge region of hIgA1 and regions of maize extensin-family of hydroxyproline-rich glycoproteins (HRGP). We propose that because of this sequence similarity, the HC hinge region of maize-expressed hIgA1 can become a substrate for posttranslational conversion of Pro to Hyp by maize prolyl-hydroxylase(s) with the subsequent arabinosylation of the Hyp residues by Hyp-glycosyltransferase(s) in the Golgi apparatus in maize endosperm tissue. The observation of up to six Pro/Hyp hydroxylations combined with extensive arabinosylation in the hIgA1 HC hinge region is well in agreement with the Pro/Hyp hydroxylation model and the Hyp contiguity hypothesis suggested earlier in literature for plant HRGP. For the first time, the extensin-like Hyp/Pro conversion and O-linked arabinosylation are described for a recombinant therapeutic protein expressed in transgenic plants. Our findings are of significance to the field of plant biotechnology and biopharmaceutical industry-developing transgenic plants as a platform for the production of recombinant therapeutic proteins.