Dietary indigestible components exert different regional effects on luminal mucin secretion through their bulk-forming property and fermentability

The purpose of this study was to examine the effects of dietary indigestible components on mucin secretion in the respective parts of the gastrointestinal tract through their physico-chemical properties. Rats were fed either a control diet or diets containing 5% polystyrene foam (PSF), 5% fructooligosaccharide (FOS), 5% PSF + 5% FOS, or 10% beet fiber for 10 d. Mucins in the small intestine and feces were greater in the PSF, PSF + FOS, and beet fiber groups than in the control and FOS groups. In the cecum, greater mucins were observed in the FOS, PSF + FOS, and beet fiber groups than in the control and PSF groups. None of the dietary treatment was effective on gastric mucins. Cecal mucins were significantly correlated with the cecal pool sizes of total short-chain fatty acids. The correlation between fecal mucins and fecal numbers was also significant. The results suggest that the effect of the bulk-forming property of the dietary indigestible component on mucin secretion is limited to the duct, while fermentability is effective only in the cecum.     

Partial replacement of waxy cornstarch by recrystallized amylose retards the development of insulin resistance in rats

We examined in rats whether or not the prolonged ingestion of recrystallized amylose (RCA) would prevent the development of insulin resistance. Rats were fed on a diet containing waxy cornstarch (WCS) as carbohydrate or a diet containing 30% RCA in place of WCS for 18 wk. Glucose tolerance test (GTT) was conducted at every four weeks. On wk 16, the plasma insulin response as assessed by the area under the curve was lower in the RCA diet group than in the WCS diet group. The fasting plasma insulin level tended to increase over time in both groups, but was lower in the RCA diet group on wk 16. An autopsy revealed that the adipose tissue mass and serum free fatty acid concentrations were significantly higher in the WCS diet group. The results suggest that prolonged ingestion of RCA had the effect of slowing the development of insulin resistance through a lower concentration of serum free fatty acids, presumably due to the prevention of adipocyte hypertrophy.     

Effectiveness of buzz pollination in Pedicularis chamissonis: significance of multiple visits by bumblebees

Pollination success of plants is highly susceptible to the frequency of visits and foraging behavior of pollinators. Pollination of the nectarless flowers of Pedicularis species depends on bumblebee workers collecting pollen by vibrating the anthers (buzz pollination). However, little is known about the efficiency of the pollination system. Foraging behavior, pollen removal from anthers and pollen deposition on stigmas of P. chamissonis were studied to assess the effectiveness of buzz pollination in an alpine snowbed population of northern Japan. Although bumblebees tended to visit most of the flowers open at a given time within inflorescences during a single visit, pollen removal rate at the first visit was about 20%, and buzzing period decreased with increasing number of previous visits, resulting in a decreasing proportion of pollen removed per visit as the number of visits increased. These trends enable plants to provide pollen for more pollinators. The number of pollen grains deposited on stigmas was not saturated during the first visit and increased with additional visits. Irrespective of weak self-compatibility, evidence of interference between self and outcross pollen was lacking for seed production. Therefore, buzz pollination in P. chamissonis acts as a mechanism that improves the chance of cross-pollination upon multiple visits if pollinator visitation is frequent.     

Restricted spacer tolerance of a zinc finger nuclease with a six amino acid linker

Zinc finger nucleases can be engineered to create highly efficient and precise changes to the genetic information within living cells. We report the investigation of an important parameter that defines the type of target site the nuclease can cleave. The active nuclease is a dimer, requiring that the DNA target site contain two zinc finger binding sites separated by a short spacer. Using a plasmid-based recombination assay in HEK 293T cells, we show that a 6 amino acid linker between the zinc finger DNA-binding domain and the FokI cleavage domain restricts nuclease activity to sites containing a 6 bp spacer. These observations concur with other recent studies, suggesting this information will be useful in the design of new potent and accurate zinc finger nucleases.     

Multifunctional Role of His159in the Catalytic Reaction of Serine Palmitoyltransferase

Serine palmitoyltransferase (SPT) belongs to the fold type I family of the pyridoxal 5′-phosphate (PLP)-dependent enzyme and forms 3-ketodihydrosphingosine (KDS) from l-serine and palmitoyl-CoA. Like other α-oxamine synthase subfamily enzymes, SPT is different from most of the fold type I enzymes in that its re face of the PLP-Lys aldimine is occupied by a His residue (His¹⁵⁹) instead of an aromatic amino acid residue. His¹⁵⁹ was changed into alanine or aromatic amino acid residues to examine its role during catalysis. All mutant SPTs formed the PLP-l-serine aldimine with dissociation constants several 10-fold higher than that of the wild type SPT and catalyzed the abortive transamination of l-serine. These results indicate that His¹⁵⁹ is not only the anchoring site for l-serine but regulates the α-deprotonation of l-serine by fixing the conformation of the PLP-l-serine aldimine to prevent unwanted side reactions. Only H159A SPT retained activity and showed a prominent 505-nm absorption band of the quinonoid species during catalysis. Global analysis of the time-resolved spectra suggested the presence of the two quinonoid intermediates, the first formed from the PLP-l-serine aldimine and the second from the PLP-KDS aldimine. Accumulation of these quinonoid intermediates indicated that His¹⁵⁹ promotes both the Claisen-type condensation as an acid catalyst and the protonation at Cα of the second quinonoid to form the PLP-KDS aldimine. These results, combined with the previous model building study (Ikushiro, H., Fujii, S., Shiraiwa, Y., and Hayashi, H. (2008) J. Biol. Chem. 283, 7542–7553), lead us to propose a novel mechanism, in which His¹⁵⁹ plays multiple roles by exploiting the stereochemistry of Dunathan''s conjecture.Coenzymes act as catalysts in biological systems, and many enzymes require coenzymes as the important catalytic group. In most cases, coenzymes can carry out the catalysis in the absence of the enzyme protein. However, the reaction rate is much lower than the rate in the system containing the enzyme protein. Furthermore, the reaction specificity is reduced in the nonenzymatic system; coenzymes without the enzyme protein tend to undergo side reactions. A remarkable example is the coenzyme pyridoxal 5′-phosphate (PLP).³ PLP is a versatile catalyst catalyzing transamination, decarboxylation, elimination, aldol cleavage, Claisen-type condensation, etc. of amino acids. Therefore, a pyridoxal enzyme is required to have a structure that enables elaborated chemical mechanism by which only a specific reaction proceeds at each catalytic step.Serine palmitoyltransferase (SPT) catalyzes the condensation reaction of l-serine and palmitoyl-CoA to produce 3-ketodihydrosphingosine (KDS) (1). This is the first step in the sphingolipid biosynthesis. SPT belongs to the PLP-dependent α-oxamine synthase subfamily containing 5-aminolevulinate synthase, 8-amino-7-oxononanoate synthase, and 2-amino-3-ketobutyrate CoA ligase (2–6). All of them have been successfully crystallized, and their three-dimensional structures have been determined (7–12). These enzymes belong to the fold type I family of the PLP-dependent enzymes according to their folding pattern (5, 6). The commonly known fold type I PLP-dependent enzymes have an aromatic amino acid residue locating at the re face of the PLP-Lys internal aldimine and stacking with the pyridine ring of PLP. On the other hand, all members of the PLP-dependent α-oxamine synthase subfamily known to date have a His residue in this position. Therefore, the His residue is expected to play unique roles in the reaction mechanism of the PLP-dependent α-oxamine synthase subfamily enzymes.Scheme 1 shows the chemical reaction mechanism of SPT (1, 13). At the active site of SPT, PLP forms an aldimine with the ϵ-amino group of Lys²⁶⁵ (internal aldimine, I). The internal aldimine undergoes transaldimination with the first substrate l-serine to yield the PLP-l-serine aldimine (external aldimine, II). After binding of the second substrate palmitoyl-CoA, α-deprotonation occurs to form the first quinonoid intermediate (III). The carbanionic Cα of III attacks palmitoyl-CoA (Claisen-type condensation) to generate a condensation product (IV), which, by decarboxylation, yields the second quinonoid intermediate (V). Protonation at Cα of V gives the external aldimine of PLP-KDS (VI). Finally, release of KDS regenerates the internal aldimine (I). For this reaction mechanism, we proposed by model building studies that His¹⁵⁹ of SPT is the anchoring site for both l-serine and palmitoyl-CoA and possibly involved in the catalytic steps (13). However, no experimental analyses have been made to confirm this proposal or to gain further insight into the function of the residue. To determine the catalytic role of His¹⁵⁹, especially its role in the reaction specificity of PLP-dependent α-oxamine synthase subfamily enzymes, we constructed mutant Sphingomonas paucimobilis SPTs, in which His¹⁵⁹ was replaced by Ala and aromatic amino acid residues, and analyzed the reaction of these mutant enzymes. The results showed that His¹⁵⁹ has at least two additional distinct functions: one as a residue that controls the reaction pathway by adjusting the conformation of the PLP-l-serine external aldimine and the other as an acid catalyst that promotes the reactions of the Claisen-type condensation and the following steps.Open in a separate windowSCHEME 1.Reaction mechanism of SPT. The results were taken from Refs. 1 and 13 with modifications.     

Glioblastoma Formation from Cell Population Depleted of Prominin1-Expressing Cells

Prominin1 (Prom1, also known as CD133 in human) has been widely used as a marker for cancer stem cells (CSCs), which self-renew and are tumorigenic, in malignant tumors including glioblastoma multiforme (GBM). However, there is other evidence showing that Prom1-negative cancer cells also form tumors in vivo. Thus it remains controversial whether Prom1 is a bona fide marker for CSCs. To verify if Prom1-expressing cells are essential for tumorigenesis, we established a mouse line, whose Prom1-expressing cells can be eliminated conditionally by a Cre-inducible DTA gene on the Prom1 locus together with a tamoxifen-inducible CreER^TM, and generated glioma-initiating cells (GICs-LD) by overexpressing both the SV40 Large T antigen and an oncogenic H-Ras^L61 in neural stem cells of the mouse line. We show here that the tamoxifen-treated GICs-LD (GICs-DTA) form tumor-spheres in culture and transplantable GBM in vivo. Thus, our studies demonstrate that Prom1-expressing cells are dispensable for gliomagenesis in this mouse model.     

Immunogenic Burkholderia pseudomallei Outer Membrane Proteins as Potential Candidate Vaccine Targets

Background

Burkholderia pseudomallei is the causative agent of melioidosis, a disease of significant morbidity and mortality in both human and animals in endemic areas. There is no vaccine towards the bacterium available in the market, and the efficacy of many of the bacterium''s surface and secreted proteins are currently being evaluated as vaccine candidates.

Methodology/Principal Findings

With the availability of the B. pseudomallei whole genome sequence, we undertook to identify genes encoding the known immunogenic outer membrane protein A (OmpA). Twelve OmpA domains were identified and ORFs containing these domains were fully annotated. Of the 12 ORFs, two of these OmpAs, Omp3 and Omp7, were successfully cloned, expressed as soluble protein and purified. Both proteins were recognised by antibodies in melioidosis patients'' sera by Western blot analysis. Purified soluble fractions of Omp3 and Omp7 were assessed for their ability to protect BALB/c mice against B. pseudomallei infection. Mice were immunised with either Omp3 or Omp7, subsequently challenged with 1×10⁶ colony forming units (cfu) of B. pseudomallei via the intraperitoneal route, and examined daily for 21 days post-challenge. This pilot study has demonstrated that whilst all control unimmunised mice died by day 9 post-challenge, two mice (out of 4) from both immunised groups survived beyond 21 days post-infection.

Conclusions/Significance

We have demonstrated that B. pseudomallei OmpA proteins are immunogenic in mice as well as melioidosis patients and should be further assessed as potential vaccine candidates against B. pseudomallei infection.