Phosphopeptide enrichment by aliphatic hydroxy acid-modified metal oxide chromatography for nano-LC-MS/MS in proteomics applications

We developed novel methods for phosphopeptide enrichment using aliphatic hydroxy acid-modified metal oxide chromatography (MOC). Titania and zirconia were successfully applied to enrich phosphopeptides with the aid of aliphatic hydroxy acids, such as lactic acid and beta-hydroxypropanoic acid, to reduce the interaction between acidic non-phosphopeptides and the metal oxides. These methods removed the vast majority of non-phosphopeptides from phosphoprotein standard digests, and large numbers of phosphopeptides could be readily identified. The methods were coupled with nano-LC-MS/MS systems without difficulty. Recovery of phosphopeptides in MOC varied greatly from peptide to peptide, ranging from a few percent to 100%, and the average was almost 50%. Repeatability and linearity were satisfactory. In an examination of the cytoplasmic fraction of HeLa cells, more than 1000 phosphopeptides were identified using lactic acid-modified titania MOC and beta-hydroxypropanoic acid-modified zirconia MOC, respectively. The overlap between phosphopeptides enriched by these two methods was 40%, and the combined results provided 1646 unique phosphopeptides. To our knowledge, this is the first successful application of a single MOC-based approach to phosphopeptide enrichment from complex biological samples such as cell lysates.     

Effects of total parenteral nutrition on rat bile canaliculi: a stereologic analysis

OBJECTIVE: To quantitate, in a stereologic manner, changes in bile canalicular morphology before and after choleretic infusion of total parenteral nutrition (TPN) and to determine whether TPN produces changes in localized regions within the hepatic lobule. STUDY DESIGN: Livers were obtained from sham-operated on normal adult male rats (control) and from rats that received intravenous TPN solution containing 20% glucose and 3.5% Molipron F. The tissues, obtained by a rigorous sampling procedure, were systematically subjected to stereologic analysis. Measurements were made on electron micrographs at two levels of magnification by point, intersection and profile counts, and then volume, surface area and length were estimated per unit parenchymal volume. RESULTS: The surface area of the canalicular wall per parenchymal volume increased significantly (from 5.33 x 10(-2) to 6.73 x 10(-2) microns 2/micron 3) after TPN treatment, as did the length of microvilli (from 0.241 to 0.267 microns/micron 3). However, the volume of bile canalicular lumina per parenchymal volume (0.306% and 0.320%), total length of bile canaliculi (1.05 x 10(-2) and 1.06 x 10(-2) microns/micron 3) and diameter of microvilli (8.73 x 10(-2) and 8.94 x 10(-2) microns) remained constant. CONCLUSION: These results indicate that changes in canalicular shape and microvillus hypertrophy may cause lowering efficiency of the bile flow rate.     

Cloning, expression, and characterization of a lipolytic enzyme gene (lip8) from Pseudomonas aeruginosa LST-03

A lipolytic enzyme gene (lip8) was cloned from organic solvent-tolerant Pseudomonas aeruginosa LST-03 and sequenced. In the sequenced nucleotides, an open reading frame consisting of 1,173 nucleotides and encoding 391 amino acids was found. Lip8 is considered to belong to the family VIII of lipolytic enzymes whose serine in the consensus sequence of -Ser-Xaa-Xaa-Lys- acts as catalytic nucleophile. The gene was expressed in Escherichia coli and purified by a combination of ammonium sulfate fractionation and hydrophobic interaction and ion-exchange chromatographies to homogeneity on SDS-PAGE analysis. The optimum temperature and heat stability of Lip8 were not as high as those of Lip3 and LST-03 lipase, two other lipolytic enzymes from the same strain. Addition of glycerol to a solution containing Lip8 stabilized this enzyme. By measuring the activities against various triacylglycerols and fatty acid methyl esters having carbon chains of different lengths, Lip8 was categorized as an esterase which has higher activities against fatty acid methyl esters with short-chain fatty acids.     

Synthesis and structure-activity relationships of 2-amino-8-hydroxyadenines as orally active interferon inducing agents

Recently, we have reported the 8-hydroxyadenine derivatives (2–4) as a novel class of interferon (IFN) inducing agents. In the present study, a series of 8-hydroxyadenines, which possess various amino moieties at the adenine C(2)-position, were synthesized and evaluated for their ability to induce endogenous IFN in comparison to the known active agent, Imiquimod. Among the compounds prepared, compound 9o possessing a 2-methoxyethylamino group at C(2)-position of adenine was found to exhibit potent IFN inducing activity in vivo. Compound 9o induced IFN from the dosage of 0.1 mg/kg, which was 30-fold potent than that of Imiquimod, and showed a good oral bioavailability (F=81%).     

Increased hepatic fructose 2,6-bisphosphate after an oral glucose load does not affect gluconeogenesis

The generally accepted metabolic concept that fructose 2,6-bisphosphate (Fru-2,6-P2) inhibits gluconeogenesis by directly inhibiting fructose 1,6-bisphosphatase is based entirely on in vitro observations. To establish whether gluconeogenesis is indeed inhibited by Fru-2,6-P2 in intact animals, a novel NMR method was developed using [U-13C]glucose and 2H2O as tracers. The method was used to estimate the sources of plasma glucose from gastric absorption of oral [U-13C]glucose, from gluconeogenesis, and from glycogen in 24-h fasted rats. Liver Fru-2,6-P2 increased approximately 10-fold shortly after the glucose load, reached a maximum at 60 min, and then dropped to base-line levels by 150 min. The gastric contribution to plasma glucose reached approximately 50% at 30 min after the glucose load and gradually decreased thereafter. Although the contribution of glycogen to plasma glucose was small, glucose formed from gluconeogenesis was substantial throughout the study period even when liver Fru-2,6-P2 was high. Liver glycogen repletion was also brisk throughout the study period, reaching approximately 30 micromol/g at 3 h. These data demonstrate that Fru-2,6-P2 does not inhibit gluconeogenesis significantly in vivo.     

Tissue-specific structure/function differentiation of the liver isoform of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase

The crystal structures of the human liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase in three different liganding states were determined and compared with those of the rat testis isozyme. A set of amino acid sequence heterogeneity from the two distinct genes encoding the two different tissue isozymes leads to both global and local conformational differences that may cause the differences in catalytic properties of the two isozymes. The sequence differences in a beta-hairpin loop in the kinase domain causes a translational shift of several hydrophobic interactions in the dimeric contact region, and its propagation to the domains interface results in a 5 degrees twist of the entire bisphosphatase domain relative to the kinase domain. The bisphosphatase domain twist allows the dimeric interactions between the bisphosphatase domains, which are negligible in the testis enzyme, and as a result, the conformational stability of the domain is increased. Sequence polymorphisms also confer small but significant structural dissimilarities in the substrate-binding loops, allowing the differentiated catalytic properties between the two different tissue-type isozymes. Whereas the polymorphic sequence at the bisphosphatase-active pocket suggests a more suitable substrate binding, a similar extent of sequence differences at the kinase-active pocket confers a different mechanism of substrates bindings to the kinase-active pocket. It includes the ATP-sensitive unwinding of the switch helix alpha5, which is a characteristic ATP-dependent conformational change in the testis form. The sequence-dependent structural difference disallows the liver kinase to follow the ATP-switch mechanism. Altogether these suggest that the liver isoform has structural features more appropriate for an elevated bisphosphatase activity, compared with that of the testis form. The structural predisposition for bisphosphatase activity in the liver isozyme is consistent with the liver-unique glucose metabolic pathway, gluconeogenesis.     

Ontogenic emergence and localization of larval skin antigen molecule recognized by adult T cells of Xenopus laevis: Regulation by thyroid hormone during metamorphosis

Results from previous studies using an inbred strain of Xenopus laevis have led to the proposition that metamorphosis includes the events by which the newly differentiating adult immune system, including T lymphocytes, recognizes and eliminates larval skin cells as 'non-self'. More recently, a larval antigen targeted by adult T cells was identified as a 59 kDa protein with a specific peptide sequence. Using antisera directed against the larval antigen and the peptide, immunohistochemistry and western blotting were done to examine expression of the 59 kDa larval antigen in the skin during larval and metamorphic periods. There was no expression before Nieuwkoop and Faber stage 53. Expression was first seen at the beginning of metamorphic stage 54, when hind limbs appear, and increased thereafter, in apical and skein cells of both trunk and tail regions. In the trunk region, expression started to decrease at stage 58, until it completely disappeared at stage 62 (metamorphic climax). In the tail skin, however, expression persisted throughout the metamorphic stages. Treatment of larvae with thyroid hormone (TH) resulted in repression of expression of the 59 kDa molecule in a dose-dependent manner. Downregulation occurred earlier in the trunk than in the tail skin. These results suggest involvement in metamorphic events of an immunological mechanism: differential expression of the larval antigen in the trunk and tail skin cells due to their differing concentration of TH results in the tail, but not the trunk skin, being selectively attacked by the newly differentiating adult-type immune system.     

Intraperitoneal immunization led to T cell hyporesponsiveness to Helicobacter pylori infection in mice

During Helicobacter pylori infection, T cell response is critical in the development of active gastritis and in protective immunity against infection. We studied gastric inflammation and T cell response in H. pylori-challenged mice following an intraperitoneal immunization, using whole H. pylori lysate (HpAg) in the absence of adjuvants. H. pylori-challenged mice without immunization developed moderate to severe gastric inflammation, and splenocytes from these mice produced Th1 polarizing cytokines in response to HpAg and Con A during the acute infection. On the other hand, immunized-challenged mice (those inoculated with H. pylori following immunization) had little or no gastric inflammation despite persistent H. pylori colonization. Our immunization primed splenocytes to produce IL-2, IFN-gamma, and IL-4 in response to HpAg and Con A before infection. However, these cells became hyporesponsive to both stimulants immediately after live bacterial challenge in terms of the production of these cytokines, especially IL-2 and IFN-gamma. CTLA-4 has been documented to be a negative regulator of IL-2 production and lymphoproliferation that induces peripheral tolerance and functions 24-72 hr after the initiation of T cell activation. Compared with challenged mice, T cells from immunized-challenged mice showed higher levels of CTLA-4 expression at 72 hr after oral challenge. These data suggested that our immunization inhibited the development of H. pylori-associated gastritis and induced T cell hyporesponsiveness to H. pylori infection, which might be mediated by the early induction of CTLA-4 following challenge.