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Abstract Background With the advent of metabolomics as a powerful tool for both functional and biomarker discovery, the identification of specific differences between complex metabolite profiles is becoming a major challenge in the data analysis pipeline. The task remains difficult, given the datasets' size, complexity, and common shifts in migration (elution/retention) times between samples analyzed by hyphenated mass spectrometry methods. Results We present a Mathematica (Wolfram Research, Inc.) package MathDAMP (Mathematica package for Differential Analysis of Metabolite Profiles), which highlights differences between raw datasets acquired by hyphenated mass spectrometry methods by applying arithmetic operations to all corresponding signal intensities on a datapoint-by-datapoint basis. Peak identification and integration is thus bypassed and the results are displayed graphically. To facilitate direct comparisons, the raw datasets are automatically preprocessed and normalized in terms of both migration times and signal intensities. A combination of dynamic programming and global optimization is used for the alignment of the datasets along the migration time dimension. The processed datasets and the results of direct comparisons between them are visualized using density plots (axes represent migration time and m/z values while peaks appear as color-coded spots) providing an intuitive overall view. Various forms of comparisons and statistical tests can be applied to highlight subtle differences. Overlaid electropherograms (chromatograms) corresponding to the vicinities of the candidate differences from any result may be generated in a descending order of significance for visual confirmation. Additionally, a standard library table (a list of m/z values and migration times for known compounds) may be aligned and overlaid on the plots to allow easier identification of metabolites. Conclusion Our tool facilitates the visualization and identification of differences between complex metabolite profiles according to various criteria in an automated fashion and is useful for data-driven discovery of biomarkers and functional genomics.

A 70-year-old woman was admitted to the hospital with incomplete defecation. Colonoscopy and abdominal computed tomography revealed a submucosal tumor of >50 mm in diameter in the lower rectum. The biopsy specimen was positive for CD34 and c-kit. Based on these findings, the tumor was diagnosed as a gastrointestinal stromal tumor(GIST). We decided to administer neoadjuvant therapy with imatinib mesylate(IM)because we considered that the tumor was too large to remove without tumor perforation. After 28 weeks of administering neoadjuvant therapy, the tumor size was reduced by 40%. Robot-assisted intersphincteric resection and temporary ileostomy were performed without tumor perforation. The patient survived without recurrence for 8 months after the operation, followed by treatment with IM as adjuvant therapy. The present case suggests that neoadjuvant therapy with IM may be useful for locally advanced GIST.

Primary biliary cirrhosis (PBC) is characterized by antimitochondrial antibodies (AMAs) that react with the lipoyl-containing E2 subunits of 2-oxoacid dehydrogenase complexes such as BCOADC and PDC. The lipoyl domains of E2 contain the major epitopes essential for immunopathology. However, the non-lipoyl-containing E1 subunits are also frequently targeted. Since anti-E1 antibodies always appear in combination with anti-E2 antibodies, the mechanisms underlying the autoimmunity against E1 may be linked to, but distinct from, those against E2. Here, we demonstrate that intermolecular and intramolecular determinant spreading underlies the autoimmunity against E1. We performed characterizations and epitope mapping for anti-BCOADC-E1α antibodies from both the intermolecular and intramolecular points of view. The antibody reactivities form a cluster against the BCOADC complex that is distinct from that against the PDC complex, and the anti-BCOADC-E1α antibodies arise as part of the cluster against the BCOADC complex. Multiple epitopes are present on the surface of the BCOADC-E1α molecule, and the major epitope overlaps with the active center. Sera with anti-BCOADC-E1α antibodies strongly inhibited the enzyme activity. These findings suggest that the E1α subunit as part of the native BCOADC complex is an immunogen, and that determinant spreading is involved in the pathogenesis of AMA production.

In biological networks, a small number of "hub" proteins play critical roles in the network integrity and functions. The cell cycle network orchestrates versatile cellular functions through interactions between many signaling modules, whose defects impair diverse cellular processes, often leading to cancer. However, the network architecture and molecular basis that ensure proper coordination between distinct modules are unclear. Here, we show that the ubiquitin ligase NIRF (also known as UHRF2), which induces G1 arrest, interacts with multiple cell cycle proteins including cyclins (A2, B1, D1 and E1), p53 and pRB, and ubiquitinates cyclins D1 and E1. Consistent with its versatility, a bioinformatic network analysis demonstrated that NIRF is an intermodular hub protein that is responsible for the coordination of multiple network modules. Notably, intermodular hubs are frequently associated with oncogenesis. Indeed, we detected loss of heterozygosity of the NIRF gene in several kinds of tumors. When a cancer outlier profile analysis was applied to the Oncomine database, loss of the NIRF gene was found at statistically significant levels in diverse tumors. Importantly, a recurrent microdeletion targeting NIRF was observed in non-small cell lung carcinoma. Furthermore, NIRF is immediately adjacent to the single nucleotide polymorphism rs719725, which is reportedly associated with the risk of colorectal cancer. These observations suggest that NIRF occupies a prominent position within the cell cycle network, and is a strong candidate for a tumor suppressor whose aberration contributes to the pathogenesis of diverse malignancies., (© 2011 Landes Bioscience)

The search for novel enzymes and enzymatic activities is important to map out all metabolic activities and reveal cellular metabolic processes in a more exhaustive manner. Here we present biochemical and physiological evidence for the function of the uncharacterized protein YihU in Escherichia coli using metabolite profiling by capillary electrophoresis time-of-flight mass spectrometry. To detect enzymatic activity and simultaneously identify possible substrates and products of the putative enzyme, we profiled a complex mixture of metabolites in the presence or absence of YihU. In this manner, succinic semialdehyde was identified as a substrate for YihU. The purified YihU protein catalyzed in vitro the NADH-dependent reduction of succinic semialdehyde to gamma-hydroxybutyrate. Moreover, a yihU deletion mutant displayed reduced tolerance to the cytotoxic effects of exogenous addition of succinic semialdehyde. Profiling of intracellular metabolites following treatment of E. coli with succinic semialdehyde supports the existence of a YihU-catalyzed reduction of succinic semialdehyde to gamma-hydroxybutyrate in addition to its known oxidation to succinate and through the tricarboxylic acid cycle. These findings suggest that YihU is a novel gamma-hydroxybutyrate dehydrogenase involved in the metabolism of succinic semialdehyde, and other potentially toxic intermediates that may accumulate under stress conditions in E. coli.

NIRF is a RING finger protein with a ubiquitin-like domain, a PHD finger, a YDG/SRA domain, and a RING finger domain. Previous study showed that NIRF is a nuclear protein expressed in association with cell proliferation. In this study, we further characterized NIRF functions in cell cycle regulation. Flow cytometric analysis showed that overexpression of NIRF induced an increase in G1 phase cells. Immunoprecipitation and immunoblotting experiments showed that NIRF bound to the inactive Cdk2-cyclin E complex. There existed phosphorylated NIRF in cells, and dephosphorylated NIRF interacted with Cdk2. NIRF was phosphorylated by Cdk2 in vitro. These results suggest that NIRF may participate in the G1/S transition regulation.

We previously reported the association of a novel Np95/ICBP90-like RING finger protein (NIRF) with a novel PEST-containing nuclear protein (PCNP). NIRF is a nuclear protein with a ubiquitin-like domain, a PHD finger, a YDG/SRA domain, Rb-binding motifs and a RING finger. In this study, we showed that NIRF has auto-ubiquitination activity, the hallmark of a ubiquitin ligase. PCNP was readily ubiquitinated in 293 and COS-7 cells, and NIRF ubiquitinated PCNP in vitro as well as in vivo. Considering that NIRF is implicated in cell cycle regulation, these findings suggest that NIRF and PCNP are a ubiquitin ligase and its substrate, respectively, and may constitute a novel signaling pathway with some relation to cell proliferation.

Background: A major mitochondrial autoantigen recognized by sera of patients with primary biliary cirrhosis (PBC) is dihydrolipoamide acetyltransferase (E2) of the pyruvate dehydrogenase complex (PDH). The alpha subunit of pyruvate decarboxylase (E1alpha) of PDH is also recognized in some E2-reactive PBC sera, suggesting that the occurrence of autoimmunity against Elalpha is subsequent to that against E2. METHODS. To investigate the mechanism inducing autoimmunity against E1alpha, we surveyed immunoreactive sequences of E1alpha by ELISA with synthesized oligopeptides, and determined minimum amino acid residues for each determinant., Results: The major determinants of E1alpha appeared to reside in its N-terminal region, apparently forming 'nested epitopes', and all E1alpha-reactive PBC sera tested recognized these regions. Minor epitopes were also found scattered throughout the entire sequence. The reactivities of these minor epitopes to individual PBC sera were proportional to those of the major epitopes. All the epitopes were located in hydrophilic regions of E1alpha, and many of them were out of the known functional domains (TPP-binding domain, subunit interaction site, and phosphorylation sites) whose structures are phylogenically well conserved. Furthermore, the sequences of many epitopes appeared to be specific to humans., Conclusion: These observations suggest that determinant spreading might underlie the autoimmunity against Elalpha.

We have developed a sensitive and specific polymerase chain reaction (PCR) method for identifying phytoplankton in cases of death by drowning, and we have designed four primer pairs, EG1, EG2, SK1 and SK2, for chlorophyll-related genes of Euglena gracilis and Skeletonema costatum, which are commonly distributed in all types of water. In order to evaluate the usefulness of this method for diagnosis of drowning, we have used this method for detection of plankton genes in non-drowned rabbits submerged after death and in decomposed drowned rabbits. Plankton DNA was identified in lung samples obtained from the non-drowned rabbits because of postmortem plankton penetration into the respiratory system, and plankton DNA was identified in liver and kidney samples obtained from the decomposed drown rabbits. The results show that our new PCR method is a useful method for diagnosing drowning.

We present a new PCR method for identifying plankton in cases of death by drowning. We designed four primer pairs for chlorophyll-related genes of Euglena gracilis (EG) and Skeletonema costatum (SK), which are commonly distributed in water. The primers were selected from sequences coding chloroplast/chlorophyll apoprotein of EG (EG1 and EG2) and fucoxanthin-chlorophyll a/c harvesting protein of SK (SK1 and SK2). With EG1 or EG2, up to 2 fg of EG-DNA was identified, and 0.2 pg of SK-DNA was detectable with SK1 or SK2. No PCR products were amplified from green vegetables (komatsuna, spinach, parsley) or human DNA with the four primer pairs. Regardless of the origin, seawater or fresh water, most diatoms were detectable with primer pairs of EG1 and EG2. With SK1, only Centrales diatoms were identified, and five diatom strains originating from seawater were detectable with SK2. EG1 and EG2 gave rise to PCR products from most water samples. By using Percoll, plankton was easily isolated from human tissue or blood samples and good results of PCR analysis were obtained in cases of death by drowning.

Through database mining, we found a novel PEST-containing nuclear protein (PCNP). To characterize PCNP, we carried out yeast two-hybrid screening for PCNP-interacting factors. A novel Np95/ICBP90-like RING finger protein (NIRF), which possessed a ubiquitin-like domain, a PHD finger, a YDG/SRA domain and a RING finger, was identified. Interaction between PCNP and NIRF was clarified by mammalian two-hybrid system, GST pull-down assay, and nuclear co-localization. RT-PCR showed that NIRF expression is high in proliferating phase but significantly low in G0/G1 phase in normal TIG-7 and WI-38 cells, while consistently high in tumoral HT-1080 and HepG2 cells, suggesting that NIRF is involved in cell-cycle regulation. The NIRF gene resides in 9p23-24.1 that is altered in numerous types of tumors at the top of frequency. Furthermore, the NIRF gene is just within small amplicons in some tumors, suggesting that PCNP and NIRF might be involved in some aspects of tumorigenesis.

Branched-chain alpha-keto acid dehydrogenase complex (BCKADH) contains decarboxylase (E1), dihydrolipoyl transacylase (E2), and dihydrolipoyl dehydrogenase (E3) as catalytic components. BCKADH purified from rainbow trout (Oncorhynchus mykiss) liver was comparable with mammalian BCKADH in various enzymatic characteristics, but less efficient in catalyzing the overall reaction. The trout E2 subunit was larger than the mammalian subunit and rather similar to the chicken one in relative molecular mass on SDS-PAGE, whereas the E1 component was similar between trout and mammalian both in relative molecular mass of its alpha and beta subunits and in the catalytic activity. Trout E2 cDNA cloning and nucleotide sequencing revealed that the mature trout E2 subunit consists of 435 residues, and possesses 14 additional residues compared with mammalian E2. Eleven of these are localized in two interdomain segments as two sequences with two and nine residues, respectively. Trout E2 was inferior to rat E2 in the capacity for binding the E1 component, similar to chicken E2. Thus, it appears that non-mammalian BCKADH E2 is distinct from that in mammals in the structure of interdomain segments, resulting in reduction of overall activity of the enzyme complex.

Branched-chain alpha-keto acid dehydrogenase complex is a macromolecule comprising three catalytic components: a dehydrogenase (E1) with alpha(2)beta(2) structure, an acyltransferase (E2) and a dihydrolipoamide dehydrogenase (E3). In the mammalian complex, the E2 component with 24 identical subunits forms a structural core, to which multiple copies of E1 and E3 bind noncovalently. We isolated cDNA clones encoding E1 alpha, E1 beta and E2 subunits from a chicken-liver cDNA library and performed nucleotide sequencing. Amino-acid sequences deduced from the nucleotide sequences revealed that chicken E1 alpha and E1 beta chains had substantially homologous sequences with the corresponding mammalian polypeptides, except for the N-terminus. Chicken E2 conserved three functional domains, a lipoyl-bearing domain, an E1/E3 binding domain and an inner-core domain, but contrasted strongly with mammalian E2 in respect of containing 11 additional residues in two interdomain linkers: nine sequential residues in one linker and two residues in the other. Replacement of many residues was also observed in the chicken linkers. When E2 activity for catalyzing the overall reaction was measured by activity reconstitution in combination with E1 and E3, chicken E2 was markedly less effective than mammalian E2. The capability of chicken E2 for binding E1 was also reduced when determined by the binding assay using sucrose density gradient centrifugation. Chicken E1 was functionally as well as structurally indistinguishable from mammalian E1. Thus the reduced catalytic activity of chicken E2 must arise from its reduced E1-binding capacity, which results from the characteristic structure of interdomain linkers in chicken E2.

We examined the effect of vascular endothelial growth factor (VEGF) on intracellular signal transduction pathways using isolated bovine microvascular endothelial cells (BREC). When cell growth was determined by [3H]thymidine incorporation, it was significantly stimulated by VEGF stimulation. In situ hybridization results also demonstrated that c-fos expression was enhanced by the stimulation. Although BREC expressed Flt-1 and Flk-1 as VEGF receptors at similar levels, VEGF stimulation preferentially enhanced the activity of Flt-1 tyrosine kinase. This stimulation initiated an increase in the level of GTP-form Ras and the activation of mitogen activated protein kinase (MAPK). On the other hand, BREC expressed the Janus kinase (Jak) family members Jak1, Jak2, and Tyk2, and the signal transducers and activators of transcription (Stat) family members Stat1, Stat3, and Stat6. These molecules were tyrosine phosphorylated under culture conditions used, and the phosphorylation of Tyk2 and Stat6 was specifically enhanced by VEGF stimulation. These results demonstrate that, in addition to Ras/MAPK pathways, the Flt-1/Tyk2/Stat6 pathway is important in VEGF signaling in BREC. These signal transduction systems may regulate the growth of retinal endothelial cells.

Objective: To characterize DNA in sera of patients with systemic lupus erythematosus (SLE) in terms of size, guanine plus cytosine (G+C) content (by percentage), CpG dinucleotide (CpG) (percentage), and effects on mononuclear cells (MNC)., Methods: Nine DNA clones were sequenced. Oligodeoxynucleotides with the characteristic CpG motif (TTCGAA or PuPuCGPyPy) were examined for their proliferative effect on MNC by [3H]thymidine incorporation, expression of HLA-DR and intercellular adhesion molecule (ICAM)-1 on monocytes by flow cytometry, and mRNA levels encoding interleukin 12 (IL-12) and interferon-gamma (IFN-gamma) by semiquantitative reverse transcription polymerase chain reaction., Results: The size of DNA clones ranged from 87 to 318 bp (mean +/- SD, 177+/-68) and enrichment in G+C and CpG ranged from 34.7 to 69.7% (48.1+/-10.7) and 0.63 to 12.8% (4.0+/-4.1), respectively. Three of 9 clones contained the characteristic CpG motif. Oligonucleotides proliferated MNC, and augmented HLA-DR and ICAM-1 expression in company with an increase of mRNA encoding IL-12 and IFN-gamma., Conclusion: Circulating CpG motif-containing DNA fragments in SLE increased mRNA encoding IL-12 and IFN-gamma, which in turn increased HLA-DR and ICAM-1 on monocytes, resulting in MNC proliferation. This mechanism could contribute to the pathogenesis of SLE.

We isolated microvascular endothelial cells from bovine retinas (BREC) by a colony isolation method, and investigated the effects of basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF) on growth as well as integrin subtype expression of the cultured BREC. Either of the growth factors stimulated growth of the cultured BREC. In non-stimulated BREC, alpha v beta 3 integrin expression was predominant over the other integrin subtypes tested (alpha v beta 5, alpha 2 beta 1 and alpha 5 beta 1), but no enhancement of the alpha v beta 3 expression occurred by either growth factor stimulation. On the other hand, bFGF addition stimulated alpha v beta 5 and alpha 5 beta 1 expression while VEGF induced the alpha 2 beta 1 expression as well. These results suggest that external stimuli such as bFGF and VEGF alter integrin expression profiles of BREC and probably influence interactions of endothelial cells with extracellular matrix during angiogenesis which must be involved in pathogenesis of ischemic retinal disorders. In this respect, this BREC culture system could be useful to study the mechanism of neovascularization in these disorders.

Aims/methods: Using purified E1 component of pyruvate dehydrogenase complex (PDC) from bovine heart, we measured the levels of anti-E1 antibodies in PBC sera using ELISA and determined the degree of inhibition that these antibodies exerted on E1 enzyme activity. We also estimated levels of anti-E2/Protein X (Pro-X) antibodies in PBC sera using purified E2 and Pro-X of PDC which were copurified with E1., Results/conclusions: Anti-E1 antibodies were detected in 87.5% (35/40) of PBC sera. Some of these sera inhibited E1 enzyme activity but inhibition did not correlate with levels of anti-E1 antibodies. A high positive correlation (r = 0.918) was found between levels of anti-E1 and anti-E2/Pro-X antibodies, suggesting that anti-PDC antibody production was stimulated by PDC itself. Levels of IgG class anti-E2/Pro-X antibodies were significantly higher in sera of symptomatic PBC patients than in those of asymptomatic PBC patients. It was also found that patients who were positive for only IgM class anti-E2/Pro-X antibodies had early-stage PBC.

The initial-velocity kinetics, optimal pH, acceptor specificity and the influence of metal ions, EDTA and urea were studied on the human seminal gamma-glytamyltransferase (GGT) in comparison with the human renal GGT. The activity was measured with glycylglycine as an acceptor and with gamma-glutamyl-4-nitroanilide or gamma-glutamyl-3-carboxy-4-nitroanilide as a donor. Because the double-reciprocal plots showed paralled lines, the reaction of seminal GGT proceeds in nonsequence (Ping Pong Bi Bi) mechanism. The acceptor Michaelis constants for the seminal GGT were about 2 times higher than those for the renal enzyme with gamma-glutamyl-3-carboxy-4-nitroanilide as well as gamma-glutamyl-4-nitroanilide as donors, which the donor michaelis constants for seminal GGT were very similar to those for renal enzyme. The optimal pH and pK values were 8.2-8.6 and about 7.7, respectively. There was little difference in the specificity for various acceptors between the seminal and renal enzyme. Glycylglycylglycine was an effective acceptor other than glycylglycine, showing 80% of the activity with glycylgycine. Various substrates including metal ions tested were practically neither inhibitory nor stimulatory for seminal and renal GGTs.

Binding characteristics among three catalytic components of rat liver branched-chain 2-oxo acid dehydrogenase complex (BCKADH) were investigated by ELISA. Dihydrolipoamide dehydrogenase (E3) was bound to solid-phase dihydrolipoamide acyltransferase (E2). The binding curve was hyperbolic giving a calculated half-maximal binding concentration of 167 ng/ml for E3. Specificity of the binding of E3 to E2 was certified by a competition experiment measuring binding of biotin-labeled E3 in the presence of unlabeled E3. The decarboxylase component (E1), which is the other catalytic component of the complex, prevented the E3 binding to E2. The specific binding between E2 and E3 was verified in the opposite direction with immobilized E3. E2 also bound to solid-phase E1 in a specific manner, and addition of E3 prevented the E2 binding to E1. No binding between E1 and E3 was observed. Thus, E1 and E3 prevented each other's binding to E2, suggesting that E1 and E3 may recognize overlapped binding sites on the E2 polypeptide or that they may, at least in part, sterically interact with each other on their binding to E2. The reconstitution experiment of the complex also supported such a mutually exclusive binding of E1 and E3 to the E2 core.

The N-terminal sequences of the E1 alpha, E1 beta and E2 subunits of the human branched-chain alpha-keto acid dehydrogenase complex have been determined by microsequencing. The N-terminal of human E1 beta and E2 subunits (Val and Gly, respectively) are identical to those of the corresponding rat and bovine subunits. However, the N-terminus of the human E1 alpha subunit (Ser) is identical to bovine, but differs from the rat E1 alpha (Phe) subunit. Comparison of the N-terminal sequences of human and rat E1 alpha subunits shows that the serine residue at the +1 position in the human sequence is replaced by a proline residue in the rat sequence. The presence of the proline residue apparently causes a 5'-shift by one residue in the cleavage site by the mitochondrial processing peptidase in the rat sequence, when compared to the human sequence. The results provide evidence that the mitochondrial processing peptidase cannot cleave an X-Pro bond, similar to trypsin, chymotrypsin and microsomal signal peptidases.

Peptidylglycine alpha-amidating activity catalyses the oxidation of a C-terminally glycine-extended peptide to a desglycine alpha-amidated peptide at the expense of ascorbate and O2 in the presence of Cu2+. The reaction involves oxidative N-dealkylation within the terminal glycine residue, with retention of the glycine N atom and release of the remainder as glyoxylate. Recent studies by us and others have revealed that the reaction consists of two steps via a carbinolamide as an intermediate (peptidyl alpha-hydroxyglycine), and also that two separate enzymes derived from a common precursor protein catalyse these steps, formation of the carbinolamide and its conversion into alpha-amide and glyoxylate. As for the mechanism of carbinolamide formation, two distinct pathways can be considered: direct mono-oxygenation at the glycine alpha-C atom and dehydrogenation leading to an imine followed by hydration. To draw a distinction between them, we carried out the reaction with D-Tyr-Val-Gly as the substrate either in the H2(18)O-enriched medium or under an atmosphere of 18O2, and isolated the alpha-hydroxylglycine intermediate. The fast-atom-bombardment mass-spectral analysis demonstrated that the hydroxy O atom comes from O2, but not from H2O, indicating that the alpha-hydroxylation should be a monooxygenase reaction.

Clofibrate administration to rats caused both the activation and induction of the branched-chain 2-oxo acid dehydrogenase complex in the liver; the former phenomenon occurred within the first 6 h after clofibrate administration whereas the latter occurred after 12 h. Essentially the same results were obtained with primary cultures of rat hepatocytes in the presence of 0.5 mM clofibrate, though about three-fourths of the enzyme complex in control cells (without clofibrate addition) was inactivated during a culture for 44 h, with little reduction of the enzyme amount. This was also confirmed by immunotitration analysis with antibodies raised against the purified decarboxylase and transacylase components of the enzyme complex. On the other hand, the activity of dihydrolipoamide dehydrogenase (a constituent of the complex) was little affected by clofibrate administration. The half lives of the decarboxylase and transacylase components in the primary cultures were estimated to be in the range of 22-26 h, and were unchanged in the presence of clofibrate, when determined with the use of cycloheximide and by a pulse-chase experiment. On the contrary, the rates of synthesis of these two enzyme components had increased to about 1.9-fold after 32 h cultivation in the presence of clofibrate. Thus, the increase in the synthesis of both the components resulted in induction of the complex.

A new form of gamma-glutamyltransferase was purified from human seminal plasma. The purified enzyme was composed of two non-identical subunits with apparent molecular masses of 150 and 95 kDa on polyacrylamide gel electrophoresis (PAGE) in the presence of sodium dodecyl sulfate (SDS), and showed a molecular mass of 500 and 250 kDa on gel filtration in the absence and presence of 1% Triton X-100, respectively. This enzyme was different from human renal gamma-glutamyltransferase not only in apparent molecular masses, but also in amino acid compositions of both the subunits to each other. Experiments with the antisera raised against the purified enzyme revealed that the enzyme was different from the renal, hepatic and testicular enzymes in reactivity to the antibody though partially related to those enzymes. Ouchterlony double diffusion analysis indicated that both human seminal plasma and prostatic extract contained two types of gamma-glutamyltransferase, one is that we purified and the other the renal type. Hence, it is most likely that gamma-glutamyltransferase accounting for most of the enzyme activity in semen results from prostata followed by secretion to seminal plasma.

Administration of glucagon, epinephrine, or dibutyryl cAMP to chicks induced cytosol-specific phosphoenolpyruvate carboxykinase in liver. In vitro translation assay with poly(A)+RNA indicated that this induction was due to the increase in phosphoenolpyruvate carboxykinase-coding mRNA synthesis which resulted from an increased level of hepatic cAMP. Either hydrocortisone or alpha-adrenergic agonist was ineffective for the induction by itself, but showed a significant effect when administered together with one of the inducing agents given above. In particular, hydrocortisone enhanced the synthesis of phosphoenolpyruvate carboxykinase-specific mRNA without changing the profile of the time courses of the induction and of hepatic cAMP level. Those observations suggest that the fundamental machinery required for induction of cytosol-specific phosphoenolpyruvate carboxykinase in liver is shared in common between rat and chick, and that the absence of appreciable induction of cytosol-specific hepatic phosphoenolpyruvate carboxykinase in starved chicks is due to neither lack nor impairment of the hormone-mediated induction mechanism, but is due to the difference in usage of the genetic information between the two animal species.

The activity of branched-chain 2-oxo acid dehydrogenase complex increased 3.0-fold in liver of rats fed on 0.1%(w/w) clofibrate. Immunotitration experiments with antibodies against the constituent enzymes of the complex revealed that this increase resulted mainly from the increased amounts of only two(a decarboxylase and a lipoate acyltransferase) of three components of the complex and that the other component(dihydrolipoamide dehydrogenase) remained unchanged in its content, irrespective of clofibrate administration. The increases of both enzyme components were associated with increases in their mRNA levels which were estimated by in vitro translation with poly(A)+ RNA.

Branched-chain alpha-keto acid dehydrogenase (BCKADH) was solubilized as an enzyme complex from rat liver mitochondria by sonic treatment. Dehydrogenase (E1) and dihydrolipoyltransacylase (E2) components of the complex were purified in an associated form and resolved into individual components in the presence of 1 M NaCl, while lipoamide dehydrogenase (E3) component was dissociated from the complex during purification. Analysis by gel electrophoresis in dodecyl sulfate revealed the E1 comprised two different subunits with apparent molecular weights of 36,000 and 45,500, presumably in an equal molar ratio, while E2 consisted of a single subunit with an apparent molecular weight of 51,000. The BCKADH complex was reconstituted by combining E1, E2, and E3, and the formation of the complex was confirmed by analysis by sucrose density gradient centrifugation. The reconstituted enzyme complex oxidized not only alpha-ketoisovalerate (KIV), alpha-ketoisocaproate (KIC), and alpha-keto-beta-methylvalerate (KMV), but also pyruvate and alpha-ketoglutarate. Apparent Km values were 10-12 microM for the branched-chain alpha-keto acids, 2.2 mM for pyruvate, and 2.5 mM for alpha-ketoglutarate.

Glycine decarboxylase, tentatively called P-protein as a constituent of the glycine cleavage system, was purified to near homogeneity from rat liver mitochondria. The purified P-protein was a homodimer with a molecular weight of about 210,000, consisting of identical subunits with a molecular weight of 105,000. In the exchange reaction of the carboxyl carbon of glycine wih CO2 catalyzed by the purified P-protein in the presence of H-protein, the pH optimum was 6.7, Km for glycine was 6.6 mM, and Km for H-protein was 7.4 microM. A specific rabbit antibody against the purified rat liver P-protein was prepared. Ouchterlony double diffusion analysis and immunoinhibition experiments using this antibody revealed immunological cross-reactivity among the P-proteins from various species of animals such as carp, frog, snake, chicken, bovine, and human, suggesting a quite conservative evolution of the glycine cleavage system.

The synthesis and in vitro activity of the 7-[O-substituted oxyiminoacetamido]cephalosporins (I) without substitution at 3-position of a cephem nucleus are described. Effect of changing the oxime O-substituents (R1) with various functional groups in the 7-acyl residue on antibacterial activity was examined. Against Gram-positive bacteria, cephems with hydrophilic functions in the R1 moiety such as hydroxyethyl, aminoethyl and carboxymethyl groups showed decrease of the activity, while cephems with lipophilic functions such as cyanomethyl, methylthiomethyl and halogenoethyl groups exhibited increase of the activity. However, influence of the substituents (R1) on activity against Gram-negative bacteria was observed to be relatively independent of the nature of their functional groups.

Chicken liver mitochondria contained two forms of phosphoenolpyruvate (PEP) carboxykinase (designated as Mt-I and Mt-II) and the chicken liver cytosol fraction contained three forms of PEP carboxykinase (designated as Sol-I, Sol-II, and Sol-III). Mt-I and Mt-II were purified to homogeneity. They both had a molecular weight of 72,000 as judged by the sodium dodecyl-sulfate-polyacrylamide gel electrophoresis method, whereas an apparent molecular weight of 42,000 was obtained for both enzymes when estimated by Sephadex G-100 gel filtration. Studies with a rabbit antibody against the purified Mt-I revealed that Mt-I, Mt-II, Sol-I, and Sol-II are immunochemically identical, whereas Sol-III is immunochemically different from any of the other proteins. Genetic analogy between Mt-I and Mt-II was also suggested by gel electrophoretic analysis of their peptide maps. The content of Sol-III in the adult chicken liver was very small, whereas the livers of chick embryo and very young chick contained a considerable amount of Sol-III. The level of Sol-III in the adult chicken, however, could be significantly increased by the administration of hydrocortisone or isoproterenol. Apparently Sol-III is a cytosol-specific PEP carboxykinase which is similar to the cytosolic PEP carboxykinases of various mammals. Sol-III showed a molecular weight of about 60,000 as estimated by gel filtration. Studies with combined use of the antibody and [3H]leucine revealed that some portion of PEP carboxykinase (Sol-I plus Sol-II) appearing in the liver cytosol corresponds to a precursor in transit to the mitochondria, although major portions of Sol-I and Sol-II appear to be accounted for by release of the mitochondrial enzymes.