Benzoate modulates renal and extrarenal nitrogen flow: metabolic mechanisms.

The mechanism by which benzoate enhances total nitrogen excretion was investigated in-situ and in separated rat renal proximal tubules. Orally administered benzoate augmented NH4+, urea and hippurate excretion 2, 1.9 and 76 fold respectively, as compared to baseline for control. Hippurate had similar effects. Benzoate augmented renal blood flow, glutamine extraction and total NH4+ production. Arterio-venous concentration differences of glutamine, glutamate, and NH4+ across the kidney, liver and gut demonstrated an increase in glutamine uptake by the kidney despite reduced release and uptake by the liver and gut, respectively; glutamate release by the kidney and gut was increased; NH4+ handling was unchanged at these three organs. Studies in separated rat renal proximal tubules demonstrated that benzoate stimulated glutamine dependent ammonia-genesis by activation of gamma-glutamyltransferase, via the synthesis of hippurate. The results demonstrate that benzoate can modulate the interorgan partitioning of nitrogen metabolites across several organs, the net effect of which is physiologically expressed as enhanced NH4+ , urea and hippurate excretion.     

Mass spectrometric quantification of endogenous beta-endorphin

Fast atom bombardment (FAB) mass spectrometry and multiple reaction monitoring (MRM) in the B/E linked-field scan mode were used to quantify endogenous beta-endorphin (BE) in individual human pituitary extracts. The experimental protocol includes the addition of a stable isotope-labeled internal standard ((2H4-Ile22)BE1-31, human) to the tissue homogenate before extraction, purification of the native BE by a combination of Sep-Pak chromatography and gradient high-performance liquid chromatography (HPLC), trypsin digestion to cleave BE into smaller peptides, and separation of the tryptic fragment BE20-24 (NAIIK) by isocratic reversed-phase HPLC. Mass spectrometric quantification is based upon recording either (a) the [M + H]+ ions of NAIIK and its deuterated analog ((2H4)NAIIK), or (b) the transitions ([NAIIK + H](+)----[NAI]+) and [((2H4)NAIIK + H](+)----[(2H4)NAI]+) using the B/E linked-field scan. Linear calibration curves were obtained using these two mass spectrometric techniques from standard solutions containing 1.25-20 micrograms of BE; each standard solution also contained 10 micrograms of (2H4)BE. The amounts (means +/- s.d.) of endogenous BE in five separate human pituitaries were found to be 156 +/- 84 [( M + H]+ method) and 169 +/- 99 pmol mg-1 protein (MRM method).     

Protein Paucimannosylation Is an Enriched N‐Glycosylation Signature of Human Cancers

While aberrant protein glycosylation is a recognized characteristic of human cancers, advances in glycoanalytics continue to discover new associations between glycoproteins and tumorigenesis. This glycomics‐centric study investigates a possible link between protein paucimannosylation, an under‐studied class of human N‐glycosylation [Man_1‐3GlcNAc₂Fuc_0‐1], and cancer. The paucimannosidic glycans (PMGs) of 34 cancer cell lines and 133 tissue samples spanning 11 cancer types and matching non‐cancerous specimens are profiled from 467 published and unpublished PGC‐LC‐MS/MS N‐glycome datasets collected over a decade. PMGs, particularly Man_2‐3GlcNAc₂Fuc₁, are prominent features of 29 cancer cell lines, but the PMG level varies dramatically across and within the cancer types (1.0–50.2%). Analyses of paired (tumor/non‐tumor) and stage‐stratified tissues demonstrate that PMGs are significantly enriched in tumor tissues from several cancer types including liver cancer (p = 0.0033) and colorectal cancer (p = 0.0017) and is elevated as a result of prostate cancer and chronic lymphocytic leukaemia progression (p < 0.05). Surface expression of paucimannosidic epitopes is demonstrated on human glioblastoma cells using immunofluorescence while biosynthetic involvement of N‐acetyl‐β‐hexosaminidase is indicated by quantitative proteomics. This intriguing association between protein paucimannosylation and human cancers warrants further exploration to detail the biosynthesis, cellular location(s), protein carriers, and functions of paucimannosylation in tumorigenesis and metastasis.     

Reactive extraction of cephalosporin antibiotics in hollow fiber membrane

Non-dispersive reactive extraction of cephalosporin antibiotics has been studied using hollow fiber membrane modules. Extraction as well as stripping has been studied using a pH swing procedure. Cephalosporin was extracted from an aqueous solution of cephalosporin having a pH above the pK_a2 value to an organic phase containing Aliquat-336 as the extractant and n-heptane as the diluent. The solute was stripped from the loaded organic phase to another aqueous phase of pH maintained well below the pK_a2 value of the cephalosporin. The extraction cum stripping relies on pH dependance of the distribution coefficient of cephalosporin in aqueous phase. Reasonably high solute recovery and mass transfer rate have been achieved in the hollow fiber module. Mass transfer performance of a single module has been evaluated and experimentally observed low value of height of transfer unit (HTU) indicates good prospect of hollow fiber membrane for the extraction duty.     

Synchronisation of oestrus of goats treated with progestagen-impregnated intravaginal sponges and PMSG,and reproductive performance following natural mating or A.I. with frozen semen

Oestrus was synchronised in 88 goats using progestagen-impregnated intravaginal sponges (60 mg Repromap; Upjohn) left in place for 17 days, followed by intramuscular injection of PMSG (300 i.u.) at sponge withdrawal. The percentages of does that came into oestrus in 0–24, 25–48, 49–72 and 73–96 h periods were 42, 30.5, 10.5 and 6.5 (cumulative 89.5%). The time of onset of oestrus ranged from 16 to 40 h. Breeding at the synchronised oestrus using intact bucks (24–96 h) or artificial insemination with frozen-thawed semen, at fixed times of 24, 48, 72 and 96 h, resulted in 71% and 81% conceptions respectively. Intrauterine deposition of semen was possible in 31.3%, 62.5%, 87.5% and 6.3% of cases at 24, 48, 72 and 96 h respectively. Twinning percentages were 54.5% and 56% for natural mating and artificial insemination. Kidding was spread over a 14-day period with mean gestation lengths of 149 days for singletons and 147 days for twins. The results indicate that use of progestagen-impregnated sponges with PMSG is an efficient method of synchronisation of oestrus and may result in increased fertility in goats and, if used with A.I. with frozen semen, may contribute to increased breeding efficiency and rate of genetic improvement of goats in Asia.     

The function and structural influence of selective relaxed constraint at functional intracellular loop3 of 5-HT(1A) serotonin-1 receptor family

Superoxide dismutases (SODs) are metalloenzymes that represent one important line of defense against reactive oxygen species (ROS). In this paper, two novel SOD genes, MdSOD1 and MdSOD2, which putatively encode 261 and 214 amino acid residues respectively were identified and characterized from the housefly Musca domestica. The high similarity of MdSOD1 and MdSOD2 with SODs from other organisms indicated that they should be two new members of the SOD family. qPCR exhibited a universal expression of MdSOD1 and MdSOD2 detected in various tissues of housefly larva, including the fat body, gut, hemocyte and epidermis. Expression profiling reveals that MdSOD1 and MdSOD2 can be induced significantly via not only heat shock and cadmium (Cd) stress but also Escherichia coli and Staphylococcus aureus challenge. The two genes were cloned into the prokaryotic expression vector pET-28a to obtain the fusion proteins rMdSOD1 and rMdSOD2. Between them, the activity of rMdSOD2 was found by visual assay methods. ESI-LC-MS/MS analysis showed that three peptide fragments of the protein rMdSOD2 were identical to the corresponding sequence of M. domestica MdSOD2. MdSOD1 and MdSOD2 in housefly larvae were abrogated by feeding bacteria expressing dsRNA. High mortalities were observed in the larvae treated with dsRNA of SODs at heat shock, Cd stress and bacterial invasion. This phenomenon indicated that MdSOD1 and MdSOD2 are related to the survival of M. domestica under stress. This may provide new insights into the role of the two SOD genes in protecting M. domestica against both stress and bacterial invasion.     

Codon usage of human hepatitis C virus clearance genes in relation to its expression

Hepatitis C virus (HCV) infection is among the leading causes of hepatocellular carcinoma and liver cirrhosis globally, with a high economic burden. The disease progression is well established, but less is known about the spontaneous HCV infection clearance. This study tries to establish the relationship between codon biasness and expression of HCV clearance candidate genes in normal and HCV infected liver tissues. A total of 112 coding sequences comprising 151 679 codons were subjected to the computation of codon indices, namely relative synonymous codon usage, an effective number of codon (Nc), frequency of optimal codon, codon adaptation index, codon bias index, and base compositions. Codon indices report of GC3s, GC12, hydropathicity, and aromaticity implicates both mutational and translational selection in the candidate gene set. This was further correlated with the differentially expressed genes among the selected genes using BioGPS. A significant correlation is observed between the gene expression of normal liver and cancerous liver tissues with codon bias (Nc). Gene expression is also correlated with relative codon bias values, indicating that CCL5, APOA2, CD28, IFITM1, and TNFSF4 genes have higher expression. These results are quite encouraging in selecting the high responsive genes in HCV clearance. However, there could be additional genes which could also orchestrate the clearance role with the above mentioned first line of defensive genes.     

Synthesis and chemical characterization of 2-methoxy-N(10)-substituted acridones needed to reverse vinblastine resistance in multidrug resistant (MDR) cancer cells

In an attempt to find clinically useful modulators of multidrug resistance (MDR), a series of 19 N(10)-substituted-2-methoxyacridone analogues has been synthesized. 2-Methoxyacridone and its derivatives (1-19) were synthesized. Compound 1 was prepared by the Ullmann condensation of o-chlorobenzoic acid and p-anisidine followed by cyclization using polyphosphoric acid. This compound undergoes N-alkylation in the presence of phase transfer catalyst (PTC). Stirring of 2-methoxy acridone with 1-bromo-3-chloropropane or 1-bromo-4-chlorobutane in a two-phase system consisting of organic phase (tetrahydrofuran) and 6N potassium hydroxide in the presence of tetrabutylammonium bromide leads to the formation of compounds 2 and 11 in good yield. N-(omega-Chloroalkyl) analogues were found to undergo iodide catalyzed nucleophilic substitution reaction with various secondary amines. Products were characterized by UV, IR, 1H and 13C NMR, mass-spectral data and elemental analysis. The lipophilicity expressed in log(10) P and pK(a) of compounds have been determined. All compounds were examined for their ability to increase the uptake of vinblastine (VLB) in MDR KBCh(R)-8-5 cells and the results showed that the compounds 7, 10, 12, and 15-19 at 100 microM caused a 1.05- to 1.7-fold greater accumulation of vinblastine than did a similar concentration of the standard modulator, verapamil (VRP). However, the effects on VLB uptake were specific because these derivatives had little effect in the parental drug sensitive line KB-3-1. Steady state accumulation of VLB, a substrate for P-glycoprotein (P-gp) mediated efflux, was studied in the MDR cell line KBCh(R)-8-5 in the presence and absence of novel MDR modulators. Results of the efflux experiment showed that VRP and each of the modulators (1-19) significantly inhibited the efflux of VLB, suggesting that they may be competitors for P-gp. From among the compounds examined, 14 except 1, 2, 4, 8, and 11, exhibited greater efflux inhibiting activity than VRP. All the 19 compounds effectively compete with [(3)H] azidopine for binding to P-gp, pointed out this transport membrane protein as their likely site of action. Cytotoxicity has been determined and the IC(50) values lie in the range 8.00-18.50 microM for propyl and 4-15 microM for butyl derivatives against KBCh(R)-8-5 cells suggesting that the antiproliferative activity increases as chain length increases from 3 to 4 carbons at N(10)-position. Compounds at IC(10) were evaluated for their efficacy to modulate the cytotoxicity of VLB in KBCh(R)-8-5 cells and found that the modulators enhanced the cytotoxicity of VLB by 5- to 35-fold. Modulators 12, 14-16, and 19 like VRP, were able to completely reverse the 24-fold resistance of KBCh(R)-8-5 cells to VLB. Examination of the relationship between lipophilicity and antagonism of MDR showed a reasonable correlation suggesting that hydrophobicity is one of the determinants of potency for anti-MDR activity of 2-methoxyacridones.     

The gene for a variant form of the polyadenylation protein CstF-64 is on chromosome 19 and is expressed in pachytene spermatocytes in mice

Many mRNAs in male germ cells lack the canonical AAUAAA but are normally polyadenylated (Wallace, A. M., Dass, B., Ravnik, S. E., Tonk, V., Jenkins, N. A., Gilbert, D. J., Copeland, N. G., and MacDonald, C. C. (1999) Proc. Natl. Acad Sci. U. S. A. 96, 6763-6768). Previously, we demonstrated the presence of two distinct forms of the M(r) 64,000 protein of the cleavage stimulation factor (CstF-64) in mouse male germ cells and in brain, a somatic M(r) 64,000 form and a variant M(r) 70,000 form. The variant form was specific to meiotic and postmeiotic germ cells. We localized the gene for the somatic CstF-64 to the X chromosome, which would be inactivated during male meiosis. This suggested that the variant CstF-64 was an autosomal homolog activated during that time. We have named the variant form "tau CstF-64," and we describe here the cloning and characterization of the mouse tauCstF-64 cDNA, which maps to chromosome 19. The mouse tauCstF-64 protein fits the criteria of the variant CstF-64, including antibody reactivity, size, germ cell expression, and a common proteolytic digest pattern with tauCstF-64 from testis. Features of mtauCstF-64 that might allow it to promote the germ cell pattern of polyadenylation include a Pro --> Ser substitution in the RNA-binding domain and significant changes in the region that interacts with CstF-77.     

Fructose, 1,6-diphosphate mitigates CCl4 suppressed nitric oxide synthase activity in rats

Effect of fructose 1,6-diphosphate (FDP) and carbon tetrachloride (CCl4) were studied individually and in combination on rat endothelial (ET) and smooth muscle cell (SMC) nitric oxide synthase (NOS) activities in vivo, inhibition of ET and SMC NOS activity in CCl4 treated rats was reversed in FDP + CCl4 treated animals. Cellular based NOS activity was significantly increased in FDP treated group of rats when compared to non treated controls. The results suggest a significant increase in NOS in rats treated with a combination of FDP + CCl4 thus overcoming the suppression of NOS exposed to CCl4 alone.