A novel pentasaccharide from immunostimulant oligosaccharide fraction of buffalo milk.

A processed oligosaccharide mixture of buffalo milk induced significant stimulation of antibody, delayed-type hypersensitivity response to sheep red blood cells in BALB/c mice. This also stimulated non-specific immune response of the animals measured in terms of macrophage migration index. A novel pentasaccharide has been isolated from the oligosaccharide containing fraction having immunostimulant activity of buffalo milk. This compound was isolated by a combination of gel filtration chromatography, silica gel column chromatography of derivatised oligosaccharides while the homogeneity was confirmed by high performance liquid chromatography. The results of structural analyses, i.e. proton nuclear magnetic resonance, fast atom bombardment mass spectrometry, chemical transformations and degradations are consistent with the following structure: GlcNAcbeta(1-->3)Galbeta(1-->4)GlcNAcbeta(1-->3)Gal beta(1-->4)Glc     

Presence of O-linked oligosaccharide on a threonine residue in the human transferrin receptor.

We have previously demonstrated that the human transferrin receptor (TfR) of approximately 90 kDa contains Ser/Thr-linked (O-linked) oligosaccharides. In the present study, we report our identification of the site of attachment of the O-linked oligosaccharides in the receptor. A 70 kDa fragment from the external domain of the TfR was generated by trypsin treatment of the [3H]glucosamine-labelled receptor purified from human K562 cells. The beta-elimination of the intact TfR, but not the 70 kDa fragment, released Gal-[3H]Gal-NAcitol, indicating that the 70 kDa fragment lacks O-linked oligosaccharides. In the remaining 20 kDa fragment there are three potential sites (Thr96, Thr104 and Ser106) for O-glycosylation in the extracellular domain. To identify which of these residues are O-glycosylated, both the [3H]Thr- and [3H]Ser-labelled TfR were directly treated with mild base to effect beta-elimination, and the radiolabelled amino acids and their derivatives were analysed. Approximately 2% of the total radiolabelled Thr, but no radiolabelled Ser, was converted to expected beta-elimination products by this treatment. These and other results demonstrate that only one O-linked oligosaccharide is present in the TfR and that it occurs on either Thr96 or Thr104. From human serum we purified the cleaved, soluble form of the TfR (s-TfR), which contains Thr104, but lacks Thr96. The s-TfR was sensitive to O-glycanase and bound to Jacalin lectin, indicating that the s-TfR contains an O-linked oligosaccharide.(ABSTRACT TRUNCATED AT 250 WORDS)     

Low frequency fatigue in human quadriceps is fatigue dependent and not task dependent.

It is well accepted that a low intensity/long duration isometric contraction induces more low frequency fatigue (LFF) compared to a high-intensity/short-duration contraction. However, previous reports examined the intensity/duration of the contraction but did not control the level of fatigue when concluding fatigue is task dependent. The purpose of this study was to determine whether a long duration/low intensity fatiguing contraction would induce greater LFF than a short duration/high-intensity contraction when the quadriceps muscle was fatigued to similar levels. Eighteen healthy male subjects performed quadriceps contractions sustained at 35% and 65% of maximal voluntary contraction (MVC) on separate days, until the tasks induced a similar amount of fatigue (force generating capacity=45% MVC). Double pulse torque to single pulse torque ratio (D/S ratio) was obtained before, immediately and 5min after fatigue along with the electromyographic (EMG) signal from vastus medialis (VM) and rectus femoris (RF). The D/S ratio significantly (p<0.05) increased by 8.7+/-8.5% (mean+/-SD) and 10.2+/-9.2% after 35% and 65% tasks, respectively, and remained elevated 5min into recovery; however, there was no significant difference in ratio between the two sessions immediately or 5min post-fatigue (p>0.05) even though the endurance time for the 35% fatigue task (124+/-39.68s) was significantly longer (p=0.05) than that of the 65% task (63+/-17.73s). EMG amplitude and median power frequency (MPF) analysis also did not reveal any significant differences between these two sessions after fatigue. These findings indicate that LFF fatigue is fatigue dependent as well as task intensity/duration dependent. These findings assist us in understanding task dependency and muscle fatigue.     

III6NeuAcLc4Cer in human SW1116 colorectal carcinoma cells: a possible oncofetal antigen that is not dependent on Lewis gene expression

Monospecific rabbit antibodies directed against the human milk sialyloligosaccharides III6NeuAcLcOse4 (sialyltetrasaccharide b), IV3NeuAcLcOse4 (sialyltetrasaccharide a), and IV6NeuAcnLc4Ose (sialyltetrasaccharide c) were used to detect their homologous haptens as gangliosides or ganglioside-derived sialyloligosaccharides from the human colorectal carcinoma cell line SW1116. III6NeuAcLc4Cer was first detected in human meconium [P. A. Prieto and D. F. Smith (1985) Arch. Biochem. Biophys. 241, 281-289], and its presence in a total ganglioside fraction of SW1116 cells together with its absence from a total lipid extract of normal human intestinal mucosa are consistent with III6NeuAcLc4Cer being a tumor-associated oncofetal antigen. IV3NeuAcLc4Cer, a ganglioside in human meconium [P. A. Prieto and D. F. Smith (1986) Arch. Biochem. Biophys. 249, 243-253], was also detected in SW1116 cells; an observation that is consistent with its being the immediate precursor to the sialyl-Lea ganglioside in SW1116 cells. Specific antisera against sialylated type 1 oligosaccharide chains whose expression is independent of the Lewis gene fucosyltransferase may be useful diagnostic reagents for oncofetal, carbohydrate antigens.     

Activation of the human immunodeficiency virus type 1 enhancer is not dependent on NFAT-1.

The function of a putative NFAT-1 site in the human immunodeficiency virus type 1 enhancer has been analyzed. Activation by the T-cell antigen receptor is minimal in Jurkat cells and is mediated by the kappa B sites. The putative NFAT-1 region is not required for the response to anti-CD3 or to mitogens in T-cell, B-cell, or monocyte/macrophage leukemia lines, nor is it a cis-acting negative regulatory element.     

Telomere shortening in human fibroblasts is not dependent on the size of the telomeric-3'-overhang

Telomeres shorten in human somatic cells with each round of DNA replication, and this shortening is thought to ultimately trigger replicative senescence. Telomere shortening is caused partly by the inability of semiconservative DNA replication to copy a linear strand of DNA to its very end. Post-replicative processing of telomeric ends, producing single-stranded G-rich 3' overhangs, has also been suggested to contribute to telomere shortening. This suggestion implies that a positive correlation should exist between the length of 3' overhangs and the rate of telomere shortening. We confirmed shortening of overhangs as human lung (MRC5) and foreskin (BJ) fibroblasts approach senescence by measuring overhang length using in-gel hybridization. However, a large study of fibroblast strains from 21 donors maintained under conditions which lead to two orders of magnitude of variation in telomere shortening rate failed to show any correlation between telomere overhang length and shortening rate, suggesting that overhang length is neither a cause nor a correlate of telomere shortening.     

A novel cell entry pathway for a DAF-using human enterovirus is dependent on lipid rafts

The glycosylphosphatidylinositol (GPI)-anchored complement regulatory protein decay-accelerating factor (DAF) is used by a number of enteroviruses as a receptor during infection. DAF and other GPI-anchored proteins can be found in cholesterol-rich ordered domains within the plasma membrane that are known as "lipid rafts." We have shown, by using drugs to specifically inhibit various endocytosis routes, that infection by a DAF-using strain of echovirus 11 (EV11) is dependent upon cholesterol and an intact cytoskeleton, whereas a non-DAF-using mutant derived from it was unaffected by these drugs. Using RNA transfection and virus-binding assays, we have shown that this requirement for cholesterol, the actin cytoskeleton, and the microtubule network occurs postbinding of the virus but prior to uncoating of the RNA, indicating a role during virus entry. Confocal microscopy of virus infection supported the role of cholesterol and the cytoskeleton during entry. In addition, [(35)S]methionine-labeled DAF-using EV11, but not the non-DAF-using EV11, could be copurified with lipid raft components during infection after Triton X-100 extraction. These data indicate that DAF usage by EV11 enables the virus to associate with lipid rafts and enter cells through this novel route.     

Hyper-mutation caused by the reml mutation in yeast is not dependent on error-prone or excision repair

The reml mutations of Saccharomyces cerevisiae confer a semi-dominant hyper-recombination/hyper-mutation phenotype. Neither reml mutant allele has any apparent meiotic affect. We have examined spontaneous mutation in reml-2 strains and demonstrate that the reml-2 mutation, like reml-1, confers an average 10-fold increase in reversion and forward mutation rates. Unlike certain yeast rad mutations with phenotypes similar to reml, strains containing reml are resistant to MMS and only slightly UV sensitive at very high doses. To understand the mutator phenotype of reml, we have used a double-mutant approach, combining the reml mutation with radiation-sensitive mutations affecting DNA repair. Double mutants of reml-2 and a mutation in the yeast error-prone repair group (rad6-1) or a mutation in excision repair (rad1-2 or rad4) maintain the hyper-mutation phenotype. Since mutation rates remain elevated in these double-mutant strains, it appears as if the mutations which occur in the presence of reml resemble spontaneous mutation since they do not require the action of a repair system.     

The effect of 3-bromopyruvate on human colorectal cancer cells is dependent on glucose concentration but not hexokinase II expression

Cancer cells heavily rely on the glycolytic pathway regardless of oxygen tension. Hexokinase II (HKII) catalyses the first irreversible step of glycolysis and is often overexpressed in cancer cells. 3-Bromopyruvate (3BP) has been shown to primarily target HKII, and is a promising anti-cancer compound capable of altering critical metabolic pathways in cancer cells. Abnormal vasculature within tumours leads to heterogeneous microenvironments, including glucose availability, which may affect drug sensitivity. The aim of the present study was to elucidate the mechanisms by which 3BP acts on colorectal cancer (CRC) cells with focus on the HKII/Akt signalling axis. High HKII-expressing cell lines were more sensitive to 3BP than low HKII-expressing cells. 3BP-induced rapid Akt phosphorylation at site Thr-308 and cell death via both apoptotic and necrotic mechanisms. Cells grown under lower glucose concentrations showed greater resistance towards 3BP. Cells with HKII knockdown showed no changes in 3BP sensitivity, suggesting the effects of 3BP are independent of HKII expression. These results emphasize the importance of the tumour microenvironment and glucose availability when considering therapeutic approaches involving metabolic modulation.     

The structural stability of an expression plasmid bearing a heterologous cloned gene depends on whether this gene is expressed or not

Summary During subculturing of recombinant Escherichia coli under conditions blocking cloned gene expression the plasmid was structurally stable for 200 generations. However, under conditions permitting foreign gene expression only 10% of plasmid containing cells had this gene functional after 100 generations.     

A novel sialylated N-acetylgalactosamine-containing oligosaccharide is the major complex-type structure present in Bowes melanoma tissue plasminogen activator.

We have employed fast atom bombardment mass spectrometry (FAB-MS) to screen the N-linked oligosaccharides of Bowes melanoma tissue plasminogen activator (mt-PA), and recombinant t-PAs produced by Chinese hamster ovary cells (rt-PA) and by a gene-enriched melanoma cell line (rmt-PA). These studies have confirmed the published structures for rt-PA, but are not in agreement with some of the structures reported for mt-PA. In the latter glycoprotein we have identified a novel structure as the major oligosaccharide attached to Asn-184 and Asn-448. This is a biantennary oligosaccharide consisting of a fucosylated trimannosyl core to which are attached two GalNAc(1----4)GlcNAc antennae, one of which carries a sialic acid linked at the 6-position of the GalNAc. Minor constituents are sialylated on both or neither antennae. The sialylated GalNAc moiety is unique in N-linked glycoproteins. The majority of complex structures in rmt-PA contain N-acetyllactosamine moieties at both the Asn-184 and Asn-448 sites with the novel oligosaccharide occurring as a minor component at the Asn-184 site. This study demonstrates the power of mass spectrometric strategies based on high-field two-sector FAB-MS for structure elucidations of natural and recombinant glycoproteins.     

A novel caspase dependent pathway is involved in apoptosis of human endothelial cells by Shiga toxins

Shiga toxins have been shown to induce apoptosis on primary cultures, but not passaged ones, of human umbilical vein endothelial cells, independent of cytokine pre-treatment. Here, a peculiar pattern of caspase activation was observed; caspase-3 and -2, but not conventional upstream caspases, were activated at the initial phase of 6 hr, whereas a broad range inhibitor of caspases, VAD-fmk, but not mono-specific ones, suppressed DNA fragmentation and cell death. These results suggest additional analogous molecules, which have yet to be delineated, are involved. The requirement of retrograde uptake of toxins was also proved by the intervening effect of brefeldin A.     

Glucose-stimulated insulin secretion is not dependent on activation of protein kinase A

The involvement of cyclic AMP-dependent protein kinase A (PKA) in the exocytotic release of insulin from rat pancreatic islets was investigated using the Rp isomer of adenosine 3',5'-cyclic phosphorothioate (Rp-cAMPS). Preincubation of electrically permeabilised islets with Rp-cAMPS (1 mM, 1 h, 4 degrees C) inhibited cAMP-induced phosphorylation of islet proteins of apparent molecular weights in the range 20-90 kDa, but did not affect basal (50 nM Ca2+) nor Ca2(+)-stimulated (10 microM) protein phosphorylation. Similarly, Rp-cAMPS (500 microM) inhibited both cAMP- (100 microM) and 8BrcAMP-induced (100 microM) insulin secretion from electrically permeabilised islets without affecting Ca2(+)-stimulated (10 microM) insulin release. In intact islets, Rp-cAMPS (500 microM) inhibited forskolin (1 microM, 10 microM) potentiation of insulin secretion, but did not significantly impair the insulin secretory response to a range of glucose concentrations (2-20 mM). These results suggest that cAMP-induced activation of PKA is not essential for either basal or glucose-stimulated insulin secretion from rat islets.     

Nuclear import defect of human immunodeficiency virus type 1 DNA flap mutants is not dependent on the viral strain or target cell type

We have previously established, using human immunodeficiency virus type 1 (HIV-1) strain LAI, that the HIV-1 central DNA Flap acts as a cis determinant of viral genome nuclear import. Although the impact of the DNA Flap on nuclear import has already found numerous independent confirmations in the context of lentivirus vectors, it has been claimed that it may be nonessential for infectious virus strains LAI, YU-2 (J. D. Dvorin et al., J. Virol. 76:12087-12096, 2002), HXB2, and NL4-3 (A. Limon et al., J. Virol. 76:12078-12086, 2002). We conducted a detailed analysis of virus infectivity using the provirus clones provided by the authors and analogous target cells. In contrast to published data, our results show that all cPPT mutant viruses exhibit reduced infectivity corresponding to a nuclear import defect irrespective of the viral genetic background or target cell.     

Polarised secretion of cytokines in primary human microvascular endothelial cells is not dependent on N-linked glycosylation

Human microvascular endothelial cells (HMVEC) grow in monolayers on Transwell filters and restrict permeability between the apical and basolateral media. We show that these cell monolayers are capable of sorting labelled endogenous proteins, including chemokines, growth factors and cytokines, to either the apical or basolateral media. IL-8 and GMCSF were secreted predominantly into the apical medium, whereas MIC-1 was secreted into the basolateral medium. This polarity did not correlate with glycosylation, as IL-8 and MIC-1 are both N-glycosylated, but were sorted to opposite sides of the cell. IL-6 is not glycosylated and did not display significant polarity in secretion. Similarly, the polarity of secretion of endogenous glycoproteins was not related to their glycosylation.     

Polar targeting of DivIVA in Bacillus subtilis is not directly dependent on FtsZ or PBP 2B

DivIVA is involved in Bacillus subtilis cell division and is located at the cell poles. Previous experiments suggested that the cell division proteins FtsZ and PBP 2B are required for polar targeting of DivIVA. By using outgrowing spores, we show that DivIVA accumulates at the cell poles independent of the presence of FtsZ or PBP 2B.