Prior myocardial infarction and presence of upper gastrointestinal conditions among patients with venous thromboembolism: prevalence, associated comorbidities and burden.

WHAT IS KNOWN AND OBJECTIVES: Venous thromboembolism (VTE), comprising deep vein thrombosis (DVT) and pulmonary embolism (PE), is a serious, life-threatening condition that often complicates treatment of individuals who are already ill and increases in risk with age. The comorbidity burden of VTE can complicate treatment; therefore, treatment should be influenced by the presence of comorbidities (Kearon 2012). The prevalence of common conditions in the VTE population is, therefore, an important subject of research. Prevalence of two common comorbid burdens, prior myocardial infarction (MI) and upper gastrointestinal (GI) conditions, was studied among survey respondents who reported DVT or PE. METHODS: Responses to the 2010 wave of the National Health and Wellness Survey (NHWS), a self-administered, internet-based questionnaire from a nationwide, demographically representative sample of adults, were evaluated. RESULTS AND DISCUSSION: Among the 814 participants reporting a history of VTE, 9·7% (n = 60) of the DVT subpopulation and 13·2% (n = 39) of the PE subpopulation also reported prior MI. In respondents with prior MI, cardiovascular, urological, and pain comorbidities were each reported as additional comorbidities by at least two thirds of respondents in both the PE and DVT subpopulations, with cardiovascular and urological conditions reported significantly (P < 0·05) more often than among respondents with no prior MI. Among the respondents reporting VTE, 48·9% (n = 302) of the subpopulation reporting DVT and 52·2% (n = 154) of those reporting PE also reported upper GI comorbidities. Cardiovascular and pain conditions in the respondents reporting upper GI comorbidities were each reported by more than three quarters of VTE patients in both the DVT and PE subpopulations and were significantly more common (P < 0·05) than among their counterparts without upper GI comorbidities. WHAT IS NEW AND CONCLUSION: The results of the NHWS indicate that VTE patients who have either of two common comorbid burdens, prior MI and concomitant upper GI conditions, also showed high levels of additional, concurrent comorbidities and generally poor health status. Clinicians must be aware of the total comorbidity profile of their patients who have experienced VTE in order to best manage them and prescribe appropriate therapy.

Oxidized low density lipoproteins induce apoptosis in human lymphocytes: involvement of mitogen-activated protein kinases.

Oxidized low density lipoproteins (oxLDL), macrophages and T-lymphocytes are present in atherosclerotic lesions. We and others have shown that oxLDL is cytotoxic for macrophages, endothelial, smooth muscle and activated T-lymphocytes and induce apoptosis. Here we demonstrate that (i) oxidized LDL (oxLDL), oxidized VLDL (oxVLDL) and hydrogen peroxide (H2O2) induce apoptosis in human T-lymphocytes and (ii) mitogen-activated protein kinases are involved in this process. Apoptosis was monitored by immunofluorescence microscopy and flow cytometry for annexin V binding, Apo 2.7 expression, the TUNEL reaction and caspase 3 activity. In the presence of oxLDL (100 microg/ml), oxVLDL (50 microg/ml) and H2O2 (5 mM), the fraction of apoptotic cells increased within 6 hours to more than 70%. Preincubation of lymphocytes with the MAPKK inhibitor PD-98059 and the p38MAPK inhibitor SB-203580 almost completely abolished these effects. Furthermore, oxLDL and H2O2 but not native LDL strongly enhanced phosphorylation of JNK, p38MAPK and p42/44MAPK. The results suggest that in the resting lymphocyte apoptosis triggered by oxidized lipoproteins and oxidative stress depends on the activation of p44/42MAPK and p38MAPK cascades.

Effects of antioxidants on glutathione levels and clinical recovery from the malnutrition syndrome kwashiorkor--a pilot study.

Kwashiorkor is a severe edematous form of malnutrition with high prevalence and lethality in many African countries, and repeatedly has been reported to be associated with oxidative stress. The therapy of kwashiorkor is still ineffective. In this pilot study, we tested the hypothesis that oral application of thiol-containing antioxidants increases glutathione status and is beneficial for the clinical recovery of kwashiorkor patients. The longitudinal clinical intervention study was carried out at St Joseph's Hospital, Jirapa, Ghana. Children with severe kwashiorkor were randomly assigned to either a standard treatment (ST) receiving a therapeutic protocol based on the recommendations of the WHO or to one of three study groups receiving in addition 2 x 600 mg reduced glutathione or 2 x 50 mg alpha-lipoic acid or 2 x 100 mg N-acetylcysteine per day. Patients were followed up clinically and biochemically for 20 days and compared with 37 healthy controls. Both glutathione and alpha-lipoic acid supplementation had positive effects on survival. Also, the blood glutathione concentrations correlated positively with survival rates. Furthermore, the initial skin lesions, glutathione and total protein concentrations were found to be strong predictors of survival. The data strongly suggest that a therapy restoring the antioxidative capacity by applying cysteine equivalents in the form of glutathione and/or alpha-lipoic acid is beneficial for biochemical and clinical recovery of kwashiorkor patients.

Adsorption kinetics of polyethylene glycol from aqueous solution onto activated carbon.

The adsorption equilibrium and kinetics of polyethylene glycol (PEG) in three aqueous systems were examined in this study. Langmuir isotherm was used to satisfactorily predict the adsorption capacity of PEG on activated carbon F-400 and applied to the investigation of adsorption kinetics. The surface diffusion, pore diffusion, and branched pore kinetics models successfully described the adsorption behavior of PEG on F-400 in the completely stirred tank reactor. The pore diffusion coefficients obtained from the pore diffusion model were compared with those computed by the experimental data of the short fixed-bed reactor combined with the assumption of non-hindered pore diffusion. In addition, the effects of initial concentrations of PEG and the relative importance of external and internal mass transfers for the adsorption were also taken into account and discussed in this study.

Biological markers in nasal secretions.

Biological markers in nasal secretions provide valuable information on nasal pathophysiology. However, published data on biomarker concentrations in nasal fluids are remarkably inconsistent, and the bias due to different sampling techniques, has not yet been systematically evaluated. Concentrations of various protein were repeatedly determined in nasal secretions of 16 healthy volunteers. The proteins were detected by using: 1) alpha2-macroglobulin as a marker for plasma contamination; 2) lactoferrin as a marker for glandular secretion; 3) lactate dehydrogenase as a marker for tissue injury; and 4) interleukin (IL)-1beta, IL-8, tumour necrosis factor-alpha, and eosinophil cationic protein and tryptase as indicators for tissue inflammation. A total of four different sampling methods, including nasal lavage (NL) and a new polyurethane foam sampler technique (PFST) were employed. Analyte concentrations in NL were approximately 10-times lower than in specimens obtained by PFST. Due to the unpredictable dilution during NL, various analytes were below the detection limit of the high sensitivity assays employed. With PFST, concentrations below the detection limit rarely occurred. The specimens did not significantly differ regarding plasma contamination, glandular secretion or tissue injury. The considerable variability of reported analyte concentrations in nasal secretions mainly results from different sampling techniques. To collect nasal secretions, samplers are considered superior to nasal lavage techniques.

Endothelin-1 synthesis and endothelin B receptor expression in human coronary artery smooth muscle cells and monocyte-derived macrophages is up-regulated by low density lipoproteins.

Endothelin-1 (ET-1) is a potent vasoconstrictive peptide exerting its effects predominantly by paracrine and autocrine mechanisms. ET-1 acts as a mitogen and co-mitogen on vascular smooth muscle cells, and accumulating evidence suggests that ET-1 is involved in the pathogenesis of atherosclerosis. Deposition of low density lipoproteins (LDL) in the vessel wall is known to play a crucial role in the development of atherosclerotic lesions. In the present study, we have investigated the effect of native LDL (nLDL) and oxidatively modified LDL (oxLDL) on ET-1 synthesis and endothelin receptor expression in cultured human coronary artery smooth muscle cells and human monocyte-derived macrophages. Native LDL and oxLDL induced a significant stimulation of ET-1 release and ET-1 mRNA expression in human coronary artery smooth muscle cells and monocyte-derived macrophages. Antibodies against the scavenger receptor CD36 significantly reduced the oxLDL-induced stimulation of ET-1 synthesis. The antioxidants trolox and probucol did not significantly inhibit the LDL-induced rise of ET-1 release. Endothelin B receptor expression was up-regulated in both cell types after incubation with nLDL and oxLDL. In coronary smooth muscle cells, endothelin A receptor expression was slightly increased by LDL, whereas endothelin A receptor was not detectable in monocyte-derived macrophages. Coronary artery smooth muscle cells secreted a more than 150-fold higher amount of immunoreactive ET-1 into the cell culture medium than monocyte-derived macrophages. In summary, the present data, demonstrating a LDL-induced up-regulation of the endothelin system in coronary smooth muscle cells and in monocyte-derived macrophages, provide further support for a pathophysiological role of endothelin in coronary atherosclerosis and suggest that ET-1 might be involved in mediating the atherogenic effects of LDL.

Mg2+-induced tRNA folding.

Mg(2+)-induced folding of yeast tRNA(Phe) was examined at low ionic strength in steady-state and kinetic experiments. By using fluorescent labels attached to tRNA, four conformational transitions were revealed when the Mg(2+) concentration was gradually increased. The last two transitions were not accompanied by changes in the number of base pairs. The observed transitions were attributed to Mg(2+) binding to four distinct types of sites. The first two types are strong sites with K(diss) of 4 and 16 microM. The sites of the third and fourth types are weak with a K(diss) of 2 and 20 mM. Accordingly, the Mg(2+)-binding sites previously classified as "strong" and "weak" can be further subdivided into two subtypes each. Fluorescent transition I is likely to correspond to Mg(2+) binding to a unique strong site selective for Mg(2+); binding to this site causes only minor A(260) change. The transition at 2 mM Mg(2+) is accompanied by substantial conformational changes revealed by probing with ribonucleases T1 and V1 and likely enhances stacking of the tRNA bases. Fast and slow kinetic phases of tRNA refolding were observed. Time-resolved monitoring of Mg(2+) binding to tRNA suggested that the slow kinetic phase was caused by a misfolded tRNA structure formed in the absence of Mg(2+). Our results suggest that, similarly to large RNAs, Mg(2+)-induced tRNA folding exhibits parallel folding pathways and the existence of kinetically trapped intermediates stabilized by Mg(2+). A multistep scheme for Mg(2+)-induced tRNA folding is discussed.

The first phytoplasma RNase P RNA provides new insights into the sequence requirements of this ribozyme.

A high variability of RNase P RNA structures is seen among members of the Mycoplasma group. To gain further insight into the structure-function relations of this ribozyme, we have searched for the RNase P RNA gene from more distant relatives, the phytoplasmas. These mycoplasma-like organisms are the aetiological agents of many severe plant diseases. We report the sequence and catalytic properties of RNase P RNA from the phytoplasma causing apple proliferation disease. The primary and postulated secondary structure of this 443 nt long RNA are most similar to those of Acholeplasma, supporting the phylogenetic position of this pathogen. Remarkably, the extremely AT-rich (73.6%) phytoplasma RNA differs from the known bacterial consensus sequence by a single base pair, which is positioned close to the substrate cleavage site in current three-dimensional models. Phytoplasma RNase P RNA functions as an efficient ribozyme in vitro. Conversion of its sequence to the full consensus and kinetic analysis of the resulting mutant RNAs suggests that neither the sequence alone, nor the type of pairing at this position is crucial for substrate binding or catalysis by the RNase P ribozyme. These results refine the bacterial consensus structure close to the catalytic core and thus improve our understanding of RNase P RNA function.

A tobacco nuclear extract supporting transcription, processing, splicing and modification of plant intron-containing tRNA precursors.

Nuclear tRNA genes are transcribed by RNA polymerase III (Pol III) and pre-tRNAs are processed into mature tRNAs via complex processes in the nucleus. We have developed an in vitro Pol III-dependent transcription system derived from tobacco cultured cells, which supports efficiently not only transcription of a variety of plant tRNA genes but also 5'-and 3'-end processing, nucleotide modification and splicing of intron-containing pre-tRNAs. The structures of in vitro transcripts have been confirmed by primer extension analysis and by RNase T1 fingerprinting. The optimal Mg2+ concentration differed for each step so that each reaction can be controlled by adjusting the Mg2+ concentration. At 1 mm Mg2+, only transcription occurs so that pre-tRNAs accumulate. The splicing reaction can be initiated by raising Mg2+ ions (> 5 mm) and enhanced by adding 1 mm hexamminecobalt chloride. Using the optimized system for the Nicotiana intron-containing tRNATyr gene, the precise initiation and termination sites of transcription and the splice sites were determined. The presence of 1 mm NAD+ in the reaction mixture leads to the removal of the 2' phosphate at the splice junction of tRNATyr, demonstrating the activity of a 2'-phosphotransferase in the tobacco nuclear extract. Many modified nucleosides such as m2G, m22G, m1A, phi27 and phi35 are introduced in either of the studied transcripts. As shown in other systems, the conversion of U35 to phi requires an intron-containing substrate.

Strong FANCA/FANCG but weak FANCA/FANCC interaction in the yeast 2-hybrid system.

Three of at least 8 Fanconi anemia (FA) genes have been cloned (FANCA, FANCC, FANCG), but their functions remain unknown. Using the yeast 2-hybrid system and full-length cDNA, the authors found a strong interaction between FANCA and FANCG proteins. They also obtained evidence for a weak interaction between FANCA and FANCC. Neither FANCA nor FANCC was found to interact with itself. These results support the notion of a functional association between the FA gene products. (Blood. 2000;95:719-720)

Lipopolysaccharide-activated macrophages stimulate the synthesis of collagen type I and C-fibronectin in cultured pancreatic stellate cells.

We have recently identified and characterized pancreatic stellate cells (PSC) in rats and humans (Gastroenterology 1998, 15:421-435). PSC are suggested to represent the main cellular source of extracellular matrix in chronic pancreatitis. Now we describe a paracrine stimulatory loop between human macrophages and PSC (rat and human) that results in an increased extracellular matrix synthesis. Native and transiently acidified supernatants of cultured macrophages were added to cultured PSC in the presence of 0.1% fetal calf serum. Native supernatants of lipopolysaccharide-activated macrophages stimulated the synthesis of collagen type I 1.38 +/- 0.09-fold of control and c-fibronectin 1.89 +/- 0.18-fold of control. Transiently acidified supernatants stimulated collagen type I and c-fibronectin 2.10 +/- 0.2-fold and 2.80 +/- 0.05-fold of control, respectively. Northern blot demonstrated an increased expression of the collagen-I-(alpha-1)-mRNA and fibronectin-mRNA in PSC 10 hours after addition of the acidified macrophage supernatants. Cell proliferation measured by bromodeoxyuridine incorporation was not influenced by the macrophage supernatants. Unstimulated macrophages released 1.97 pg TGFbeta1/microgram of DNA over 24 hours and lipopolysaccharide-activated macrophages released 6.61pg TGFbeta1/microgram of DNA over 24 hours. These data together with the results that, in particular, transiently acidified macrophage supernatants increased matrix synthesis, identify TGFbeta as the responsible mediator. In conclusion, our data demonstrate a paracrine stimulation of matrix synthesis of pancreatic stellate cells via TGFbeta1 released by activated macrophages. We suggest that macrophages might play a pivotal role in the development of pancreas fibrosis.

Enhancement of RNA self-cleavage by micellar catalysis.

It has been reported recently that naturally occurring catalytic RNAs like hammerhead and hairpin ribozyme do not require metal ions for efficient catalysis. It seems that the folded tertiary structure of the RNA contributes more to the catalytic function than was initially recognized. We found that a highly specific self-cleavage reaction can occur within a small bulge loop of four nucleotides in a mini-substrate derived from Arabidopsis thaliana intron-containing pre-tRNA(Tyr) in the absence of metal ions. NH(4)(+) cations and non-ionic or zwitter-ionic detergents at or above their critical micelle concentration are sufficient to catalyze this reaction. The dependence on micelles for the reaction leads to the assumption that physical properties, i.e. the hydrophobic interior of a micelle, are essential for this self-cleavage reaction. We suggest that NH(4)(+)-ions play a crucial role for the entry of the negatively charged RNA into the hydrophobic interior of a detergent micelle. A change of the pattern of hydration or hydrogen bonds caused by the hydrophobic surrounding enhances the reaction by a factor of 100. These findings suggest that highly structured RNAs may shift pK(a) values towards neutrality via the local environment and thereby enhance their ability to perform general acid-base catalysis without the participation of metal ions.

A SECIS binding protein (SBP) is distinct from selenocysteyl-tRNA protecting factor (SePF).

In mammals, most of the selenium contained in their body is present as an unusual amino acid, selenocysteine (Sec), whose codon is UGA. Because the UGA codon is normally recognized as a translational stop signal, it is intriguing how cells recognize and distinguish the UGA Sec codon from the UGA stop codon. In eukaryotic selenoprotein mRNAs, it has been proposed that a conserved stem-loop structure designated Sec insertion sequence (SECIS) located in the 3'-untranslated regions is required for recognition of UGA as a Sec codon. Although some proteins (SBPs) have been reported to bind to SECIS, it is not clear how the SECIS element can mediate Sec insertion at UGA. Eukaryotic Sec-tRNA(Sec) is not recognized by elongation factor EF-1alpha, but is recognized specifically by a Sec-tRNA(Sec) protecting factor, SePF, in bovine liver extracts. In this study, we provide evidence that SePF is distinct from SBP by chromatography. Upon UV irradiation, the SECIS RNA was cross-linked to a 47.5 kDa protein, a likely candidate of SBP, that is contained in the complex with a molecular mass of 150 kDa. These results suggest that SBP and SePF play different roles for the Sec incorporation. To our knowledge, this is the first demonstration that SBP is discriminated from the factor which directly recognizes Sec-tRNA(Sec), providing a novel clue to the mechanism of selenocysteine decoding in eukaryotes.

Depending on their concentration oxidized low density lipoproteins stimulate extracellular matrix synthesis or induce apoptosis in human coronary artery smooth muscle cells.

Various lines of evidence indicate that oxidative stress resulting in lipid peroxidation and protein modification is involved in the pathogenesis of atherosclerosis and coronary heart disease. We have investigated the effect of modified (oxidized) low-density lipoproteins (oxLDL) on collagen and fibronectin synthesis in cultured human coronary artery smooth muscle cells (HCA-SMC). As shown by immunofluorescence microscopy and time-resolved fluorescence immunoassay, oxLDL dose-dependently stimulated collagen type I and fibronectin synthesis in cultured HCA-SMC. The effect on matrix synthesis was biphasic, with a maximum effect at concentrations between 1 and 10 microg/ml oxLDL. Higher oxLDL concentrations (>25 microg/ml) were cytotoxic. Beside oxLDL, malondialdehyde-modified LDL also stimulated extracellular matrix synthesis. In the presence of 100 microg/ml ascorbic acid, 25, 50 and 100 microg/ml oxLDL induced apoptosis within 6-8 hours (demonstrated by TUNEL-reaction, annexin-V binding and APO-2.7-expression). Apoptosis was not induced by normal (unmodified) LDL and malondialdehyde-modified LDL. The radical scavengers and antioxidants TROLOX and probucol and the hydrogen peroxide eliminator catalase significantly reduced oxLDL-induced apoptosis. Our results demonstrate that low concentrations of oxLDL are profibrogenic by stimulating extracellular matrix synthesis, whereas higher oxLDL concentrations induce oxidative stress and apoptosis in coronary artery smooth muscle cells. The profibrogenic effect might be relevant in the formation of atherosclerotic plaques, and the proapoptotic effect might contribute to an increased plaque vulnerability.

Coordination of tRNA nuclear export with processing of tRNA.

Eukaryotic tRNAs are synthesized in the nucleus and need to be exported to the cytoplasm where they function in translation. tRNA export is mediated by exportin-t, which binds tRNA directly and with high affinity. tRNAs are initially synthesized as precursor molecules. Maturation to functional tRNA takes place in the nucleus, precedes export, and includes trimming of the 5' and 3' ends, posttranscriptional addition of the 3' CCA end, nucleoside modifications, and in some cases splicing. Here we address the question of how tRNA maturation is coordinated with export and thus how cytoplasmic accumulation of inactive maturation intermediates is avoided. This could, in principle, be achieved by nuclear retention of immature tRNA or by selective export of the fully mature form. We show that exportin-t has a strong preference for tRNA with correctly processed 5' and 3' ends and nucleoside modification. tRNA recognition by exportin-t can thus be considered as a quality control mechanism for these maturation steps prior to tRNA export. Surprisingly however, exportin-t can efficiently bind unspliced tRNA and intron-containing tRNA is exported when the rate of splicing is slow. During characterization of the exportin-t/tRNA interaction we found that exportin-t recognizes features in the tRNA that are conserved between prokaryotic and eukaryotic tRNAs. Our data suggest that correct tRNA shape, the 5' and 3' terminal ends, and the TpsiC loop are critical for exportin-t binding.

Age-dependent altered proportions in subpopulations of tonsillar lymphocytes.

Age-related changes in functional subsets of lymphocytes may influence the potential to build up immune responses. In particular, the capacity of tonsillar lymphocytes to counter infections may be altered during ageing. In order to address this question we investigated the proportional distribution of several subsets of tonsillar T and B cells with regard to ageing. Tonsils were derived from 119 patients between 2 and 65 years of age. Lymphocyte subsets were monitored by three-colour fluorescence of relevant CD markers in flow cytometry. As a general tendency the percentage of CD3+ T cells steadily increased whereas that of CD19+ B cells decreased at the same time. No significant differences were observed between lymphocytes of patients with and without inflammatory history of the tonsils. The percentage of CD8+ T cells declined whereas that of CD4+ T cells increased during the same time span. CD45RA+ T cells increased during the first two decades of life and gradually decreased thereafter. In contrast, CD45RO+ T cells showed an opposite trend. No differences were seen in the population of CD3-/CD56+ natural killer (NK) cells. The mature B cell marker CD40 showed no significant changes during ageing. However, CD38+ B cells, representing B cells of late maturation stages, dramatically declined up to the age of 65. In a similar manner the CD5+ subpopulation of B cells decreased during ageing. Substantial changes in major tonsillar T and B cell populations as shown in this study may have an impact on the ageing process of the immune system.

Heterogeneous spectrum of mutations in the Fanconi anaemia group A gene.

Fanconi anaemia (FA) is a genetically heterogeneous autosomal recessive disorder associated with chromosomal fragility, bone-marrow failure, congenital abnormalities and cancer. The gene for complementation group A (FAA), which accounts for 60-65% of all cases, has been cloned, and is composed of an open reading frame of 4.3 kb, which is distributed among 43 exons. We have investigated the molecular pathology of FA by screening the FAA gene for mutations in a panel of 90 patients identified by the European FA research group, EUFAR. A highly heterogeneous spectrum of mutations was identified, with 31 different mutations being detected in 34 patients. The mutations were scattered throughout the gene, and most are likely to result in the absence of the FAA protein. A surprisingly high frequency of intragenic deletions was detected, which removed between 1 and 30 exons from the gene. Most microdeletions and insertions occurred at homopolymeric tracts or direct repeats within the coding sequence. These features have not been observed in the other FA gene which has been cloned to date (FAC) and may be indicative of a higher mutation rate in FAA. This would explain why FA group A is much more common than the other complementation groups. The heterogeneity of the mutation spectrum and the frequency of intragenic deletions present a considerable challenge for the molecular diagnosis of FA. A scan of the entire coding sequence of the FAA gene may be required to detect the causative mutations, and scanning protocols will have to include methods which will detect the deletions in compound heterozygotes.

Functional groups of sialic acids involved in binding to siglecs (sialoadhesins) deduced from interactions with synthetic analogues.

The siglecs, formerly called sialoadhesins, are a family of I-type lectins binding to sialic acids on the cell surface. Five members of this family have been identified: sialoadhesin, myelin-associated glycoprotein (MAG), Schwann cell myelin protein (SMP), CD22 and CD33. We have investigated the relevance of substituents at position C-9 and in the N-acetyl group of N-acetylneuraminic acid, using a series of synthetic sialic-acid analogues either on resialylated human erythrocytes or as free alpha-glycosides in hapten inhibition. All five siglecs require the hydroxy group at C-9 for binding, suggesting hydrogen bonding of this substituent with the binding site. Remarkable differences were found among the proteins in their specificity for modifications of the N-acetyl group. Whereas sialoadhesin, MAG and SMP do not tolerate a hydroxy group as in N-glycolylneuraminic acid, they bind to halogenated acetyl residues. In the case of MAG, N-fluoroacetylneuraminic acid is bound about 17-fold better than N-acetylneuraminic acid. In contrast, human and murine CD22 both show good affinity for N-glycolylneuraminic acid, but only human CD22 bound the halogenated compounds. In conclusion, our data indicate that interactions of the hydroxy group at position 9 and the N-acyl substituent contribute significantly to the binding strength.

Abnormal surface expression of sialoglycans on B lymphocyte cell lines from patients with carbohydrate deficient glycoprotein syndrome I A (CDGS I A).

The carbohydrate-deficient glycoprotein syndromes (CDGS) are genetic, multisystemic diseases characterized by deficiencies in the glycosylation of many secretory glycoproteins, lysosomal enzymes, and possibly cell surface glycoproteins resulting in central nervous system abnormalities and frequent early death by infection. Here we examined whether membranous glycoconjugates of lymphocytes are affected by this disorder. For this, we analyzed cell surface-expressed sialoglycans of Epstein Barr virus (EBV)-transformed B cell lines derived from peripheral B lymphocytes of several patients with CDGS I A. These CDG-LCL (lymphoblastoid cell lines) expressed differentiation markers comparable to those of other EBV-transformed B cell lines. No apparent defects in the gross glycosylation process of defined complex glycosylated proteins such as the surface-expressed major histocompatibility complex class I glycoprotein or secreted immunoglobulin (IgM) were identified. However, using a novel flow cytometric enzyme assay to measure cell surface alpha2,6 sialylation on live cells we found that CDG-LCL express less alpha2,6 sialylated glycans in comparison to other EBV-transformed B cell lines. Also, CDG-LCL bound less of the B lymphocyte lectin CD22, specific for alpha2,6 sialylated lactosamines and known to modulate B cell receptor mediated signaling, as demonstrated by using a soluble CD22-immunoglobulin fusion protein in flow cytometry. CDG-LCL showed stronger surface staining with the monoclonal antibody 1B2 which detects a distinct group of surface-expressed lactosaminyl epitopes. After pretreatment with neuraminidase of Newcastle disease virus (NDVN) it became apparent that in CDG-LCL a significantly larger portion of the 1B2 epitopes was sialylated in alpha2,3 linkage as compared to other B cell lines. Intracellular alpha2,6 sialyltransferase activity as well as polymerase chain reaction products specific for four different sialyltransferases did not significantly differ in CDG-LCL as compared to other EBV-B cell lines. Differences in sialylation may be caused by the respective oligosaccharide core structures available for alpha2,6 or alpha2,3 sialylation in CDG-LCL. Therefore, lymphocytes derived from CDGS patients have distinct deviations in their surface-expressed lactosaminoglycan structures which may affect functions as exemplified by reduced interactions of CD22 with its ligands.

Minimal tRNA(Ser) and tRNA(Sec) substrates for human seryl-tRNA synthetase: contribution of tRNA domains to serylation and tertiary structure.

The recognition process of tRNA(Ser) and tRNA(Sec) by human seryl-tRNA synthetase (SerRS) was studied using T7 transcripts representing defined regions of human tRNA(Ser) or tRNA(Sec) and the influence of the tRNA elements on serylation and tertiary structure was elucidated. The anticodon arms of both tRNAs showed no contribution to serylation in contrast to the acceptor stems and the long extra arms. D and T arms were only involved in formation of the L-shaped tRNA structure, not in the recognition process between tRNAs and SerRS. This is the first report of microhelices adapted from human tRNAs being aminoacylated by their homologous synthetase.