Comparison of N-glycosides of fetuins from different species and human alpha 2-HS-glycoprotein.

Complex type N-glycosides of commercial bovine fetuin preparations from pooled fetal calf serum have been shown to contain comparable amounts of Gal4,4,4TRI (see structure A below) and Gal4,4,3TRI (structure B) as major asialo-structures. To investigate whether there is a clear genetic specificity for synthesis of these oligosaccharides, N-glycosides from two preparations of bovine fetuin, each from a single calf, were examined. Both of these structures were present in each calf, and there was only a subtle quantitative difference in the ratio of these two structures between the calves. Thus, a specific galactosyltransferase, presumably required for the biosynthesis of the Gal4,4,3TRI structure, may exist in both of these individual calves. Comparison of fetuin N-glycosides was also extended to sheep, pig, and human alpha 2-HS-glycoprotein, the human counterpart of bovine fetuin, using high-pH anion-exchange chromatography of the reducing oligosaccharides as well as HPLC of their pyridinylamino derivatives. The N-glycosides of ovine fetuin also have both Gal4,4,4TRI and Gal4,4,3TRI structures in a ratio similar to that of bovine fetuin. However, the major N-glycoside of porcine fetuin is of a fucosyl biantennary complex type structure (structure C below) and human alpha 2-HS-glycoprotein has an N-glycoside which is almost exclusively a nonfucosylated biantennary structure (structure D). This species-specific presence of N-glycosides of fetuins and comparison with N-glycosides of other glycoproteins suggest that the polypeptide sequence of a glycoprotein may affect its N-glycan structure by regulating the activity of specific glycosyltransferases. [formula: see text]     

Isolation and characterization of the major mannose-binding protein in chicken serum

Mannose-binding activity is abundantly present in chicken serum. The major mannose-binding protein has been isolated from chicken serum by affinity chromatography and gel filtration. The protein consists of two subunits of 75 000 and 26 500 daltons. Unlike hepatic lectins or other mannose-binding proteins, this protein does not require calcium for binding mannose-containing glycoconjugates. The chicken serum mannose-binding protein is immunochemically distinct from the chicken hepatic lectin and rabbit serum mannose-binding protein.     

Effect of oregano essential oil and carvacrol on Cryptosporidium parvum infectivity in HCT-8 cells

Cryptosporidium parvum is the second leading cause of persistent diarrhea among children in low-resource settings. This study examined the effect of oregano essential oil (OEO) and carvacrol (CV) on inhibition of C. parvum infectivity in vitro. HCT-8 cells were seeded (1 × 10⁶) in 96-well microtiter plates until confluency. Cell viability and infectivity were assessed by seeding HCT-8 cell monolayers with C. parvum oocysts (1 × 10⁴) in two modalities: 1) 4 h co-culture with bioactive (0–250 μg/mL) followed by washing and incubation (48 h, 37 °C, 5% CO₂) in bioactive-free media; and 2) 4 h co-culture of C. parvum oocysts followed by washing and treatment with bioactive (0–250 μg/mL) during 48-h incubation. Cell viability was tested using Live/Dead? assay whereas infectivity was measured using C. parvum-specific antibody staining via immunofluorescence detection. Loss of cell viability was observed starting at 125 μg/mL and 60 μg/mL for OEO and CV, respectively. Neither OEO nor CV modulated the invasion of C. parvum sporozoites in HCT-8 cells. Treatment with bioactive after invasion reduced relative C. parvum infectivity in a dose-dependent manner to 55.6 ± 10.4% and 45.8 ± 4.1% at 60 and 30 μg/mL of OEO and CV, respectively. OEO and CV are potential bioactives to counteract C. parvum infection in children.     

Codon bias and plasticity in immunoglobulins

Immunoglobulin genes experience Darwinian evolution twice. In addition to the germline evolution all genes experience, immunoglobulins are subjected, upon exposure to antigen, to somatic hypermutation. This is accompanied by selection for high affinity to the eliciting antigen and frequently results in a significant increase in the specificity of the responding population. The hypermutation mechanism displays a strong sequence specificity. Thus arises the opportunity to manipulate codon bias in a site-specific manner so as to direct hypermutation to those parts of the gene that encode the antigen-binding portions of the molecule and away from those that encode the structurally conserved regions. This segregation of mutability would clearly be advantageous; it would enhance the generation of potentially useful variants while keeping mutational loss to acceptably low levels. But it is not clear that the advantage gained would be large enough to produce a measurable effect within the background stochasticity of the evolutionary process. I have performed a pair of statistical tests to determine whether site- specific codon bias in human immunoglobulin genes is correlated with the sequence specificity of the somatic mutation mechanism. The sequence specificity of the mutator was determined by analysis of a database of published immunoglobulin intron sequences that had experienced somatic mutation but not selection. The site-specific codon bias was determined by analysis of published sequences of human germline immunoglobulin V genes. Both tests strongly suggest that evolution has acted to enhance the plasticity of immunoglobulin genes under somatic hypermutation.      

Specificity of chicken liver carbohydrate binding protein

Chicken hepatic lectin was isolated with affinity chromatography by using neoglycoproteins of bovine serum albumin (BSA) to which n moles of glycosides has been attached by amidination (Glycn-AI-BSA) [Lee, Y. C., Stowell, C. P., & Krantz, M. J. (1976) Biochemistry 15, 3956-3963] attached to Sepharose 4B. The same protein could be isolated from Man-, GlcNAc-, and Glc-AI-BSA-Sepharose columns and was identical with the protein previously reported [Kawasaki, T., & Ashwell, G. (1977) J. Biol. Chem. 252, 6536-6543]. The sugar specificity for binding to the isolated chicken hepatic lectin examined with Glycn-AI-BSA showed the order of potency for binding Glycn-AI-BSA to be D-GlcNAc greater than D-Glc, D-Man, L-Fuc greater than D-Gal, and the estimated Ki's for binding GlcNAc36-AI-BSA, Glc37-AI-BSA, Man33-AI-BSA, and L-Fuc28-AI-BSA were (6-20) X 10(-11), (2-3) X 10(-8), (3-9) X 10(-8), and 5 X 10(-8) M, respectively. The binding requirements of the binding protein were studied with a wide variety of Glycn-BSA's with different sugars and aglyconic linkages, as well as simple sugars and glycosides. It was concluded that (1) GlcNAc is the most potent sugar for binding, (2) the requirement for C-2 substituents is flexible, (3) an equatorial OH group at C-3 and C-4 must be present, (4) the 5-CH2OH group is not required for binding, (5) the lectin cannot accommodate a negative charge at C-6, and (6) D-Man and L-Fuc bind equally well.(ABSTRACT TRUNCATED AT 250 WORDS)     

Binding and endocytosis of glycoproteins by isolated chicken hepatocytes

The binding and endocytosis of glycoproteins containing different terminal sugars by isolated chicken hepatocytes were studied. At 2 degrees C, where there is no endocytosis, the hepatocyte surface bound 30 800 GlcNAc44-AI-BSA molecules [a bovine serum albumin (BSA) derivative which contains 44 residues of N-octylglucosamine (GlcNAc)] [Lee, Y.C., Stowell, C.P., & Krantz, M.J. (1976) Biochemistry 15, 3956-3963] and 32 900 asialoagalactoorosomucoid (AGOR) molecules per cell with estimated dissociation constants of 5 X 10(-10) and 4 X 10(-9) M, respectively. In the presence of digitonin or Triton X-100, each hepatocyte bound 7-18 times more ligand than in the absence of these detergents. Bound 125I-AGOR could be dissociated from the cell surface by 5.5 X 10(-5) M GlcNAc44-AI-BSA with a t 1/2 of 30 min, while GlcNAc (10 mM) or ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (4 mM) could dissociate over 98% of the surface-bound radioactivity within 10 min. Several neoglycoproteins inhibited the binding of 125I-AGOR, requiring for 50% inhibition 2.1 X 10(-9), 4.0 X 10(-7), 1.6 X 10(-6), and 2 X 10(-6) M for GlcNAc44-, Glc37-, Man43-, and L-Fuc28-AI-BSA, respectively. The bound AGOR and neoglycoproteins were internalized and degraded at 37 degrees C. [125I]Iodide was the only labeled degradation product found. When the hepatocytes were exposed to 250 nM AGOR at 37 degrees C, ca. 100 000 molecules of AGOR were associated with the cell surface at the steady state of endocytosis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Subcellular localization of rat kidney phosphate independent glutaminase

The phosphate independent glutaminase is contained in the brush border membrane of the rat kidney proximal tubule cells. This glutaminase activity cofractionates with the brush border membrane marker activities, alkaline phosphatase and γ-glutamyltranspeptidase, during differential centrifugation. About 30% of these activities are recovered with the mitochondrial fraction, the remainder is pelleted in the heavy microsomal fraction. The phosphate independent glutaminase in both fractions bands, during isopycnic centrifugation, with a mean density of 1.16–1.17 and is coincident with both brush border membrane marker activities. The isolation of intact, individual kidney cells was accomplished by initial perfusion of the kidneys in situ with a collagenase-papain solution followed by a brief incubation in the same enzyme solution. Incubation of isolated cells with a higher concentration of papain results in selective release of the phosphate independent glutaminase. The fact that this occurs without appreciable release of a cytoplasmic marker activity, lactate dehydrogenase, suggests that the phosphate independent glutaminase may be localized on the external surface of the kidney cells.     

An intraspecific gene duplication polymorphism of the urate oxidase gene of Drosophila virilis: a genetic and molecular analysis

Nineteen strains of Drosophila virilis from diverse geographic locations were examined by genetic and molecular analyses, revealing (a) 12 strains with a single copy of the urate oxidase (UO) gene per haploid genome and (b) 7 strains with a tandem duplication of the UO locus. The D. virilis strains with the UO duplication appear to have identical restriction maps of this region, implying either a single origin for the duplication or several similar events occurring at a hot spot. On the basis of the location of the duplication breakpoints and the restriction sites flanking these breakpoints, this duplication probably arose through nonhomologous recombination involving either a breakage and rejoining event or replication slippage. because documented cases of intraspecific gene duplication polymorphism are rare, the D. virilis UO duplication will be useful in identifying the molecular event giving rise to a gene duplication.      

7th SOSORT consensus paper: conservative treatment of idiopathic & Scheuermann's kyphosis

Abstract

Thoracic hyperkyphosis is a frequent problem and can impact greatly on patient's quality of life during adolescence. This condition can be idiopathic or secondary to Scheuermann disease, a disease disturbing vertebral growth. To date, there is no sound scientific data available on the management of this condition. Some studies discuss the effects of bracing, however no guidelines, protocols or indication's of treatment for this condition were found. The aim of this paper was to develop and verify the consensus on managing thoracic hyperkyphosis patients treated with braces and/or physiotherapy.

Methods

The Delphi process was utilised in four steps gradually modified according to the results of a set of recommendations: we involved the SOSORT Board twice, then all SOSORT members twice, with a Pre-Meeting Questionnaire (PMQ), and during a Consensus Session at the SOSORT Lyon Meeting with a Meeting Questionnaire (MQ).

Results

There was an unanimous agreement on the general efficacy of bracing and physiotherapy for this condition. Most experts suggested the use of 4-5 point bracing systems, however there was some controversy with regards to physiotherapeutic aims and modalities.

Conclusion

The SOSORT panel of experts suggest the use of rigid braces and physiotherapy to correct thoracic hyperkyphosis during adolescence. The evaluation of specific braces and physiotherapy techniques has been recommended.