Comparative aspects of infectious salmon anemia virus,an orthomyxovirus of fish,to influenza viruses

Infectious salmon anaemia (ISA) is a viral disease that was first recorded in 1984 in farmed Atlantic salmon. The infectious salmon anaemia virus (ISAV) is classified as the type species of the genus Isavirus in the Orthomyxoviridae family and is evolutionary remote to the influenza viruses. The genome consists of eight negative single-stranded RNA segments, and it utilises the same mechanisms as influenza viruses to enter and exit cells. Although a common ancestor of ISAV and other genera of Orthomyxoviruses could be dated back several millions of years, there are still many similarities between ISAV and the influenza viruses regarding morphology, replication cycles and interactions with their respective hosts.     

DNA sequence profiles of the colorectal cancer critical gene set KRAS-BRAF-PIK3CA-PTEN-TP53 related to age at disease onset

The incidence of colorectal cancer (CRC) increases with age and early onset indicates an increased likelihood for genetic predisposition for this disease. The somatic genetics of tumor development in relation to patient age remains mostly unknown. We have examined the mutation status of five known cancer critical genes in relation to age at diagnosis, and compared the genomic complexity of tumors from young patients without known CRC syndromes with those from elderly patients. Among 181 CRC patients, stratified by microsatellite instability status, DNA sequence changes were identified in KRAS (32%), BRAF (16%), PIK3CA (4%), PTEN (14%) and TP53 (51%). In patients younger than 50 years (n = 45), PIK3CA mutations were not observed and TP53 mutations were more frequent than in the older age groups. The total gene mutation index was lowest in tumors from the youngest patients. In contrast, the genome complexity, assessed as copy number aberrations, was highest in tumors from the youngest patients. A comparable number of tumors from young (<50 years) and old patients (>70 years) was quadruple negative for the four predictive gene markers (KRAS-BRAF-PIK3CA-PTEN); however, 16% of young versus only 1% of the old patients had tumor mutations in PTEN/PIK3CA exclusively. This implies that mutation testing for prediction of EGFR treatment response may be restricted to KRAS and BRAF in elderly (>70 years) patients. Distinct genetic differences found in tumors from young and elderly patients, whom are comparable for known clinical and pathological variables, indicate that young patients have a different genetic risk profile for CRC development than older patients.     

Stereospecific pharmacokinetics and toxicodynamics of ketorolac after oral administration of the racemate and optically pure enantiomers to the rat

To determine the stereospecific pharmacokinetics and gastrointestinal permeability (GI) changes (surrogate measures of toxicity) in the rat following oral administration of S, R, and racemic ketorolac (KT), optically pure enantiomers (S and R 2.5 mg/kg), and racemic KT (5 mg/kg) were administered orally to male Sprague-Dawley rats and plasma samples were collected for 6 h post-dose for pharmacokinetic assessments. KT-induced changes in GI permeability were assessed using sucrose and 51Cr-EDTA as markers of gastroduodenal and distal intestinal permeability, respectively. After the racemate, R-KT was predominant in plasma (AUC S/R, 0.45). No significant differences in pharmacokinetic indices were evident following administration of the racemate as compared with individual enantiomers. In plasma, there was only negligible S-KT after administration of R-KT. After S-KT, on the other hand, AUC of R-KT was found to be 6.7% of that of S-KT. Both permeability markers showed considerable interanimal variability. Gastroduodenal permeability was significantly increased from baseline by the racemate but not by either of the two enantiomers administered alone. Permeability to 51Cr-EDTA was not significantly increased above baseline for any of the treatments. The plasma concentration of R-KT found after administration of S-KT may be from the < 2% chiral impurity which appears magnified due to its slower clearance as compared with its antipode. There is no evidence of a pharmacokinetic interaction between the enantiomers. Since 2.5 mg/kg S-KT is somewhat less toxic on the gastroduodenum than 5 mg/kg racemate, it may be a safer alternative to the latter, at least in the rat model.     

Naive T cell recruitment to nonlymphoid tissues: a role for endothelium-expressed CC chemokine ligand 21 in autoimmune disease and lymphoid neogenesis

Naive T cells are usually excluded from nonlymphoid tissues. Only when such tertiary tissues are subjected to chronic inflammation, such as in some (but not all) autoimmune diseases, are naive T cells recruited to these sites. We show that the CCR7 ligand CC chemokine ligand (CCL)21 is sufficient for attracting naive T cells into tertiary organs. We performed intravital microscopy of cremaster muscle venules in T-GFP mice, in which naive T cells express green fluorescent protein (GFP). GFP(+) cells underwent selectin-dependent rolling, but no firm adherence (sticking). Superfusion with CCL21, but not CXC chemokine ligand 12, induced integrin-dependent sticking of GFP(+) cells. Moreover, CCL21 rapidly elicited accumulation of naive T cells into sterile s.c. air pouches. Interestingly, a second CCR7 ligand, CCL19, triggered T cell sticking in cremaster muscle venules, but failed to induce extravasation in air pouches. Immunohistochemistry studies implicate ectopic expression of CCL21 as a mechanism for naive T cell traffic in human autoimmune diseases. Most blood vessels in tissue samples from patients with rheumatoid arthritis (85 +/- 10%) and ulcerative colitis (66 +/- 1%) expressed CCL21, and many perivascular CD45RA(+) naive T cells were found in these tissues, but not in psoriasis, where CCL21(+) vessels were rare (17 +/- 1%). These results identify endothelial CCL21 expression as an important determinant for naive T cell migration to tertiary tissues, and suggest the CCL21/CCR7 pathway as a therapeutic target in diseases that are associated with naive T cell recruitment.     

A simple combined method for determination of β-1,3-glucan and cell wall polysaccharides in diatoms

A new method is described for the combined determination of -1,3-glucan and cell wall polysaccharides in diatoms, representing total cellular carbohydrate. The glucan is extracted by 0.05 mol l^–1 H₂SO₄ at 60 °C for 10 min, and the cell wall polysaccharides are subsequently hydrolyzed by 80% H₂SO₄ at 0–4 °C for 20 h. Each carbohydrate fraction is determined by the phenol-sulphuric acid method. The method has been demonstrated for axenic cultures of the marine diatom Skeletonema costatum and natural marine phytoplankton populations dominated by diatoms. Cellular glucan and cell wall polysaccharides were determined with standard deviations of 1–3% and 2–5%, respectively.     

MOBILIZATION OF β-1,3-GLUCAN AND BIOSYNTHESIS OF AMINO ACIDS INDUCED BY NH4+ ADDITION TO N-LIMITED CELLS OF THE MARINE DIATOM SKELETONEMA COSTATUM (BACILLARIOPHYCEAE)

Mobilization of the reserve β-1,3-glucan (chrysolaminaran) in N-limited cells of the marine diatom Skeletonema costatum (Grev.) Cleve (Bacillariophyceae) was investigated. The diatom was grown in pH-regulated batch cultures with a 14:10-h light:dark cycle until N depletion. In a pulse-chase experiment, the cells were first incubated in high light (200 μmol photons·m ^{− 2}·s ^{− 1}) with ¹⁴C-bicarbonate until dissolved inorganic carbon was exhausted. Unlabeled bicarbonate (1 mM) was then added, and the cells were incubated in the dark and subsequently in low light (20 μmol photons·m ^{− 2}·s ^{− 1}) with additions of 40 μM NH₄ ⁺ . In the ¹⁴C pulse phase with high light and N depletion, β-1,3-glucan accumulated and accounted for 85% of incorporated ¹⁴C. In the subsequent ¹⁴C chase phases, added NH₄ ⁺ was assimilated at an N-specific rate of 0.11 h ^{− 1} in both the dark and low light, and in both cases it caused a significant mobilization of β-1,3-glucan (dark, 26%; low light, 19%). Biochemical fractionation of organic ¹⁴C showed that free amino acids were most rapidly labeled in the early stage of NH₄ ⁺ assimilation, whereas proteins and polysaccharides were labeled more rapidly after 1.2 h. Analysis of the cellular free amino acids strongly indicated that de novo biosynthesis was occurring, with a Gln:Glu ratio increasing from 0.4 to 10 within 1.2 h. After the NH₄ ⁺ was exhausted, the cellular pools of glucan and amino acids became constant or slowly decreased. In another experiment, N-limited cells were first incubated in high light until dissolved inorganic carbon was exhausted and were further incubated in high light with 150 μM NH₄ ⁺ under inorganic carbon limitation. Added NH₄ ⁺ was assimilated at an N-specific rate of 0.023 h ^{− 1}, and cellular β-1,3-glucan decreased by 15% within 6 h. Hence, β-1,3-glucan was mobilized during NH₄ ⁺ assimilation, even though inorganic carbon was modifying the metabolic rates. The results provide new evidence of β-1,3-glucan supplying essential precursors for biosynthesis of amino acids and other components in S. costatum in both the dark and subsaturating light and even saturating light under inorganic carbon limitation.     

Biosynthesis of dermatan sulphate. Assay and properties of the uronosyl C-5 epimerase.

During biosynthesis of dermatan sulphate D-glucuronate (GlcA) residues are converted to L-iduronate (IdoA) residues via the reaction [Formula: see text]. The reaction occurs on the polymer level and is catalysed by a C-5 uronosyl epimerase. The reversible release of the C-5 hydrogen was utilized as a measure of the enzyme activity with 5-3H-labelled chondroitin as a substrate. 3H released during incubation was distilled and quantified by liquid-scintillation counting. The epimerase has a low pH optimum (5.6) and requires divalent cations, Mn2+ being the most efficient for activity. The Km for chondroitin is 1.2 x 10(-4) M. The epimerase is largely associated with the microsomal fractions (90%). Two-thirds of the activity can be solubilized by detergents. Microsomes from cultured fibroblasts contain two different uronosyl epimerases, one for the biosynthesis of heparan sulphate and one for that of dermatan sulphate. The two epimerases have different cofactor and pH requirements.     

Bi-directional chiral inversion of ketoprofen in CD-1 mice

The R enantiomers of some of the 2-arylpropionic acid non-steroidal antiinflammatory drugs (NSAIDs) are known to undergo metabolic chiral inversion to their more pharmacologically active antipodes. This process is drug and species dependent and usually unidirectional. The S to R chiral inversion, on the other hand, is rare and has been observed, in substantial extents, only for ibuprofen in guinea pigs and 2-phenylpropionic acid in dogs. After i.p. administration of single doses of racemic ketoprofen or its optically pure enantiomers to male CD-1 mice and subsequent study of the concentration time-course of the enantiomers, we noticed substantial chiral inversion in both directions. Following racemic doses, no stereoselectivity in the plasma-concentration time courses was observed. After dosing with optically pure enantiomer, the concentration of the administered enantiomer predominated during the absorption phase. During the terminal elimination phase, however, the enantiomers had the same concentrations. Our observation is suggestive of a rapid and reversible chiral inversion for ketoprofen enantiomers in mice. Chirality 9:29–31, 1997. © 1997 Wiley-Liss, Inc.     

Effective depletion of albumin using a new peptide-based affinity medium

Blood plasma and serum are very useful samples for the detection, identification and quantitation of proteins associated with both health and disease. However, analysis of plasma and serum is a challenge because traces of interesting polypeptides and proteins can be dominated by the very high concentration of albumin present. Albumin may be depleted by adsorption to immunoaffinity columns or to columns containing dyes such as Cibacron Blue, or by ultrafiltration, but these methods are far from ideal. We describe a new peptide-based affinity medium which is effective for removing albumin and is very specific. The albumin-binding capacity is at least 14 mg per mL of gel. The material may be reused hundreds of times after a simple regeneration step involving NaOH, with full retention of specificity and capacity. The material was tested with human and monkey plasma and serum and rat serum, and has been used to deplete litre volumes of human plasma. The development of other peptide-based affinity media to deplete abundant proteins is briefly discussed.