Counterion binding and hydration of hyaluronate and chondroitin in solution: An 17O, 23Na,and 25Mg nuclear-magnetic-relaxation study

Nuclear magnetic relaxation rates of H₂¹⁷O, ²³Na⁺, and ²⁵Mg²⁺ have been measured in aqueous hyaluronate solutions. The dependence on solution pH of the relaxation rates has been investigated, as well as the competition behavior of Na⁺ with Ca²⁺ and Mg²⁺. H₂¹⁷O and ²³Na⁺ relaxation rates in chondroitin and hyaluronate solutions have been compared in the interval, 2 ? pH ? 12.5. The ion binding of hyaluronate can be fully accounted for by Coulomb interactions, with no need to involve chemical specificity. The hydration is only slighly pH dependent, and is comparable in magnitude to hydration of synthetic polyelectrolytes and monosaccharides. Ion-binding and hydration properties of hyaluronate and chondroitin are quite similar, except at elevated pH. At alkaline pH, an increase in charge density with pH is seen in hyaluronate and, to a much lesser degree, in chondroitin, possibly due to the titration of hydroxy groups. H₂¹⁷O data indicate an alkali-induced transition in both glycosaminoglycans.     

Immunotherapy of human colon cancer by antibody-targeted superantigens

T lymphocytes generally fail to recognize human colon carcinomas, suggesting that the tumour is beyond reach of immunotherapy. Bacterial superantigens are the most potent known activators of human T lymphocytes and induce T cell cytotoxicity and cytokine production. In order to develop a T-cell-based therapy for colon cancer, the superantigen staphylococcal enterotoxin A (SEA) was given tumour reactivity by genetic fusion with a Fab fragment of the monoclonal antibody C242 reacting with human colon carcinomas. The C242Fab-SEA fusion protein targeted SEA-reactive T cells against MHC-class-II-negative human colon carcinoma cells in vitro at nanomolar concentrations. Treatment of disseminated human colon carcinomas growing in humanized SCID mice resulted in marked inhibition of tumour growth and the apparent cure of the animals. Therapeutic efficiency was dependent on the tumour specificity of the fusion protein and human T cells. Immunohistochemistry demonstrated massive infiltration of human T cells in C242Fab-SEA-treated tumours. The results merit further evaluation of C242Fab-SEA fusion proteins as immunotherapy in patients suffering from colon carcinoma.     

Crystal structure of the HIV-2 neutralizing Fab fragment 7C8 with high specificity to the V3 region of gp125

7C8 is a mouse monoclonal antibody specific for the third hypervariable region (V3) of the human immunodeficiency virus type 2 (HIV-2)-associated protein gp125. The three-dimensional crystal structure of the Fab fragment of 7C8, determined to 2.7 Å resolution, reveals a deep and narrow antigen-binding cleft with architecture appropriate for an elongated epitope. The highly hydrophobic cleft is bordered on one side by the negatively charged second complementarity determining region (CDR2) and the unusually long positively charged CDR3 of the heavy chain and, on the other side, by the CDR1 of the light chain. Analysis of 7C8 in complex with molecular models of monomeric and trimeric gp125 highlights the importance of a conserved stretch of residues FHSQ that is localized centrally on the V3 region of gp125. Furthermore, modeling also indicates that the Fab fragment neutralizes the virus by sterically impairing subsequent engagement of the gp125 trimer with the co-receptor on the target cell.     

Chylomicron formation and glucagon-like peptide 1 receptor are involved in activation of the nutritional anti-inflammatory pathway

Enteral administration of lipid-enriched nutrition effectively attenuates inflammation via a cholecystokinin (CCK)-mediated vagovagal anti-inflammatory reflex. Cholecystokinin release and subsequent activation of the vagus are dependent on chylomicron formation and associated with release of additional gut peptides. The current study investigates the intestinal processes underlying activation of the CCK-mediated vagal anti-inflammatory pathway by lipid-enriched nutrition. Rats and mice were subjected to hemorrhagic shock (HS) or endotoxemia, respectively. Prior to the experimental procedures, animals were fasted or fed lipid-enriched nutrition. Pluronic L-81 (L-81) was added to the feeding to investigate involvement of chylomicron formation in activation of mesenteric afferent fibers and the immune-modulating potential of lipid-enriched nutrition. Ob/Ob mice and selective receptor antagonists were used to study the role of leptin, glucagon-like peptide 1 and peptide YY in activation of the nutritional reflex. Electrophysiological analysis of mesenteric afferents in mice revealed that lipid-enriched nutrition-mediated neural activation was abrogated by L-81 (P<.05). L-81 blunted the beneficial effects of lipid-enriched nutrition on systemic inflammation and intestinal integrity in both species (all parameters, P<.01). Ob/Ob mice required a higher dose of nutrition compared with wild-type mice to attenuate plasma levels of TNF-α and ileum-lipid binding protein, a marker for enterocyte damage (both P<.01), suggesting a higher stimulation threshold in leptin-deficient mice. Administration of a glucagon-like peptide 1-receptor antagonist, but not leptin or peptide YY antagonists, suppressed the effects of lipid-enriched nutrition. These data indicate that chylomicron formation is essential and activation of the glucagon-like peptide 1-receptor is involved in activation of the nutritional anti-inflammatory pathway by lipid-enriched nutrition.     

Effects of training at simulated altitude on performance and muscle metabolic capacity in competitive road cyclists

Differences between the effects of training at sea level and at simulated altitude on performance and muscle structural and biochemical properties were investigated in 8 competitive cyclists who trained for 3-4 weeks, 4-5 sessions/week, each session consisting of cycling for 60-90 min continuously and 45-60 min intermittently. Four subjects, the altitude group (AG), trained in a hypobaric chamber (574 torr = 2300 m above sea level), and the other four at sea level (SLG). Before and after training work capacity was tested both at simulated altitude (574 torr) and at sea level, by an incremental cycle ergometer test until exhaustion. Work capacity was expressed as total amount of work performed. Venous blood samples were taken during the tests. Leg muscle biopsies were taken at rest before and after the training period. AG exhibited an increase of 33% in both sea level and altitude performance, while SLG increased 22% at sea level and 14% at altitude. Blood lactate concentration at a given submaximal load at altitude was significantly more reduced by training in AG than SLG. Muscle phosphofructokinase (PFK) activity decreased with training in AG but increased in SLG. All AG subjects showed increases in capillary density. In conclusion, work capacity at altitude was increased more by training at altitude than at sea level. Work capacity at sea level was at least as much improved by altitude as by sea level training. The improved work capacity by training at altitude was paralleled by decreased exercise blood lactate concentration, increased capillarization and decreased glycolytic capacity in leg muscle.     

Naming progastrin-derived peptides

The antral hormone gastrin continues to be in focus, because its hormonal and growth promoting effects are essential both for the function of the normal stomach and for the pathogenesis of major dyspeptic and neoplastic diseases. Deduction of the progastrin structure has improved the insight in the cellular synthesis of gastrin, but has also revealed that the biosynthetic machinery is complex, and, accordingly, that progastrin is processed to a multitude of more or less bioactive fragments. The naming of these fragments has, however, become inconsistent and confusing. Therefore, we propose a systematic nomenclature for progastrin-derived peptides of which there are three classes: (I) The gastrins with the evolutionary preserved tetrapeptide amide (Trp-Met-Asp-PheNH₂) at the C-terminus, which ensures high-affinity binding to the gastrin (CCK-B) receptor. Among the gastrins, gastrin-34 and gastrin-17 constitute the primary forms. (II) Processing intermediates, which are early products of progastrin that contain the structure of the primary gastrins within their sequence, but still cannot bind the gastrin receptor due to insufficient processing at their C-terminus. (III) Flanking fragments from the N- and C-termini of progastrin that do not contain any primary gastrin in their sequence, but nevertheless may undergo posttranslational processing. Each fragment can be specified with suffixes corresponding to the derived sequence in progastrin.     

Inflammation in neurodegenerative disease--a double-edged sword

Inflammation is a defense reaction against diverse insults, designed to remove noxious agents and to inhibit their detrimental effects. It consists of a dazzling array of molecular and cellular mechanisms and an intricate network of controls to keep them in check. In neurodegenerative diseases, inflammation may be triggered by the accumulation of proteins with abnormal conformations or by signals emanating from injured neurons. Given the multiple functions of many inflammatory factors, it has been difficult to pinpoint their roles in specific (patho)physiological situations. Studies of genetically modified mice and of molecular pathways in activated glia are beginning to shed light on this issue. Altered expression of different inflammatory factors can either promote or counteract neurodegenerative processes. Since many inflammatory responses are beneficial, directing and instructing the inflammatory machinery may be a better therapeutic objective than suppressing it.     

Combined Therapeutic Effect of Dietary Selenium and Vitamin Ε on Manifested Vesd Syndrome in Weaned Pigs

By using a therapeutic dietary supplementation in pigs, which had developed the vitamin Ε and selenium deficiency (VESD) syndrome, the same amounts of α-tocopheryl acetate and selenium were found to be effective as under prophylactic conditions. The experiment thus supported the conclusions that the addition of 5 mg DL-α-tocopheryl acetate/kg and 135 μg selenium/kg to a diet, which contained only traces of vitamin Ε and selenium, represents a level of minimal requirement. Glutathione peroxidase activity in blood serum was used to evaluate the selenium status in pigs. A modified method for determination of tocopherol in fat tissue was described. The addition of 15 mg α-tocopheryl acetate/kg diet was demonstrated to be sufficient to maintain the tocopherol stores in body fat at an unchanged level.     

5/6th Nephrectomy in Combination with High Salt Diet and Nitric Oxide Synthase Inhibition to Induce Chronic Kidney Disease in the Lewis Rat

Chronic kidney disease (CKD) is a global problem. Slowing CKD progression is a major health priority. Since CKD is characterized by complex derangements of homeostasis, integrative animal models are necessary to study development and progression of CKD. To study development of CKD and novel therapeutic interventions in CKD, we use the 5/6th nephrectomy ablation model, a well known experimental model of progressive renal disease, resembling several aspects of human CKD. The gross reduction in renal mass causes progressive glomerular and tubulo-interstitial injury, loss of remnant nephrons and development of systemic and glomerular hypertension. It is also associated with progressive intrarenal capillary loss, inflammation and glomerulosclerosis. Risk factors for CKD invariably impact on endothelial function. To mimic this, we combine removal of 5/6th of renal mass with nitric oxide (NO) depletion and a high salt diet. After arrival and acclimatization, animals receive a NO synthase inhibitor (NG-nitro-L-Arginine) (L-NNA) supplemented to drinking water (20 mg/L) for a period of 4 weeks, followed by right sided uninephrectomy. One week later, a subtotal nephrectomy (SNX) is performed on the left side. After SNX, animals are allowed to recover for two days followed by LNNA in drinking water (20 mg/L) for a further period of 4 weeks. A high salt diet (6%), supplemented in ground chow (see time line Figure 1), is continued throughout the experiment. Progression of renal failure is followed over time by measuring plasma urea, systolic blood pressure and proteinuria. By six weeks after SNX, renal failure has developed. Renal function is measured using ''gold standard'' inulin and para-amino hippuric acid (PAH) clearance technology. This model of CKD is characterized by a reduction in glomerular filtration rate (GFR) and effective renal plasma flow (ERPF), hypertension (systolic blood pressure>150 mmHg), proteinuria (> 50 mg/24 hr) and mild uremia (>10 mM). Histological features include tubulo-interstitial damage reflected by inflammation, tubular atrophy and fibrosis and focal glomerulosclerosis leading to massive reduction of healthy glomeruli within the remnant population (<10%). Follow-up until 12 weeks after SNX shows further progression of CKD.