Reactions of ferrous neuroglobin and cytoglobin with nitrite under anaerobic conditions

Recent evidence suggests that the reaction of nitrite with deoxygenated hemoglobin and myoglobin contributes to the generation of nitric oxide and S-nitrosothiols in vivo under conditions of low oxygen availability. We have investigated whether ferrous neuroglobin and cytoglobin, the two hexacoordinate globins from vertebrates expressed in brain and in a variety of tissues, respectively, also react with nitrite under anaerobic conditions. Using absorption spectroscopy, we find that ferrous neuroglobin and nitrite react with a second-order rate constant similar to that of myoglobin, whereas the ferrous heme of cytoglobin does not react with nitrite. Deconvolution of absorbance spectra shows that, in the course of the reaction of neuroglobin with nitrite, ferric Fe(III) heme is generated in excess of nitrosyl Fe(II)-NO heme as due to the low affinity of ferrous neuroglobin for nitric oxide. By using ferrous myoglobin as scavenger for nitric oxide, we find that nitric oxide dissociates from ferrous neuroglobin much faster than previously appreciated, consistently with the decay of the Fe(II)-NO product during the reaction. Both neuroglobin and cytoglobin are S-nitrosated when reacting with nitrite, with neuroglobin showing higher levels of S-nitrosation. The possible biological significance of the reaction between nitrite and neuroglobin in vivo under brain hypoxia is discussed.     

On peptide bond formation, translocation, nascent protein progression and the regulatory properties of ribosomes. Derived on 20 October 2002 at the 28th FEBS Meeting in Istanbul.

High-resolution crystal structures of large ribosomal subunits from Deinococcus radiodurans complexed with tRNA-mimics indicate that precise substrate positioning, mandatory for efficient protein biosynthesis with no further conformational rearrangements, is governed by remote interactions of the tRNA helical features. Based on the peptidyl transferase center (PTC) architecture, on the placement of tRNA mimics, and on the existence of a two-fold related region consisting of about 180 nucleotides of the 23S RNA, we proposed a unified mechanism integrating peptide bond formation, A-to-P site translocation, and the entrance of the nascent protein into its exit tunnel. This mechanism implies sovereign, albeit correlated, motions of the tRNA termini and includes a spiral rotation of the A-site tRNA-3' end around a local two-fold rotation axis, identified within the PTC. PTC features, ensuring the precise orientation required for the A-site nucleophilic attack on the P-site carbonyl-carbon, guide these motions. Solvent mediated hydrogen transfer appears to facilitate peptide bond formation in conjunction with the spiral rotation. The detection of similar two-fold symmetry-related regions in all known structures of the large ribosomal subunit, indicate the universality of this mechanism, and emphasizes the significance of the ribosomal template for the precise alignment of the substrates as well as for accurate and efficient translocation. The symmetry-related region may also be involved in regulatory tasks, such as signal transmission between the ribosomal features facilitating the entrance and the release of the tRNA molecules. The protein exit tunnel is an additional feature that has a role in cellular regulation. We showed by crystallographic methods that this tunnel is capable of undergoing conformational oscillations and correlated the tunnel mobility with sequence discrimination, gating and intracellular regulation.     

Biomechanical investigations of different osteosynthesis procedures in the proximal tibia-introduction of a new measuring system]

In this article a new measuring system for biomechanical studies of various osteosynthesis procedures on the tibia is described. The paired tibias are osteotomized and embedded in PMMA in accordance with a randomization protocol. Testing is then carried out under nonaxial loading at 350 N, 600 N and 900 N, uniaxial bending applying a force of 12 Nm, and torsion at a force of 5 Nm in a universal pneumatic testing machine. Ultrasonic sensors pick up movement at the fracture gap under external loading. Testing of the measuring setup has been successful, and relevant results were obtained.     

Expression and characterization of delayed rectifying K+ channels in anterior rat taste buds

Delayed rectifying K⁺ (DRK) channels in taste cells have been implicated in the regulation of cell excitability and as potential targets for direct and indirect modulation by taste stimuli. In the present study, we have used patch-clamp recording to determine the biophysical properties and pharmacological sensitivity of DRK channels in isolated rat fungiform taste buds. Molecular biological assays at the taste bud and single-cell levels are consistent with the interpretation that taste cells express a variety of DRK channels, including members from each of the three major subfamilies: KCNA, KCNB, and KCNC. Real-time PCR assays were used to quantify expression of the nine DRK channel subtypes. While taste cells express a number of DRK channels, the electrophysiological and molecular biological assays indicate that the Shaker Kv1.5 channel (KCNA5) is the major functional DRK channel expressed in the anterior rat tongue. transduction     

Development of humanized antibodies as cancer therapeutics

Recent success in the development of monoclonal antibody-based anti-cancer drugs has largely benefitted from the advancements made in recombinant technologies and cell culture production. These reagents, derived from the antibodies of mouse origin, while maintaining the exquisite specificity and affinity to the tumor antigens, have low immunogenicity and toxicity in human. High-level expressing cell clones are generated and used to produce large quantities of the recombinant antibodies in bioreactors in order to meet the clinical demand for therapeutic applications. In this report, the systems and general methodologies developed by us to construct and produce humanized antibodies from the parent mouse antibodies are described. Once the humanized antibodies are available, they can be applied in three principal forms for cancer therapy: (1) naked antibodies, (2) drug- or toxin conjugates, and (3) radioconjugates. Using the humanized anti-CD22 (epratuzumab) and anti-carcinoembryonic antigen (ant-CEA; labetuzumab) antibody prototypes, clinical applications of naked and radiolabeled humanized monoclonal antibodies are described.     

Modulation of carbohydrate metabolism and peptide hormones by soybean isoflavones and probiotics in obesity and diabetes

Soybean and its isoflavones have been shown to have beneficial effects on carbohydrate and lipid metabolism and on renal function. Probiotics may potentiate the beneficial effects of isoflavones by converting the inactive isoflavone glycoside to aglycones, which are biologically active, thereby producing a synergistic effect. We therefore studied the effects of soybean isoflavones in the presence and absence of probiotics on glucose and triglyceride metabolism and the peptide hormones involved in their metabolism. Lean and obese SHR/N-cp rats were fed AIN-93 diets containing 0.1% soybean isoflavone mixture, 0.1% probiotics mixture or both. Plasma was analyzed for glucose, triglycerides, parameters of renal function and peptide hormones -- insulin, leptin, glucagon and ACTH -- that are involved in glucose and lipid metabolism. Isoflavones given alone lowered plasma glucose in both phenotypes while triglyceride was decreased only in lean animals. Isoflavones also lowered aspartate amino transferase and alanine amino transferase in both phenotypes. Isoflavones had significant effect on plasma insulin, leptin and glucagon in lean rats but not in obese rats. Thus, our data show that in lean animals, isoflavones have hypoglycemic and hypolipidemic effect, and the effect is mediated by changes in peptide hormones. When lipid levels are very high as in obese rats, isoflavones fail to lower plasma triglyceride levels. Probiotics do not appear to enhance the effect of isoflavones.     

EB1627: a soluble prodrug of the potent anticancer cyanoguanidine CHS828

To overcome pharmacokinetic and solubility problems observed in early clinical trials with the potent anticancer compound CHS828, we synthesised a series of prodrugs with improved properties. The best compound obtained was EB1627, with a tetraethyleneglycol moiety attached to the parent drug via a carbonate linkage. This compound was found soluble enough to be given i.v. and the drug was rapidly released in vivo exerting a very potent inhibitory activity alone and in combination with known cytostatics (etoposide) in animal models in vivo.