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[Evoked potentials in the reticular formation of the rabbit midbrain during formation of a dominant focus in it].

During creation of a dominant focus in the midbrain reticular formation (RF) by its multiple stimulation with a high-frequency current (stimulation frequency 200 Hz, pulse duration 0.1-0.5 ms, voltage 1-3 V, duration 5 s) a statistically significant increase of the amplitude of the evoked potential (EP) in RF to light flashes was revealed in comparison with background data. Significant increase of EP amplitude was also observed in RF in response to the same stimuli applied in successive experiments without RF stimulation, which pointed to the existence of a latent dominant focus in the CNS.

Animals↗

Formation of glycine conjugate and (-)-(R)-enantiomer from (+)-(S)-2-phenylpropionic acid suggesting the formation of the CoA thioester intermediate of (+)-(S)-enantiomer in dogs.

It has been proposed that the chiral inversion of the 2-arylpropionic acids is due to the stereospecific formation of the (-)-R-profenyl-CoA thioesters which are putative intermediates in the inversion. Accordingly, amino acid conjugation, for which the CoA thioesters are obligate intermediates, should be restricted to those optical forms which give rise to the (-)-R-profenyl-CoA, i.e., the racemates and the (-)-(R)-isomers. We have examined this problem in dogs with respect to 2-phenylpropionic acid(2-PPA). Regardless of the optical configuration of 2-phenylpropionic acid administered, the glycine conjugate was the major urinary metabolite and this was shown to be exclusively the (+)-(S)-enantiomer by chiral HPLC. Both (-)-(R)- and (+)-(S)-2-phenylpropionic acid were present in plasma after the administration of either antipode, and further evidence of the chiral inversion of both enantiomers was provided by the presence of some 25% of the opposite enantiomer in the free 2-phenylpropionic acid and its glucuronide excreted in urine after administration of (-)-(R)- and (+)-(S)-2-phenylpropionic acid. The (+)-(S)-enantiomer underwent chiral inversion to the (-)-(R)-antipode when incubated with dog hepatocytes. These data suggests that both enantiomers of 2-phenylpropionic acid are substrates for canine hepatic acyl CoA ligase(s) and thus undergo chiral inversion, but that the CoA thioester of only (+)-(S)-2-phenylpropionic acid is a substrate for the glycine N-acyl transferase. These studies are presently being extended to the structure and species specificity of the reverse inversion and amino acid conjugation of profen NSAIDs.

Animals↗

High-throughput synthesis of conopeptides: a safety-catch linker approach enabling disulfide formation in 96-well format.

Conotoxins exhibit a high degree of selectivity and potency for a range of pharmacologically relevant targets. The rapid access to libraries of conotoxin analogues, containing multiple intramolecular disulfide bridges for use in drug development, can be a very labor intensive, multi-step task. This work describes a high-throughput method for the synthesis of cystine-bridged conopeptides. Peptides were assembled on a peptide synthesizer employing the Fmoc solid-phase strategy using a safety-catch amide linker (SCAL). Side-chain protecting groups were removed on solid phase before SCAL activation with ammonium iodide in TFA, finally releasing the peptide into the TFA solution. Disulfide bond formation was performed in the cleavage mixture employing DMSO. This improved method allows mixtures of oxidized peptides to be obtained in parallel directly from a peptide synthesizer. A single HPLC purification of the resulting crude oxidized material produced peptides of > 95% purity.

Acrylic Resins↗

Competition for factors and cellular resources as a principle of pattern formation in Hydra. I. Increase of the potentials for head and bud formation and rescue of the regeneration-deficient mutant reg-16 by treatment with diacylglycerol and arachidonic acid.

The mutant strain reg-16 of Hydra magnipapillata has less capacity to regenerate a head than the wild-type strain 105 when examined in a standard test. The present comparative study with various strains of Hydra shows: (1) The head-forming potential of reg-16 and also of a widely used strain of Hydra vulgaris is severely impaired by the presence of a just-emerging or latent bud. (2) Periodic treatment with dioctanoylglycerol (DAG), a known activator of protein kinase C (PKC), plus arachidonic acid (AA), the mother compound of the eicosanoid signal substances, enabled reg-16 and H. vulgaris to insert additional tentacles into their original whorl while the export of cells by budding was reduced. (3) On the other hand, during the period of daily treatment with DAG+AA, potentials for head and bud formation were stored. Gastric segments excised from pretreated animals formed more tentacles than untreated wild-type 105 and even formed supernumerary head structures; in addition, segments excised from all body regions quickly resumed budding in spite of starvation and while they regenerated a head. (4) With continued treatment supernumerary tentacles were elicited in all strains although with high frequency only in Hydra magnipapillata, wt105. However, while wt105 formed supernumerary head structures preferentially at ectopic sites, reg-16, H. vulgaris, and Hydra oligactis instead increased the number of tentacles in the apical head, which occasionally split into two heads. (5) The treatment with DAG+AA caused the budding zone to shift closer to the foot end. (6) In H. vulgaris a pulse treatment with a high dose of DAG frequently caused a mirror image duplication of the budding zone; in wt105 mere excessive feeding over 2-3 weeks may have the same effect. The hypothesis is proposed that a regenerating head and a beginning bud compete for hormone-like factors, which enable the cells to increase positional value, and for precursor cells. Periodic treatment with activators of PKC plus AA leads to an augmentation of these resources, and head structures and buds can be produced simultaneously. Traditional terms are reinterpreted correspondingly: the high level of "head inhibition" in reg-16 is interpreted as a low level of resources, in particular, of head-promoting factors, and the low head activation level as a low ability to make use of resources.

Animals↗

Formation of hydrogen bonds precedes the rate-limiting formation of persistent structure in the folding of ACBP.

A burst phase in the early folding of the four-helix two-state folder protein acyl-coenzyme A binding protein (ACBP) has been detected using quenched-flow in combination with site-specific NMR-detected hydrogen exchange. Several of the burst phase structures coincide with a structure consisting of eight conserved hydrophobic residues at the interface between the two N and C-terminal helices. Previous mutation studies have shown that the formation of this structure is rate limiting for the final folding of ACBP. The burst phase structures observed in ACBP are different from the previously reported collapsed types of burst phase intermediates observed in the folding of other proteins.

Amides↗

Nitrogen oxides and hydroxyguanidines: formation of donors of nitric and nitrous oxides and possible relevance to nitrous oxide formation by nitric oxide synthase.

The involvement of nitric oxide in numerous biological functions has led to the intense study of nitric oxide (NO) generation by the nitric oxide synthases (NOS) responsible. In addition to NO, nitric oxide synthases produce N(G)-hydroxy-L-arginine, superoxide anion and, indirectly, NOx species such as peroxynitrite and, possibly, nitrous oxide (N2O). Consequently, the interactions of N(G)-hydroxy-L-arginine with NO and other oxides of nitrogen (NOx) are of considerable interest. N(G)-Hydroxy-L-arginine and other monosubstituted hydroxyguanidines react with aqueous aerobic NO, peroxynitrite, and various NOx and nitrosating agents to form compounds that subsequently release NO and N2O. Spectrometric data indicate that the nitrosation product of N(G)-hydroxy-L-arginine is of the same N-nitroso-N-hydroxy/diazeniumdiolate (formerly "NONOate") structure as previously found for the nitrosation products of other model hydroxyguanidines. These decompose in aqueous solution in a pH-dependent manner to yield mainly NO and ureas at low pH, N2O and cyanamides at basic pH, and what appear to be primary nitrosamines/ nitrosoimines. Studies on purified iNOS using a mass spectrometer with a gas-permeable membrane inlet identified both NO and N2O (or 15NO and 15N15NO with 15N-labeled L-arginine as substrate) as products of NOS activity. These experiments suggest that much more NO than N2O is produced under the conditions studied and that N2O formation can be rationalized via the reaction of NOx species with N(G)-hydroxy-L-arginine.

Arginine↗

A contribution to the formation mechanism of calcium oxalate urinary calculi. III. On the role of magnesium in the formation of oxalate calculi.

The influence of magnesium in vitro on the precipitation of calcium oxalate was investigated. Even at maximum physiological magnesium concentrations a litholytic effect could not be observed, but the retardation of the calcium oxalate crystallization caused by magnesium might be decisive for a reduction in calculi formation. The enlargement of the calcium oxalate crystals and aggregates caused by the retardation of crystallization, however, should be regarded as a contraindicating factor for Mg therapy in oxalate calculous disease. It is safe to say that high magnesium concentrations prevent the conversion into Whewellite of the calcium oxalate calculi substance primarily formed as Weddellite.

Calcium↗

Prevention and treatment of linear scar formation in the scalp: basic principles of the mechanism of scar formation.

Linear scar formation in the scalp after suturing an incision has been considered unavoidable. It was not known why scars formed even if the hair bulb was left intact. The authors developed a subcutaneous tissue-shaving method for radical treatment of bromidrosis and studied the process of hair regeneration by using thick-tissue specimens. They suggest that stem cells (lower) are located not only in the lower end of the telogen hair follicles but also in the sebaceous isthmus at the secretory opening of the sebaceous gland (upper stem cells). They found that linear scars can be prevented and existing linear scars can be surgically treated by using a relaxed suture on a scalp incision to avoid excessive pressure on the upper stem cells.

Adult↗

Chemiluminescent measurement of increased free radical formation after ischemia/reperfusion. Mechanisms of free radical formation in the liver.

It has been proposed that xanthine oxidase-derived superoxide mediates reperfusion injury in the liver; however, there is a little direct evidence to support this hypothesis. In this paper we describe a model system to directly and noninvasively measure oxyradical formation and hepatic injury in isolated perfused rat liver. Using this sensitive chemiluminescent technique, we clearly demonstrate the theorized burst in oxygen radical production upon reperfusion of previously ischemic liver, without perturbing the system with chemical luminescence enhancers. This increase in chemiluminescence (CL) upon reperfusion was diminished by the free radical scavengers trolox and ascorbate, as well as N-2-mercaptoproprionyl-glycine (MPG), thereby confirming the oxyradical nature of this signal. Additionally, superoxide dismutase and the xanthine oxidase inhibitor allopurinol, but not catalase, attenuated the reperfusion effect, providing the most direct evidence so far that XOD derived superoxide anion is formed during liver reperfusion. Hepatic injury (AST release) did not appear to relate to increased CL, supporting the notion that the oxyradical flux may serve as a signal for other events leading to tissue injury. Further studies using this sensitive chemiluminescent technique should aid in delineating the detailed mechanism(s) of reperfusion injury.

Animals↗

Experimental glomerular lesions induced by chronic immune complex formation. I. Formation and elimination of the immune complex (relationship between the immune status and the glomerular changes).

Chronic immune complex formation was induced in rabbits by daily administration of 12.5 g bovine serum. In good antibody producer animals immediate immune complex production and elimination from the circulation were demonstrable. This was followed within a few minutes by the appearance of free 125I in fairly large amounts in blood, as a sign of immediate phagocytosis and disintegration of the 125I-labelled immune complexes. Phagocytic activity decreased in the host animal during chronic heteroprotein administration in every case. The earliest glomerular changes were those of exudative glomerulonephritis, the extent of which depended on the antibody productivity of the animal. Persistent immunocomplexaemia induced by administration of the antigen over 60 and 100 days, respectively, resulted in mesangioproliferative glomerulonephritis in 7, in membranoproliferative glomerulonephritis in 3, and in membraneous glomerulonephritis in 1 out of 11 laboratory animals.

Animals↗

Ammonia formation by the reduction of nitrite/nitrate by FeS: ammonia formation under acidic conditions.

One issue for the origin of life under a non-reducing atmosphere is the availability of the reduced nitrogen necessary for amino acids, nucleic acids, etc. One possible source of this nitrogen is the formation of ammonia from the reduction of nitrates and nitrites produced by the shock heating of the atmosphere and subsequent chemistry. Ferrous ions will reduce these species to ammonium, but not under acidic conditions. We wish to report results on the reduction of nitrite and nitrate by another source of iron (II), ferrous sulfide, FeS. FeS reduces nitrite to ammonia at lower pHs than the corresponding reduction by aqueous Fe+ 2. The reduction follows a first order decay, in nitrite concentration, with a half-life of about 150 min (room temperature, CO2, pH 6.25). The highest product yield of ammonia measured was 53%. Under CO2, the product yield decreases from pH 5.0 to pH 6.9. The increasing concentration of bicarbonate, at higher pH, interferes with the reaction. Comparing experiments under N2 CO2 shows the interference of bicarbonate. The reaction proceeds well in the presence of such species as chloride, sulfate, and phosphate, though the yield drops significantly with phosphate. FeS also reduces nitrate and, unlike with Fe+ 2, the reduction shows more reproducibility. Again, the product yield decreases with increasing pH, from 7% at pH 4.7 to 0% at pH 6.9. It appears that nitrate is much more sensitive to the presence of added species, perhaps not competing as well for binding sites on the FeS surface. This may be the cause of the lack of reproducibility of nitrate reduction by Fe+ 2 (which also can be sensitive to binding by certain species).

Ammonia↗

A reexamination of the melanin formation assay of tyrosinase and an extension to estimate phaeomelanin formation.

This paper presents some modifications of the melanin formation assay for tyrosinase from the point of view of both eu- and phaeomelanosynthesis. On the one hand, eumelanosynthesis can be estimated using neutral paper filters, such as the 3MM Whatman filters so far employed. The main advantages of this sort of paper are the very low blank values obtained in the absence of tyrosinase and its greater mechanical resistance in the successive washing steps. It is shown that the sensitivity of the assay can be enhanced by the addition of 1 mM Ni(II) to the incubation mixture or of NaOH to stop the enzymatic reaction and allow the incorporation of indolic intermediates into the polymer. Furthermore, the accuracy is also enhanced by the proposed modifications, since all reactions from dopaquinone are standardized, and the assay becomes only dependent on the tyrosinase activity. On the other hand, phaeomelanosynthesis cannot be estimated using neutral paper because of the slow rate of polymerization of the intermediates and the poor absorption of thiol-dopa conjugates to this kind of paper. It is shown that synthesis of this type of melanin can be estimated in the presence of glutathione by means of a cationic filter paper and by washing the excess of the radioactive substrate with distilled water instead of acidic media. Thus, the assay may be adapted to measure eu- or phaeomelanosynthetic activity by introducing slight modifications. This assay must be used with caution if detergent-solubilized tyrosinase is used, because detergents strongly inhibit melanin absorption to paper filters.

Animals↗

Berberine inhibits rat vascular smooth muscle cell proliferation and migration in vitro and improves neointima formation after balloon injury in vivo. Berberine improves neointima formation in a rat model.

Berberine, an alkaloid isolated from Chinese medicinal herbs, long been known for its anti-microbial activity and used to treat various infectious disorders in traditional Chinese medicine. In the present study, we have tested the hypothesis that berberine could inhibit vascular smooth muscle cell (VSMC) proliferation as it did in endothelial cells or cancer cells. Our results show that berberine significantly inhibits growth factor, mainly angiotensin II (AngII) and heparin binding epidermal growth factor (HB-EGF), induced VSMC proliferation and migration in vitro, and this effect is achieved by delaying or partially suppressing activation of Akt pathway rather than ERK pathway. Furthermore, we have examined its effect in vivo using a rat carotid artery injury model. A 28 days of chronic berberine treatment using an osmotic pump (100 microg kg(-1)d(-1), 2 weeks before and 2 weeks after the injury) improved neointima formation. The Neointima/Media ratio for control group and berberine treated group were 1.14+/-0.11 and 0.85+/-0.06 (p<0.05), respectively, and the reduction was approximately 25%. The result of the present study suggests a possibility of berberine being a potent agent to control restenosis after balloon angioplasty and warrants further study to gain a more complete understanding of its underlying mechanisms at a cellular level.

Angioplasty, Balloon↗

Characterisation of complex formation between members of the Mycobacterium tuberculosis complex CFP-10/ESAT-6 protein family: towards an understanding of the rules governing complex formation and thereby functional flexibility.

We have previously shown that the secreted M. tuberculosis complex proteins CFP-10 and ESAT-6 form a tight, 1:1 complex, which may represent their functional form. In the work reported here a combination of yeast two-hybrid and biochemical analysis has been used to characterise complex formation between two other pairs of CFP-10/ESAT-6 family proteins (Rv0287/Rv0288 and Rv3019c/Rv3020c) and to determine whether complexes can be formed between non-genome paired members of the family. The results clearly demonstrate that Rv0287/Rv0288 and Rv3019c/3020c form tight complexes, as initially observed for CFP-10/ESAT-6. The closely related Rv0287/Rv0288 and Rv3019c/Rv3020c proteins are also able to form non-genome paired complexes (Rv0287/Rv3019c and Rv0288/Rv3020c), but are not capable of binding to the more distantly related CFP-10/ESAT-6 proteins.

Antigens, Bacterial↗

Inhibitory activity of kinetin on free radical formation of activated platelets in vitro and on thrombus formation in vivo.

Kinetin has been shown to have anti-aging effects on several different systems, including plants and human cells. Recently, we demonstrated that kinetin markedly inhibited platelet aggregation in washed human platelets. In the present study, an electron spin resonance (ESR) method was used to further evaluate the scavenging activity of kinetin on the free radicals formed. Kinetin (70 and 150 microM) concentration dependently reduced the ESR signal intensity of hydroxyl radicals in collagen (1 microg/ml)-activated platelets. Furthermore, kinetin was effective in reducing the mortality of ADP-induced acute pulmonary thromboembolism in mice when administered intravenously at doses of 4 and 6 mg/kg. In addition, intravenous injection of kinetin (4 and 6 mg/kg) significantly prolonged the bleeding time by approximately 1.9- and 2.1-fold as compared with normal saline in severed mesenteric arteries of rats. A continuous infusion of kinetin (0.6 mg/kg/min) for 10 min also significantly increased the bleeding time by about 2.3-fold, and the bleeding time returned to baseline within 120 min after cessation of kinetin infusion. Platelet thrombi formation was induced by irradiation of mesenteric venules with filtered light in mice pretreated intravenously with fluorescein sodium. When kinetin was administered at 13 and 14 mg/kg in mice pretreated with fluorescein sodium (5 mg/kg), the occlusion time was significantly prolonged. In conclusion, these results suggest that kinetin has effective free radical-scavenging activity in vitro and antithrombotic activity in vivo. Treatment with kinetin may lower the risk of thromboembolic-related disorders. Therefore, kinetin may be a potential therapeutic agent for arterial thrombosis, but its toxicity must be further assessed.

Adenine↗

Trace elements and urinary stone formation: new aspects of the pathological mechanism of urinary stone formation.

The urinary stone, serum and 24-hour urine concentrations of 14 trace elements were determined in urinary stone patients by inductively coupled plasma atomic-emission spectroscopy. The data obtained for 25 active stone patients and 32 whose last stone episode had occurred at least 12 months previously were compared with those of 25 healthy individuals. Urinary nickel, manganese and lithium excretion, and serum nickel, manganese and cadmium concentrations were statistically significantly lower for active stone patients compared to those with previous stone episodes and healthy individuals. No difference in the concentrations of trace elements could be found, however, for patients with previous stone episodes and healthy individuals. Nickel, manganese, lithium and cadmium could be of significance in the pathological mechanism of stone formation, not from mineralogical or crystallographic viewpoints but for the smooth flow of enzymatic reactions in biological systems.

Adult↗

Secondary organic aerosol formation by glyoxal hydration and oligomer formation: humidity effects and equilibrium shifts during analysis.

Glyoxal is a significant atmospheric aldehyde formed from both anthropogenic aromatic compounds and biogenic isoprene emissions. The chemical behavior of glyoxal relevant to secondary organic aerosol (SOA) formation and analysis is examined in GC-MS, electrospray ionization (ESI)-MS, and particle chamber experiments. Glyoxal oligomers are shown to rapidly decompose to glyoxal in GC injection ports at temperatures > or = 120 degrees C. Glyoxal dihydrate monomer is dehydrated at temperatures > or = 140 degrees C during GC analysis but shows only oligomers (n < or = 7) upon ESI-MS analysis. Thus both of these analytical techniques will cause artifacts in speciation of glyoxal in SOA. In particle chamber experiments, glyoxal (at -0.1 Torr) condensed via particle-phase reactions when relative humidity levels exceeded a threshold of -26%. Both the threshold humidity and particle growth rates (-0.1 nm/min) are consistent with a recent study performed at glyoxal concentrations 4 orders of magnitude below those used here. This consistency suggests a mechanism where the surface water layer of solid-phase aerosol becomes saturated with glyoxal dihydrate monomer, triggering polymerization and the establishment of an organic phase.

Aerosols↗

Alpha,2-, alpha,3-, and alpha,4-dehydrophenol radical anions: formation, reactivity, and energetics leading to the heats of formation of alpha,2-, alpha,3-, and alpha,4-oxocyclohexadienylidene.

We have regiospecifically generated the alpha,2-, alpha,3-, and alpha,4-dehydrophenoxide anions by collisional activation of o-, m-, and p-nitrobenzoate. The alpha,2 and alpha,4 isomers also were synthesized by reacting o-benzyne radical anion with carbon dioxide and electron ionization of p-diazophenol. All three dehydrophenol radical anions were differentiated from each other and identified by probing their chemical reactivity with several reagents. Each isomer was converted to phenoxide and its corresponding quinone as well. Thermochemical measurements were carried out on all three radical anions and their hydrogen-atom affinities, proton affinities, and electron binding energies are reported. These measured quantities are combined in thermodynamic cycles to derive the heats of formation of each of the radical anions and their corresponding carbenes (i.e., alpha,2-, alpha,3-, and alpha,4-dehydrophenol). These results are compared to MCQDPT2, G3, G2+(MP2), and B3LYP calculations and experimental data for appropriate reference compounds.

Journal Article↗