The prognostic significance of micrometastases in node-negative squamous cell carcinoma of the vulva.

Nodal involvement is one of the most significant prognostic factors in squamous cell carcinoma (SCC) of the vulva. We conducted a retrospective analysis of 31 women with histologically node-negative SCC from a population-based cohort of Grampian women. Median follow-up was 42 months after radical vulvectomy with groin node dissection. In total, 13 women (42%) were found to have micrometastases on immunohistochemistry. The risk of recurrence was almost 20-fold higher in those with micrometastases compared to those without (hazard ratio=19.6 (95% CI 2.3-171).

Challenges and approaches to the robotic detection of enantioenrichment on Saturn's moon, Titan.

Saturn's moon, Titan, is one of the most interesting locations for organic chemistry in the solar system. As a possible follow up to the Cassini-Huygens mission, which will conduct the first in situ analysis of the Titan atmosphere in 2004, studies are underway for missions to explore in detail the chemical composition of Titan's atmosphere, surface, and oceans. In order to seek out and explore complex chemical systems that may represent steps on the pathway to life, instruments for detection of enantioenrichment should be included on such a mission. The formidable challenges of robotic measurement of enantioenrichment on Titan are summarized, and experimental approaches to this problem are reviewed. In addition, some speculations are offered concerning locations on Titan where complex fractionation of organic materials may have occurred.

Formation of highly enantioenriched microenvironments by stochastic sorting of conglomerate crystals: a plausible mechanism for generation of enantioenrichment on the prebiotic earth.

Abiotic generation of local areas of enantioenrichment is to be expected whenever one deals with the 5-10% of organic solids that crystallize as conglomerates. Since an individual crystal of a conglomerate contains only a single enantiomer, simple sorting processes involving winds, waves, or similar forces can act to deposit individual crystals into unique environments. Subsequent dissolution may afford nearly enantiopure solutions. Therefore, in contrast to common perception, enantioenrichment is not a unique signature of living systems, it is simply evidence of a certain degree of chemical complexity.

Improved method for rapid evaluation of chiral stationary phase libraries.

[figure: see text] An improved method for rapid LC/MS screening of chiral stationary phases based on the use of isotopically labeled enantiomers is reported.

Possible role of salsolinol quinone methide in the decrease of RCSN-3 cell survival.

The endogenous dopamine-derived neurotoxin salsolinol was found to decrease survival in the dopaminergic neuronal cell line RCSN-3, derived from adult rat substantia nigra in a concentration-dependent manner (208 microM salsolinol induced a 50% survival decrease). Incubation of RCSN-3 cells with 100 micro;M dicoumarol and salsolinol significantly decreased cell survival by 2.5-fold (P < 0.001), contrasting with a negligible effect on RCHT cells, which exhibited nearly a 5-fold lower nomifensine-insensitive dopamine uptake. The levels of catalase and glutathione peroxidase mRNA were decreased when RCSN-3 cells were treated with 100 microM salsolinol alone or in the presence of 100 microM dicoumarol. In vitro oxidation of salsolinol to o-quinone catalyzed by lactoperoxidase gave the quinone methide and 1,2-dihydro-1-methyl-6,7-isoquinoline diol as final products of salsolinol oxidation as determined by NMR analysis. Evidence of the formation of salsolinol o-semiquinone radical has been provided by ESR studies during one-electron oxidation of salsolinol catalyzed by lactoperoxidase.

New chiral crown ether stationary phase for the liquid chromatographic resolution of alpha-amino acid enantiomers.

A new chiral stationary phase (CSP) for the liquid chromatographic separation of enantiomers was prepared by bonding a novel enantiopure (diphenyl-substituted 1,1'-binaphthyl) crown ether to 5 microm silica gel. The resulting CSP was applied to the separation of the enantiomers of various natural and unnatural alpha-amino acids. All alpha-amino acids tested were resolved very well on the new CSP, with the exception of proline, which does not contain a primary amino group. The resolution of alpha-amino acid enantiomers on this new CSP was found to be dependent on the type and amounts of organic and acidic modifiers, and on column temperature.

Identification of an N-hydroxyguanidine reducing activity of xanthine oxidase.

A guanoxabenz [1-(2,6-dichlorobenzylideneamino)-3-hydroxyguanidine; an N-hydroxyguanidine] reducing enzymatic activity of rat spleen cytosol was investigated. By means of protein purification and N-terminal amino acid sequencing, the reducing activity was shown to reside in xanthine oxidase. The action of the enzyme on guanoxabenz resulted in the formation of guanabenz [1-(2,6-dichlorobenzylidene-amino)-3-guanidine]; the product formation could be monitored by HPLC and its identity was confirmed by NMR analysis. The reduction of guanoxabenz required xanthine or NADH as reducing substrates, while the process could be blocked by allopurinol, a selective inhibitor of xanthine oxidase. By using bovine milk xanthine oxidase, the guanoxabenz reducing activity of the enzyme was also verified. We conclude that guanoxabenz is a novel electron acceptor structure for xanthine oxidase.

Characterization of the enzymatic activity for biphasic competition by guanoxabenz (1-(2,6-dichlorobenzylidene-amino)-3-hydroxyguanidine) at alpha2-adrenoceptors. II. Description of a xanthine-dependent enzymatic activity in spleen cytosol.

The mechanism for formation of high affinity binding of guanoxabenz (1-(2,6-dichlorobenzylidene-amino)-3-hydroxyguanidine) to alpha2-adrenoceptors by the rat spleen cytosol was studied. We report here that the spleen cytosolic fraction mediated the reduction of guanoxabenz to guanabenz (1-(2,6-dichlorobenzylidene-amino)-3-guanidine), the latter having an almost 100-fold higher affinity for rat alpha2A-adrenoceptors than guanoxabenz itself. The reaction product could be separated by high-performance liquid chromatography and its identity as guanabenz confirmed by nuclear magnetic resonance. The spleen cytosolic activity could be separated into high and low molecular weight components, the high molecular weight component requiring low molecular weight factors for maximal activity. Xanthine oxidase seems to be the most likely candidate responsible for the activity, as the guanoxabenz-reducing activity of the high molecular weight component could be sustained by exogenously applied xanthine, while it was potently blocked by allopurinol. The conversion of guanoxabenz by the cytosolic activity was also quite potently blocked by DWO1, 1-(3,4-dimethoxybenzylideneamino)3-hydroxyguanidine, a hydroxyguanidine analogue to guanoxabenz.

Metabolic activation of dopamine o-quinones to o-semiquinones by NADPH cytochrome P450 reductase may play an important role in oxidative stress and apoptotic effects.

In this study, it is shown that considerable evidence for the possible pathway by which dopamine o-quinone, o-quinone and aminochrome can be activated metabolically by NADPH cytochrome P450 reductase to high reactive semiquinones. These findings were discussed from a mechanistic standpoint as well as in terms of potential physiological implications of dopamine o-quinones and o-semiquinones' concerted action in oxidative stress and apoptotic events.

Alphitol, a phenolic substance from Alphitonia zizyphoides which inhibits prostaglandin biosynthesis in vitro.

The new phenolic compound, 3,5-dihydroxy-4-methoxy phenethyl alcohol, named alphitol, and betulinic acid were from the bark of Alphitonia zizyphoides. The chemical structure of alphitol was determined by mass spectrometry in combination with one and two dimensional NMR, including HMBC. Both compounds inhibited prostaglandin biosynthesis in vitro, alphitol with an IC50 value of 0.66mM, which is of the same magnitude as acetyl salicylic acid.

Design, synthesis, and biological activities of four angiotensin II receptor ligands with gamma-turn mimetics replacing amino acid residues 3-5.

Disulfide cyclization is a powerful method for reducing the conformational space of a peptide. This in turn may enable the study of its bioactive conformation. Several analogues of angiotensin II (Ang II) containing a disulfide bridge between amino acids 3 and 5 have been reported. Among these the cyclic octapeptides c[Hcy3,5]-Ang II, c[Cys3,5]-Ang II, and c[Pen 3,5]-Ang II showed significant activity at Ang II receptors. We have performed conformational analysis studies using theoretical calculations and 1H-NMR spectroscopy on tripeptide model compounds of these cyclic octapeptides which show that the cyclic moieties of c[Cys3,5]-Ang II and c[Pen3,5]-Ang II preferentially assume an inverse gamma-turn conformation. On the basis of these results, we substituted amino acid residues 3-5 in Ang II with two different gamma-turn mimetics giving four diastereomeric Ang II analogues. Interestingly, two of these are equipotent to Ang II in binding to AT1 receptors. In the contractile test using rabbit aorta rings, one of the analogues is an agonist with full contractile activity approximately equipotent to c[Pen3,5]-Ang II but 300-fold less potent than Ang II. This low potency may suggest that Ang II does not adopt a gamma-turn in the 3-5 region when interacting with the receptor.

Human class Mu glutathione transferases, in particular isoenzyme M2-2, catalyze detoxication of the dopamine metabolite aminochrome.

Human glutathione transferases (GSTs) were shown to catalyze the reductive glutathione conjugation of aminochrome (2, 3-dihydroindole-5,6-dione). The class Mu enzyme GST M2-2 displayed the highest specific activity (148 micromol/min/mg), whereas GSTs A1-1, A2-2, M1-1, M3-3, and P1-1 had markedly lower activities (<1 micromol/min/mg). The product of the conjugation, with a UV spectrum exhibiting absorption peaks at 277 and 295 nm, was 4-S-glutathionyl-5,6-dihydroxyindoline as determined by NMR spectroscopy. In contrast to reduced forms of aminochrome (leucoaminochrome and o-semiquinone), 4-S-glutathionyl-5, 6-dihydroxyindoline was stable in the presence of molecular oxygen, superoxide radicals, and hydrogen peroxide. However, the strongly oxidizing complex of Mn3+ and pyrophosphate oxidizes 4-S-glutathionyl-5,6-dihydroxyindoline to 4-S-glutathionylaminochrome, a new quinone derivative with an absorption peak at 620 nm. GST M2-2 (and to a lower degree, GST M1-1) prevents the formation of reactive oxygen species linked to one-electron reduction of aminochrome catalyzed by NADPH-cytochrome P450 reductase. The results suggest that the reductive conjugation of aminochrome catalyzed by GSTs, in particular GST M2-2, is an important cellular antioxidant activity preventing the formation of o-semiquinone and thereby the generation of reactive oxygen species.

The flavonoid constituents of two Polypodium species (Calaguala) and their effect on the elastase release in human neutrophils.

Five flavonoid compounds were isolated from two Polypodium species (P.decumanum and P.triseriale) with the common name Calaguala. Structure elucidation was carried out using different NMR techniques and revealed the presence of one new glycoside (kaempferol 3-O-beta-D-xylopyranosyl-(1-2)-beta-D-arabinopyranoside) (1), two known flavonoid glycosides, rutin and kaempferol 3-O-alpha-D-arabinopyranoside (2,3), the trimeric proanthocyanidin, selligueain (4), and the coumarinic acid derivative, melilotoside (5). The compounds were tested for their activity in PAF induced exocytosis in human neutrophils but none of the compounds showed PAF specific activity. Instead, they showed more general effects on the neutrophil including inhibition of the spontaneous elastase release (5) and potentiation of the release induced by PAF (1). Selligueain was found to inhibit the proteolytic enzyme, elastase in vitro.

Extended length heterobifunctional coupling agents for protein conjugations.

A series of extended length heterobifunctional coupling agents is described. The successive aminocaproic acid homologation of succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate, a known 9-atom long maleimide active ester linker, yielded 16-, 23-, and 30-atom long maleimide active ester homologues. The performance study of these coupling agents in automated microparticle enzyme immunoassays showed that, in the alpha fetoprotein assay, in which the linkers were employed in the construction of the alkaline phosphatase-antibody conjugates, the signal increased 64% when the length of the linker was incremented from 9 atoms to 23 atoms and 82% for the 30-atom long linker as compared with the 9-atom homologue. Similar improvements were observed in the performance of carbohydrate antigen, marker of ovarian cancer (CA-125), immunoassay where the linkers were used for conjugation of the capture antibody anti-CA-125 to the microparticle. Thus, a 300% signal improvement resulted when a 30-atom linker was used instead of the 9-atom homologue. The observed differences in the performance of the conjugates are interpreted as resulting from improved antibody binding and lowering of the steric hindrance of the complementarity-determined region of the antibody when longer coupling agents were used.

A versatile strategy for the O4-protection and modification of the lactam function of uridine and uridylic acid.

A set of O4-aryl groups has been proposed which can both protect the lactam functions of uridine from electrophilic attacks during nucleic acid synthesis, and also serve as a good leaving group for site-specific modification at C4, under a nucleophilic reaction condition.

The application of 15N-NMR spectroscopy in the problems of nucleoside and nucleotide chemistry.

15N-NMR spectroscopy has been shown to be useful to understand the chemical nature of nitrogen in a biomolecule. We herein report the use of 15N-NMR as a tool for the study of exact location of a substitution on the guanine or uracil moieties in a nucleoside, to understand the implication of a protecting group on the reactivity and the basicity of nitrogen atoms in pyrimidines or in purines and to assign the structure of isomeric N7 and N9 substituted purines.