Microquantification of proteins with low chromophore content

The advantages of the intrinsic fluorescence of the tryptophan and the absorbance of the methionine residues of the 18 kDa-hsp - a recombinant protein from Mycobacterium leprae - was exploited here to develop a sensitive and low costs method for protein assaying. They presented linearity between 3 and 1000 g of protein. The correlations between intrinsic fluorescence or absorbance at 230 nm and protein contents were both superiors to 0.99. These methods can be extended to others proteins with low aromatic residue contents.     

MIB-1 and S-phase cell fraction predict survival in non-Hodgkin's lymphomas

The monoclonal antibody anti-Ki67 is used to detect proliferating cells, but its main limitation is the requirement of fresh-frozen material. On a series of patients with non-Hodgkin's lymphoma, we used a Ki67 equivalent monoclonal antibody, the recently proposed MIB-1, on formalin-fixed histopathological material using microwave antigen retrieval. MIB-1 expression was analysed in relation to other proliferation indices, such as autoradiographic ³H-thymidine labelling index (³HTL1) and flow cytometric S-phase cell fraction (FCM-S) and to pathological status. Moreover, the prognostic relevance of the cell kinetic indices was defined in uni- and multivariate analyses including histology and tumour stage. The relationship between MIB-1 index and the other proliferation indices was statistically significant even though the correlation coefficient was around 0.6. The MIB-1 index was also related to the REAL (Revised European American Lymphoma) classification, but not to the Ann Arbor stage classification. Univariate analysis showed that the MIB-1 index was a significant predictor of 6-year survival in the overall series and in distinctly analysed low-grade and high-grade lymphoma subgroups. With regard to S-phase indices, ³HTLI was a powerful prognosticator in patients with high-grade histologies and FCM-S in patients with low-grade histologies. Multivariate analyses revealed that MIB-1 indiex, ³HTLI and FCM-S retained their prognostic significance independent of histology. In conclusion, the MIB-1 antibody provides prognostic information in non-Hodgkin's lymphomas and has the main advantage that it can be used in formalin-fixed, paraffin-embedded specimens.     

Distribution of transposable elements in neotropical species of Drosophila

The phylogenetic distribution of transposable families, P, gypsy, hobo, I, and mariner has been analyzed in 33 species of 11 groups of neotropical Drosophila and a Drosophilidae species Zygotrica vittimaculosa, using squash blot and dot blot. Genomic DNA of almost all neotropical species tested hybridized with gypsy probe and some species showed a particularly strong hybridization signal, as D. gaucha, D. virilis, and species of flavopilosa group. The hobo element was restricted to melanogaster group and some strains of D. willistoni. Only D. simulans DNA showed hybridization to mariner probe in all species tested and D. simulans and D. melanogaster showed hybridization with I element probe. P element homologous sequence was present in D. melanogaster and all species and strains of the willistoni and saltans groups tested. The presence of at least one P-homologous sequence was detected in Drosophila mediopunctata. This one was the only P-bearing species of all six tested from the tripunctata group. Four different pairs of primers homologous to segments of the canonical sequence of D. melanogaster's P were used to amplify specific sequences from D. mediopunctata DNA, showing the occurrence of seemingly well-conserved P-homologous sequences. This revised version was published online in July 2006 with corrections to the Cover Date.     

Hemagglutination properties of Enterococcus

In total, 86 enterococcal strains including representatives of most of the described species were tested for the ability to agglutinate human, sheep, and rabbit erythrocytes. Five strains did not react with any of the erythrocytes tested, and 81 (94.2%) strains agglutinated only rabbit erythrocytes. The hemagglutination titers ranged from 2 to 64. Loss of the hemagglutination activity was observed when rabbit erythrocytes were treated with trypsin or neuraminidase. Trypsin treatment of the bacterial suspensions also caused loss of the agglutination ability. On the other hand, heat treatment of bacterial suspensions increased the efficiency of the interactions, and higher titers were obtained. Assays for inhibition of hemagglutination were performed with -d-fucose, -d-galactose, -d-galactose, d-glucose, N-acetyl-galactosamine, N-acetyl-glucosamine, N-acetylneuraminic acid, N-acetylneuraminic acid-lactose, and fetuin. Only fetuin was able to inhibit the hemagglutination reactions. The results showed that hemagglutination properties are common to the different enterococcal species tested. They also suggest that enterococci possess hemagglutinins of proteic and non-proteic nature that are involved in the attachment to sialic acid-containing receptors on the surface of rabbit erythrocytes.     

Hemispheric asymmetries in cortical electrical activity during sleep. I

The hypothesis of a predominance of the right hemisphere in stage REM as compared to NREM has been tested through a spectral analysis of the EEG recorded from left (T3) and right (T4) temporal sites in 5 young healthy right-handed male subjects. Variations in the asymmetry coefficient R - L/R + L in different sleep stages have been analyzed by one way ANOVAs and Sheffé's tests. The hypothesis of a progressive increase in left hemisphere activity throughout different REM cycles as one approaches final awakenings have been investigated by comparing variations in the asymmetry coefficient for epochs of REM and stage 2 NREM sampled in different phases of the REM cycle. EEG results do not support either the hypothesized stage dependent or cycle dependent variation in EEG activity during sleep. We question whether variations in EEG amplitude and synchronization can be used as indices of hemispheric asymmetries during sleep.     

Exudate-feeding byCallithrix jacchus penicillata in semideciduous woodland (Cerradão) in central Brazil

We report the exudate feeding behavior of two groups of marmosets (Callithrix jacchus penicillata) living permanently in Cerradão, a common woodland formation of Central Brazil. Cerradão is an open canopy formation and marmosets must occasionally descend to the ground in order to move from tree to tree. Even in atypical habitat, exudate eating is the predominant foraging activity. Marmosets are engaged in exudate collection over 70% of the total time spent feeding. They were observed gnawing on seven species of trees, and consumed exudates from four of these species. We compared the degree of utilization of the exudate sources, and examined a number of different characteristics of the exudates. Morphological adaptations that allow for the exploitation of the “exudate-eater niche” may be an important component of the adaptability ofCallithrix marmosets.     

Distribution of intrinsic nerve cell bodies and axons which take up aromatic amines and their precursors in the small intestine of the guinea-pig

Summary The sites of uptake, decarboxylation and retention of 1-dopa and the uptake and retention of dopamine and 6-hydroxytryptamine in the small intestine of the guinea-pig have been localised histochemically with a fluorescence technique for arylethylamines. In segments of ileum from untreated guinea-pigs only noradrenergic axons are fluorescent; these axons were eliminated by surgical denervation (crushing nerves running to the intestine through the mesentery) or by chemical denervation with 6-hydroxydopamine. In denervated segments of ileum, cell bodies and processes of intrinsic neurons become fluorescent after the injection of 1-dopa, dopamine or 6-hydroxytryptamine and the inhibition of monoamine oxidase, as do cells of Brunner's glands and Paneth cells. About 11% of the nerve cell bodies in the submucous plexus and 0.4% of those in the myenteric plexus become fluorescent. Varicose intrinsic axons which take up amines are found amongst the nerve cell bodies of the myenteric and submucous plexuses. They also ramify in the principal connections of the plexuses, in the tertiary strands of the myenteric plexus, in the deep muscular plexus and contribute sparse supplies of axons to arterioles in the submucosa and to the lamina propria of the mucosa. The axons are resistant to the degenerative actions of 6-hydroxydopamine.It is suggested that the intrinsic amine handling axons are more likely to utilise an indolamine related to 5-hydroxytryptamine than they are to utilise a catecholamine as a neurotransmitter.     

On the nature of chromophore in pig kidney diamine oxidase.

The nature of the 500-nm chromophore in pig kidney diamine oxidase was investigated by absorption spectroscopy and fluorescence in the presence of various chelating or carbonyl-specific reagents. From the spectroscopic measurements the following conclusions can be drawn. First, the 500-nm absorption band is not due to copper, the reduction of which is not related to the disappearance of this band. Second, phenylhydrazine and cycloserine give rise, upon reaction with the enzyme, to absorptions very similar to those of a pyridoxal enzyme, aspartate aminotransferase. Third, these enzyme derivatives are unexpectedly non-fluorescent. Copper removal, obtained after prolonged incubation of cycloserine-treated enzyme in the presence of reducing and chelating agents, leads to a fluorescence similar to that of cycloserine-aspartate transminase. It is proposed that copper is coordinated to the postulated pyridoxal phosphate of diamine oxidase through the pyridine nitrogen.     

Alpha-adrenergic receptors on human platelets.

[³H] dihydroergocyrptine, an α-adrenergic antagonist, binds specifically to sites on human platelet membranes. Prostaglandin E₁ (PGE₁) stimulates the production of cyclic AMP (cAMP) in human platelets. Alpha-adrenergic agonists, 1-epinephrine and 1-norepinephrine, and antagonists, phentolamine, phenoxybenzamine, and dihydroergocyrptine inhibit the binding of [³H] dihydroergocryptine. The α-adrenergic agonists inhibit PGE₁-stimulated cAMP production and the α-adrenergic antagonists phentolamine and dihydroergocryptine reverse this inhibition. The β-adrenergic agonist 1-isoproterenol and the β-adrenergic antagonists d1-propranolol and 1-alprenolol do not significantly alter binding or PGE₁-stimulated cAMP production. Clonidine, dopamine, and serotonin inhibit binding, but clonidine and dopamine are weak inhibitors of PGE₁-stimulated cAMP production, and serotonin is without effect. Tyramine, an amine without direct adrenergic activity fails to inhibit binding. Alpha-adrenergic agonists decrease the apparent affinity of a PGE₁-receptor activating cAMP production. The inhibition of PGE₁-stimulated cAMP production is a physiological measure of α-adrenergic agonist binding to the α-receptor.