Fourth-derivative spectrophotometry of proteins

Fourth-derivative spectrophotometry offers several advantages over classical absorption or difference spectrophotometry in examining the characteristics of aromatic amino acids in proteins. The basic principles of the technique and its applications are outlined.     

Induction of the blue form of bacteriohodopsin by low concentrations of sodium dodecyl sulfate

The effects produced on bacteriorhodopsin by low concentrations of several detergents have been studied by absorption and fourth-derivative spectrophotometry. Sodium dodecyl sulfate induces the appearance of the blue form of bacteriorhodopsin (λ_max = 600 nm) at pH values up to 7.0 in a reversible manner. The apparent pK of the purple-to-blue transition raised with increasing concentration of SDS. Of the other detergents tested, only sodium dodecyl-N-sarcosinate showed a slight red-shift of the absorption band to 580 nm, whereas sodium taurocholate, Triton X-100 and cetyltrimethylammonium bromide did not favour the appearance of the blue form. The effect of SDS was found to be consistent with a localized conformational change that moves away the counter-ion of the protonated Schiff base.     

Seasonal study of the fungal biota of the fur of dogs

During a one year period, 944 dogs from the Municipal kennel of Barcelona were examined to detect animals with suspected dermatophytosis. Only a few animals (1.8%) presented skin lesions but none of them had dermatophytosis. A representative number of dogs without visible skin lesions (n=172), selected at random, were used to carry out a seasonal study of the mycobiota of their fur. Fifteen isolates belonging to the genera Microsporum and Trichophyton were isolated from 14 of the 172 (8.1%) dogs without lesions. The identity of these fungi was Microsporum gypseum (6/15), Trichophyton terrestre (4/15), M. canis (2/15), M. cookei (2/15) and Trichophyton ajelloi (1/15) (one strain each of M. gypseum and T. ajelloi were isolated from one dog). Species of Penicillium (% prevalence=89.5%), Alternaria (86.6%), Cladosporium (84.9%), Aspergillus (77.3%), Scopulariopsis (65.7%) and Chrysosporium (64.5%) were the most prevalent. No significant differences in the fungal biota were observed with respect to age, gender, hair length or between mixed and pure breed dogs. A large number of isolates, including species belonging to the genera Beauveria, Chrysosporium, Malbranchea and Scopulariopsis, that macroscopically and/or microscopically resemble dermatophytes and may be mistaken for them, produced a red color change in Dermatophyte Test Medium. No significant seasonal difference was detected among the isolates belonging to the the most frequently encountered genera, with the exception of Scopulariopsis (higher in summer and autumn) and Chrysosporium (higher in summer). Species from other genera, with lower occurrence also presented significant differences in their seasonal distribution. Arthrinium, Aureobasidium, Chaetomium and Phoma spp. presented maximum prevalence peaks in spring, Fusarium, Paecilomyces, Phoma and Rhizopus spp. in summer and Geotrichum and Mucor spp. in autumn. The Microsporum and Trichophyton species were more frequently isolated in summer.     

Dyes as fungal inhibitors: effect on colony diameter.

The effects of a wide range of concentrations of 13 dyes on the colony diameters of nine fungal strains (including members of the Deuteromycetes and Zygomycetes) were evaluated. Auramine at a concentration of 50 ppm (50 micrograms/ml), methylene blue at a concentration of 500 ppm, gentian violet at a concentration of 5 ppm, and phenol red at a concentration of 50 ppm performed as well as the commonly used dyes dichloran at a concentration of 2 ppm and rose bengal at a concentration of 50 ppm in that they allowed adequate colony development of the Deuteromycetes strains tested and controlled rapidly spreading fungi.     

Mechanism of p38 MAPK–induced EGFR endocytosis and its crosstalk with ligand-induced pathways

Ligand binding triggers clathrin-mediated and, at high ligand concentrations, clathrin-independent endocytosis of EGFR. Clathrin-mediated endocytosis (CME) of EGFR is also induced by stimuli activating p38 MAPK. Mechanisms of both ligand- and p38-induced endocytosis are not fully understood, and how these pathways intermingle when concurrently activated remains unknown. Here we dissect the mechanisms of p38-induced endocytosis using a pH-sensitive model of endogenous EGFR, which is extracellularly tagged with a fluorogen-activating protein, and propose a unifying model of the crosstalk between multiple EGFR endocytosis pathways. We found that a new locus of p38-dependent phosphorylation in EGFR is essential for the receptor dileucine motif interaction with the σ2 subunit of clathrin adaptor AP2 and concomitant receptor internalization. p38-dependent endocytosis of EGFR induced by cytokines was additive to CME induced by picomolar EGF concentrations but constrained to internalizing ligand-free EGFRs due to Grb2 recruitment by ligand-activated EGFRs. Nanomolar EGF concentrations rerouted EGFR from CME to clathrin-independent endocytosis, primarily by diminishing p38-dependent endocytosis.     

Unusual Role of the 3-OH Group of Oligosaccharide Substrates in the Mechanism of Bacillus 1,3-1,4-β-glucanase

Abstract

In the mechanism of retaining β-glycosidases, the 2-hydroxyl group of the substrate in the monosaccharyl unit involved in catalysis (subsite -1) is beleived to play an important role through hydrogen bonding interactions with protein residues that are optimized at the transition state. Commonly, removal of the 2-OH group of the substrate results in a 10–12 kcal·mol^-1 transition state destabilization. However, this effect seems not to be general as reported here for Bacillus 1,3-1,4-β-glucanase, a family 16 retaining endo-glycosidase. A p-nitrophenol 2-deosxy tetrasaccharide substrate was synthesized to probe the involvement of the 2-OH group in catalysis. Comparative kinetics with wild-type and subsite +1 mutants show that the 2-deoxy analog is a better substrate than the corresponding 2-hydroxy substrate. It is tentatively proposed that the 2-deoxy analog adopts a different conformation upon binding that compensates for the lack of the 2-OH substituent.     

Reduced striatal ecto-nucleotidase activity in schizophrenia patients supports the “adenosine hypothesis”

Schizophrenia (SZ) is a major chronic neuropsychiatric disorder characterized by a hyperdopaminergic state. The hypoadenosinergic hypothesis proposes that reduced extracellular adenosine levels contribute to dopamine D₂ receptor hyperactivity. ATP, through the action of ecto-nucleotidases, constitutes a main source of extracellular adenosine. In the present study, we examined the activity of ecto-nucleotidases (NTPDases, ecto-5′-nucleotidase, and alkaline phosphatase) in the postmortem putamen of SZ patients (n = 13) compared with aged-matched controls (n = 10). We firstly demonstrated, by means of artificial postmortem delay experiments, that ecto-nucleotidase activity in human brains was stable up to 24 h, indicating the reliability of this tissue for these enzyme determinations. Remarkably, NTPDase-attributable activity (both ATPase and ADPase) was found to be reduced in SZ patients, while ecto-5′-nucleotidase and alkaline phosphatase activity remained unchanged. In the present study, we also describe the localization of these ecto-enzymes in human putamen control samples, showing differential expression in blood vessels, neurons, and glial cells. In conclusion, reduced striatal NTPDase activity may contribute to the pathophysiology of SZ, and it represents a potential mechanism of adenosine signalling impairment in this illness.