Improved Stability of Methanolic Wright's Stain with Additive Reagents

Additive reagents have been investigated to improve the stability of methanolic Wright's stain. The addition of ammonium halides, monoalkyiamine hydrochlorides, dialkylamine hydrochlorides or trialkylamine hydrochlorides to methanolic Wright's stain was found to enhance the stability of stain components in methanol. No change in performance is observed with these additives present. Random precipitation in the stain solution was still observed with the addition of ammonium halides and monoalkyiamine hydrochlorides. No precipitation was found in stain solutions containing hydrochlorides of most dialkylamines and trialkylamines. Of the compounds evaluated, 0.6% diethylamine hydrochloride added to methanolic stain solutions produced the most desirable overall results. Mechanisms of stabilization and precipitation in these stain solutions are proposed, Essentially, separation of the thiazine-eosinate ion pair through interaction with an appropriate additive increases stain stability. The solubilities of thiazine-eosinate or additive cation-eosinate ion pairs in methanol determine the formation of precipitate in such stain solutions.     

Immunochemical studies on human monoclonal macroglobulins with specificities for 3,4-pyruvylated D-galactose and 4,6-pyruvylated D-glucose

Four of six human monoclonal IgM proteins were found to react best with Klebsiella polysaccharides containing 3,4py beta DGal (pyruvic acetalated D-galactopyranose), one with Klebsiella polysaccharides with 4,6pyDGlc; the sixth is uncharacterized. The combining sites of two of these (IgMWEA and IgMNAE) were essentially indistinguishable by quantitative precipitin studies at varying pH and by quantitative precipitin inhibition assays, but the other two differed in specificity of their combining sites from these and from each other. These differences were detected by precipitin inhibition assays with 3,4py beta DGal-containing oligosaccharide alditols, the R and S isomers of methyl 4,6py alpha DGal, the R isomer of methyl 4,6py beta DGal, or the R and S isomers of methyl 4,6py alpha DGlc, and -beta DGlc. In all of these except the S isomer of methyl 4,6pyDGal and R isomer of methyl 4,6pyDGlc, the carboxyl group is axial to the plane of the acetal ring. Their specificity appears to be determined by the nonreducing ends of chains and is considered to be cavity-type.     

Leaf traits of natural populations of <Emphasis Type="Italic">Adiantum reniforme</Emphasis> var. <Emphasis Type="Italic">sinensis</Emphasis>, endemic to the Three Gorges region in China

Leaf mass per unit area (LMA), carbon and nitrogen contents, leaf construction cost, and photosynthetic capacity (P _max) of Adiantum reniforme var. sinensis, an endangered fern endemic to the Three Gorges region in southwest China, were compared in five populations differing in habitat such as soil moisture and irradiance. The low soil moisture and high irradiance habitat population exhibited significantly higher LMA, area-based leaf construction (CC_A), and carbon content (C_A), but lower leaf nitrogen content per unit dry mass (N_M) than the other habitat populations. The high soil moisture and low irradiance habitat populations had the lowest CC_A, but their cost/benefic ratios of CCA/P _max were similar to the medium soil moisture and irradiance habitat population due to their lower leaf P _max. Hence A. reniforme var. sinensis prefers partially shaded, moist but well-drained, slope habitats. Due to human activities, however, its main habitats now are cliffs or steeply sloped bare rocks with poor and thin soil. The relatively high energy requirements and low photosynthetic capacity in these habitats could limit the capability of the species in extending population or interspecific competition and hence increase its endangerment.     

Metarhizium robertsii Produces an Extracellular Invertase (MrINV) That Plays a Pivotal Role in Rhizospheric Interactions and Root Colonization

As well as killing pest insects, the rhizosphere competent insect-pathogenic fungus Metarhizium robertsii also boosts plant growth by providing nitrogenous nutrients and increasing resistance to plant pathogens. Plant roots secrete abundant nutrients but little is known about their utilization by Metarhizium spp. and the mechanistic basis of Metarhizium-plant associations. We report here that M. robertsii produces an extracellular invertase (MrInv) on plant roots. Deletion of MrInv (⊿MrInv) reduced M. robertsii growth on sucrose and rhizospheric exudates but increased colonization of Panicum virgatum and Arabidopsis thaliana roots. This could be accounted for by a reduction in carbon catabolite repression in ⊿MrInv increasing production of plant cell wall-degrading depolymerases. A non-rhizosphere competent scarab beetle specialist Metarhizium majus lacks invertase which suggests that rhizospheric competence may be related to the sugar metabolism of different Metarhizium species.     

Quantitative Förster resonance energy transfer analysis for kinetic determinations of SUMO-specific protease

Förster resonance energy transfer (FRET) technology has been widely used in biological and biomedical research, and it is a very powerful tool for elucidating protein interactions in either dynamic or steady state. SUMOylation (the process of SUMO [small ubiquitin-like modifier] conjugation to substrates) is an important posttranslational protein modification with critical roles in multiple biological processes. Conjugating SUMO to substrates requires an enzymatic cascade. Sentrin/SUMO-specific proteases (SENPs) act as an endopeptidase to process the pre-SUMO or as an isopeptidase to deconjugate SUMO from its substrate. To fully understand the roles of SENPs in the SUMOylation cycle, it is critical to understand their kinetics. Here, we report a novel development of a quantitative FRET-based protease assay for SENP1 kinetic parameter determination. The assay is based on the quantitative analysis of the FRET signal from the total fluorescent signal at acceptor emission wavelength, which consists of three components: donor (CyPet–SUMO1) emission, acceptor (YPet) emission, and FRET signal during the digestion process. Subsequently, we developed novel theoretical and experimental procedures to determine the kinetic parameters, k_cat, K_M, and catalytic efficiency (k_cat/K_M) of catalytic domain SENP1 toward pre-SUMO1. Importantly, the general principles of this quantitative FRET-based protease kinetic determination can be applied to other proteases.     

Near-infrared time-resolved optical absorption studies of the reaction of fully reduced cytochrome c oxidase with dioxygen

Electron transfer during the reaction of fully reduced bovine heart cytochrome oxidase with dioxygen has been studied at 24 degrees C in the near-infrared region following photolysis of the fully reduced CO-bound complex. The transient spectral changes and kinetics were followed on microsecond to millisecond time scales at nine different wavelengths between 597 and 935 nm and were analyzed using singular value decomposition and global exponential fitting. Four apparent lifetimes, 14 micros, 40 micros, 86 micros, and 1.1 ms, were resolved. The near-infrared spectra of the intermediates are extracted on the basis of a previously proposed mechanism [Sucheta et al. (1998) Biochemistry 37, 17905-17914] and compared to model spectra of the postulated intermediates. The data provide a comprehensive picture of the spectral contributions of the different redox centers in their respective oxidation or ligation states in the near-infrared region and strongly support that Cu(A) is partially (2/3), but not fully, oxidized in the 3-electron-reduced ferryl intermediate.