Analysis of the Antigenic Composition of Trypanosoma brucei brucei Bloodstream and Culture Forms by the Quantitative Direct Fluorescent Antibody Methods*

SYNOPSIS The quantitative direct fluorescent antibody (QDFA) methods were employed for the antigenic analysis of bloodstream forms and culture procyclics of 2 variants, TRUM (Trypanosome Research University of Massachusetts) 106 and TRUM 107, of Trypanosoma brucei brucei. Intact and trypsinized trypanosomes were studied. It was demonstrated that: (A) The specific variant antigens are localized in the surface coat of bloodstream trypomastigotes. (B) In addition to the common antigens shared by bloodstream forms and culture procyclics, there are also certain antigens unique to these latter stages. (C) Still another group of antigens. not found in the culture procyclics, appears to be shared by the bloodstream forms, irrespective of their variant-specific antigens. These antigens may be present in part in the coat or on the cell membrane and in part within the cytoplasm. (D) Irrespective of the bloodstream-form variant from which they are derived, the procyclics are antigenically the same. The QDFA results are analyzed statistically and discussed in the light of the available literature.     

Determination of K forms in K-fertilized soils by electro-ultrafiltration

We confirmed the suitability of electro-ultrafiltration (EUF) for (a) determination of the distribution of potassium fertilizer among the various forms of potassium in soils with a predominance of micaceous minerals in their clay fraction, and (b) investigated the effects of the degree of openness of the dominant micaceous mineral and of incubation time on the kinetics of the EUF extraction of K from these soils.Samples of illitic, mixed-layer and vermiculitic soils from Galicia (N.W. Spain) were incubated at field capacity for 450 days with 0 (blank), 5,15 or 25 mg K (as KCl) per 100 g dry soil. After 1, 30, 150 and 450 days, subsamples were removed and repeatedly extracted using electro-ultrafiltration at low (20° C/200 V) and then high (80° C/400 V) temperature/voltage (6 and 10 five-minute extractions, respectively). Five different pools of K were identified: solution K (K_s), surface and internal K (collectively, K_p), slowly exchangeable K (K_e) and non-exchangeable K (K_i). The effects of increasing the incubation time depended on the dominant clay mineralogy: after 450 days, the K added to illitic soils was mostly solution K, whereas that added to vermiculitic soils was mostly internal K.For both low and high temperature/voltage EUF experiments, the extraction-time data were best fitted by the Elovich equation (extracted K=a+b ln t). The kinetic coefficient b depended on the incubation time and dominant clay mineral, and for given soil and incubation time increased linearly with the dose of added K.Abbreviations EUF Electroultrafiltration - K_s Solution potassium - K_p Easily exchangeable (surface + internal) potassium - K_e Slowly exchangeable potassium - K_i Non-exchangeable potassium     

Molecular forms of lipases and their localization in the fungus Rhizopus microsporus by immuno-electron microscopy

Several lipases differing in their molecular masses (24, 32, 43, 66 and 98 kDa and 28, 40, 45 and 69 kDa) were found in Rhizopus microsporus UzLT-4B and UzLT-5C, respectively. The lipases in each strain were immunologically related. Strain UzLT-5C grown on a medium with lipid substrate secreted lipases of 32, 66 and 98 kDa whereas strain UzLT-4B produced lipases of 45 and 69 kDa on the same medium. Immuno-electron microscopy indicated that the intracellular lipases were in peripheral zones of the hyphae, primarily in the periplasm and adjacent vesicles. Immobilization in Ca-alginate gel revealed unusual structures in the cell wall. These structures accumulated lipases and, apparently, exported the enzymes out of the cell.The authors are with the Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, 142292, Pushchino, Moscow Region, Russia     

Biological species and morphological characteristics ofArmillaria mellea complex in Hokkaido:A. sinapina and two new species,A. jezoensis andA. singula

Three intersterility groups ofArmillaria mellea sensu lato were discovered by examining all pairwise combinations of monosporous isolates of basidiomes collected in Hokkaido. One of them, group IV, was identified asA. sinapina by mating it with tester strains. Two new species, groups III and V, were namedA. jezoensis andA. singula, respectively. Their morphological forms and the ecology of their basidiomes are described.     

The functional role of molecular forms of acetylcholinesterase in neuromuscular transmission

The severity of poisoning following acetylcholinesterase (AChE) inhibition correlates weakly with total AChE activity. This may be partly due to the existence of functional and non-functional pools of AChE. AChE consists of several molecular forms. The aim of the present study was to investigate which of these forms will correlate best with neuromuscular transmission (NMT) remaining after partial inhibition of this enzyme. Following sublethal intoxication of rats with the irreversible AChE inhibitor soman, diaphragms were isolated after 0.5 or 3 h. It appeared that at 3 h after soman poisoning the percentage of G₁ increased, while those of G₄ and A₁₂ decreased. NMT was inhibited more strongly than in preparations obtained from the 0.5 h rats with the same level of AChE inhibition, but with a normal ratio of molecular forms. NMT correlated positively with G₄ as well as with A₁₂, but inversely with G₁. In vitro inhibition with the charged inhibitors DEMP and echothiophate resulted in higher levels of total AChE, relatively less G₁ and more G₄ and A₁₂ than after incubation with soman, but led to less NMT. Treatment of soman-intoxicated rats with the reactivating compound HI-6 resulted in preferential reactivation of A₁₂, persisting low levels of G₁ and concurrent recovery of NMT as compared with saline-treated soman controls with equal total AChE activity. Apparently, in rat diaphragm G₄ and A₁₂ are the functional AChE forms.     

Neuropathology, biochemistry, and biophysics of alpha-synuclein aggregation

Aggregation of alpha-synuclein, an abundant and conserved pre-synaptic brain protein, is implicated as a critical factor in several neurodegenerative diseases. These diseases, known as synucleinopathies, include Parkinson's disease, dementia with Lewy bodies (LBs), diffuse LB disease, the LB variant of Alzheimer's disease, multiple system atrophy, and neurodegeneration with brain iron accumulation type I. Although the precise nature of in vivoalpha-synuclein function remains elusive, considerable knowledge has been accumulated about its structural properties and conformational behavior. alpha-Synuclein is a typical natively unfolded protein. It is characterized by the lack of rigid, well-defined, 3-D structure and possesses remarkable conformational plasticity. The structure of this protein depends dramatically on its environment and it accommodates a number of unrelated conformations. This paper provides an overview of the biochemistry, biophysics, and neuropathology of alpha-synuclein aggregation.     

Mechanisms of attenuation of ischemic heart injury by means of a modified reperfusion

Mechanisms of attenuation of membrane injury and metabolic impairments in postischemic cardiomyocytes have been studied on a model of ischemic and reperfusion stress of rat heart using a modified early reperfusion. Optimization of the reperfusion infusate composition augmented recovery of cardiac pump and contractile function. This was accompanied by reduced release of lactate dehydrogenase activity and systems generating short-living reactive oxygen species into myocardial effluent and was associated with more efficient oxidative metabolism recovery and decreased losses of intracellular total creatine and amino acids pools. The results indicate perspectives of postischemic functional and metabolic myocardial injury correction by means of the controlled reperfusion.     

Analysis of Yarrowia lipolytica extracellular lipase Lip2p glycosylation

Wild-type (WT) Yarrowia lipolytica strain secretes a major extracellular lipase Lip2p which is glycosylated. In silico sequence analysis reveals the presence of two potential N-glycosylation sites (N113IS and N134NT). Strains expressing glycosylation mutant forms were constructed. Esterase activities for the different forms were measured with three substrates: p-nitrophenol butyrate (p-NPB), tributyrin and triolein. Sodium dodecyl sulfate polacrylamide gel electrophoresis analysis of supernatant indicated that the suppression of the two sites of N-glycosylation did not affect secretion. S115V or N134Q mutations led to lipase with similar specific activity compared with WT lipase while a T136V mutation reduced specific activity toward p-NPB and tributyrin. Electrospray ionization MS of the WT entire protein led to an average mass of 36 950 Da, higher than the mass deduced from the amino acid sequence (33 385 Da) and to the observation of at least two different mannose structures: Man(8)GlcNAc(2) and Man(9)GlcNAc(2). LC-tandem MS analysis of the WT Lip2p after trypsin and endoproteinase Asp-N treatments led to high coverage (87%) of protein sequence but the peptides containing N113 and N134 were not identified. We confirmed that the presence of N-glycosylation occurred at both N113 and N134 by MS of digested proteins obtained after enzymatic deglycosylation or from mutant forms.