Characterization of two extracellular proteinases from Aspergillus terreus and their role in the formation of low molecular weight endoglucanases

Two extracellular proteolytic activities from the wood degrading fungus Aspergillus terreus have been characterized. Proteinase I (serine thiol-dependent enzyme) was active over a broad pH range (7·0–10·0) and at 55°C. The second proteinase (metalloproteinase) showed optimal activity at pH 6·0–7·0 and at 65–70°C. Both proteins had isoelectric points at acid pH and contained carbohydrate moieties. The metalloproteinase possessed a uniquely high content of serine and threonine and an extremely low percentage of glutamate and aspartate. The metalloproteinase was involved in the formation of the low molecular mass endoglucanases of A. terreus.     

Covalent and non-covalent inhibitors of the phosphate transporter of sarcoplasmic reticulum.

The sarcoplasmic reticulum (SR) of skeletal muscle contains a Pi transporter which transports Pi into the lumen of the SR, increasing the level of accumulation of Ca2+ by SR by forming insoluble salts with Ca2+. Phosphonocarboxylic acids inhibit the transport of Pi by the transporter, phosphonoformic acid itself being transported into the SR increasing the level of accumulation of Ca2+. Phenylphosphonic acid also inhibits Pi transport, distinguishing the Pi transporter of SR from the Na+/Pi transporter of brush-border membranes. Oxalate transport is also inhibited by the phosphono-carboxylic acids, consistent with the suggestion that oxalate and phosphate are carried on the same transporter. The effects of maleate are, however, not inhibited, suggesting a separate carrier for the dicarboxylic acids. Acetic anhydride and phenylglyoxal inhibit the transporter, Pi providing protection against the effects of acetic anhydride, suggesting the presence of a lysine residue at the Pi binding site. ATP provides protection against the effects of acetic anhydride and phenylglyoxal, suggesting the presence of an ATP binding site on the transporter.     

Estimation of male and female body segment parameters of the Bulgarian population using a 16-segmental mathematical model

Based on a large-scale anthropometric measurements of 5290 individuals (2435 males and 2855 females) of the Bulgarian population aged between 30 and 40 years, we present 16-segmental 3D geometrical model of the human body of the average Bulgarian male and female and calculate mass, volume, location of the mass center and moments of inertia for all the segments for both genders. This study extends current anthropometrical data pool of Caucasian. Wherever possible, the comparison between our model results and data reported in literature for other Caucasian shows an overall good agreement, thus supporting the validity of the described method.     

The molecular tweezer CLR01 reduces aggregated,pathologic, and seeding-competent α-synuclein in experimental multiple system atrophy

Multiple system atrophy (MSA) is a fatal, adult-onset neurodegenerative disorder that has no cure and very limited treatment options. MSA is characterized by deposition of fibrillar α-synuclein (α-syn) in glial cytoplasmic inclusions in oligodendrocytes. Similar to other synucleinopathies, α-syn self-assembly is thought to be a key pathologic event and a prominent target for disease modification in MSA. Molecular tweezers are broad-spectrum nanochaperones that prevent formation of toxic protein assemblies and enhance their clearance. The current lead compound, CLR01, has been shown to inhibit α-syn aggregation but has not yet been tested in the context of MSA. To fill this gap, here, we conducted a proof-of-concept study to assess the efficacy of CLR01 in remodeling MSA-like α-syn pathology in the PLP-α-syn mouse model of MSA. Six-month-old mice received intracerebroventricular CLR01 (0.3 or 1 mg/kg per day) or vehicle for 32 days. Open-field test revealed a significant, dose-dependent amelioration of an anxiety-like phenotype. Subsequently, immunohistochemical and biochemical analyses showed dose-dependent reduction of pathological and seeding-competent forms of α-syn, which correlated with the behavioral phenotype. CLR01 treatment also promoted dopaminergic neuron survival in the substantia nigra. To our knowledge, this is the first demonstration of an agent that reduces formation of putative high-molecular-weight oligomers and seeding-competent α-syn in a mouse model of MSA, supporting the view that these species are key to the neurodegenerative process and its cell-to-cell progression in MSA. Our study suggests that CLR01 is an attractive therapeutic candidate for disease modification in MSA and related synucleinopathies, supporting further preclinical development.     

Spread and competitiveness of Plum Pox Virus: Rec and ‐D strains in experimental plum orchard

Sharka disease caused by Plum pox virus (PPV) is endemic in Bulgaria. The speed and mode of spread of PPV‐Rec and PPV‐D isolates, molecularly characterised in advance and introduced in an experimental plum orchard to serve as permanent sources of infection was studied from 2008 to 2015. The trees were visually inspected for symptoms development and tested by DASI‐ELISA twice each year. The positive samples were serologically (‐M and ‐D specific MAbs) and molecularly (IC‐RT‐PCR) strain‐typed, and sequenced in (Cter)NIb‐(Nter)CP coding region. Artificially inoculated trees were found infected in the spring of 2009. After 7 years 43 trees (11.2%) became naturally infected. The majority of the trees were infected by PPV‐Rec (65%), while only one tree (2.5%) by PPV‐D. Nevertheless PPV‐M was not introduced in the orchard, this strain was found in single and mixed infections with PPV‐Rec strain. None of the isolates in naturally infected trees was identical to PPV‐Rec and PPV‐D inoculums, while several of them were a 100% identical to the respective PPV‐Rec, PPV‐D or PPV‐M isolates from neighbourhood‐infected trees. After the initial random pattern of PPV spread, aggregation of the new infections around the trees infected in previous years was observed. The presence of only Phorodon humuli and Hyalopterus pruni complex, known as PPV vectors and their poor abundance (5.0% from all caught aphids), together with some specific features of the landscape influenced the slow natural development of PPV in the experimental orchard.     

Immobilization of Bacillus licheniformis cells, producers of thermostable α-amylase, on polymer membranes

Cells of Bacillus licheniformis 44MB82-G immobilized on different polymer membranes were used for production of thermostable α-amylase. The α-amylase yields of the membrane-immobilized cells were affected by the reactive chemical groups of the carriers and the spacer size. Formaldehyde-activated polysulphone membranes (PS-FA) were the most suitable for effective immobilization. The highest amylase yield (62% increase of the control) and operational stability (97% residual activity after 480 h repeated batch cultivation) were obtained with this system. This was confirmed by scanning electron micrographs. An additional increase of α-amylase production by PS-FA-membrane immobilized cells was achieved in a fluidized-bed reactor. Received 20 March 1997/ Accepted in revised form 08 January 1998     

Stereoselective binding of ketoprofen enantiomers to human serum albumin studied by high-performance liquid affinity chromatography

A chiral stationary phase based on immobilized human serum albumin (HSA) was used to study the stereoselective binding of ketoprofen enantiomers by means of high-performance liquid affinity chromatography. The technique of zonal elution was applied together with a novel mathematical approach describing attachment to more than one type of binding site. Phenylbutazon (PBZ) and diazepam (DAZ) were used as markers for the major believed binding regions on HSA. Both R- and S-ketoprofen (KTR and KTS) display high affinity to the primary PBZ- and DAZ-binding sites and low-affinity to the secondary DAZ sites. The binding to high-affinity regions is accepted to be a stepwise process initiated by the binding to the primary DAZ sites and followed by the attachment to the primary PBZ sites. The chiral recognition is attributed to the high-affinity PBZ-binding sites and to the low-affinity DAZ-binding sites.     

Interactions between biochar and mycorrhizal fungi in a water-stressed agricultural soil

Biochar may alleviate plant water stress in association with arbuscular mycorrhizal (AM) fungi but research has not been conclusive. Therefore, a glasshouse experiment was conducted to understand how interactions between AM fungi and plants respond to biochar application under water-stressed conditions. A twin chamber pot system was used to determine whether a woody biochar increased root colonisation by a natural AM fungal population in a pasture soil (‘field’ chamber) and whether this was associated with increased growth of extraradical AM fungal hyphae detected by plants growing in an adjacent (‘bait’) chamber containing irradiated soil. The two chambers were separated by a mesh that excluded roots. Subterranean clover was grown with and without water stress and harvested after 35, 49 and 63 days from each chamber. When biochar was applied to the field chamber under water-stressed conditions, shoot mass increased in parallel with mycorrhizal colonisation, extraradical hyphal length and shoot phosphorus concentration. AM fungal colonisation of roots in the bait chamber indicated an increase in extraradical mycorrhizal hyphae in the field chamber. Biochar had little effect on AM fungi or plant growth under well-watered conditions. The biochar-induced increase in mycorrhizal colonisation was associated with increased growth of extraradical AM fungal hyphae in the pasture soil under water-stressed conditions.