Construction of a novel lipolytic fusion biocatalyst GDEst-lip for industrial application

The gene encoding esterase (GDEst-95) from Geobacillus sp. 95 was cloned and sequenced. The resulting open reading frame of 1497 nucleotides encoded a protein with calculated molecular weight of 54.7 kDa, which was classified as a carboxylesterase with an identity of 93–97% to carboxylesterases from Geobacillus bacteria. This esterase can be grouped into family VII of bacterial lipolytic enzymes, was active at broad pH (7–12) and temperature (5–85 °C) range and displayed maximum activity toward short acyl chain p-nitrophenyl (p-NP) esters. Together with GD-95 lipase from Geobacillus sp. strain 95, GDEst-95 esterase was used for construction of fused chimeric biocatalyst GDEst-lip. GDEst-lip esterase/lipase possessed high lipolytic activity (600 U/mg), a broad pH range of 6–12, thermoactivity (5–85 °C), thermostability and resistance to various organic solvents or detergents. For these features GDEst-lip biocatalyst has high potential for applications in various industrial areas. In this work the effect of additional homodomains on monomeric GDEst-95 esterase and GD-95 lipase activity, thermostability, substrate specificity and catalytic properties was also investigated. Altogether, this article shows that domain fusing strategies can modulate the activity and physicochemical characteristics of target enzymes for industrial applications.     

Usefulness of the diagnostic kit Enteroplast for identification of Enterobacteriaceae

Comparative evaluation of biochemical properties determined by ENTEROPLAST kit (P.P.Z. Plastomed) and biochemical set of tests (conventional method) of 140 strains representing 12 genera of Enterobacteriaceae family was undertaken. Consistent results positive or negative (in 12 biochemical tests) were obtained in 93%. The highest percentage of inconsistent results of biochemical reactions (positive in conventional method and negative in ENTEROPLAST kit) were observed in following tests: growth on Simmons medium--17.8%, MR--7.8%, fermentation of raffinose--7.2%. Significantly lower percentage of inconsistent results was found in the case of false positive reactions (negative in conventional method and positive in ENTEROPLAST kit). In summary, it seems that Enteroplast kit can be used for routine diagnostic examinations for identification of rods of Enterobacteriaceae family, basing on their biochemical properties.     

Characterization of an activity-dependent, neuronal surface proteoglycan identified with monoclonal antibody Cat-301

Monoclonal antibody Cat-301 was previously shown to recognize a surface-associated antigen on subsets of mammalian CNS neurons whose expression is regulated by neuronal activity early in an animal's postnatal life. We now present the partial purification and characterization of the Cat-301 antigen and demonstrate that it is a chondroitin sulfate proteoglycan. Extracellular localization of the Cat-301 epitope is demonstrated by staining live, intact neurons in situ. Extraction of the antigen from membranes in the absence of detergent indicates that it is either a peripheral membrane protein or a component of an extracellular matrix. The Cat-301 antigen migrates on Western blots of SDS gels with a molecular weight of integral of 680,000 dalton and is purified by DEAE chromatography and Sepharose gel filtration in 8 M urea (pH 4.9) buffer. The antigen is sensitive to chondroitinase ABC, indicating that it is a chondroitin sulfate proteoglycan. Furthermore, we provide strong evidence that the biochemically characterized antigen is indeed the histologically detected species by using a second antibody, Cat-304, that produces immunohistological staining patterns identical to those of Cat-301 and reacts with the purified antigen, but at a distinct epitope. Our earlier developmental findings and the present localization and biochemical results suggest that the antigen may play a role in the maturation of functional connections between neurons, perhaps through stabilization of axosomatic and axodendritic synapses.     

A nonenzymatic method for the determination of picomole amounts of lactate using HPLC: its application to single muscle fibers

A method for the determination of lactate is described in which the esterification of lactate with the uv-absorbing compound alpha-p-dibromoacetophenone is followed by separation and quantitation of the ester by reversed-phase HPLC with detection at 254 nm. The reproducibility, detection limit, and precision of the method are comparable to those of conventional methods which use enzymatic cycling for enhanced performance. The applicability of this rapid and simple method is illustrated with the determination of picomole amounts of lactate in resting and stimulated single muscle fibers.     

A rare subclinical or mild type of Becker muscular dystrophy caused by a single exon 48 deletion of the dystrophin gene

In the material of 227 families with Becker muscular dystrophy (BMD), we found nine non-consanguineous families with 17 male individuals carrying a rare mutation—a single exon 48 deletion of the dystrophin gene—who were affected with a very mild or subclinical form of BMD. They were usually detected thanks to accidental findings of elevated serum creatine phosphokinase (sCPK). A thorough clinical analysis of the carriers, both children (12) and adults (5), revealed in some of them muscle hypotonia (10/17) and/or very mild muscle weakness (9/17), as well as decreased tendon reflexes (6/17). Adults, apart from very mild muscle weakness and calf hypertrophy in some, had no significant abnormalities on neurological assessments and had good exercise tolerance. Parents of the children carriers of the exon 48 deletion are usually unaware of their children being affected, and possibly at risk of developing life-threatening cardiomyopathy. The same concerns the adult male carriers. Therefore, the authors postulate undertaking preventive measures such as cascade screening of the relatives of the probands. Newborn screening programmes of Duchenne muscular dystrophy (DMD)/BMD based on sCPK marked increase may be considered.     

Left atrial strain - an early marker of left ventricular diastolic dysfunction in patients with hypertension and paroxysmal atrial fibrillation

Background

2D strain imaging of the left atrium (LA) is a new echocardiographic method which allows us to determine contractile, conduit and reservoir functions separately. This method is particularly useful when changes are subtle and not easily determined by traditional parameters, as it is in arterial hypertension and atrial fibrillation (AF). The aims of our study were: to determine LA contractile, conduit and reservoir function by 2D strain imaging in patients with mild arterial hypertension and paroxysmal AF; to assess LA contractile, conduit and reservoir functions’ relation with LV diastolic dysfunction (DD) parameters.

Methods

LA contractile, conduit and reservoir functions together with echocardiographic signs of LV DD were assessed in 63 patients with arterial hypertension and paroxysmal AF. Patients were grouped according to number of signs showing LV DD (annular e’ velocity: septal e’?<?7 cm/s, lateral e’?<?10 cm/s, average E/e’ ratio?>?14, LA volume index >?34 ml/m², peak tricuspid regurgitation velocity?>?2.8 m/s) present. Number of patients with 0 signs – 17, 1 sign – 26, 2 signs – 19. Contractile, conduit and reservoir functions were compared between the groups.

Results

Mean contractile, conduit and reservoir strains in all the patients were???14.14 (± 5.83) %, 15.98 (± 4.85) % and 31.03 (± 7.64) % respectively. Contractile strain did not differ between the groups. Conduit strain was higher in patients with 0 signs compared with other groups (p =?0.016 vs 1 sign of LV DD and p =?0.001 vs 2 signs of LV DD). Reservoir strain was higher in patients with 0 signs compared with other groups (p =?0.014 vs 1 sign of LV DD and p <?0.001 vs 2 signs of LV DD).

Conclusions

The patients with paroxysmal AF and primary arterial hypertension have decreased reservoir, conduit and pump LA functions even in the absence of echocardiographic signs of LV DD. With increasing number of parameters showing LV DD, LA conduit and reservoir functions decrease while contractile does not change. LA conduit and reservoir functions decrease earlier than the diagnosis of LV DD can be established according to the guidelines in patients with primary arterial hypertension and AF.