[Open, non-comparative phase III clinical study to evaluate the efficacy and safety of sapropterin in patients with phenylketonuria and hyperphenylalaninemia].

BACKGROUND: Phenylketonuria (PKU) is an autosomal recessive inherited disease associated with impaired metabolism of the amino acids phenylalanine (Phe) and tyrosine. The main criterion for diagnosis of PKU is high blood Phe level determined during neonatal screening. In case where PKU patient is responsive to tetrahydrobiopterin treatment, sapropterin restores the impaired activity of the enzyme phenylalanine hydroxylase, resulting in the stimulation of normal Phe metabolism and thereby enhancing patient tolerance to natural products. AIM: The present open, non-comparative clinical study was initiated to assess the degree and frequency of response after 8-day sapropterin administration and assess the safety of 6-week sapropterin treatment in patients with PKU and hyperphenylalaninemia. PATIENTS AND METHODS: The study enrolled 90 patients with PKU. The criterion of response to 8-day sapropterin therapy was the reduction of Phe blood levels ≥ 30% compared with the baseline value. RESULTS: Positive response to treatment was observed in 30 (33.3%) patients (95% CI 23.7-44.1). The mean percentage change in Phe blood levels after the 8-day response test period compared to Phe levels prior to dosing was 14.1 ± 28.4% in the overall subject population (95% CI 8.2-20.1) and 44.3 ± 15.1% in the subpopulation of patients with a positive response (95% CI 38.6-49.9). During the study, adverse events were reported in 24 (26.7%) patients in the overall population in 16 (53.3%) patients in the subpopulation who had a response. CONCLUSION: The study results confirmed the efficacy and safety of sapropterin therapy in patients with PKU, which is consistent with international clinical trials data.

[Activity dynamics of potential marker enzymes of Serratia marcescens cytoplasm and periplasm].

Activity dynamics of glucose-6-phosphate dehydrogenase, alkaline phosphatase, beta-galactosidase and beta-lactamase in the course of growth and development of Gram-negative bacteria Serratia marcescens was studied. Glucose-6-phosphate dehydrogenase can serve as a marker of cytoplasm and be also used as a marker of plasma membrane continuity. Alkaline phosphatase is a marker ofperiplasm. Glucose-6-phosphate dehydrogenase, beta-lactamase and beta-galactosidase can be additionally used as markers of the outer membrane continuity of microbial cells.

[Nuclease biosynthesis and growth of Serratia marcescens in the presence of 2-(p-aminobenzenesulfonamide)-thiazole].

The biosynthesis of nuclease in Serratia marcescens has been studied under the conditions of purine synthesis inhibition with 2-(p-aminobenzenesulfonamide)-thiazole. The addition of this sulfonamide to S. marcescens at different growth stages is found to inhibit both culture growth and nuclease synthesis.

Study of the mechanism of action of p-chloromercuribenzoate on endonuclease from the bacterium Serratia marcescens.

The mechanism of action of p-chloromercuribenzoate (PCMB) on Serratia marcescens nuclease was investigated. The analysis showed that PCMB forms complexes with DNA. Binding of C7H5O2Hg+ to DNA changes the secondary structure of the DNA. These changes alter the enzymatic activity of S. marcescens nuclease, which was previously found to be sensitive to the secondary structure of the substrates. The nuclease activity was either suppressed or stimulated in the presence of PCMB depending on the C7H5O2Hg+ to nucleotide equivalent ratio. Binding of C7H5O2Hg+ to DNA did not form an abortive enzyme-substrate complex. Binding of Mg2+ to the C7H5O2Hg-DNA complex caused appropriate changes in secondary structure of the substrate. Since Mg2+ and C7H5O2Hg+, though differing in the type of metal cation, are similar in their mechanisms of influence on enzymatic activity of S. marcescens nuclease, the identity of other metal-containing effectors in their mechanism of action on Serratia marcescens nuclease is assumed.

[Polydispersity of Serratia marcescens nuclease at optimal pH values]. / Polidispersnost' nukleazy Serratia marcescens pri optimal'nom znachenii pH.

Treatment with dimethyl suberimidate, a cross-linking bifunctional agent, showed that Sm1 and Sm2 nucleases of Serratia marcescens B10M1 are polydisperse in solution and consist of monomers and dimers at the level of pH optimal for the enzyme activity. The data suggest that nucleases from the strain B10M1 and any other strain are polydisperse at pH optimum if their amino acid sequences are identical.

[Identification of marker chromosomes and translocations in man using a computerized diagnostic database and fluorescent in situ hybridization]. / Identifikatsiia markernykh khromosom i translokatsii cheloveka s pomoshch'iu komp'iuternoi diagnosticheskoi bazy dannykh i fliuorestsentnoi gibridizatsii in situ.

Cytogenetic analysis revealed five carriers of supernumerary marker chromosomes and two carriers of unbalanced chromosome translocations in patients of the medical genetic consultation clinic. A new method for exact identification of such chromosome rearrangements requiring additional molecular cytogenetic diagnosis was proposed. The method involves computer analysis of abnormal phenotypic traits with the use of diagnostic databases and fluorescent in situ hybridization (FISH) to DNA probes specific for the most probable computer-selected chromosome syndromes. On average, the method allowed the number of necessary DNA probes to be decreased four times. In the tested patients, supernumerary marker chromosomes were shown to be derived from chromosomes 2, 9, and 15, and translocations were identified as dic(Y;18) and ins(6;21). Limited possibilities of using the method when (1) a chromosome syndrome is not clearly defined in a diagnostic system or (2) phenotypic expression of a marker chromosome is not significant are discussed. Presumably, the method will allow a reliable estimation of the efficiency of various diagnostic systems.

Isoforms of Serratia marcescens nuclease. The role of Mg2+ in the hydrolysis mechanism.

Structural and functional differences between isoforms Sm1 and Sm2, a lack of influence of free Mg2+ on the isoform structures, formation of DNA-magnesium complex serving with great probability as a real substrate for the nuclease has been summarized on the basis of experimental data. Mg2+ forming a complex with phosphate groups of DNA are supposed to further increase the electrophilicity of the phosphorus atoms besides causing a conformational change of the substrate.

[Serratia marcescens nucleases. I. Comparison of native and recombinant nucleases using electrospray mass spectrometry]. / Nukleazy Serratia marcescens. I. Sravnenie prirodnoi i rekombinantnoi nukleaz s ispol'zovaniem élektrosprei-mass-spektrometrii.

Isoforms of the natural and recombinant nucleases of Serratia marcescens were characterized by their molecular mass, which was determined by electrospray mass spectrometry. The natural nuclease was isolated from the S. marcescens B10M1 culture, whereas the recombinant nuclease was obtained from Escherichia coli MT102 cells carrying plasmid p403-SD2 with the nuclease gene nuc. The primary structure for each of the isoforms isolated from the nuclease preparations was determined by comparing its molecular mass with that of known amino acid sequence, which was determined from the nucleotide sequence of the nuc gene. Both preparations included identical nuclease variants with N-terminal amino acid residues removed. The number of isoforms in the natural nuclease was, however, significantly greater than in the recombinant nuclease. The structures of some of the isoforms were confirmed by N-terminal analysis.

[Serratia marcescens nucleases. II. Primary structure analysis by peptide mapping combined with plasma-desorption mass spectrometry]. / Nukleazy Serratia marcescens. II. Analiz pervichnykh struktur putem peptidnogo kartirovaniia v kombinatsii s plazmenno-desorptsionnoi mass-spektrometriei.

The primary structures of nucleases Sm1, Sm2, and Sm3 produced by Serratia marcescens were completely characterized using plasma desorption mass spectrometry (PDMS) of proteolytic peptides isolated by reverse-phase HPLC. The isoforms were separated by anion-exchange chromatography on DEAE cellulose and subjected to hydrolysis by the lysine-specific endoproteinase Lys-C. Comparative analysis of the peptides identified by PDMS showed that all three nucleases are N-terminal variants of the same protein: Sm2 represents a "mature" protein form, whereas Sm1 and Sm3 lack three and one N-terminal amino acid residues, respectively.

[The classification of pneumoconioses]. / O klassifikatsii pnevmokoniozov.

The article deals with analysis of scientific data concerning etiology, pathogenesis, clinical and roentgenologic manifestations, morphologic appearances and other aspects to precise and refine the former idea of pneumoconioses. The authors present the main principles for improved classification of pneumoconioses.

Kinetic studies of the Serratia marcescens extracellular nuclease isoforms.

Kinetic studies on the two major isoforms of Serratia marcescens nuclease, Sm2 and Sm1, have revealed them to be functionally equivalent. Both isoforms display marked substrate inhibition by DNA and RNA. They both require magnesium for optimal activity, but retain low catalytic activity in its absence. Both are moderately inhibited by mononucleotides including 5'-ATP, 5'-AMP, 5'-TTP and 3'5'-pTp. The two strongest mononucleotide inhibitors studied, 5'-ATP and 5'-AMP, display inhibition constants, KI, on the order of 10(-5) M. In assessing the strength of mononucleotide inhibition the type of nucleotide base appears to be more important than the number of phosphate moieties.

[The isolation of nucleases Sm1 and Sm2 in preparative amounts and a study of their antigenicity]. / Vydelenie nukleaz Sm1 i Sm2 v preparativnykh kolichestvakh i izuchenie ikh antigennosti.

Two enzyme forms of endonuclease (Sm 1 and Sm 2) strain B10M1 in 60 and 100 mg respectively have been isolated from the culture fluid Serratia marcescens. The chromatographic and electrophoretic properties and N-terminal amino acid residues are different for both enzymes. The purification procedure consists of dialysis and ion-exchange chromatography on DEAE- and phosphocellulose. The yield of nucleases Sm1 and Sm2 are 14% and 28% respectively. The antigenic differences of nucleases Sm1 and Sm2 have been found by cross immunoenzyme analysis.

[Isolation and characteristics of intracellular nuclease isoforms from Serratia marcescens]. / Vydelenie i kharakteristika izoform vnekletochnoi nuklezy Serratia marcescens.

Two enzyme forms were isolated from the commercial preparation of extracellular endonuclease of Serratia marcescens strain B10 M1. The chromatographic and electrophoretic properties, isoelectric points and N-terminal amino acid residues are different for both enzymes. At the final step of the purification procedure including ion-exchange chromatography on phospho- and DEAE-cellulose columns the yields of nucleases Sm1 and Sm2 were 13% and 25%, respectively. No significant differences were found in the specific activities of nucleases Sm1 and Sm2 (3.6 x 10(6) and 4.0 x 10(6) un. act./mg of protein). A comparative analysis of tryptic nuclease hydrolysate peptides was carried out. The amino acid sequences of some polypeptide segments of the proteins were determined. The structural similarity of the enzyme was established and the amino terminal regions of the proteins were identified. The localization of the disulfide bonds in the molecules of the both nucleases was determined. The similarity of nucleases Sm1 and Sm2 strain B10 M1 to S. marcescens endonucleases obtained from other strains was demonstrated.

[Serratia marcescens endonuclease. Properties of the enzyme]. / Endonukleaza Serratia marcescens. Kharakteristika fermenta.

Some physico-chemical properties of endonuclease (EC 3.1.4.9) from Serratia marcescens were studied and the amino acid composition of the enzyme was determined. The protein molecule was shown to contain one SH-group and one S-S-bond, which renders it different from the well studied nuclease (EC 3.1.4.7) from Staph. pyogenes. The conditions for reconstitution of the S-S-bond by dithioerythritol for quantitative estimation of cysteine residues of the endonuclease molecule were selected. The N-terminal amino acid was found to be threonine. The UV spectra for the enzyme are typical for proteins; A 0,1% 1cm,280nm is 1.46, epsilon 25 degrees 280nm,pH7,4 is 47292 M-1 cm-1. The sedimentation coefficient in phosphate buffer sW, 20 degrees is 3.4 S, pI is 6.5 and 7.5.

[Isolation and physico-chemical properties of homogenous nuclease from Serratia marcescens]. / Poluchenie nukleazy Serratia marcescens v gomogennom sostoianii i izuchenie fiziko-khimicheskikh svoistv fermenta.

A simplified procedure for purification of nuclease from Serratia marcescens, including chromatography on DEAE- and phosphocellulose in a stationary regime has been developed. The method described results in a physically homogenous enzyme, which does not contain phosphatase, phosphodiesterase or proteinase admixtures. The molecular weight of the enzyme as determined by polyacrylamide gel electrophoresis is 33 000 +/- 10%. p-Chloromercurybenzoate (10(-2) M) completely inactivates the enzyme, while beta-mercaptoethanol (0,64 M) in the presence of 2 M urea causes only partial inactivation of the enzyme. Urea (4 or 7 M), when added to the reaction mixture, increases the enzyme activity 2,2-, 1,7- and 1,4-fold as compared to native, denaturated DNA and RNA, respectively.