Structures of Burkholderia thailandensis nucleoside kinase: implications for the catalytic mechanism and nucleoside selectivity.

The nucleoside kinase (NK) from the mesophilic Gram-negative bacterium Burkholderia thailandensis (BthNK) is a member of the phosphofructokinase B (Pfk-B) family and catalyzes the Mg(2+)- and ATP-dependent phosphorylation of a broad range of nucleosides such as inosine (INO), adenosine (ADO) and mizoribine (MZR). BthNK is currently used in clinical practice to measure serum MZR levels. Here, crystal structures of BthNK in a ligand-free form and in complexes with INO, INO-ADP, MZR-ADP and AMP-Mg(2+)-AMP are described. The typical homodimeric architecture of Pfk-B enzymes was detected in three distinct conformational states: an asymmetric dimer with one subunit in an open conformation and the other in a closed conformation (the ligand-free form), a closed conformation (the binary complex with INO) and a fully closed conformation (the other ternary and quaternary complexes). The previously unreported fully closed structures suggest the possibility that Mg(2+) might directly interact with the ß- and γ-phosphates of ATP to maintain neutralization of the negative charge throughout the reaction. The nucleoside-complex structures also showed that the base moiety of the bound nucleoside is partly exposed to the solvent, thereby enabling the recognition of a wide range of nucleoside bases. Gly170 is responsible for the solvent accessibility of the base moiety and is assumed to be a key residue for the broad nucleoside recognition of BthNK. Remarkably, the G170Q mutation increases the specificity of BthNK for ADO. These findings provide insight into the conformational dynamics, catalytic mechanism and nucleoside selectivity of BthNK and related enzymes.

Outbreak of late-onset Group B Streptococcal disease with serotype Ib in a Neonatal Intensive Care Unit.

BACKGROUND: Hospital outbreaks of invasive group B streptococcus (GBS) infection are rare. There are only a few published reports of late-onset GBS outbreaks in neonatal intensive care units (NICUs). We report here three cases of late-onset GBS in our NICU. CASE: Three preterm very low birth weight (VLBW) infants born at 24 -27 weeks gestation developed lateonset GBS sepsis within four weeks. Two asymptomatic GBS carriers were identified in the NICU prior to the outbreak. Tests of maternal rectovaginal GBS colonization were negative in all three cases; as such, vertical transmission was unlikely. All three GBS isolates were capsular serotype 1b, with comparable antibiotic susceptibility profiles. CONCLUSION: Preterm delivery and VLBW are associated with an increased risk of invasive late-onset GBS infection. This report underscores the ongoing risk of nosocomial transmission of GBS in the NICU.

Enzymatic characterization of an amine oxidase from Arthrobacter sp. used to measure phosphatidylethanolamine.

Ethanolamine oxidase was screened with the aim of using it to establish a novel enzymatic phosphatidylethanolamine assay. Ethanolamine oxidase activity was detected in the crude extract of Arthrobacter sp., and the enzyme was purified more than 15-fold in three steps with a 54% yield. SDS-PAGE revealed the presence of only one band, which migrated, with an apparent molecular mass of 70 kDa. Biochemical characterization of the enzyme showed phenylethylamine to be the preferred substrate, with the highest kcat/Km value. The primary structure, determined by sequencing the cloned gene, showed a high degree of identity to Cu-containing phenylethylamine oxidase (64%). When heterologously overexpressed in Escherichia coli, the enzyme exhibited only a trace of amine oxidase activity, but high levels of activity emerged after exposure to Cu2+, as is typical of recombinant copper amine oxidases. Preliminary application of this enzyme coupled with phospholipase D for determination of phosphatidylethanolamine is also described. This is the first enzymatic method for the measurement of phosphatidylethanolamine.

Subjective evaluation of a peer support program by women with breast cancer: A qualitative study.

AIM: The aim of this study was to determine the subjective evaluation of a breast cancer peer support program based on a survey of the participants who completed the program. METHODS: Semistructured interviews were held with 10 women with breast cancer. The responses were subject to a qualitative inductive analysis. RESULTS: Women with breast cancer who participated in the breast cancer peer support program evaluated the features of the program and cited benefits, such as "Receiving individual peer support tailored to your needs," "Easily consulted trained peer supporters," and "Excellent coordination." Also indicated were benefits of the peer support that was received, such as "Receiving peer-specific emotional support," "Obtaining specific experimental information," "Re-examining yourself," and "Making preparations to move forward." The women also spoke of disadvantages, such as "Strict management of personal information" and "Matching limitations." CONCLUSIONS: In this study, the subjective evaluation of a peer support program by women with breast cancer was clarified . The women with breast cancer felt that the program had many benefits and some disadvantages. These results suggest that there is potential for peer support-based patient-support programs in medical services that are complementary to the current support that is provided by professionals.

Sphingomyelinase C from Streptomyces sp. A9107: unusual primary structure for bacterial sphingomyelinase C.

A sphingomyelinase C (SMase) was identified in the culture supernatant of Streptomyces sp. A9107 (S-SMase). Although S-SMase seems to be a typical bacterial SMase, the primary structure of S-SMase was unusual for known bacterial SMase. The gene was functionally overexpressed in the culture medium of recombinant Rhodococcus erythropolis.

Enzymatic assay method for measuring mizoribine levels in serum.

A sensitive and specific method for assaying serum mizoribine levels that can be applied to general automatic clinical analyzers was developed. Regression analysis of the enzymatic assay (y) vs. the HPLC method (x) produced the following relation: y=0.964x+0.090 (n=262, Sy, x=6.37 ng/mL).

A novel nucleoside kinase from Burkholderia thailandensis: a member of the phosphofructokinase B-type family of enzymes.

The genome of the mesophilic Gram-negative bacterium Burkholderia thailandensis contains an open reading frame (i.e. the Bth_I1158 gene) that has been annotated as a putative ribokinase and PFK-B family member. Notably, although the deduced amino acid sequence of the gene showed only 29% similarity to the recently identified nucleoside kinase from hyperthermophilic archaea Methanocaldococcus jannaschii, 15 of 17 residues reportedly involved in the catalytic activity of M. jannaschii nucleoside kinase were conserved. The gene was cloned and functionally overexpressed in Rhodococcus erythropolis, and the purified enzyme was characterized biochemically. The substrate specificity of the enzyme was unusually broad for a bacterial PFK-B protein, and the specificity extended not only to purine and purine-analog nucleosides but also to uridine. Inosine was the most effective phosphoryl acceptor, with the highest k(cat)/K(m) value (80 s(-1).mm(-1)) being achieved when ATP served as the phosphoryl donor. By contrast, this enzyme exhibited no activity toward ribose, indicating that the recombinant enzyme was a nucleoside kinase rather than a ribokinase. To our knowledge, this is the first detailed analysis of a bacterial nucleoside kinase in the PFK-B family.

A novel enzymatic method for measuring mizoribine 5'-monophosphate levels in serum.

Inosine 5'-monophosphate dehydrogenases (IMPDH) from Oceanobacillus iheyensis and Bacillus subtilis were biochemically characterized with the aim of establishing a mizoribine 5'-monophosphate (MZR-P) assay. MZR-P is the active metabolite of mizoribine, which has been successfully used as an immunosuppressive agent. A sensitive method for measuring the MZR-P concentration in serum, based on its ability to inhibit IMPDH, was developed. The method was applied to an automatic clinical analyzer, enabling measurement of MZR-P levels in a set of serum samples. MZR-P at concentrations of 0.1-13.6 (microg/ml (0.3-40 microM) in the samples could be measured with 0.7% within-run coefficients of variation.