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Abstract Introduction Scedosporium apiospermum is increasingly recognized as a cause of localized and disseminated mycotic infections in near-drowning victims. Case presentation We report the case of a 59-year-old Japanese woman who was a survivor of a tsunami in northeastern Japan and who had lung and brain abscesses caused by S. apiospermum. Initially, an aspergillus infection was suspected, so she was treated with micafungin. However, computed tomography scans of her chest revealed lung abscesses, and magnetic resonance images demonstrated multiple abscesses in her brain. S. apiospermum was cultured from her bronchoalveolar lavage fluid, and antimycotic therapy with voriconazole was initiated. Since she developed an increase in the frequency of premature ventricular contractions, an adverse drug reaction to the voriconazole was suspected. She was started on a treatment of a combination of low-dose voriconazole and liposomal amphotericin B. After combination therapy, further computed tomography scans of the chest and magnetic resonance images of her brain showed a demarcation of abscesses. Conclusions Voriconazole appeared to have a successful record in treating scedosporiosis after a near drowning but, owing to several adverse effects, may possibly not be recommended. Thus, a combination treatment of low-dose voriconazole and liposomal amphotericin B may be a safe and effective treatment for an S. apiospermum infection. Even though a diagnosis of scedosporiosis may be difficult, a fast and correct etiological diagnosis could improve the patient's chance of recovery in any case.

Background: Ovarian vein thrombosis (OVT) may cause maternal mortality by inducing pulmonary thromboembolism (PTE). However, the prevalence, etiology, risk factors, prognosis, and optimal treatments for asymptomatic OVT during and after pregnancies are unclear, which therefore requires a high clinical index of suspicion for certain diagnoses due to its vague presentation. We herein present a case of asymptomatic postpartum OVT that extended toward the inferior vena cava (IVC), resulting in a potential risk of PTE., Case Presentation: A 30-year-old postpartum woman presented with slight dyspnea after an uneventful vaginal delivery at 40 weeks of gestation. We checked her laboratory data to exclude lethal thrombosis; D-dimer levels were 85.6 μg/mL. We performed computed tomography (CT) to search the presence of PTE and deep vein thrombosis (DVT); although no signs of PTE and DVT in her legs were detected, CT and trans-abdominal ultrasonography (TAUS) revealed a right OVT. Heparin was administered, and D-dimer levels decreased; warfarin at a dose of 2 mg/day was subsequently administered to control anti-coagulopathy. However, D-dimer was re-elevated despite adequate anticoagulation treatment, and extension of the right OVT to the IVC was detected by CT and TAUS. With warfarin administration, CT and TAUS showed the disappearance of right OVT. The patient was discharged from the hospital 17 days after delivery., Conclusions: Even asymptomatic postpartum OVT may lead to PTE. Universal screening guidelines and optimal treatment strategies for asymptomatic OVT in pregnant and postpartum women should be established through future studies., (© 2022. The Author(s).)

We studied the detection of drug-metabolizing enzyme inhibitiors using column-switching high performance liquid chromatography with tris(2,2'-bipyridine)ruthenium(II) (Ru(bpy)(3)(2+))-electrogenerated chemiluminescence detection. This can be applied to evaluate the genetic diversity concerning the ability of cytochrome P450 (CYP) 2D6 to metabolize drug in vitro. We demonstrated the ability of CYP2D6 to enable us to examine drugs metabolizing enzyme inhibition with high performance and sensitivity. This method can be applied to investigate metabolite inhibitors of CYP2D6 in vitro and in vivo. Thus, Metixene was found to be a potential CYP2D6 inhibitor., (© 2011 Pharmaceutical Society of Japan)

A new series of zinc porphyrin-fullerenes bridged by flexible oligosilane chains ZnP-[Sin]-C60 (n = 1-5) was synthesized, and the photophysical properties of these molecules were investigated using steady-state and time-resolved spectroscopic methods. The spectral observations can be well explained by assuming the coexistence of extended conformers and folded conformers, that is, the observed emissions are from the extended conformers while the folded conformers form very short lifetime nonfluorescent excited-state charge-transfer (CT) complexes. Time-resolved transient absorption spectra suggest the generation of intramolecular radical-ion pairs that have sub-microsecond lifetimes. With the number of silicon atoms of the bridged oligosilane, the lifetimes of the radical-ion pairs do not vary regularly, indicating that intramolecular collision of the radical-cation moiety with the radical-anion moiety controls the charge-recombination rate. The attenuation factor of the electron transfer of the silicon chain was evaluated by the bridge-length dependence of charge-separation rate to be 0.16 A-1 on the basis of the oligosilane chain-length dependence of fluorescence lifetimes. This is the first evaluation of the attenuation factor for the one-dimensional Si-Si chain to the best of our knowledge.

The monomeric multimetal-binding beta-galactosidase of Saccharopolyspora rectivirgula (srbg), a glycosyl hydrolase family-2 enzyme, has a unique sequence consisting of 192 amino acid residues with no similarity to known proteins. This 192-residue sequence (termed the "iota [iota] sequence") appears to be inserted into a sequence homologous to the active-site domain of the Escherichia coli lacZ enzyme (lacZbg). To assess the effects of the t sequence at specific sites of beta-galactosidase on the catalytic functioning and molecular properties of beta-galactosidase, deletion or insertion mutants of beta-galactosidases were constructed, expressed in LacZ- E. coli strains, and characterized: srbgdelta in which the iota sequence was deleted from srbg, and lacZbgI, in which the 192-residue iota sequence was inserted into the corresponding position (between Asp591 and Phe592) in the active-site domain of lacZbg. srbgdelta was a catalytically inactive, dimeric protein which retained multimetal-binding characteristics, suggesting that the iota sequence is very important for maintaining the structure necessary for the catalytic functioning and the monomeric structure of srbg but is not responsible for the unique metal ion requirements of srbg. On the other hand, lacZbgI existed as a mixture of a monomer, a tetramer, and higher multimers. The monomeric species was inactive, whereas the tetramer and other multimers were catalytically active (V(max )K(m) value, 25% of that of lacZbg) and highly specific for beta-D-galactoside. The tetrameric lacZbgI was activated by Mg2+ and Mn2+ with lowered metal affinities, and the stoichiometry of metal binding was unchanged from that of lacZbg. These results, along with the published stereo structure of lacZbg, suggest that, in lacZbgI, the inserted 192-residue iota peptide could fold independently of the lacZbg domains into a "sub-domain," lying distant from the active site and subunit interfaces.

Kexstatin I is a potent proteinaceous inhibitor of Kex2 proteinase (EC 3.4.21.61). In the present study we show the molecular cloning, primary structure determination and expression of the gene encoding kexstatin I. We also demonstrate its enhanced activity and specificity for Kex2 proteinase inhibition by rational mutagenesis. The cloned kexstatin I gene encoded a protein of 145 amino acid residues, including the 35-residue signal sequence for secretion. The amino acid sequence showed 52% identity with those of the Streptomyces subtilisin inhibitors (SSIs). Thus kexstatin I is the first SSI-family member that can inhibit Kex2 proteinase. The reactive site of the inhibitor was determined to be -Thr(69)-Lys(70) downward arrowGlu(71)-, where downward arrow indicates the reactive site. Because Kex2 proteinase generally shows the highest affinity for substrates with basic amino acid residues at the P(1) and P(2) sites, conversion of the Thr(69)-Lys(70) segment of the inhibitor into dibasic motifs was expected to result in enhanced inhibitory activities. Thus we constructed kexstatin I mutants, in which the Thr(69)-Lys(70) sequence was replaced by the Thr(69)-Arg(70), Lys(69)-Lys(70) and Lys(69)-Arg(70) sequences using PCR-based mutagenesis, and analysed them kinetically. Among these mutants, the Lys(69)-Arg(70) mutant was the most potent inhibitor. The K(i) for Kex2 proteinase was 3.2x10(-10) M, which was 140-fold lower than that of the inhibitor with the Thr(69)-Lys(70) sequence. Although kexstatin I could also inhibit subtilisin, the enhancement of inhibitory activity upon such mutations was specific for Kex2 proteinase inhibition.

Bacillus sp. SAM1606 alpha-glucosidase catalyzes the transglucosylation of sucrose to produce three regioisomers of the glucosylsucroses, with theanderose (6-O(G)-glucosylsucrose) as the most abundant transfer product. To find the active-site amino acid residues which can affect the reactivity and regiospecificity of the glucosyl transfer, 16 mutants with amino acid substitutions near the active site were allowed to react with 1.75 M sucrose at 60 degrees C, pH 6.0, and the course of transglucosylation as well as the product specificity were analyzed. The sites of the amino acid substitutions were selected by comparing the conserved amino acid sequences located near the active site of the SAM1606 enzyme with those of the Bacillus oligo-1,6-glucosidases (O16G), which have very high amino acid sequence similarities near the active site but have a distinct substrate specificity. The results showed that, among the mutated SAM1606 enzymes examined, only the mutants with substitution of Gly273 with Pro showed an altered reactivity and specificity of transglucosylation; these mutants exhibited a significantly enhanced initial velocity of glucosyl transfer, yielding isomelezitose (6-O(F)-glucosylsucrose) instead of theanderose as the major transfer product. These results indicate that the substitution of Gly273 with Pro critically governs the enhanced reactivity and altered specificity of the transglucosylation. The notion that the amino acid residue at this position is the determinant of the glucosyl-transfer specificity was further confirmed by observation that the Bacillus cereus O16G, which has a proline at the corresponding position, produced isomelezitose as the major transfer product during transglucosylation with sucrose.

To examine the role of His452 of the Saccharopolyspora rectivirgula beta-galactosidase in the binding of a tightly bound, catalytically important Mn2+ (i.e., class II Mn2+) ion, His452 was replaced with Phe or Glu and the respective site-directed mutants, H452F and H452E, were characterized. Neither mutant contained Mn2+ in an Mn2+-free buffer and both were virtually inactive in the absence of Mn2+ (their relative activities being less than 0.03% that of the fully activated wild-type enzyme). When Mn2+ was added, however, the mutants were activated to 3% (for H452F) and 0.8% (for H452E) of the full activity of the wild type. The Mn2+ concentrations needed for half-maximal activation of H452F and H452E were, respectively, 15,000 and 5000 times higher than the reported dissociation constant (2 nM) of the class II Mn2+, suggesting that His452 plays a key role in the binding of this catalytically important Mn2+. Activation of the mutants by Mn2+, albeit very weak, contrasts with a lack of any such metal activation previously observed with the two corresponding mutants of Escherichia coli lacZ beta-galactosidase.

We found a novel proteinaceous Kex 2 proteinase inhibitor, named kexstatin, in the culture supernatant of Streptomyces platensis Q268. The purified kexstatin was homogeneous by SDS-PAGE and the molecular weight was estimated to be 13,000. The N-terminal amino acid sequence of kexstatin has high similarity to Streptomyces subtilisin inhibitor (SSI), suggesting that kexstatin belongs to the SSI family. Kexstatin was a strong inhibitor of Kex 2 proteinase and subtilisin but not thermolysin, trypsin, or chymotrypsin. The IC50 value of kexstatin against 1 microgram of Kex 2 proteinase was 1.4 micrograms.

To understand the relationship between the structure and functions of the peroxidase of Arthromyces ramosus, a novel taxon of hyphomycete, and the evolutionary relationship of the A.ramosus peroxidase (ARP) with the other peroxidases, we isolated complementary and genomic DNA clones encoding ARP and characterized them. The sequence analyses of the ARP and cDNA coding for ARP showed that a mature ARP consists of 344 amino acids with a N-terminal pyroglutamic acid preceded by a signal peptide of 20 amino acid residues. The amino acid sequence of ARP was 99% identical to that of the peroxidase of Coprinus cinereus, a basidiomycete, and also had very high similarities (41-43% identity) to those of basidiomycetous lignin peroxidases, although we could find no lignin peroxidase activities for ARP when assayed with lignin model compounds. We could identified His184 and His56 as proximal and distal ligands to heme, respectively, and Arg52 as an essential Arg. Comparison of the sequences of complementary and genomic DNAs found that protein-encoding DNA is interrupted by 14 intervening sequences. The ARP cDNA was expressed in the yeast Saccharomyces cerevisiae under the promoter of the glyceraldehyde 3-phosphate dehydrogenase gene, yielding 0.02 units/ml of a secreted active peroxidase.

We cloned an alpha-glucosidase gene from thermophilic Bacillus sp. SAM1606 to overexpress it in Escherichia coli transformants. Deletion of the 5'-noncoding region as well as expression of the alpha-glucosidase gene under the control of the icp promotor of the insecticidal crystal protein gene from Bacillus thuringiensis subsp. sotto enhanced the enzyme productivity to 23.5 U/ml, which was 12,000-fold higher than that obtained by the strain SAM1606. The open reading frame corresponding to the alpha-glucosidase encoded 587 amino acid residues including a residue coded by the initiation codon TTG, and the molecular mass of the alpha-glucosidase from N-terminal serine was calculated to be 68,886 Da. Sequence analysis revealed that the SAM1606 alpha-glucosidase belonged to the alpha-amylase family. The SAM1606 alpha-glucosidase showed extremely high sequence identity (62-65%) to the Bacillus cereus and Bacillus thermoglucosidasius oligo-1,6-glucosidases, which were 72% identical to each other. Sequence identity in the suggested active site regions were essentially the same (80-82%) among these three enzymes. However, the substrate specificity of the SAM1606 alpha-glucosidase was significantly different from those of the oligo-1,6-glucosidases. The thermostability of these three alpha-glucosidases could be correlated with the increase in the number of proline residues, whose occurrence was predicted at beta turns and coils in the enzymes.

Fusaric acid-resistance genes (fus) were isolated from Pseudomonas cepacia. The nucleotides of the 5437 base pairs containing the fus genes were sequenced.

A human MDR1 cDNA was introduced into yeast cells. Immunoblot analysis and indirect immunostaining showed that some of the P-glycoprotein produced was situated in its native orientation in the yeast plasma membrane. Drug-binding activities of the recombinant P-glycoproteins were markedly decreased compared to that of the authentic P-glycoprotein. To identify the bases of decreased binding we studied the effects of membrane component sterols on the azidopine binding and found that ergosterol, which is the main sterol in the yeast membrane, and calciferol, which is produced from ergosterol by UV irradiation, inhibited azidopine binding. These sterols in yeast membrane probably inhibit the function of human P-glycoprotein as a multidrug transporter in yeast cells, because expression of P-glycoprotein in yeast cells did not confer resistance to doxorubicin.

The insecticidal crystal protein (ICP) gene, icp, from a 68-kb plasmid derived from Bacillus thuringiensis subsp. sotto was cloned in Escherichia coli. The icp expression in E. coli cells was confirmed by both immunological and insect-toxicity assays of the cell extract. The entire icp gene resides in the 6.6-kb PstI fragment, which codes for a 144-kDal peptide identical to the intact ICP, as determined by its size and reaction with anti-ICP antibody. Deletion analysis further revealed that the 2.8-kb region within the 6.6-kb PstI fragment codes for ICP. Analysis of the nucleotide sequence indicated that a peptide of 934 amino acid residues truncated at the C-terminal end is encoded by this 2.8-kb fragment. A unique feature of this truncated ICP is the abundance of cysteine and lysine residues within its C-terminal region.

The float-polishing technique has been studied to determine its suitability for producing supersmooth surfaces on optical materials, yielding a roughness of <2 A rms. An attempt was made to polish six different materials including fused quartz, Zerodur, and sapphire. The low surface roughness was achieved on fused quartz, Zerodur, and Corning experimental glass-ceramic materials, and a surface roughness of <1 A rms was obtained on O-cut single-crystal sapphire. Presumably, similar surface finishes can also be obtained on CerVit and ULE quartz, which could not be polished satisfactorily in this set of experiments because of a mismatch between sample mounting and machine configuration.