Genomic rearrangement at 10q24 in non-syndromic split-hand/split-foot malformation

Split-hand/split-foot malformation (SHFM) is a congenital limb malformation characterized by a median cleft of hand and/or foot due to the absence of central rays. Five loci for syndromic and non-syndromic SHFM, termed SHFM1-5, have been mapped to date. Recently, a 0.5 Mb tandem genomic duplication was found at chromosome 10q24 in SHFM3 families. To refine the minimum duplicated region and to further characterize the SHFM3 locus, we screened 28 non-syndromic SHFM families for tandem genomic duplication of 10q24 by Southern blot and sequence analysis of the dactylin gene. Of 28 families, only two showed genomic rearrangements. Representative patients from the two families exhibit typical SHFM, with symmetrically affected hands and feet. One patient is a familial case with a 511,661 bp tandem duplication, whereas the second is a sporadic case arising from a de novo, 447,338 bp duplication of maternal origin. The smaller duplication in the second patient contained the LBX1, BTRC, POLL, and DPCD genes and a disrupted extra copy of the dactylin gene, and was nearly identical to the smallest known duplicated region of SHFM3. Our results indicate that genomic rearrangement of SHFM3 is rare among non-syndromic SHFM patients and emphasize the importance of screening for genomic rearrangements even in sporadic cases of SHFM.     

Development of Neospora caninum cultured with human serum in vitro and in vivo

Because there has been no report of symptomatic Neospora caninum infection in humans, we examined the effect of human serum on the parasite's growth in either a bovine angioendothelial cell or Caco-2 cell culture in vitro and in immunocompromised mice in vivo. There was no difference in intracellular parasite numbers between cells incubated with human serum at 24 hr after challenge and those incubated with fetal bovine serum (FBS), which has no titer for the anti-N. caninum agglutination antibody test. Serum of sheep infected with N. caninum, which has the anti-N. caninum antibody, reduced the numbers of the intracellular parasite significantly. We also showed that there was no inhibitory effect on the intracellular multiplication of the parasite in cells incubated with human serum through incorporation of 3H-uracil. CB-17 scid mice administered human serum daily and challenged with N. caninum died on day 20 or 22 after challenge, when large numbers of parasite clusters were found in the brain, oviduct, adrenal gland, lung, stomach, spleen, skeletal muscle, pancreas, and mesenteric lymph nodes. Scid mice administered FBS survived until the end of the experiment. These results suggest that adult human serum may have no inhibitory effect on the development of N. caninum in vitro and in vivo.     

Inference of S-system models of genetic networks using a cooperative coevolutionary algorithm

MOTIVATION: To resolve the high-dimensionality of the genetic network inference problem in the S-system model, a problem decomposition strategy has been proposed. While this strategy certainly shows promise, it cannot provide a model readily applicable to the computational simulation of the genetic network when the given time-series data contain measurement noise. This is a significant limitation of the problem decomposition, given that our analysis and understanding of the genetic network depend on the computational simulation. RESULTS: We propose a new method for inferring S-system models of large-scale genetic networks. The proposed method is based on the problem decomposition strategy and a cooperative coevolutionary algorithm. As the subproblems divided by the problem decomposition strategy are solved simultaneously using the cooperative coevolutionary algorithm, the proposed method can be used to infer any S-system model ready for computational simulation. To verify the effectiveness of the proposed method, we apply it to two artificial genetic network inference problems. Finally, the proposed method is used to analyze the actual DNA microarray data.     

Mediated spectroelectrochemical titration of proteins for redox potential measurements by a separator-less one-compartment bulk electrolysis method

One-compartment bulk electrolysis and simultaneous spectroscopic measurements are realized in a conventional spectroscopic cuvette without separator by using a mesh-type working electrode with extremely large surface area and a wire-type counter electrode with very small surface area. Spectrophotometric monitoring revealed complete electrolysis in a first-order kinetics. This technique was applied to mediated titration of cytochrome c and bilirubin oxidase for determining their redox potentials. Kinetics for the solution redox reaction between protein and mediator is described. The subtraction of spectral background due to mediator adsorption is very easy because of high reproducibility. The experiments can be done under completely anaerobic conditions. Low-absorbance protein samples (of low concentrations or small absorption coefficients) and hydrophobic proteins (such as membrane-bound proteins) are acceptable for measurements.     

Two extracellular proteins with alkaline peroxidase activity, a novel cytochrome c and a catalase-peroxidase, from Bacillus sp. No.13

A novel cytochrome c and a catalase-peroxidase with alkaline peroxidase activity were purified from the culture supernatant of Bacillus sp. No.13 and characterized. The cytochrome c exhibited absorption maxima at 408 nm (Soret band) in its oxidized state, and 550 (alpha-band), 521 (beta-band), and 415 (Soret band) nm in its reduced state. The native cytochrome c with a relative molecular mass of 15,000 was composed of two identical subunits. The cytochrome c showed over 50 times higher peroxidase activity than those of known c-type cytochromes from various sources. The optimum pH and temperature of the peroxidase activity were about 10.0 and 70 degrees C, respectively. The peroxidase activity is stable in the pH range of 6.0 to 10.8 (30 degrees C, 1-h treatment), and at temperatures up to 80 degrees C (pH 8.5, 20-min treatment). The heme content was determined to be 1 heme per subunit. The amino acid sequence of the cytochrome c showed high homology with those of the c-type cytochromes from Bacillus subtilis and Bacillus sp. PS3. The catalase-peroxidase showed high catalase activity and considerable peroxidase activity, the specific activities being 55,000 and 0.94 micromol/min/mg, respectively. The optimum pH and temperature of the peroxidase activity were in the range of 6.4 to 10.1 and 60 degrees C, respectively. The catalase-peroxidase showed a lower K(m) value (0.67 mM) as to H(2)O(2) than known catalase-peroxidases.     

6-S-cysteinyl flavin mononucleotide-containing histamine dehydrogenase from Nocardioides simplex: molecular cloning, sequencing, overexpression, and characterization of redox centers of enzyme

Histamine dehydrogenase from Nocardioides simplex is a homodimeric enzyme and catalyzes oxidative deamination of histamine. The gene encoding this enzyme has been sequenced and cloned by polymerase chain reactions and overexpressed in Escherichia coli. The sequence of the complete open reading frame, 2073 bp coding for a protein of 690 amino acids, was determined on both strands. The amino acid sequence of histamine dehydrogenase is closely related to those of trimethylamine dehydrogenase and dimethylamine dehydrogenase containing an unusual covalently bound flavin mononucleotide, 6-S-cysteinyl-flavin mononucleotide, and one 4Fe-4S cluster as redox active cofactors in each subunit of the homodimer. The presence of the identical redox cofactors in histamine dehydrogenase has been confirmed by sequence alignment analysis, mass spectral analysis, UV-vis and EPR spectroscopy, and chemical analysis of iron and acid-labile sulfur. These results suggest that the structure of histamine dehydrogenase in the vicinity of the two redox centers is almost identical to that of trimethylamine dehydrogenase as a whole. The structure modeling study, however, demonstrated that a putative substrate-binding cavity in histamine dehydrogenase is quite distinct from that of trimethylamine dehydrogenase.     

Effects of Type II diabetes on capillary hemodynamics in skeletal muscle

Microcirculatory red blood cell (RBC) hemodynamics are impaired within skeletal muscle of Type I diabetic rats (Kindig CA, Sexton WL, Fedde MR, and Poole DC. Respir Physiol 111: 163-175, 1998). Whether muscle microcirculatory dysfunction occurs in Type II diabetes, the more prevalent form of the disease, is unknown. We hypothesized that Type II diabetes would reduce the proportion of capillaries supporting continuous RBC flow and RBC hemodynamics within the spinotrapezius muscle of the Goto-Kakizaki Type II diabetic rat (GK). With the use of intravital microscopy, muscle capillary diameter (d(c)), capillary lineal density, capillary tube hematocrit (Hct(cap)), RBC flux (F(RBC)), and velocity (V(RBC)) were measured in healthy male Wistar (control: n = 5, blood glucose, 105 +/- 5 mg/dl) and male GK (n = 7, blood glucose, 263 +/- 34 mg/dl) rats under resting conditions. Mean arterial pressure did not differ between groups (P > 0.05). Sarcomere length was set to a physiological length ( approximately 2.7 mum) to ensure that muscle stretching did not alter capillary hemodynamics; d(c) was not different between control and GK rats (P > 0.05), but the percentage of RBC-perfused capillaries (control: 93 +/- 3; GK: 66 +/- 5 %), Hct(cap), V(RBC), F(RBC), and O(2) delivery per unit of muscle were all decreased in GK rats (P < 0.05). This study indicates that Type II diabetes reduces both convective O(2) delivery and diffusive O(2) transport properties within muscle microcirculation. If these microcirculatory deficits are present during exercise, it may provide a basis for the reduced O(2) exchange characteristic of Type II diabetic patients.     

Novel FAD-dependent glucose dehydrogenase for a dioxygen-insensitive glucose biosensor

A novel FAD-dependent glucose dehydrogenase (FAD-GDH) was found and its enzymatic property for glucose sensing was characterized. FAD-GDH oxidized glucose in the presence of some artificial electron acceptors, except for O2, and exhibited thermostability, high substrate specificity and a large Michaelis constant for glucose. FAD-GDH was applied to an amperometric glucose sensor with Fe(CN)6(3-) as a soluble mediator. The use of a relatively high concentration of Fe(CN)6(3-) resulted in a good linearity between the current response and the glucose concentration, taking into account a large Michaelis constant for Fe(CN)6(3-). The glucose sensor was completely insensitive to O2 and responded linearly to glucose up to 30 mM. Compared to glucose, the response to other saccharides was negligible. The sensor can be stored at room temperature in a desiccator for at least one month without any change in the response or activity.     

Genetically engineered Bifidobacterium animalis expressing the Salmonella flagellin gene for the mucosal immunization in a mouse model

BACKGROUND: A critical component of the host defense against enteric infections is the immunological response of the mucosal membrane, a major starting point of infectious disease, such as typhoid fever. The mucosal immune system consists of an integrated network of lymphoid tissues, mucous membrane-associated cells, and effector molecules. In the present study, we developed a recombinant Bifidobacterium animalis (B. animalis) genetically modified with the Salmonella flagellin gene for mucosal immunization as an oral typhoid vaccine. METHODS: We constructed an oral vaccine against Salmonella typhimurium, consisting of recombinant B. animalis containing the flagellin gene of Salmonella. The recombinant B. animalis was administered orally to mice every other day for 6 weeks. Anti-flagellin antibodies in the serum and stools were measured by enzyme-linked immunosorbent assay (ELISA). RESULTS: We detected significantly higher levels of flagellin-specific IgA in the serum and stools of the mice treated with the recombinant B. animalis containing the flagellin gene than was seen in those treated with parental B. animalis. CONCLUSIONS: Our findings suggest that an oral vaccination using recombinant B. animalis genetically modified with the flagellin gene of Salmonella may be effective against Salmonella infections.     

Transplantation and differentiation of donor cells in the cloned pigs

The application of nuclear transfer technology is an interesting approach to investigate stem and progenitor cell transplantation therapy. If stem cells are used as a nuclear donor, donor cells can engraft into cloned animals without histocompatible problems. However, it is still uncertain whether donor cells can engraft to cloned animal and differentiate in vivo. To address this problem, we transplanted donor cells to dermal tissues of cloned pigs developed by using preadipocytes as donor cells. Preadipocytes are adipocytic progenitor which can differentiate to mature adipocytes in vitro. We showed that the donor preadipocytes were successfully transplanted into the cloned pigs without immune rejection and they differentiated into mature adipocytes in vivo 3 weeks after transplantation. In contrast, allogenic control preadipocytes, which can differentiate in vitro, did not differentiate in vivo. These results indicate that donor progenitor cells can differentiate in cloned animal.