Kinetics of Escherichia coli destruction by microwave irradiation.

The kinetics of destruction of Escherichia coli cells suspended in a solution by microwave irradiation with a microwave oven were studied. During radiation at several powers, the temperature of 0.01 M phosphate buffer (PB), pH 7.0, in a glass beaker increased linearly at a rate of A (degrees Centigrade per second) according to the exposure time. When E. coli cells suspended in PB were exposed in the same beaker, the number of viable cells decreased according to the exposure time and the power used. The survival curve was approximated to a set of three linear parts. For each part, a rate constant of destruction (k) and an extrapolated starting temperature (T0) at several powers were estimated. Thereafter, the relationships between A and k and between A and T0 were studied. When a flat petri dish was used, the A value of exposed PB was lower and bacterial destruction was inhibited; the survival curve was similar to a curve predicted from the A value by using the relationships between the parameters. As the concentration of salt in the solution increased (from 0 to 1.35 M), the A value decreased and bacterial destruction was more suppressed. No remarkable difference between the destruction profiles for microwave exposure and conventional heating, which had the potential to generate an equal A value, was detected. These results showed that the parameter A of an irradiated solution is essential when kinetics of bacterial destruction by microwave exposure are studied and that the destruction profile can be interpreted mostly by means of thermal effects.     

Ultrastructural preservation of plasma membranes by non-lethal slow freezing to liquid nitrogen temperature

Secondary hyphae of Lyophyllum ulmarium were shown to tolerate slow freezing, which allowed extracellular freezing, to -196 degrees C. A freeze-fracture study showed that under this non-lethal freezing condition, the plasma membrane of the secondary hyphae did not show any ultrastructural changes as compared with the control, except gross cellular shrinkage. Tertiary hyphae of Lyophyllum ulmarium, on the other hand, were completely injured by slow freezing to -196 degrees C, and the plasma membrane showed distinct intramembrane particle aggregation as a result of direct membrane contact caused by severe cellular deformation. It is suggested that the absence of freezing injury in the secondary hyphae was due to ultrastructural preservation of the plasma membrane, which resulted from avoidance of severe cellular deformation, while occurrence of freezing injury in the tertiary hyphae is considered to be due to ultrastructural changes in the plasma membrane caused by severe cellular deformation.     

Tailing of thermal inactivation curve of Aspergillus niger spores.

The nonlinear thermal inactivation of Aspergillus niger spores in phosphate-citrate buffer was studied. The thermal inactivation pattern of the spore consisted of a shoulder, an accelerated decline, and a tail at various constant temperatures around 60 degrees C. The pattern fitted a thermotolerant subpopulation model. In the model, we postulated that some spores in the initial population had become thermotolerant at a certain ratio during heating. The model parameters including the rate coefficients, the time lag, and the existence ratio of thermotolerant cells were analyzed at various temperatures. The tailing was not observed at an initial concentration below 10(3) cells per ml. Cells cultured from thermotolerant cells showed an inactivation pattern similar to that of the original cells. Also, cells at the second heating showed the same thermotolerance as or were slightly more thermosensitive than the original cells. Intermittent heating was found to be effective to inactivate cells at a high concentration.     

Characterization of a novel missense mutation in the prodomain of GDF5, which underlies brachydactyly type C and mild Grebe type chondrodysplasia in a large Pakistani family

All TGF-beta family members have a prodomain that is important for secretion. Lack of secretion of a TGF-beta family member GDF5 is known to underlie some skeletal abnormalities, such as brachydactyly type C that is characterized by a huge and unexplained phenotypic variability. To search for potential phenotypic modifiers regulating secretion of GDF5, we compared cells overexpressing wild type (Wt) GDF5 and GDF5 with a novel mutation in the prodomain identified in a large Pakistani family with Brachydactyly type C and mild Grebe type chondrodyslplasia (c527T>C; p.Leu176Pro). Initial in vitro expression studies revealed that the p.Leu176Pro mutant (Mut) GDF5 was not secreted outside the cells. We subsequently showed that GDF5 was capable of forming a complex with latent transforming growth factor binding proteins, LTBP1 and LTBP2. Furthermore, secretion of LTBP1 and LTBP2 was severely impaired in cells expressing the Mut-GDF5 compared to Wt-GDF5. Finally, we demonstrated that secretion of Wt-GDF5 was inhibited by the Mut-GDF5, but only when LTBP (LTBP1 or LTBP2) was co-expressed. Based on these findings, we suggest a novel model, where the dosage of secretory co-factors or stabilizing proteins like LTBP1 and LTBP2 in the microenvironment may affect the extent of GDF5 secretion and thereby function as modifiers in phenotypes caused by GDF5 mutations.     

Common and Distinct Clinical Features in Adult Patients with Anti-Aminoacyl-tRNA Synthetase Antibodies: Heterogeneity within the Syndrome

Objective

To identify similarities and differences in the clinical features of adult Japanese patients with individual anti-aminoacyl-tRNA synthetase antibodies (anti-ARS Abs).

Methods

This was a retrospective analysis of 166 adult Japanese patients with anti-ARS Abs detected by immunoprecipitation assays. These patients had visited Kanazawa University Hospital or collaborating medical centers from 2003 to 2009.

Results

Anti-ARS Ab specificity included anti-Jo-1 (36%), anti-EJ (23%), anti-PL-7 (18%), anti-PL-12 (11%), anti-KS (8%), and anti-OJ (5%). These anti-ARS Abs were mutually exclusive, except for one serum Ab that had both anti-PL-7 and PL-12 reactivity. Myositis was closely associated with anti-Jo-1, anti-EJ, and anti-PL-7, while interstitial lung disease (ILD) was correlated with all 6 anti-ARS Abs. Dermatomyositis (DM)-specific skin manifestations (heliotrope rash and Gottron’s sign) were frequently observed in patients with anti-Jo-1, anti-EJ, anti-PL-7, and anti-PL-12. Therefore, most clinical diagnoses were polymyositis or DM for anti-Jo-1, anti-EJ, and anti-PL-7; clinically amyopathic DM or ILD for anti-PL-12; and ILD for anti-KS and anti-OJ. Patients with anti-Jo-1, anti-EJ, and anti-PL-7 developed myositis later if they had ILD alone at the time of disease onset, and most patients with anti-ARS Abs eventually developed ILD if they did not have ILD at disease onset.

Conclusion

Patients with anti-ARS Abs are relatively homogeneous. However, the distribution and timing of myositis, ILD, and rashes differ among patients with individual anti-ARS Abs. Thus, identification of individual anti-ARS Abs is beneficial to define this rather homogeneous subset and to predict clinical outcomes within the “anti-synthetase syndrome.”     

An In Situ Hybridization Study of Perlecan,DMP1, and MEPE in Developing Condylar Cartilage of the Fetal Mouse Mandible and Limb Bud Cartilage

The main purpose of this in situ hybridization study was to investigate mRNA expression of three bone/cartilage matrix components (perlecan, DMP1, and MEPE) in developing primary (tibial) and secondary (condylar) cartilage. Perlecan mRNA expression was first detected in newly formed chondrocytes in tibial cartilage at E13.0, but this expression decreased in hypertrophic chondrocytes at E14.0. In contrast, at E15.0, perlecan mRNA was first detected in the newly formed chondrocytes of condylar cartilage; these chondrocytes had characteristics of hypertrophic chondrocytes, which confirmed the previous observation that progenitor cells of developing secondary cartilage rapidly differentiate into hypertrophic chondrocytes. DMP1 mRNA was detected in many chondrocytes within the lower hypertrophic cell zone in tibial cartilage at E14.0. In contrast, DMP1 mRNA expression was only transiently detected in a few chondrocytes of condylar cartilage at E15.0. Thus, DMP1 may be less important in the developing condylar cartilage than in the tibial cartilage. Another purpose of this study was to test the hypothesis that MEPE may be a useful marker molecule for cartilage. MEPE mRNA was not detected in any chondrocytes in either tibial or condylar cartilage; however, MEPE immunoreactivity was detected throughout the cartilage matrix. Western immunoblot analysis demonstrated that MEPE antibody recognized two bands, one of 67 kDa and another of 59 kDa, in cartilage-derived samples. Thus MEPE protein may gradually accumulate in the cartilage, even though mRNA expression levels were below the limits of detection of in situ hybridization. Ultimately, we could not designate MEPE as a marker molecule for cartilage, and would modify our original hypothesis.Key words: Mandibular condylar cartilage, perlecan, DMP1; MEPE, in situ hybridization     

Dynamics of cell wall components of Magnaporthe grisea during infectious structure development

Oligosaccharides derived from cell wall of fungal pathogens induce host primary immune responses. To understand fungal strategies circumventing the host plant immune responses, cell wall polysaccharide localization was investigated using fluorescent labels during infectious structure differentiation in the rice blast fungus Magnaporthe grisea . α-1,3-glucan was labelled only on appressoria developing on plastic surfaces, whereas it was detected on both germ tubes and appressoria on plant surfaces. Chitin, chitosan and β-1,3-glucan were detected on germ tubes and appressoria regardless of the substrate. Major polysaccharides labelled at accessible surface of infectious hyphae were α-1,3-glucan and chitosan, but after enzymatic digestion of α-1,3-glucan, β-1,3-glucan and chitin became detectable. Immunoelectron microscopic analysis showed α-1,3-glucan and β-1,3-glucan intermixed in the cell wall of infectious hyphae; however, α-1,3-glucan tended to be distributed farther from the fungal cell membrane. The fungal cell wall became more tolerant to chitinase digestion upon accumulation of α-1,3-glucan. Accumulation of α-1,3-glucan was dependent on the Mps1 MAP kinase pathway, which was activated by a plant wax derivative, 1,16-hexadecanediol. Taken together, α-1,3-glucan spatially and functionally masks β-1,3-glucan and chitin in the cell wall of infectious hyphae. Thus, a dynamic change of composition of cell wall polysaccharides occurs during plant infection in M. grisea .