Flux Analysis of Glucose Metabolism in Rhizopus oryzae for the Purpose of Increasing Lactate Yields

A flux analysis of glucose metabolism in the filamentous fungus Rhizopus oryzae was achieved using a specific radioactivity curve-matching program, TFLUX. Glycolytic and tricarboxylic acid cycle intermediates labeled through the addition of extracellular [U-14C]glucose were isolated and purified for specific radioactivity determinations. This information, together with pool sizes and the rates of glucose utilization and end product production, provided input for flux maps of the metabolic network under two different experimental conditions. Based upon the flux analysis of this system, a mutant of R. oryzae with higher lactate and lower ethanol yields than the parent was sought for and found.     

The RHC21 gene of budding yeast, a homologue of the fission yeast rad21 +gene, is essential for chromosome segregation

The Saccharomyces cerevisiae gene RHC21 is a homologue of the fission yeast rad21 ⁺gene, which affects the sensitivity of cells to γ-irradiation and is essential for cell growth in S. pombe. Disruption of the RHC21 gene showed that it is also essential in S. cerevisiae. To examine its function in cell growth further, we have isolated temperature-sensitive mutants for the RHC21 gene and characterized one of them, termed rhc21-sk16. When this mutant was incubated at 36° C, the percentage of large-budded cells was increased. Most of the large-budded cells had aberrant nuclear structures, such as unequally extended nuclear DNA with incompletely elongated spindles across the mother-daughter neck or only in a mother cell. Furthermore, a circular minichromosome is more unstable in the mutant than in the wild-type, even at 25° C. Flow cytometry showed that the bulk of DNA replication takes place normally at the restrictive temperature in the mutant. These results indicated that the RHC21 gene is required for proper segregation of the chromosomes. In addition, we found that the mutant is sensitive not only to UV radiation and γ-rays but also to the antimicrotubule agent nocodazole at 25° C. This suggests that the RHC21 gene is involved in the microtubule function. We discuss how the RHC21 gene product may be involved in chromosome segregation and microtubule function. Received: 10 March 1997 / Accepted: 1 September 1997     

Heparan sulfate proteoglycan synthesis in CHO DG44 and HEK293 cells

Chinese hamster ovary (CHO) and human embryonic kidney 293 (HEK293) cells are the most popular host cells for transient gene expression (TGE) of therapeutic proteins. These host cells require high transfection efficiency in order to enhance TGE. Heparan sulfate proteoglycan (HSPG) at the cell surface is known to regulate endocytosis for gene delivery. The HSPG expression in CHO DG44 and HEK293E cells was investigated in an effort to enhance the TGE. Immunostaining of HSPGs followed by confocal microscopy and flow cytometry analyses revealed that CHO DG44 cells possessed a higher amount of cell-surface and intracellular HSPGs than HEK293E cells. The mRNA levels of the representative enzymes involved in the HSPG biosynthesis in CHO DG44, which were determined by quantitative real time PCR, were quite different from those in HEK293E cells. Taken together, the results obtained here would be useful in improving TGE in CHO DG44 and HEK293E cells through genetic engineering of HSPG synthesis.     

Genome scan linkage analysis identifies quantitative trait loci affecting serum clinical–chemical traits in Korean native chicken

Alterations in robustness- and health-related traits lead to physiological changes, such as changes in the serum clinical chemical parameters in individuals. Therefore, clinical–chemical traits can be used as biomarkers to examine the health status of chickens. The aim of the present study was to detect the quantitative trait loci (QTLs) influencing eight clinical–chemical traits (glucose, total protein, creatinine, high-density lipoprotein cholesterol, total cholesterol, glutamic oxaloacetic transaminase, glutamic pyruvic transaminase, and α-amylase) in an F₁ nuclear families comprising 83 F₀ founders and 585 F₁ progeny of Korean native chickens. Genotypic data on 135 DNA markers representing 26 autosomes have been generated for this resource pedigree. The total length of the map was 2729.4 cM. We used a multipoint variance component linkage approach to identify QTLs for the traits. A significant QTL affecting serum α-amylase levels was identified on chicken chromosome (GGA) 7 [logarithm of odds (LOD) = 3.02, P value = 1.92 × 10^?4]. Additionally, we detected several suggestive linkage signals for the levels of total cholesterol, glutamic oxaloacetic transaminase, glutamic pyruvic transaminase, and creatinine on GGA 4, 12, 13, and 15. In this study, serum α-amylase levels related significant QTL was mapped on GGA7 and cholesterol, glutamic oxaloacetic transaminase, glutamic pyruvic transaminase, and creatinine traits related suggestive QTLs were detected on GGA4, 12, 13 and 15, respectively. Further verification and fine mapping of these identified QTLs can provide valuable information for understanding the variations of clinical chemical trait in chickens.     

Growth of mycobacteria on carbon monoxide and methanol

Several mycobacterial strains, such as Mycobacterium flavescens, Mycobacterium gastri, Mycobacterium neoaurum, Mycobacterium parafortuitum, Mycobacterium peregrinum, Mycobacterium phlei, Mycobacterium smegmatis, Mycobacterium tuberculosis, and Mycobacterium vaccae, were found to grow on carbon monoxide (CO) as the sole source of carbon and energy. These bacteria, except for M. tuberculosis, also utilized methanol as the sole carbon and energy source. A CO dehydrogenase (CO-DH) assay, staining by activity of CO-DH, and Western blot analysis using an antibody raised against CO-DH of Mycobacterium sp. strain JC1 (formerly Acinetobacter sp. strain JC1 [J. W. Cho, H. S. Yim, and Y. M. Kim, Kor. J. Microbiol. 23:1-8, 1985]) revealed that CO-DH is present in extracts of the bacteria prepared from cells grown on CO. Ribulose bisphosphate carboxylase/oxygenase (RubisCO) activity was also detected in extracts prepared from all cells, except M. tuberculosis, grown on CO. The mycobacteria grown on methanol, except for M. gastri, which showed hexulose phosphate synthase activity, did not exhibit activities of classic methanol dehydrogenase, hydroxypyruvate reductase, or hexulose phosphate synthase but exhibited N,N-dimethyl-4-nitrosoaniline-dependent methanol dehydrogenase and RuBisCO activities. Cells grown on methanol were also found to have dihydroxyacetone synthase. Double immunodiffusion revealed that the antigenic sites of CO-DHs, RuBisCOs, and dihydroxyacetone synthases in all mycobacteria tested are identical with those of the Mycobacterium sp. strain JC1 enzymes.     

Photoautotrophic growth response of in vitro cultured coffee plantlets to ventilation methods and photosynthetic photon fluxes under carbon dioxide enriched condition

Effects of two ventilation methods (forced and natural) and two photosynthetic photon fluxes (PPF, 150 and 250 μmol m⁻² s⁻¹) on the photoautotrophic growth of in vitro cultured coffee (Coffea arabusta) plantlets were investigated. Number of air exchanges was 2.7, 5.9 and 3.9 h⁻¹ for forced low rate, forced high rate and natural ventilation, respectively. Single node cuttings of in vitro cultured coffee plantlets were cultured on Florialite, a mixture of vermiculite and cellulose fibers with high air porosity, emerged in liquid half strength basal MS medium, without sucrose, vitamins and plant growth regulators. The study included 40 days in the in vitro stage and 10 days in the ex vitro stage. Mean fresh and dry weights, leaf area, shoot and root lengths and net photosynthetic rate per plantlet were significantly greater in forced high rate treatments compared with those in natural and forced low rate treatments. PPF had a distinct effect on shoot length suppression and root elongation of coffee plantlets in forced high rate treatments. The control of carbon dioxide concentration inside the culture box according to the plant demand when growing was easy with the forced ventilation method in photoautotrophic micropropagation. This revised version was published online in June 2006 with corrections to the Cover Date.     

Macro- to Microscale Strain Transfer in Fibrous Tissues is Heterogeneous and Tissue-Specific

Mechanical deformation applied at the joint or tissue level is transmitted through the macroscale extracellular matrix to the microscale local matrix, where it is transduced to cells within these tissues and modulates tissue growth, maintenance, and repair. The objective of this study was to investigate how applied tissue strain is transferred through the local matrix to the cell and nucleus in meniscus, tendon, and the annulus fibrosus, as well as in stem cell-seeded scaffolds engineered to reproduce the organized microstructure of these native tissues. To carry out this study, we developed a custom confocal microscope-mounted tensile testing device and simultaneously monitored strain across multiple length scales. Results showed that mean strain was heterogeneous and significantly attenuated, but coordinated, at the local matrix level in native tissues (35–70% strain attenuation). Conversely, freshly seeded scaffolds exhibited very direct and uniform strain transfer from the tissue to the local matrix level (15–25% strain attenuation). In addition, strain transfer from local matrix to cells and nuclei was dependent on fiber orientation and tissue type. Histological analysis suggested that different domains exist within these fibrous tissues, with most of the tissue being fibrous, characterized by an aligned collagen structure and elongated cells, and other regions being proteoglycan (PG)-rich, characterized by a dense accumulation of PGs and rounder cells. In meniscus, the observed heterogeneity in strain transfer correlated strongly with cellular morphology, where rounder cells located in PG-rich microdomains were shielded from deformation, while elongated cells in fibrous microdomains deformed readily. Collectively, these findings suggest that different tissues utilize distinct strain-attenuating mechanisms according to their unique structure and cellular phenotype, and these differences likely alter the local biologic response of such tissues and constructs in response to mechanical perturbation.