Stimulation of glucose utilization and lactate production in cultured human fibroblasts by thyroid hormone

Human dermal fibroblasts were obtained by harvesting outgrowing cells from the dermal tissue explants and cultured in Dulbecco's modified Eagle medium containing 10% fetal calf serum. After the cells reached confluency, culture was continued in the medium containing calf serum which was deprived of thyroid hormone by the treatment with activated charcoal. These fibroblasts were responsive to exogeneously added thyroid hormone (triiodothyronine) at physiological concentrations, resulting in enhanced utilization of glucose and production of lactate. This stimulation by thyroid hormone was dependent upon the length of exposure to the hormone and its concentration.The hormone did not show any effects on cellular DNA and protein content. The experimental system described above seems to be easy to reconstitute and should be useful for the elucidation of the mechanism of thyroid hormone action.     

Studies on aspartase. III. Alteration of enzymatic properties upon trypsin-mediated activation.

Highly purified aspartase (L-aspartate ammonia-lyase, EC 4.3.1.1) from Escherichia coli, already of full activity, is further activated 3.3-fold by limited treatment with trypsin. The activation requires a few minutes to attain maximum level, and hereafter the activity gradually decreases to complete inactivation. Prior or intermediate addition of soybean trypsin inhibitor results in an immediate cessation of any further change in the enzyme activity. Upon trypsin-mediated activation no appreciable change is detected in the molecular weight of the enzyme subunits as judged from sodium dodecyl sulfate polyacrylamide gel electrophoresis, nor in the pH vs. activity profile in the presence of added metal ions. However, S0.5 and hill coefficient for L-aspartate considerably increase upon activation. As the trypsin-mediated activation proceeds, a marked absorbance difference spectrum of the trypsin-treated aspartase vs. untreated aspartase appears with negative absorbance maxima at 278 and 285 nm. When the trypsin-activated enzyme is denatured in 4 M guanidine-HCl, followed by removal of the denaturant by dilution, the enzyme activity is readily restored to as much as 1.5 times that of the native enzyme, indicating that the trypsin-activated enzyme is rather a stable molecule.     

N-type Calcium Channel in the Pathogenesis of Experimental Autoimmune Encephalomyelitis

One of the family of voltage-gated calcium channels (VGCC), the N-type Ca²⁺ channel, is located predominantly in neurons and is associated with a variety of neuronal responses, including neurodegeneration. A precise mechanism for how the N-type Ca²⁺ channel plays a role in neurodegenerative disease, however, is unknown. In this study, we immunized N-type Ca²⁺ channel α_1B-deficient (α_1B^−/−) mice and their wild type (WT) littermates with myelin oligodendrocyte glycoprotein 35–55 and analyzed the progression of experimental autoimmune encephalomyelitis (EAE). The neurological symptoms of EAE in the α_1B^−/− mice were less severe than in the WT mice. In conjunction with these results, sections of the spinal cord (SC) from α_1B^−/− mice revealed a reduction in both leukocytic infiltration and demyelination compared with WT mice. No differences were observed in the delayed-type hypersensitivity response, spleen cell proliferation, or cytokine production from splenocytes between the two genotypes. On the other hand, Western blot array analysis and RT-PCR revealed that a typical increase in the expression of MCP-1 in the SC showed a good correlation with the infiltration of leukocytes into the SC. Likewise, immunohistochemical analysis showed that the predominant source of MCP-1 was activated microglia. The cytokine-induced production of MCP-1 in primary cultured microglia from WT mice was significantly higher than that from α_1B^−/− mice and was significantly inhibited by a selective N-type Ca²⁺ channel antagonist, ω-conotoxin GVIA or a withdrawal of extracellular Ca²⁺. These results suggest that the N-type Ca²⁺ channel is involved in the pathogenesis of EAE at least in part by regulating MCP-1 production by microglia.     

Genetic characteristics and possible introduced origin of the paradise fish <Emphasis Type="Italic">Macropodus opercularis</Emphasis> in the Ryukyu Archipelago,Japan

It has hitherto been unknown whether the paradise fish Macropodus opercularis that inhabits the Ryukyu Archipelago, Japan, is native to the region or was introduced. This study examined the genetic identity of fish from five islands in the Ryukyu Archipelago (Okinoerabu, Okinawa, Yagaji, Kume, and Minamidaito islands) and compared it with those from Taiwan Island, mainland China, and Hainan Island. Analyses of the mtDNA control (760 bp) and cytb (660 bp) regions showed that haplotypes of specimens from the Ryukyu Archipelago were the same as or were very similar (with a 1- or 2-bp difference) to those from Taiwan. In addition, haplotypes from the Ryukyu Archipelago also showed lower genetic diversity than those from Taiwan Island, mainland China, and Hainan Island. These results suggest a high likelihood that the fish in the Ryukyu Archipelago were artificially introduced from Taiwan. However, the possibility that the fish is indigenous to the Ryukyu Archipelago cannot be completely ruled out, because some haplotypes and a clade from the Ryukyu Archipelago have not been found in the other areas. Regardless of its origin, we emphasize the importance of the conservation of the paradise fish in the Ryukyu Archipelago as an indicator of the threatened wetland environment as well as for its cultural value.     

Variant of Penicillinase Mediated by an R Factor in Escherichia coli

The penicillinase from an Escherichia coli strain harboring an R factor R(GN823) was purified and its properties were compared with those of a known type I penicillinase mediated by R factors. The molecular weight and S(20,w) of the enzyme were 22,600 and 2.42S, respectively. The isoelectric point of the enzyme was 6.9. These values are clearly different from those of type I penicillinase. The specific activity of the enzyme was 84,700 units per mg of the purified enzyme protein, which is about 20 times higher than that of the type I penicillinase. However, similarities were observed between the enzyme and the type I-penicillinase at optimal pH (6.5 to 7.0), optimal temperature (40 to 45C), substrate specificity, Michaelis constants for penicillins and cephaloridine, and effect of inhibitors. Furthermore, antiserum against type I penicillinase showed cross-reaction against this enzyme. The enzyme was named type Ib penicillinase, and the original type I penicillinase was renamed type Ia-penicillinase.     

Purification and characterization of thermostable aspartase from Bacillus sp. YM55-1.

A thermostable aspartase was purified from a thermophile Bacillus sp. YM55-1 and characterized in terms of activity and stability. The enzyme was isolated by a 5-min heat treatment at 75 degrees C in the presence of 11% (w/v) ammonium sulfate and 100 mM aspartate, followed by Q-Sepharose anion-exchange and AF-Red Toyopearl chromatographies. The native molecular weight of aspartase determined by gel filtration was about 200,000, and this enzyme was composed of four identical monomers with molecular weights of 51,000 determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Unlike Escherichia coli aspartase, the enzyme was not activated by the presence of magnesium ion at alkaline pH. At the optimum pH, the Km and Vmax were 28.5 mM and 700 units/mg at 30 degrees C and 32.0 mM and 2200 units/mg at 55 degrees C, respectively. The specific activity was four and three times higher than those of E. coli and Pseudomonas fluorescens enzymes at 30 degrees C, respectively. Eighty percent of the activity was retained after a 60-min incubation at 55 degrees C, and the enzyme was also resistant to chemical denaturants; 80% of the initial specific activity was detected in assay mixtures containing 1.0 M guanidine hydrochloride. The purified enzyme shared a high sequence homology in the N-terminal region with aspartases from other organisms.