Purification and characterization of M3 protein expressed on the surface of group A streptococcal type 3 strain C203

Abstract Monoclonal antibodies (mAbs) have been produced by immunizing BALB/C mice with whole M⁺ bacteria in incomplete Freund adjuvant and the resulting mAbs for M3 protein have been selected by an indirect immuno-fluorescent technique using formaldehyde-fixed M⁺ and M⁻ bacteria. Four mAbs reacted with a 65 kDa protein in an extract obtained from the cell wall of M⁺ bacteria after treatment with N -acetyl muramidase and lysozyme. The purified 65 kDa protein neutralized the phagocytic activity of rabbit anti-M3 antibody. The N-terminal amino acid sequence of the 65 kDa protein was identical with that of protein generated by the M3 gene which has been previously cloned and sequenced. The evidence indicates that the 65 kDa protein is M3 protein. The M3 protein bound not only human fibrinogen but also human serum albumin (HSA). When the M3 protein was purified by gel-filtration and ion-exchange chromatography in the absence of phenylmethyl sulfonyl fluoride (PMSF), four fragments (35 kDa, 32 kDa, 30 kDa, and 25 kDa) in addition to the intact molecule appeared. N-terminal amino acid sequence analysis showed that 35 kDa and 25 kDa fragments were ANAAD and DARSV, respectively, being identical at positions 1–5 and 198–202 to the M3 gene derived protein. Therefore, the 35 kDa and 25 kDa fragments, which were presumed to be cleavage products, may be derived from the C-terminal part and N-terminal part of the intact molecule, respectively. When the effect of purified M3 protein in the bactericidal activity of normal human blood in the presence of M⁻ bacteria was investigated, the M3 protein was responsible for the organism's resistance to attack by phagocytic cells.     

Cellular and Vacuolar Fusion of Protoplasts Electrofused Using Platinum Microelectrodes

Protoplasts isolated from cultured cells of Coptis japonicaand Euphorbia millii were electrically fused using platinummicroelectrodes. The process involved two stages, cellular andvacuolar fusion, which are characterized respectively by transientwrinkling of the membrane and the formation of a dark-red precipitate. (Received June 12, 1987; Accepted October 13, 1987)     

Microbiological leaching of a chalcopyrite concentrate by Thiobacillus ferrooxidans

The microbiological leaching of a chalcopyrite concentrate has been investigated using a pure strain of Thiobacillus ferrooxidans. The optimum leaching conditions regarding pH, temperature, and pulp density were found to be 2.3, 35°C, and 22%, respectively. The energy of activation was calculated to be 16.7 kcal/mol. During these experiments the maximum rate of copper dissolution was about 215 mg/liters/hr and the final copper concentration was as high as 55 g/liter. This latter value is in the range of copper concentrations which may be used for direct electrorecovery of copper. Jarosite formation was observed during the leaching of the chalcopyrite concentrate. When the leach residue was reground to expose new substrate surface, subsequent leaching resulted in copper extractions up to about 80%. On the basis of this experimental work, a flow sheet has been proposed for commercial scale biohydrometallurgical treatment of high-grade chalcopyrite materials.     

Methotrexate chronotherapy is effective against rheumatoid arthritis

Methotrexate (MTX) is the most important drug for treating rheumatoid arthritis (RA). It has been stated that cytokines play an important role in the pathogenesis of RA, and that cytokine levels increase and show 24-h rhythms in RA patients. Previously, we found that arthritis was relieved after the administration of MTX at specific times in synchronization with the 24-h rhythm of tumor necrosis factor (TNF)-α in collagen-induced arthritis (CIA) animals. Based on our findings in an earlier study of the dosing time-dependent effects of MTX in MRL/lpr mice, which develop autoimmune disorders that share similarities with human RA, we examined here the utility of MTX chronotherapy in Japanese RA patients. In an initial animal modeling study, we collected blood from MRL/lpr mice at different times (2, 6, 10, 14, 18, or 22 hours after the light was turned on [HALO]), and we measured TNF-α mRNA expression in leukocytes. MTX was administered to the mice at two different dosing times (6 or 18 HALO), and various blood parameters were measured to estimate arthritis activity. TNF-α mRNA levels showed a clear 24-h rhythm with a peak at 22 HALO and a trough at 18 HALO after RA had developed. In these MRL/lpr mice, inflammation and TNF-α were markedly reduced when the MTX dosing time was matched to the time (18 HALO) when the TNF-α level began to increase. We then applied these findings to Japanese RA patients by switching them from the standard MTX three times/wk (day 1: after breakfast and supper; day 2: after breakfast schedule), to chronotherapy, in which the dose and number of doses/wk were not changed but MTX was administered once-a-day at bedtime. Disease Activity Score (DAS)28, modified health assessment questionnaire (MHAQ), and adverse effects were assessed. With MTX chronotherapy, DAS28, which is commonly used to quantitatively assess RA symptoms, was significantly improved at all follow-up clinical visit times compared with the baseline (vs. 1 mo: p?=?.0197, 2 mos: p?=?.0107, 3 mos: p?=?.0087). Significant symptom recovery was observed in 41.2% of patients, and 23.5% of patients achieved clinical remission during the 3 mos of follow-up. Functional capacity of RA patients, as indicated by the MHAQ, was markedly improved by chronotherapy. There were no severe adverse effects. Thus, we demonstrated (i) inflammation and plasma TNF-α concentrations were significantly reduced in MRL/lpr mice treated with MTX at 18 HALO, the time when TNF-α mRNA level began to increase; and (ii) MTX bedtime chronotherapy was safe, markedly reduced disease activity, and improved the functional capacity of RA patients. The findings on RA patients show that bedtime MTX chronotherapy can improve RA symptoms compared to the current standard dosing methods.     

Radical-Mediated Cyclization of A 6-Chloro-9-(2-Deoxy--d erythro-pent-1-enofuranosyl)-8-(2,2-dibromovinyl) purine

Abstract

A vinyl radical generated from a 6-chloro-9-(2-deoxy--d eryrhro-pent-l-enofuranosyl)-8-(2,2-dibromovinyl) purine effected cyclization either at the 1′-or at the 2′-position. The result is discussed in comparison with our previous study of the corresponding uracil derivative.     

Acute and long-lasting effects of peripheral injection of caerulein and CCK-8 on the central GABAergic system in mice

Acute and long-lasting effects of peripheral injection of caerulein (CLN) and cholecystokinin octapeptide (CCK-8) on the gamma-aminobutylic acid (GABA) content and the GABA accumulation by aminooxyacetic acid (AOAA) in the discrete brain regions of mice were examined. The content and accumulation of GABA in the striatum, hypothalamus, and frontal cortex was measured with high performance liquid chromatography with electrochemical detection (HPLC-ECD). The GABA content slightly decreased in the striatum 60 min after CLN and CCK-8 were administered, whereas it slightly increased in the hypothalamus and frontal cortex. Moreover, with CLN and CCK-8, the GABA accumulation after AOAA treatment decreased in the striatum and hypothalamus 30 min after injection. Meanwhile, when administering CLN, the GABA content as well as the GABA accumulation after AOAA treatment increased in the striatum and frontal cortex 1 day after injection, and continued to increase the second and third day in the striatum. These results showed that peripheral injection of CLN and CCK-8 had effects on the central GABAergic system with local specific actions, and also the long-lasting and time-dependent biphasic effects of CLN.