Absorption of a novel prodrug of L-dopa, L-3-(3-hydroxy-4-pivaloyloxyphenyl)alanine (NB-355). In vitro and in situ studies

Absorption mechanism and absorption site of a prodrug of L-DOPA, L-3-(3-hydroxy-4-pivaloyloxyphenyl)alanine (NB-355, 1) was investigated using rats. Prodrug 1 (0.5 mM) was taken up by intestinal tissue segments time-dependently in vitro at pH 6.0. However, the rate of uptake was less than that of L-dopa. Inhibitors of the amino acid active transport system (L-Phe, dinitrophenol, ouabain) had no effect on the uptake of prodrug 1. In the intestinal tissue segments, prodrug 1 was extensively hydrolyzed by diisopropylfluorophosphate-sensitive esterase(s). To characterize the absorption site, gastrointestinal tracts were ligated to make acute loops in situ and prodrug 1 or L-dopa was injected into the loops. L-dopa disappeared rapidly from the lumen of the jejunum. In contrast, prodrug 1 disappeared rapidly from the ileum rather than the duodenum or jejunum. From these results, it was suggested that prodrug 1 was slowly absorbed primarily from the lower small intestine.     

Suppression of Gl Arrest and Enhancement of G2 Arrest by Inhibitors of Poly(ADP-ribose) Polymerase: Possible Involvement of Poly(ADP-ribosyl)ation in Cell Cycle Arrest Following γ-Irradiation

Low-dose γ-irradiation of mouse embryonic fibroblast C3D2F1 3T3-a cells caused Gl arrest along with G2 arrest and inhibition of replicative DNA synthesis. When the cells were cultured in the presence of inhibitors of poly(ADP-ribose) polymerase [EC 2.4.2.30], such as 3-aminobenzamide, benzamide and luminol, Gl arrest of C3D2F1 3T3-a cells was suppressed and enhancement of G2 arrest was observed. In contrast, 3-aminobenzoic acid, a non-inhibitory analog of 3-aminobenzamide, did not suppress Gl arrest following γ-irradiation. These results suggest that the poly(ADP-ribosyDation reaction is critical for the pathway of Gl arrest and is also involved in the pathway of G2 arrest.     

Serum thymidine kinase,a possible marker for monitoring the effect of bone marrow transplant treatment in early recovery phase

We measured serum thymidine kinase (TK) activity with a radioenzyme assay system employing [I-125]-iododeoxyuridine as the tracer on serial specimens from five bone marrow transplant (BMT) patients before and after transplantation. The serum level of TK activity in the 4 patients with effective BMT treatment ranged from 3.0 to 16.9 U/L (mean, 7.80 U/L) before transplantation and from 27.3 to 236.1 U/L (mean, 82.95 U/L) after the BMT treatment. Mean serum TK activity increased 13.17-fold (range, 1.68 to 29.14-fold). In contrast, the activity in the patient with ineffective BMT treatment was not significantly different during, before, or after BMT treatment. In addition, serum TK activity in BMT patients was well correlated with the change in the number of leukocytes before and after BMT treatment [r = +0.709 (p less than 0.01), y = 0.012 x +0.87]. We conclude that the determination of serum TK activity in BMT patients is very useful in monitoring the course of bone marrow transplantation in the early recovery phase.     

Long-Term Follow-Up of the Changes in Circulating Cytokines, Soluble Cytokine Receptors, and White Blood Cell Subset Counts in Patients with Rheumatoid Arthritis (RA) After Monoclonal Anti-TNFα Antibody Therapy

To investigate the mechanism of the long-lasting efficacy of chimeric monoclonal anti-TNF antibody (cA2) therapy for rheumatoid arthritis (RA), eight patients with refractory RA were treated with a single infusion of cA2 and the changes in circulating cytokines (IL-1, IL-6, TNF, and IL-10), soluble cytokine receptors (TNF-RI, RII, and sIL-6R) and peripheral white blood cell (WBC) subset counts were followed up long-term (12 weeks) after cA2 therapy in them. Significant clinical responses (>20% improvement according to Paulus' criteria) were observed just after cA2 infusion and lasted more than 4 weeks in all patients, as reported elsewhere. Moreover, five of the eight patients showed prolonged clinical responses (>12 weeks). The elevated serum IL-6 and sTNF-RI (or RII) levels before treatment rapidly decreased after treatment. The serum IL-10 levels also significantly elevated before treatment. The elevations of serum IL-10 levels were augmented after treatment and stayed higher than the baseline in four patients with prolonged clinical responses. No significant TNF, IL-1 and -, or sIL-6R were detected in the sera of the patients before treatment and during the whole study period. On the other hand, peripheral lymphocytes as well as total WBC and neutrophils increased for 4 weeks after treatment. However, thereafter, only the lymphocyte count decreased gradually and stayed below the baseline long-term (12 weeks). FACS analysis revealed the predominance of T lymphocytes in the decrease in lymphocyte counts. These results suggest that the augmentation of IL-10 production and the decrease in T cells might partly contribute to the long-lasting efficacy of cA2 treatment in RA.     

Study of monoclonal gammopathy in Kagawa Prefectural Central Hospital

We investigated 361 patients with monoclonal gammopathy in whom immunoelectrophoresis was performed (1,037 tests) between 1986 and 2002 at Kagawa Prefectural Central Hospital. In this study, we identified 222 patients with monoclonal gammopathy of undetermined significance (MGUS). Malignant transformation of MGUS to multiple myeloma occurred in 15 patients (6.8%). No significant differences were observed in the means of total protein (TP), albumin (Alb), albumin/globulin ratio (A/G ratio), IgG, IgA, or IgM level in the initial examination between the patients who remained as MGUS and patients with malignant transformation of MGUS. However, the rate of progression to malignancy was high when the levels of normal immunoglobulins other than M protein were below the normal range. Since the number of MGUS cases detected and the number of protein fractionation performed were proportionate, and MGUS was found by protein fractionation in routine tests, protein fractionation is essential for detection of MGUS, and it is necessary to add serum protein fractionation to routine initial examination. In addition, long-term follow-up of patients with monoclonal gammopathy and preparation of a database of patient information are useful for monitoring the outcome.     

Expression profiles of the duplicated matrix metalloproteinase-9 genes suggest their different roles in apoptosis of larval intestinal epithelial cells during Xenopus laevis metamorphosis.

Matrix metalloproteinases (MMPs) play a pivotal role in development and/or pathogenesis through degrading extracellular matrix (ECM) components. We have previously shown that Xenopus MMP-9 gene is duplicated. To assess possible roles of MMP-9 and MMP-9TH in X. laevis intestinal remodeling, we here analyzed their expression profiles by in situ hybridization and show that their expression is transiently up-regulated during thyroid hormone-dependent metamorphosis. Of interest, MMP-9TH mRNA is strictly localized in the connective tissue and most highly expressed just beneath the larval epithelium that begins to undergo apoptosis. On the other hand, cells expressing MMP-9 mRNA become first detectable in the connective tissue and then, after the start of epithelial apoptosis, also in the larval epithelium. These results strongly suggest that MMP-9TH is responsible in the larval epithelial apoptosis through degrading ECM components in the basal lamina, whereas MMP-9 is involved in the removal of dying epithelial cells during amphibian intestinal remodeling.     

An Immunohistochemical Study

C cell proliferation of the thyroid, which was designated as primary C cell hyperplasia (PCCH), was demonstrated in aged Fischer 344 rats in high incidence using the immunohistochemical method for calcitonin. It developed from mild to severe PCCH and resulted in nodular PCCH and tumor formation. The combined incidence of PCCH and nodular PCCH was increased with age and appeared in 60.7% of 7–15 month old group and 92.7% of 16–24 month old group possibly as a preneoplastic C cell lesion. It is almost an equivalent C cell lesion reported in the human thyroid with familial C cell carcinoma and therefore this Fischer 344 rat can provide a useful animal model to study familial C cell tumor of the thyroid.     

Immunocytochemistry of keratan sulfate proteoglycan and dermatan sulfate proteoglycan in porcine tooth-germ dentin

Keratan sulfate proteoglycan and dermatan sulfate proteoglycan have been reported to inhibit collagen fibrillogenesis. We investigated their distribution in order to evaluate the role of proteoglycan in dentinogenesis. Specimens of porcine tooth-germ dentin and erupted teeth were the materials on which antibodies to keratin sulfate and dermatan sulfate proteoglycan were used. Predentin was found to be positive for both antibodies and the reaction ceased in the calcification front. Uniformly thick collagen fibrils (30-70 nm in diameter) were distributed in the predentin matrix, which would become intertubular dentin in the future. Both antibodies reacted positively along these fibrils. In contrast, along the surface layer of dentin in the tooth germ and that in erupted teeth, collagen fibrils of 10-300 nm in diameter were noted occasionally in dentinal tubules whose odontoblastic processes had disappeared and these heterogeneous fibrils were negative for both antibodies. Our findings suggest that keratan sulfate proteoglycan and dermatan sulfate proteoglycan distributed in the predentin inhibit calcification of collagen fibrils in the uncalcified matrix and disappear in the calcification front. It is further suggested that keratan sulfate proteoglycan and dermatan sulfate proteoglycan distributed along collagen fibrils in the predentin matrix maintain uniform thickness, whereas collagen fibrils in dentinal tubules varied in thickness because of the absence of involvement of both proteoglycans. Therefore, keratan sulfate proteoglycan and dermatan sulfate proteoglycan were thought to be involved in both calcification and matrix formation.