Sequence-dependent Termination of Mammalian DNA Polymerase Reaction by a New Platinum Compound, (–)-(R)-2-Aminomethylpyrrolidine(1,1-cyclobutane-dicarboxylato)-2-platinum(II) Monohydrate

We examined the mechanisms of the inhibition of DNA synthesis by a new platinum compound, (-)-( R )-2-aminomethylpyrrolidine(1,1-cyclobutane-dicarboxylato)-2-platinum(II) monohydrate (DWA-2114R), a derivative of the antitumor drug cis- diamminedichloroplatinum(II) (CDDP), using prokaryotic and eukaryotic DNA polymerases. Preincubating activated DNA with CDDP or DWA-2114R reduced its template activity for prokaryotic and eukaryotic DNA polymerases in a dose-dependent manner. DWA2114R required six times greater drug concentration and two times longer incubation time to show the same decrease of the template activity compared to CDDP. Treatment of primed pUC118 ssDNA templates with the two drugs followed by second-strand synthesis by prokaryotic and eukaryotic DNA polymerases revealed that DWA2114R bound to DNA in a similar manner to CDDP and these adducts blocked DNA elongation by DNA polymerases of eukaryotes as well as of prokaryotes. With these two drugs, the elongations by E. coli DNA polymerase I (Klenow fragment), T7 DNA polymerase and calf thymus DNA polymerase α were strongly arrested at guanine-guanine sequences (GG). Stop bands were also observed at adenine-guanine sequences (AG) guanine-adenine-guanine sequences (GAG) and mono-guanine sequence (G). Calf testis DNA polymerase β was also arrested efficiently at AG, GAG and G, but much more weakly at GG. This pattern was common to DWA2114R and CDDP.     

Silicone-Coated Polypropylene Hollow-Fiber Oxygenator: Experimental Evaluation and Preliminary Clinical Use

Background. A membrane oxygenator consisting of a microporous polypropylene hollow fiber with a 0.2-μm ultrathin silicone layer (cyclosiloxane) was developed. Animal experimental and preliminary clinical studies evaluated its reliability in bypass procedures.

Methods. Five 24-hour venoarterial bypass periods were conducted on dogs using the oxygenator (group A). In 5 controls, bypass periods were conducted using the same oxygenator without silicone coating (group B). As a preliminary clinical study, 14 patients underwent cardiopulmonary bypass with the silicone-coated oxygenator.

Results. Eight to 16 hours (mean, 12.2 hours) after initiation of bypass, plasma leakage occurred in all group B animals, but none in group A. The O₂ and CO₂ transfer rates after 24 hours in group A were significantly higher than at termination of bypass in group B (p < 0.005 and p < 0.03, respectively). Scanning electron microscopy of silicone-coated fibers after 24 hours of bypass revealed no damage to the silicone coating of the polypropylene hollow fibers. In the clinical study, the oxygenator showed good gas transfer, acceptable pressure loss, low hemolysis, and good durability.

Conclusions. This oxygenator is more durable and offers greater gas transfer capabilities than the previous generation of oxygenators.     

Regional difference in the appearance of apoptotic cell death in the ligamentum flavum of the human cervical spine

Ossification or calcification of the ligamentum flavum (LF) is relatively common in the middle and lower cervical, thoracic, and lumbar spine but extremely rare in the upper cervical region. This clinical fact suggests that there exist local factors promoting or preventing ossification or calcification of LF. However, little is known about the differences in the ultrastructure and cellular alterations of the LF between the different spinal levels, even in the cervical spine. With electron microscopy, we examined samples of LF collected surgically from the upper and lower cervical spine regions; we then studied the apoptotic appearance of ligament cells using a preferential labeling method. We found direct evidence of apoptosis of ligament cells in the LF. Apoptosis was more apparent in the upper region samples than in the lower region samples. The spaces around the normal fibroblasts were filled with thick collagen fibrils, but the collagen fibrils disappeared around the apoptotic bodies and thin fibrils were formed. The difference of the level of apoptosis may correlate to the ultrastructual difference of LF, and our data will benefit further investigations seeking to clarify the mechanism of various pathological conditions in the human LF.     

Effects of nerve growth factor and glucocorticoid on cultured human pheochromocytoma cells

Primary cell cultures of two human pheochromocytomas (PC) that were associated with high serum levels of adrenaline and noradrenaline were developed to study the effects of nerve growth factor (NGF) and dexamethasone on the morphology and function of PC cells in vitro. By phase-contrast microscopy, cultured cells were small and hyperchromatic on the first day of culture; neurite-like processes that extended to other cells developed several days later and were maintained for more than 3 months. NGF (100ng/ml), dexamethasone (10^–5M), or NGF + dexamethasone were added to the culture media 2 weeks after the cultured cells had stabilized. Catecholamine concentrations in the medium were maintained at higher levels after addition of NGF, dexamethasone, or NGF + dexamethasone as compared to control cells. In the presence of NGF, extension of neurite-like processes was clearly accelerated, while high levels of dexamethasone inhibited growth of processes. These in vitro studies showed that the addition of NGF or the removal of dexamethasone induces differentiation of adrenal neurons present in pheochromocytomas, suggesting that adrenocortical steroid hormones influence the morphological control of adrenal medullary cells.     

Effect of post-transplant double filtration plasmapheresis on recurrent focal and segmental glomerulosclerosis in renal transplant recipients

In the present study, we reviewed the effect of post-transplant double filtration plasmapheresis (DFPP) on recurrent focal segmental glomerulosclerosis (FSGS) in the transplanted kidney allograft. Sixteen patients with post-transplant recurrent FSGS were enrolled in this study. Out of 16 patients with recurrent FSGS after transplantation, five did not receive DFPP and lost their grafts, while 11 did receive DFPP and four of these patients lost their grafts. Seven patients were able to maintain normal renal function for an average observation period of 57.1 +/- 40.7 months (range 7-125 months). In five patients who had a significant reduction in urinary protein after DFPP, the urinary protein level decreased from 26.60 +/- 23.05 g/day (range 3.34-62.6 g/day) to 2.95 +/- 3.42 g/day (range 0.02-8.64 g/day) and renal function was maintained. The beneficial effects of DFPP on graft outcome were more likely to occur if the patients experienced a marked drop in urinary excretion. Thus, post-transplant DFPP appears to be effective for reducing urinary protein levels and improving long-term graft survival. With the small numbers in this trial, however, none of the findings were statistically significant. We recommend the use of post-transplant DFPP to prevent the progression of recurrent FSGS.     

Monoclonal and polyclonal immunoglobulin G deposits on tubular basement membranes of native and pretransplant kidneys: A retrospective study

Monoclonal tubular basement membrane immune deposits (TBMID) are associated with progression of interstitial injury in renal allograft. However, the significance of monoclonal and polyclonal TBMID in the native kidney remains unclear. We retrospectively analyzed 1894 native kidney biopsies and 1724 zero-hour biopsies performed between 2008 and 2018 in our institution. The rate of immunoglobulin G (IgG) TBMID was found to be 8.4% among native kidney biopsies and 0.4% among zero-hour biopsies. Polyclonal TBMID is common in IgG4-related tubulointerstitial nephritis (37.5%), diabetic nephropathy (31.3%) and lupus nephritis (25.5%). Monoclonal IgG TBMID was identified in seven cases, including three zero-hour biopsies. The combination of IgG1κ was observed in two cases, IgG1λ in three, and IgG2κ in two. Electron microscopy revealed powdery electron-dense deposits in all cases. Monoclonal gammopathy of undetermined significance was diagnosed in one case. Although one patient with focal segmental glomerulosclerosis developed renal failure, all others exhibited stable renal function. Monoclonal IgG TBMID in the native kidney is not associated with renal prognosis. However, this may be an interesting immunopathological finding that would help clarify the pathogenesis of TBM immune deposits. Further study for both monoclonal and polyclonal TBMID is required in the future.     

Association between sarcopenia and higher-level functional capacity in daily living in community-dwelling elderly subjects in Japan

This study aimed to determine the association between sarcopenia, defined by muscle mass, muscle strength, and physical performance, and higher-level functional capacity in community-dwelling Japanese elderly people. Subjects were 1158 elderly, community-dwelling Japanese people aged 65 or older. We used bioelectrical impedance analysis to measure muscle mass, grip strength to measure muscle strength, and usual walking speed to measure physical performance. Sarcopenia was characterized by low muscle mass, plus low muscle strength or low physical performance. Subjects without low muscle mass, low muscle strength, and low physical performance were classified as "normal." Examination of higher-level functional capacity was performed using the Tokyo Metropolitan Institute of Gerontology Index of Competence (TMIG-IC). The TMIG-IC is a 13-item questionnaire completed by the subject; it contains five questions on self-maintenance and four questions each on intellectual activity and social role. Sarcopenia was identified in 11.3% and 10.7% of men and women, respectively. The percentage of disability for instrumental activities of daily living (IADL) was 39.0% in men with sarcopenia and 30.6% in women with sarcopenia. After adjustment for age, in men, sarcopenia was significantly associated with IADL disability compared with intermediate and normal subjects. In women, sarcopenia was significantly associated with every subscale of the TMIG-IC disability compared with intermediate and normal subjects. This study revealed that sarcopenia, defined by muscle mass, muscle strength, and physical performance, had a significant association with disability in higher-level functional capacity in elderly Japanese subjects. Interventions to prevent sarcopenia may prevent higher-level functional disability among elderly people.