rho-mediated protein tyrosine phosphorylation in lysophosphatidic-acid-induced tumor-cell invasion

Rat ascites hepatoma cells (MMI) invade a mesothelial cell monolayer in vitro in assay medium containing serum, but not in serum-free medium. Serum could be completely replaced by I-oleoyl lysophosphatidic acid (LPA) in inducing invasion. LPA-induced invasion was inhibited by genistein, a tyrosine-kinase inhibitor. Protein tyrosine phosphorylation in response to LPA was thus analyzed in order to determine the molecular mechanism of invasion. LPA of invasion-inducible concentrations evoked a transient increase in tyrosine phosphorylation, mainly of 110- to 130-kDa proteins in MMI cells but not in mesothelial cells. These concentrations of LPA were over 10 times higher (10 to 25 μM) than those necessary to produce a variety of biological actions, such as tyrosine phosphorylation in fibroblasts, neurite retraction and platelet aggregation. Protein tyrosine phosphorylation and invasion by MMI cells induced by LPA are largely regulated by rho p21, because both were inhibited by Clostridium botulinum C3 exo-enzyme, which is known to specifically inactivate rho p21. Invasion of MCL by MMI cells induced by serum and that by B16FE7 cells induced by LPA were inhibited by genistein or C3 as well. By immunoprecipitation, we detected p125 focal adhesion kinase (FAK) as a major protein of 110- to 130-kDa tyrosine phosphorylated in response to LPA. Tyrosine phosphorylation of paxillin by LPA was also detected. © 1996 Wiley-Liss, Inc.     

Prediction of human pharmacokinetics for low‐clearance compounds using pharmacokinetic data from chimeric mice with humanized livers

Development of low‐clearance (CL) compounds that are slowly metabolized is a major goal in the pharmaceutical industry. However, the pursuit of low intrinsic CL (CL_int) often leads to significant challenges in evaluating the pharmacokinetics of such compounds. Although in vitro–in vivo extrapolation is widely used to predict human CL, its application has been limited for low‐CL_int compounds because of the low turnover of parent compounds in metabolic stability assays. To address this issue, we focused on chimeric mice with humanized livers (PXB‐mice), which have been increasingly reported to accurately predict human CL in recent years. The predictive accuracy for nine low‐CL_int compounds with no significant turnover in a human hepatocyte assay was investigated using PXB‐mouse methods, such as single‐species allometric scaling (PXB‐SSS) approach and a novel physiologically based scaling (PXB‐PBS) approach that assumes that the CL_int per hepatocyte is equal between humans and PXB‐mice. The percentages of compounds with predicted CL within 2‐ and 3‐fold ranges of the observed CL for low‐CL_int compounds were 89% and 100%, respectively, for both PXB‐SSS and PXB‐PBS approaches. Moreover, the predicted CL was mostly consistent among the methods. Conversely, the percentages of compounds with predicted CL within 2‐ and 3‐fold ranges of the observed CL for low‐CL_int compounds were 50% and 63%, respectively, for multispecies allometric (MA) scaling. Overall, these PXB‐mouse methods were much more accurate than conventional MA scaling approaches, suggesting that PXB‐mice are useful tools for predicting the human CL of low‐CL_int compounds that are slowly metabolized.

Study Highlights

• WHAT IS THE CURRENT KNOWLEDGE ON THE TOPIC?

It is important to identify low‐clearance (CL) compounds that are slowly metabolized during the drug discovery process, but the ability of in vitro–in vivo extrapolation to predict human CL decreases as the value of intrinsic CL (CL_int) decreases. Although chimeric mice with humanized livers (PXB‐mice) have been reported to be useful for predicting human CL, their applicability to low‐CL_int compounds with no significant turnover in human hepatocyte assays has not been clarified.

• WHAT QUESTION DID THIS STUDY ADDRESS?

This study examined the predictive accuracy of PXB‐mouse methods for low‐CL_int compounds.

• WHAT DOES THIS STUDY ADD TO OUR KNOWLEDGE?

In addition to the previously reported single‐species allometric scaling approach, we proposed a novel physiologically based scaling approach. Both methods displayed much greater predictive accuracy for low‐CL_int compounds than conventional multispecies allometric scaling approaches.

• HOW MIGHT THIS CHANGE CLINICAL PHARMACOLOGY OR TRANSLATIONAL SCIENCE?

The findings should improve the accuracy of human pharmacokinetic prediction and enable efficient and safe first‐in‐human studies.     

Ankle arthrodesis using a retrograde intramedullary rod with fins for highly destructive joints in elderly patients with rheumatoid arthritis: A report of two cases

Herein, we present two cases of older adult patients with highly destructive changes in ankle joints (Larsen grade IV) who underwent retrograde intramedullary ankle nail fixation with fins. In both patients, bony union was achieved, and full weight‐bearing was attained at 3 months after surgery.     

Young woman with repeated late stent thrombosis: a case report

A 34-year-old woman with hypertension, obesity, and history of smoking presented with unstable angina and severe stenosis of the proximal left anterior descending artery. Percutaneous coronary intervention was performed with stent implantation resulting in adequate expansion. She was treated with aspirin and ticlopidine for 1 month, then only aspirin for 1 month. One year after stenting, she presented with acute myocardial infarction and total occlusion at the stent. Balloon angioplasty was performed. She took ticlopidine and aspirin for 9 months. Two months later, she presented with acute myocardial infarction with reocclusion at the stent. Blood examination showed no manifest collagen disease or thrombophilia. This case of repeated late stent thrombosis occurred in a young woman not treated by brachytherapy.     

Critical role of the IgM Fc receptor in IgM homeostasis,B-cell survival,and humoral immune responses

IgM antibodies have been known for decades to enhance humoral immune responses in an antigen-specific fashion. This enhancement has been thought to be dependent on complement activation by IgM–antigen complexes; however, recent genetic studies render this mechanism unlikely. Here, we describe a likely alternative explanation; mice lacking the recently identified Fc receptor for IgM (FcμR) on B cells produced significantly less antibody to protein antigen during both primary and memory responses. This immune deficiency was accompanied by impaired germinal center formation and decreased plasma and memory B-cell generation. FcμR did not affect steady-state B-cell survival but specifically enhanced the survival and proliferation induced by B-cell receptor cross-linking. Moreover, FcμR-deficient mice produced far more autoantibodies than control mice as they aged, suggesting that FcμR is also required for maintaining tolerance to self-antigens. Our results thus define a unique pathway mediated by the FcμR for regulating immunity and tolerance and suggest that IgM antibodies promote humoral immune responses to foreign antigen yet suppress autoantibody production through at least two pathways: complement activation and FcμR.     

Renal cell carcinoma (RCC) and telomerase activity: relationship to stage

Limited information is available on the correlation of telomerase activity and the clinical and pathological characteristics, in patients with renal cell carcinoma (RCC). Telomerase repeat amplification protocol (TRAP) was used to measure telomerase activity in frozen RCC specimens from partial/radical nephrectomies performed between 1987 and 1991. Presence of tumor tissue was verified by a pathologist using hematoxylin and eosin stained sections. RNA was measured to ensure the presence of intact protein necessary for telomerase expression. Data on demographics, tumor type, and stage at presentation, local recurrence, distant metastasis, disease-free survival (DFS), and overall survival (OS) was collected, and telomerase activity was correlated with each of these variables. Forty-nine of 67 patients (73%) were telomerase positive (+ve). Gender and stage were the only variables that appeared to be associated with telomerase positivity. Tumors were telomerase +ve in 12/21 females (57 %) vs. 37/46 males (80%) (P = 0.07). Tumors were telomerase +ve in 85% of Stage IV, 76% of Stage III, and 70% of Stage I/II patients (P = 0.12). Five-year survival was 0% for Stage IV, 57% for Stage III, and 77% for Stage I/II patients (P < 0.001), DFS 54% for stage III and 84% for Stage I/II patients (P = 0.05). Telomerase activity, however, was not related to survival in either univariate or multivariate analysis. In patients with telomerase +ve tumors 5-year survival was 55%, and with telomerase −ve tumors 58% (P = 0.56). Stage was the only variable associated with OS or DFS in clear cell RCC patients. In patients with advanced disease, there is a high incidence of telomerase positivity was found, within this limited sample, however, no correlation with survival was found.     

Case of Mycobacterium haemophilum misdiagnosed as Mycobacterium intracellulare due to one base insertion in the bacterial genome

Mycobacterium haemophilum is a slow‐growing, non‐tuberculous mycobacteria that causes cutaneous infection. We describe a case of cutaneous infection in a 68‐year‐old Japanese man with polymyositis. This was caused by M. haemophilum harboring one base insertion in gene sequence. At first, the causal microorganism was misidentified as M. intracellulare by COBAS^® TaqMan^® MAI test. However, poor growth on Ogawa media and growth enhancement on 7H11C agar around a hemin‐containing disk prompted us to reinvestigate the causal microorganisms, which were revealed to be M. haemophilum. Amplified polymerase chain reaction products were sequenced, and the 16S rRNA gene, rpoB, hsp65 and internal transcribed spacer region sequences showed a 100%, 100%, 99.66% and 99.7% match, respectively, with the corresponding regions of M. haemophilum, but it harbored a novel gene sequence in hsp65. The sequences determined by gene analysis of the M. haemophilum strain were deposited into the International Nucleotide Sequence Database. Although numerous cases of M. haemophilum infection have been reported in other countries, only six cases have been reported in Japan to date. It could be possible that this novel mutation lead to misdiagnosis. As M. haemophilum prefers a lower growth temperature (30–32°C) and it requires iron in the culture medium, M. haemophilum could be misidentified or overlooked. Accordingly, a M. haemophilum infection should be considered in cases of cutaneous infection of the body sites, of which surface temperature is low.