Calcium and the replication of human vascular smooth muscle cells: studies on the activation and translocation of extracellular signal regulated kinase (ERK) and cyclin D1 expression

Since the precise role of sarcoplasmic reticular Ca2+ in mediating vascular smooth muscle cells (VSMC) proliferation is unknown, the effect of pre-incubation with thapsigargin on extracellular signal regulated kinase (ERK) activation, the translocation of activated of ERK 1/2 to the nucleus, cyclin D1 expression, the onset of S phase and cytosolic Ca2+ levels were studied. Human saphenous vein VSMCs (hVSMC) were incubated with 10 nM thapsigargin for 24 h followed by stimulation with fetal calf serum and the activation of ERK1/2 and cyclin D1 assessed by western blotting, the intracellular distribution of ERK1/2 using indirect immunofluorescence, the onset of S-phase with the incorporation of bromodeoxyuridine and sarcoplasmic reticular Ca2+ status using FURA-2. Thapsigargin had a marginal effect on ERK1/2 activation only at 5 min and 10 min after stimulation with fetal calf serum. In contrast, the rapid translocation of ERK1/2 to the nucleus was completely blocked by thapsigargin. S phase was delayed by 8 h by thapsigargin which co-incided with the recovery of cytosolic Ca2+ levels and cyclin D1 expression. It is concluded that the inhibitory effect of thapsigargin (depletion of Ca2+ pools) on hVSMC replication is mediated through the inhibition of translocation of activated ERK1/2 to the nucleus and not to the phosphorylation of ERK, per se, which in turn prevents cyclin D1 expression and thus progression of the cell cycle.     

A comparison of fixed-bearing and mobile-bearing total knee arthroplasty at a minimum follow-up of 4.5 years

BACKGROUND: Durable long-term independent results with the Low Contact Stress rotating-platform (mobile-bearing) and the Insall Burstein-II (fixed-bearing) total knee prostheses have been reported, but no studies describing either the mid-term or long-term results and comparing the two prostheses are available, to our knowledge. METHODS: Thirty-two patients who had bilateral arthritis of the knee with similar deformity and preoperative range of motion on both sides and who agreed to have one knee replaced with a mobile-bearing total knee design and the other with a fixed-bearing design were prospectively evaluated. Comparative analysis of both designs was done at a mean follow-up period of six years, minimizing patient, surgeon, and observer-related bias. Clinical and radiographic outcome, survival, and complication rates were compared. RESULTS: Patients with osteoarthritis had better function scores and range of motion compared with patients with rheumatoid arthritis. However, with the numbers available, no benefit of mobile-bearing over fixed-bearing designs could be demonstrated with respect to Knee Society scores, range of flexion, subject preference, or patellofemoral complication rates. Radiographs showed no difference in prosthetic alignment. Two knees with a mobile-bearing prosthesis required a reoperation: one had an early revision because of bearing dislocation and another required conversion to an arthrodesis to treat a deep infection. CONCLUSIONS: We found no advantage of the mobile-bearing arthroplasty over the fixed-bearing arthroplasty with regard to the clinical results at mid-term follow-up. The risk of bearing subluxation and dislocation in knees with the mobile-bearing prosthesis is a cause for concern and may necessitate early revision. LEVEL OF EVIDENCE: Therapeutic Level II.     

Histone h3 modifications in rat hepatic stellate cells by ethanol

AIMS: Hepatic stellate cells (HSCs) play critical roles in the development of hepatic fibrosis caused by various agents including alcohol. Ethanol causes post-translational modification in histone. The goal of this study is to investigate whether ethanol affected acetylation and methylation of histone H3 in rat HSCs. METHODS: We isolated and separated HSCs using collagenase perfusion of liver followed by Nycodenz density gradient centrifugation. HSCs were divided and treated with different concentrations of ethanol for various times. Histone was isolated using acid extraction method. Acetylation and methylation of histone H3 at Lys9 was analysed by both western blot and fluorescein isothiocyanate (FITC) immunochemical stain. Acetylation of histone H3 at Lys9 (Ac-H3-lys9), Lys14 (Ac-H3-Lys14), Lys18 (Ac-H3-lys18), or Lys23 (Ac-H3-lys23) was checked by western blotting. RESULTS: At lysine 9, ethanol caused dose-dependent increase of Ac-H3 up to 200 mM. Ac-H3-lys9 increased with a maximum of 86-fold at 72 h and 200 mM ethanol treatment, and decreased thereafter. This increase was confirmed by both western blotting and FITC stain. At high dose, ethanol increased acetylation of histone H3 at Lys23 (Ac-H3-lys23), but it had no effect on Ac-H3-lys14 or Ac-H3-lys18. The intensity of the FITC-labelled dimethyl-histone H3 at Lys9 (Me-H3-lys9) antibody appeared to decrease slightly with increasing dose of ethanol. But this did not appear to change when monitored by western blotting. CONCLUSIONS: Ethanol caused dose and time-dependent increase in acetylation of histone H3 at Lys9, but not at Lys14 or Lys18. Compared with hepatocytes the Ac-H3-lys9 in HSCs required longer ethanol exposure. Levels of Me-H3-lys9 seemed to remain unaltered. Thus increase in Ac-H3-lys9 represents a nuclear-chromatin modification event in HSCs exposed to ethanol.     

Copper, zinc, and Cu/Zn ratio in carcinoma of the gallbladder

INTRODUCTION: The exact role of copper and zinc in the etiology of carcinoma of the gallbladder is unclear. Some studies suggest the Cu/Zn ratio is a good indicator of the extent and prognosis in carcinoma of the gastrointestinal tract. The aim of the present study is to estimate the micronutrient profile and Cu/Zn ratio in the serum, tissues, and bile of patients with benign and malignant gallbladder diseases. METHODS: The present study was carried out in 60 patients comprising 30 each of carcinoma of the gallbladder and cholelithiasis, and 30 age and sex matched controls. Copper and zinc levels were estimated in blood, bile, and tissue using a Perkin Elmer Model 2380 Atomic absorption Spectrophotometer. RESULTS: The mean serum zinc levels were significantly lower in patients with carcinoma of the gallbladder than in patients with cholelithiasis and in healthy controls (P < 0.001). The mean serum copper levels were significantly higher in patients with carcinoma of the gallbladder as compared to patients with cholelithiasis and healthy controls (P < 0.001). Biliary and tissue zinc levels were significantly lower in patients with carcinoma of the gallbladder than in patients with cholelithiasis. Biliary and tissue copper levels were higher in patients with carcinoma of the gallbladder than in patients with cholelithiasis. The serum Cu/Zn ratio showed a gradual and significant increase from 1.11 in healthy controls to 1.35 in patients with cholelithiasis and 2.12 in patients with carcinoma of the gallbladder. The biliary and tissue Cu/Zn ratios were also significantly increased in patients with carcinoma of the gallbladder than in patients with cholelithiasis (P < 0.001). CONCLUSIONS: Our data support an association between lower zinc levels and consequently an increased Cu/Zn ratio and carcinoma of the gallbladder. Whether zinc supplementation has a protective effect in preventing carcinoma of the gallbladder needs to be studied further.     

Dietary agents in the chemoprevention of prostate cancer

Nutritional factors have been estimated to contribute 20-60% of cancers around the globe, and almost one-third of deaths are being reported in Western countries. According to estimates by the American Cancer Society, during the year 2005 about 232,090 new cases of prostate cancer will be diagnosed alone in the United States and 30,350 men will die of this disease. The high incidence and long latency period of prostate cancer offer plenty of time to pursue strategies toward prevention and/or treatment to suppress or revert this disease. Epidemiological evidence suggests that plant-based dietary agents decrease the risk of some types of human cancer, including prostate cancer. Intake of 400-600 g/day of fruits and vegetables is associated with reduced risk of several cancers. The use of micronutrients and/or other phenolic agents in the diet or synthetic exogenous supplements to prevent neoplastic transformation of normal cells or to slow the progression of established malignant changes in cancer cells is termed "chemoprevention." Considerable attention has been devoted to identify plant-based dietary agents that may serve as natural inhibitors of prostate carcinogenesis. Much progress has been made in the last decade in this area of investigation through identification of pathways that play important roles in prostate tumorigenesis. This article summarizes epidemiological, clinical, and mechanistic studies and the significance of plant-derived dietary agents such as flavonoids, indoles, isothiocyanates, phenolics, monoterpenes, and complementary and alternative agents in the management of prostate cancer with recommendations for future studies to advance this area of research.     

Selection of tumor-targeting agents on freshly excised human breast tumors using a phage display library

The selective delivery of therapeutic agents to tumor site without harming rest of the body is a major challenge in clinical oncology today. Phage display approach has been used for searching ligands for cell-surface macromolecules on cancer cells so that they can be employed as drug targeting agents. Although basic protocols for biopanning cells are available, they are not as such suitable for screening highly complex and diverse target as whole tumor. Present study is an attempt to select peptide ligands specific to whole tumors. The cells from freshly collected human breast tumors were biopanned with phage displayed disulfide-constrained random heptapeptide library, following subtraction on normal human breast cells. Comparative analysis of amino acid frequencies in tumor-selected peptides and in random peptides from unselected library showed that selection was not random. The binding assessment of tumor-selected clones, using highly sensitive chemiluminescence ELISA, demonstrated that 47-75% of selected clones, depending on a tumor, bound to tumor cells they were panned on. Furthermore, several clones bound exclusively or preferentially to tumor cells in comparison to normal breast cells. It was interesting to note that insert sequences of tumor-binding clones from different tumors shared significant motifs. It shows the possibility of identifying ligands that may bind to tumor-specific targets common in certain tumors. The results of this investigation on seven human breast tumors demonstrated that, using procedures developed in the present study, whole tumors can be panned successfully with phage displayed library and tumor-binding ligands can be identified rapidly in high throughput manner. This is an important enabling step in identifying lead molecules for developing novel, specific, and effective agents that can be used for the diagnosis and treatment of cancer.