Comparison of DNA polymerase activities between recombinant feline immunodeficiency and leukemia virus reverse transcriptases

In this study, we present enzymatic differences found between recombinant RTs derived from feline leukemia virus and feline immunodeficiency virus. Firstly, FIV RT showed low steady state K(m) values for dNTPs compared to FeLV RT. Consistent with this, FIV RT synthesized DNA more efficiently than FeLV RT at low dNTP concentrations. We observed similar concentration-dependent activity differences between other lentiviral (HIV-1 and SIV) and non-lentiviral (MuLV and AMV) RTs. Second, FeLV RT showed less efficient misincorporation with biased dNTP pools and mismatch primer extension capabilities, compared to FIV RT. In M13mp2 lacZalpha forward mutation assays, FeLV RT displayed approximately 11-fold higher fidelity than FIV RT. Finally, FeLV RT was less sensitive to 3TCTP and ddATP than FIV RT. This study represents the comprehensive enzymatic characterization of RTs from a lentivirus and a non-lentivirus retrovirus from the same host species. The data presented here support enzymatic divergences seen among retroviral RTs.     

Molecular targets of dietary polyphenols with anti-inflammatory properties

There is persuasive epidemiological and experimental evidence that dietary polyphenols have anti-inflammatory activity. Aspirin and other non-steroidal anti-inflammatory drugs (NSAIDs) have long been used to combat inflammation. Recently, cyclooxygenase (COX) inhibitors have been developed and recommended for treatment of rheumatoid arthritis (RA) and osteoarthritis (OA). However, two COX inhibitors have been withdrawn from the market due to unexpected side effects. Because conventional therapeutic and surgical approaches have not been able to fully control the incidence and outcome of many inflammatory diseases, there is an urgent need to find safer compounds and to develop mechanism-based approaches for the management of these diseases. Polyphenols are found in many dietary plant products, including fruits, vegetables, beverages, herbs, and spices. Several of these compounds have been found to inhibit the inflammation process as well as tumorigenesis in experimental animals; they can also exhibit potent biological properties. In addition, epidemiological studies have indicated that populations who consume foods rich in specific polyphenols have lower incidences of inflammatory disease. This paper provides an overview of the research approaches that can be used to unravel the biology and health effects of polyphenols. Polyphenols have diverse biological effects, however, this review will focus on some of the pivotal molecular targets that directly affect the inflammation process.     

Isolated full-thickness cervical stromal invasion warrants post-hysterectomy pelvic radiotherapy in FIGO stages IB-IIA uterine cervical carcinoma

OBJECTIVE: To evaluate the potential benefit of postoperative radiotherapy (PORT) in women with isolated full-thickness cervical stromal invasion (FTSI) as an unfavorable pathological finding after radical hysterectomy and pelvic lymph node dissection (PLND) in FIGO stages IB-IIA cervical carcinoma. METHODS: A total of 1868 patients with stages IB-IIA cervical carcinoma underwent radical hysterectomy and PLND between January 1982 and December 2002. Seventy-four of these patients had isolated FTSI without any other unfavorable pathological finding, such as lymph node metastasis, microscopic parametrial involvement, involved resection margin, lympho-vascular space invasion, or large clinical tumor diameter (>4 cm). Forty-one of these patients had no adjuvant treatment (S group) and 33 received PORT (PORT group). Patients with isolated FTSI who received chemotherapy were excluded. Treatment outcomes in the PORT and S groups were compared. RESULTS: Ten-year disease-free survival (DFS) and pelvic-failure-free survival (PFFS) of S group vs. PORT group were 73.2% vs. 92.4% (P=0.038) and 79.8% vs. 97.0% (P=0.044), respectively. According to a Cox proportional hazards model developed by forward, stepwise regression incorporating all prognostic variables, only PORT was marginally significant for DFS (RR 0.234; 95% CI 0.051-1.067; P=0.061) and significant for PFFS (RR 0.055; 95% CI 0.005-0.620; P=0.019). A grade 4 late complication developed in two patients (6%) in PORT group. CONCLUSION: PORT administered to patients with isolated FTSI after radical hysterectomy and PLND improves pelvic control in FIGO stages IB-IIA cervical carcinoma with acceptable morbidity.     

Optical approach for monitoring the periodontal ligament changes induced by orthodontic forces around maxillary anterior teeth of white rats

Structural variations of the periodontal ligament (PDL) induced by orthodontic forces have been evaluated by optical coherence tomography (OCT) and compared to images obtained by conventional radiography. Here, two specially designed orthodontic appliances were installed on the maxillary anterior teeth of white rats for applying different magnitudes of orthodontic forces. Constant distraction force magnitudes of 0, 5, 10, and 30 gf were given to four respective rats over a period of 5 days. At the end of the treatment period, the rats were sacrificed and the maxillaries were extracted for X-ray and OCT imaging. The PDL variations, proportional to the force magnitude, were clearly indicated in the OCT measurements. The OCT images further showed that the ligament was torn for a constant orthodontic force of 30 gf. These results support the clinical dental application of OCT for monitoring the ligament changes during orthodontic procedures. The real-time imaging capability of OCT, together with its high resolution, has the potential to help dentists with in vivo orthodontic treatments in human subjects as well.     

Frataxin is reduced in Friedreich ataxia patients and is associated with mitochondrial membranes

Friedreich ataxia is a progressive neurodegenerative disorder caused by loss of function mutations in the frataxin gene. In order to unravel frataxin function we developed monoclonal antibodies raised against different regions of the protein. These antibodies detect a processed 18 kDa protein in various human and mouse tissues and cell lines that is severely reduced in Friedreich ataxia patients. By immunocytofluorescence and immunocytoelectron microscopy we show that frataxin is located in mitochondria, associated with the mitochondrial membranes and crests. Analysis of cellular localization of various truncated forms of frataxin expressed in cultured cells and evidence of removal of an N-terminal epitope during protein maturation demonstrated that the mitochondrial targetting sequence is encoded by the first 20 amino acids. Given the shared clinical features between Friedreich ataxia, vitamin E deficiency and some mitochondriopathies, our data suggest that a reduction in frataxin results in oxidative damage.      

Identification of MEN1 gene mutations in sporadic carcinoid tumors of the lung

Lung carcinoids occur sporadically and rarely in association with multiple endocrine neoplasia type 1 (MEN1). There are no well defined genetic abnormalities known to occur in these tumors. We studied 11 sporadic lung carcinoids for loss of heterozygosity (LOH) at the locus of the MEN1 gene on chromosome 11q13, and for mutations of the MEN1 gene using dideoxy fingerprinting. Additionally, a lung carcinoid from a MEN1 patient was studied. In four of 11 (36%) sporadic tumors, both copies of the MEN1 gene were inactivated. All four tumors showed the presence of a MEN1 gene mutation and loss of the other allele. Observed mutations included a 1 bp insertion, a 1 bp deletion, a 13 bp deletion and a single nucleotide substitution affecting a donor splice site. Each mutation predicts truncation or potentially complete loss of menin. The remaining seven tumors showed neither the presence of a MEN1 gene mutation nor 11q13 LOH. The tumor from the MEN1 patient showed LOH at chromosome 11q13 and a complex germline MEN1 gene mutation. The data implicate the MEN1 gene in the pathogenesis of sporadic lung carcinoids, representing the first defined genetic alteration in these tumors.      

Interferon-γ induces cellular senescence through p53-dependent DNA damage signaling in human endothelial cells

Cellular senescence is a stress-response phenomenon in which cells lose the ability to proliferate; it is induced by telomere shortening, activation of oncogenes or tumor suppressor genes, or exposure to a sub-lethal dose of DNA damaging agents or oxidative stresses. cDNA microarray analysis reveals that the levels of interferons (IFNs) and IFN-inducible genes were altered during replicative senescence in human umbilical vascular endothelial cells (HUVECs). However, the role of IFNs in cellular senescence of HUVECs remains unidentified. This study demonstrated that prolonged treatment with IFN-γ induced cellular senescence in HUVECs, as confirmed by G0/G1 cell cycle arrest, up-regulation of p53 and p21 protein levels, increased SA-β-gal staining, and the accumulation of phospho-H₂AX foci. IFN-γ-induced cellular senescence was observed only in p16-knockdown cells or p16-null mouse embryonic fibroblasts (MEFs), but not in p53-knockdown cells or p53-null MEFs. IFN-γ treatment increased ROS production, and an antioxidant, N-acetylcysteine, inhibited IFN-γ-induced cellular senescence. Knockdown of ATM kinase or IFI16 rescued IFN-γ-induced cellular senescence. Therefore, these results suggest that IFN-γ might play an important role in cellular senescence through a p53-dependent DNA damage pathway and contribute to the pathogenesis of atherosclerosis via its pro-senescent activity.     

Aberrant gene expression associated with recurrent pregnancy loss

Recent studies indicate that a number of factors including chromosomal abnormalities, immunological feto-maternal rejection, hormonal irregulation and anatomical factors are involved in provoking recurrent pregnancy loss (RPL). This indicates that normal cellular regulation of these factors is required for maintaining normal pregnancy. In addition, it is expected that biological processes for maintaining normal pregnancy require a series of differential gene expression. As expected, our previous investigations revealed that there are >/=30 genes showing different levels of expression between normal and RPL patients. In addition, other research groups have also identified a number of genes that are expressed aberrantly in pregnancy failure. In this review, recent study on aberrant expression levels of genes, which are grouped as immunity-related, angiogenesis-related, apoptosis-related and other groups of genes, will be discussed.     

Aqueous N,N-diethylnicotinamide (DENA) solution as a medium for accelerated release study of paclitaxel

N,N-Diethylnicotinamide (DENA) was identified as an excellent hydrotropic agent for paclitaxel (PTX) in our previous studies. The aqueous solubility of PTX was increased by several orders of magnitude in the presence of DENA. Because of such a high hydrotropic property, DENA was used as a release medium providing a sink condition for the release of PTX from poly(lactic-co-glycolic acid) (PLGA) matrices. The release profiles of PTX from PLGA matrices into DENA, serum and phosphate-buffered saline (PBS) were compared. The stability of PTX in DENA and the degradation of PLGA molecules in DENA were examined. The degradation rate constant of PTX in 2 M DENA was similar to those in other aqueous solutions. The use of 2 M DENA as a release medium allowed differentiation of the release profiles of PTX from PLGA matrices made of different PLGA compositions. The PTX release from PLGA matrices was much faster in DENA solution than in serum or PBS, and the concentration of DENA affected the PTX release rate. The presence of DENA in the release medium increased the hydrolysis rate of PLGA polymers. The faster release of PTX from PLGA matrices in DENA solution may be due to the high PTX solubility and faster degradation of PLGA polymers in the presence of DENA. Our study suggests that the aqueous DENA solution can be used for the accelerated release study of PTX from PLGA matrices.