A correlation between membrane cholesterol level, cell adhesion and tumourigenicity of polyoma virus transformed cells

We have investigated the implications of the rise in membrane cholesterol levels on several in vitro and in vivo properties of polyoma virus transformed rat fibroblasts (PyF), with a special emphasis on alpha5beta1 integrin functions. We show that increased membrane cholesterol causes the PyF cells to change their shape and become more bipolar in appearance. These cells also show significantly higher adhesion to the cell-binding domain of fibronectin, increased localization of alpha5beta1 integrin and talin molecules in focal adhesions and a more robust actin cytoskeleton organization. PyF cells with increased membrane cholesterol show reduced growth in vitro and tumours caused by these cells in nude mice are slow growing. These changes in the growth properties of PyF cells are reversible when the cholesterol levels of PyF cells become normal. Our results suggest that changes in membrane cholesterol levels influence the growth and morphological properties of transformed cells, which can be exploited in controlling the growth of tumours in vivo.     

Immunocontraceptive potential of recombinantly expressed minimized chicken riboflavin carrier protein (mini-RCP) in rodents

Chicken riboflavin carrier protein (RCP; 219 AA) harbours four linear epitopes, constituted by the peptide residues 3-23, 64-83, 130-147 and 200-219. Antibodies to these sequences bioneutralize maternal RCP and provide protection from pregnancy in rodents. In order to overcome the major histocompatibility complex-dependent variability in immune response often encountered with use of single peptides for vaccination in genetically outbred populations, we have assembled a novel synthetic gene, incorporating in tandem the nucleotide sequences coding for all the four neutralizing epitopes of chicken RCP and expressed in Escherichia coli. The gene product, mini-RCP has been characterized for its immunogenic properties and contraceptive potential in rodents. Immunization of rabbits and rats led to generation of antibodies against individual peptide components, as determined by enzyme-linked-immunosorbent assay (ELISA). However, immunized rats carried pregnancy to term and delivered healthy offsprings. Antisera from these rats exhibited decreased affinity of binding to the native protein. These findings suggest that the prospects of covalently-linked epitope peptides need to be cautiously evaluated during the design and development of peptide-based vaccines.     

Phosphorylation of profilin regulates its interaction with actin and poly (L-proline)

Activation of bovine platelets with thrombin and phorbol 12,13-dibutyrate (PDBu) resulted in phosphorylation of profilin on serine. The phosphorylation was inhibited when platelets were pretreated with the PI 3-kinase inhibitor, LY294002, indicating that profilin phosphorylation is a downstream event with respect to PI 3-kinase activation. Phosphorylation of profilin resulted in significant decrease in actin polymerization (16.5%), indicating an increased affinity of phosphoprofilin towards actin. The critical actin monomer concentration (Cc) increased to 260 nM in the presence of phosphoprofilin in comparison with 200 nM in the presence of profilin. The interaction of phosphoprofilin with phosphatidylinositol 4,5-bisphosphate [PI (4,5)-P2] and poly (L-proline) (PLP) was examined by monitoring the quenching of tryptophan fluorescence. Scatchard plot and binding isotherm data obtained revealed no difference in PI (4,5)-P2 binding between profilin and phosphoprofilin (Kd=20.4 microM), while poly (L-proline)-binding studies indicated a sixfold decrease (27.34 microM for profilin and 4.73 microM for phosphoprofilin) in Kd with phosphoprofilin. In vivo studies with platelets indicated an increased association of p85alpha, the regulatory subunit of PI 3-kinase with phosphoprofilin over profilin. Overall, the data presented conclude that profilin phosphorylated under in vivo conditions and phosphorylation depends upon activation of PI 3-kinase. Phosphoprofilin exhibited increased affinity to poly (L-proline) sequences both in vitro and in vivo.     

Tackling the plant proteome: practical approaches, hurdles and experimental tools

The study of complex biological questions through comparative proteomics is becoming increasingly attractive to plant biologists as the rapidly expanding plant genomic and expressed sequence tag databases provide improved opportunities for protein identification. This review focuses on practical issues associated with comparative proteomic analysis, including the challenges of effective protein extraction and separation from plant tissues, the pros and cons of two-dimensional gel-based analysis and the problems of identifying proteins from species that are not recognized models for functional genomic studies. Specific points are illustrated using data from an ongoing study of the tomato and pepper fruit proteomes.     

The progressive changes of nasal symmetry and growth after nasoalveolar molding: a three-year follow-up study

The purpose of this study was to assess the progressive changes of nasal symmetry, growth, and relapse after presurgical nasoalveolar molding and primary cheiloplasty in unilateral complete cleft lip/palate infants. Twenty-five consecutive complete unilateral cleft lip/palate infants were included. All the infants underwent nasoalveolar molding before primary cheiloplasty. Standard 1:1 ratio basilar photographs were taken before and after nasoalveolar molding, 1 week after cheiloplasty, and yearly for 3 years. Linear measurements were made directly on the photographs. The results of this study revealed that the nasal asymmetry was significantly improved after nasoalveolar molding and was further corrected to symmetry after primary cheiloplasty. After the primary cheiloplasty, the nasal asymmetry significantly relapsed in the first year postoperatively and then remained stable and well afterward. The relapse was the result of a significant differential growth between the cleft and noncleft sides in the first year postoperatively. To compensate for relapse and differential growth, the authors recommend (1) narrowing down the alveolar cleft as well as possible by nasoalveolar molding, (2) overcorrecting the nasal vertical dimension surgically, and (3) maintaining the surgical results using a nasal conformer.     

Upper extremity limb salvage with microvascular reconstruction in patients with advanced sarcoma

Limb salvage is a viable alternative to amputation in many cases of advanced sarcoma. The authors examined their experience with microvascular reconstruction of upper extremity defects after sarcoma resection, focusing on oncologic and functional outcomes. A retrospective analysis yielded 17 patients who underwent 18 free flap procedures and met the inclusion criteria. Most patients (71 percent, n = 12) had recurrent sarcoma at presentation to the authors' institution. Malignant fibrous histiocytoma was the most common pathologic subtype (n = 6). High-grade tumors were present in 94 percent of patients (n = 16). The free flap survival rate was 100 percent. The rectus abdominis flap was the most common free flap used (39 percent; n = 7). Local recurrence occurred in nine flaps (50 percent), and five patients ultimately required amputations. Six patients (35 percent) had distant recurrence. The mean Enneking score for limb function was 73 percent of the maximum (21.9 of 30). The 5-year disease-specific survival rate was 61.3 percent. In select patients with advanced upper extremity sarcoma undergoing limb salvage, microvascular flap reconstruction can provide reliable, safe coverage with reasonable preservation of function.     

5Alpha-reductase inhibition activity of steroids isolated from marine soft corals

The aim of this study is to determine the 5alpha-reductase inhibitory activity of several new steroidal compounds PR-01-PR-07 by measuring the conversion of [3H]T to[3H]DHT in Penicillium crustosum broths. These compounds were obtained from marine soft corals collected on the coasts of Andaman and Nicobar at Hori, Natkal and Kalipur (Diglipur) Islands and identified as Sinularia grandilobata Verseveldt, Sinularia crassa Tixier- Durivault, Sinularia gravis Tixier- Durivault, Sinularia sp., Lobophytum sp., Lobophytum crassum and Cladiella sp. PR-01-PR-04 significantly inhibited the conversion of [3H]T to [3H]DHT (P < 0.05) whereas PR-05 and PR-06 did not show an appreciable difference (P > 0.05) in this model. On the other hand PR-07 stimulated (P < 0.05) the enzymatic reaction.     

Hypoglycemic activity of inorganic constitutents in Eugenia jambolana seed on streptozotocin-induced diabetes in rats

Medicinal herbs used in indigenous medicines for the management of diabetes mellitus contain both organic and inorganic constituents. Some of these inorganic trace elements possess antidiabetic properties, which accounts for the activity of medicinal herbs. The aim of this study was to analyze the inorganic trace elements present in Eugenia jambolana seeds and to evaluate the hypoglycemic activity of the inorganic part of E. jambolana seeds on streptozotocin-induced diabetes. The seeds of E. jambolana seeds were reduced to ash and the inorganic elements present were assayed. The hypoglycemic efficacy of the inorganic part was tested by the glucose tolerance test on streptozotocin-induced diabetes. Elements such as zinc, chromium, vanadium, potassium, and sodium, possessing hypoglycemic activity, were present in the seed. The E. jambolana seed ashtreated diabetic rats exhibited normoglycemia and better glucose tolerance. The conclusion that the inorganic constituents might play a important role in the antidiabetic nature E. jambolana seeds was reached.     

Artificial neural network as an alternative to multiple regression analysis in optimizing formulation parameters of cytarabine liposomes

The objective of the study was to optimize the formulation parameters of cytarabine liposomes by using artificial neural networks (ANN) and multiple regression analysis using 3(3) factorial design (FD). As model formulations, 27 formulations were prepared. The formulation variables, drug (cytarabine)/lipid (phosphatidyl choline [PC] and cholesterol [Chol]) molar ratio (X1), PC/Chol in percentage ratio of total lipids (X2), and the volume of hydration medium (X3) were selected as the independent variables; and the percentage drug entrapment (PDE) was selected as the dependent variable. A set of causal factors was used as tutorial data for ANN and fed into a computer. The optimization was performed by minimizing the generalized distance between the predicted values of each response and the optimized one that was obtained individually. In case of 3(3) factorial design, a second-order full-model polynomial equation and a reduced model were established by subjecting the transformed values of independent variables to multiple regression analysis, and contour plots were drawn using the equation. The optimization methods developed by both ANN and FD were validated by preparing another 5 liposomal formulations. The predetermined PDE and the experimental data were compared with predicted data by paired t test, no statistically significant difference was observed. ANN showed less error compared with multiple regression analysis. These findings demonstrate that ANN provides more accurate prediction and is quite useful in the optimization of pharmaceutical formulations when compared with the multiple regression analysis method.