Current Status for High Titre Poxvirus Stock Preparation in CEF Under Serum-Free Medium Conditions: Implication for Vaccine Development

In light of the recent detection of BSE in North America and its endemic nature in other regions of the world, there is a real need to employ cell culture conditions that do not require any animal-derived material. Here we report the use of an ultra-low protein serum-free medium (VP-SFM, Invitrogen) for the amplification of poxviruses in primary chicken embryo fibroblasts (CEF). We compared the amplification of four different poxviruses (canarypox, modified Ankara Virus (MVA), vaccinia virus strain Copenhagen and myxoma strain Lausanne) in three different media: DMEM 10%, DMEM 2% and serum-free medium VP-SFM. VP-SFM is a serum-free, ultra-low protein medium containing no proteins or peptides of human or animal origin designed to support the replication of viruses and the production of recombinant proteins and monoclonal antibodies. Our results show that high titre poxvirus stocks can be prepared in VP-SFM equivalent to that prepared in serum containing medium.     

Green Tea Polyphenol Epigallocatechin Gallate Inhibits Adipogenesis and Induces Apoptosis in 3T3‐L1 Adipocytes

Objective: Green tea catechins have been shown to promote loss of body fat and to inhibit growth of many cancer cell types by inducing apoptosis. The objective of this study was to determine whether epigallocatechin gallate (EGCG), the primary green tea catechin, could act directly on adipocytes to inhibit adipogenesis and induce apoptosis. Research Methods and Procedures: Mouse 3T3‐L1 preadipocytes and mature adipocytes were used. To test the effect of EGCG on viability, cells were incubated for 3, 6, 12, or 24 hours with 0, 50, 100, or 200 μM EGCG. Viability was quantitated by MTS assay. To determine the effect of EGCG on apoptosis, adipocytes were incubated for 24 hours with 0 to 200 μM EGCG, then stained with annexin V and propidium iodide and analyzed by laser scanning cytometry. Both preadipocytes and adipocytes were also analyzed for apoptosis by terminal deoxynucleotidyl transferase dUTP nick‐end labeling assay. To determine the effect of EGCG on adipogenesis, maturing preadipocytes were incubated during the 6‐day induction period with 0 to 200 μM EGCG, then stained with Oil‐Red‐O and analyzed for lipid content. Results: EGCG had no effect on either viability or apoptosis of preconfluent preadipocytes. EGCG also did not affect viability of mature adipocytes; however, EGCG increased apoptosis in mature adipocytes, as demonstrated by both laser scanning cytometry and terminal deoxynucleotidyl transferase dUTP nick‐end labeling assays. Furthermore, EGCG dose‐dependently inhibited lipid accumulation in maturing preadipocytes. Discussion: These results demonstrate that EGCG can act directly to inhibit differentiation of preadipocytes and to induce apoptosis of mature adipocytes and, thus, could be an important adjunct in the treatment of obesity.     

Change in Vascular Adhesion Protein‐1 and Metabolic Phenotypes after Vertical Banded Gastroplasty for Morbid Obesity

Objective: The association between circulating vascular adhesion protein‐1 (VAP‐1) and metabolic phenotypes has been shown to be inconsistent. The current study explored whether the changes in serum VAP‐1 levels correlate with the changes in metabolic phenotypes after weight reduction surgery. Research Methods and Procedures: Clinical characteristics and serum VAP‐1 levels in 20 morbidly obese subjects (mean BMI 38.84 kg/m²) were measured before and after vertical banded gastroplasty. Results: Before surgery, serum VAP‐1 levels correlated positively with fasting plasma glucose (γ = 0.56, p = 0.01) and negatively with insulin levels (γ = ?0.51, p = 0.021). After surgery, the changes in serum VAP‐1 levels were negatively correlated with the changes in waist circumference (γ = ?0.57, p = 0.011), diastolic blood pressure (DBP) (γ = ?0.56, p = 0.015), and mean arterial pressure (γ = ?0.46, p = 0.055). In multivariate regression, serum VAP‐1 levels were negatively correlated with waist circumference (β = ?2.36, p = 0.014) and DBP (β = ?3.02, p = 0.017) after adjusting for age and gender. The change in DBP was negatively correlated with the change in VAP‐1 levels after adjusting for age, gender, and steady‐state plasma glucose. Discussion: The results suggest that VAP‐1 levels are correlated with fasting glucose and insulin levels in morbidly obese subjects. After surgery, the changes in VAP‐1 levels were associated with changes in visceral adiposity and DBP. Serum VAP‐1 might modulate DBP independently from the changes in insulin resistance in morbidly obese people.     

Weight loading young chicks inhibits bone elongation and promotes growth plate ossification and vascularization.

The mechanical stimuli resulting from weight loading play an important role in mature bone remodeling. However, the effect of weight loading on the developmental process in young bones is less well understood. In this work, chicks were loaded with bags weighing 10% of their body weight during their rapid growth phase. The increased load reduced the length and diameter of the long bones. The average width of the bag-loaded group's growth plates was 75 +/- 4% that of the controls, and the plates showed increased mineralization. Northern blot analysis, in situ hybridization, and longitudinal cell counting of mechanically loaded growth plates showed narrowed expression zones of collagen types II and X compared with controls, with no differences between the relative proportions of those areas. An increase in osteopontin (OPN) expression with loading was most pronounced at the bone-cartilage interface. This extended expression overlapped with tartarate-resistant acid phosphatase staining and with the front of the mineralized matrix in the chondro-osseous junction. Moreover, weight loading enhanced the penetration of blood vessels into the growth plates and enhanced the gene expression of the matrix metalloproteinases MMP9 and MMP13 in those growth plates. On the basis of these results, we speculate that the mechanical strain on the chondrocytes in the growth plate causes overexpression of OPN, MMP9, and MMP13. The MMPs enable penetration of the blood vessels, which carry osteoclasts and osteoblasts. OPN recruits the osteoclasts to the cartilage-bone border, thus accelerating cartilage resorption in this zone and subsequent ossification which, in turn, contributes to the observed phenotype of narrower growth plate and shorter bones.     

Increase in the Thermic Effect of Food in Women by Adrenergic Amines Extracted from Citrus Aurantium

Objective: To compare the thermic response to a meal between men and women of varied body composition and to determine whether adrenergic amines extracted from citrus aurantium (CA) induce an increase in metabolic rate and enhance the thermic response to the meal. Research Methods and Procedures: In 30 healthy weight‐stable subjects (17 women, 13 men; BMI: 20 to 42 kg/m²), body composition was determined by bioimpedance analysis followed by resting energy expenditure for 20 minutes, and the thermic effect of food (TEF) of a 1.7‐MJ, 30‐gram protein meal was determined intermittently for 300 minutes by indirect calorimetry. In a subset of 22 subjects, the TEFs of CA alone and when added to the same 1.7‐MJ meal were determined. Blood pressure and pulse before and throughout the studies and catecholamine excretion were determined. Results: TEF was significantly lower in women than men (152 ± 7 vs. 190 ± 12 kJ and 8.8 ± 0.4% vs. 11.0 ± 0.7% of meal), independently of age and magnitude of adiposity. The thermic response to CA alone was higher in men, but, when added to the meal, CA increased TEF only in women and to values no longer different from men. CA had no effect on blood pressure and pulse rate but increased epinephrine excretion by 2.4‐fold. Discussion: A 20% lower TEF in women suggests a diminished sympathetic nervous system response to meals, because with CA, TEF increased by 29% only in women. However, this acute response may not translate into a chronic effect or a clinically significant weight loss over time.     

Profile of Overweight and Obese People in a Mediterranean Region

Objective: To assess the prevalence of excess weight (obesity and overweight) and the related environmental risk factors in a Mediterranean population. Self‐perception of body weight, attitudes toward weight‐control behaviors, and the associated factors were also examined. Research Methods and Procedures: A cross‐sectional nutritional survey was carried out in the Balearic Islands between 1999 and 2000. A random sample (n = 1200) of the adult population (20 to 60 years old) was interviewed. Dietary questionnaires and a global questionnaire incorporating questions related to sociodemographic and lifestyle variables were utilized. Anthropometric measurements were also obtained. Results: Nearly one‐half of the population of the Balearic Islands is above the normal weight range (BMI > 25). The predictors of overweight and obesity in the Balearic Islands are to be over 40 years old, to be married, to form part of the lowest educational levels, and to have a sedentary lifestyle. A large percentage of excess‐weight individuals tend to underestimate their BMI, are not concerned about their weight status, tend to snack more often, and have never dieted. Discussion: Although the likely causes of the rise in obesity prevalence are difficult to elucidate from this study, we have identified the profile of individuals with excess weight in the Balearic Islands as well as their attitudes toward their body image.     

Modeling of the composition of materials for soft X-ray sources used in research on inertial confinement fusion

A method for calculating and optimizing the composition of materials for soft X-ray sources used in research on inertial confinement fusion is described. For a target-converter, a material composition is determined with which the conversion of laser light into X radiation is highly efficient. A comparative analysis is carried out of the efficiencies of generation of soft X-ray emission in the plasmas of some composite materials of thin conductors (wires) used as loads in X-and Z-pinches. Numerical calculations of the optical plasma properties are reported whose results make it possible to judge the emissivity of plasmas of different materials. The results obtained are compared to the data from other studies.     

Kinetic Modeling of Amino Acid Oxidation Catalyzed by a New D‐Amino Acid Oxidase from Arthrobacter protophormiae

Amino acid oxidases, which enantiospecifically catalyze the oxidative deamination of either D‐ or L‐amino acids, belong to the class of oxidoreductases functioning with a tightly bound cofactor. This cofactor favors industrial applications of D‐amino acid oxidases (D‐AAO). Hence, the enzyme is very important for the industrial application in the purification and determination of certain amino acids. In developing the enzyme‐catalyzed reaction for large‐scale production, modeling of the reaction kinetics plays an important role. Therefore, the subject of this study was the kinetics of the oxidative deamination, a very complex reaction system, which is catalyzed by D‐AAO from Arthrobacter protophormiae using its natural substrate D‐methionine and the aromatic amino acid 3,4‐dihydroxyphenyl‐D‐alanine (D‐DOPA). The kinetic parameters determined by the measurement of the initial rate and nonlinear regression were verified in batch reactor experiments by comparing calculated and experimental concentration‐time curves. It was found that the enzyme is highly specific towards D‐methionine (K_m = 0.24 mM) and not as specific to D‐DOPA as a substrate (K_m = 9.33 mM). The enzyme activity towards D‐methionine ( = 3.01 U/mL) was approx. seven times higher than towards D‐DOPA ( = 20.01 U/mL). The enzyme exhibited no activity towards L‐methionine and L‐DOPA. Batch and repetitive batch experiments were performed with both substrates in the presence and in the absence of catalase for hydrogen peroxide decomposition. Their comparison made it possible to conclude that hydrogen peroxide has no negative influence on the enzyme activity.