The fate of medial edge epithelial cells during palatal fusion in vitro: an analysis by DiI labelling and confocal microscopy.

Fusion of bilateral shelves, to form the definitive mammalian secondary palate, is critically dependent on removal of the medial edge cells that constitute the midline epithelial seam. Conflicting views suggest that programmed apoptotic death or epithelial-mesenchymal transformation of these cells is predominantly involved. Due in part to the potentially ambiguous interpretation of static images and the notable absence of fate mapping studies, the process by which this is achieved has, however, remained mechanistically equivocal. Using an in vitro mouse model, we have selectively labelled palatal epithelia with DiI and examined the fate of medial edge epithelial (MEE) cells during palatal fusion by localisation using a combination of conventional histology and confocal laser scanning microscopy (CLSM). In dynamic studies using CLSM, we have made repetitive observations of the same palatal cultures in time-course investigations. Our results concurred with the established morphological criteria of seam degeneration; however, they provided no evidence of MEE cell death or transformation. Instead we report that MEE cells migrate nasally and orally out of the seam and are recruited into, and constitute, epithelial triangles on both the oral and nasal aspects of the palate. Subsequently these cells become incorporated into the oral and nasal epithelia on the surface of the palate. We hypothesize an alternative method of seam degeneration in vivo which largely conserves the MEE population by recruiting it into the nasal and oral epithelia.     

Serial passage of tobacco rattle virus under different selection conditions results in deletion of structural and nonstructural genes in RNA 2.

The RNA genome of tobacco rattle virus (TRV) is bipartite. RNA 2 of the nematode-transmissible TRV isolate PPK20 encodes the viral coat protein (cp) and proteins with molecular weights of 29,400 and 32,800 (29.4K and 32.8K proteins). When this isolate was serially passaged in tobacco by using phenol-extracted RNA as the inoculum in each transfer, defective interfering (DI) RNAs rapidly accumulated. A number of these DI RNAs were cloned. Six DI RNAs had single internal deletions in RNA 2 that removed most of the cp gene, the 29.4K gene, and the 5' half of the 32.8K gene. The borders of the deletions in these DI RNAs were found to be flanked in the genomic RNA 2 by short nucleotide repeats or sequences resembling the 5' end of TRV genomic and subgenomic RNAs. Two DI RNAs were found to be recombinants containing a 5' sequence derived from RNA 2 and a 3' sequence derived from RNA 1. When serial passage of TRV isolate PPK20 was carried out by using leaf homogenates as inocula in each transfer, accumulation of a DI RNA (designated D7) with a functional cp gene was observed. The deletion in D7 covered the 3' end of the cp gene, the 29.4K gene, and the 5' half of the 32.8K gene. An infectious cDNA clone of D7 RNA was made. In mixed infections, D7 RNA rapidly outcompeted RNA 2 but did not compete with RNA 1. The deletion in D7 RNA abolished the nematode transmissibility of the PPK20 isolate. These results may explain the observation that many laboratory isolates of tobraviruses have lost their nematode transmissibility and contain RNA 2 molecules of widely different lengths.     

Effect of enzyme dehydration on alcalase‐catalyzed dipeptide synthesis in near‐anhydrous organic media

The effect of enzyme dehydration by molecular sieves on the coupling of phenylalanine amide and the carbamoylmethyl ester of N‐protected phenylalanine in near‐anhydrous tetrahydrofuran was investigated. This coupling was catalyzed by Alcalase covalently immobilized onto macroporous acrylic beads (Cov); these immobilized enzymes were hydrated prior to use. The dehydration kinetics of Cov by molecular sieve powder were determined by incubating Cov with different amounts of molecular sieve powder for different periods of time (0–80 h). Subsequently, the remaining coupling activity of Cov was measured. Dehydration‐induced inactivation of Cov by molecular sieve powder was found to occur in three phases: (1) an initial, rapid, major dehydration‐induced inactivation that takes place during the first activity measurement, (2) a phase of first‐order inactivation, and (3) a plateau phase in activity. These dehydration kinetics were incorporated into a previously found reaction kinetics model. The resulting model was then used to fit progress curve data of the coupling in the presence of different amounts of molecular sieve powder. Upon establishment of parameter values, the model was used to predict independent data sets and found to work well. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:870–875, 2013     

Aerobic exercise alone results in clinically significant weight loss for men and women: Midwest exercise trial 2

Exercise is recommended by public health agencies for weight management; however, the role of exercise is generally considered secondary to energy restriction. Few studies exist that have verified completion of exercise, measured the energy expenditure of exercise, and prescribed exercise with equivalent energy expenditure across individuals and genders.

Objective:

The objective of this study was to evaluate aerobic exercise, without energy restriction, on weight loss in sedentary overweight and obese men and women.

Design and Methods:

This investigation was a randomized, controlled, efficacy trial in 141 overweight and obese participants (body mass index, 31.0 ± 4.6 kg/m²; age 22.6 ± 3.9 years). Participants were randomized (2:2:1 ratio) to exercise at either 400 kcal/session or 600 kcal/session or to a nonexercise control. Exercise was supervised, 5 days/week, for 10 months. All participants were instructed to maintain usual ad libitum diets. Because of the efficacy design, completion of ≥90% of exercise sessions was an a priori definition of per protocol, and these participants were included in the analysis.

Results:

Weight loss from baseline to 10 months for the 400 and 600 kcal/session groups was 3.9 ± 4.9 kg (4.3%) and 5.2 ± 5.6 kg (5.7%), respectively, compared with weight gain for controls of 0.5 ± 3.5 kg (0.5%) (P < 0.05). Differences for weight loss from baseline to 10 months between the exercise groups and differences between men and women within groups were not statistically significant.

Conclusions:

Supervised exercise, with equivalent energy expenditure, results in clinically significant weight loss with no significant difference between men and women.