A Paradigm of Uphill Running

The biomechanical management of bioenergetics of runners when running uphill was investigated. Several metabolic and mechanical variables have been studied simultaneously to spread light on the locomotory strategy operated by humans for effective locomotion. The studied variables were: heart rate, heart rate variability, oxygen intake and blood lactate, metabolic cost, kinematics, ground reaction force and muscular activity. 18 high-level competitive male runners ran at 70% VO_2max on different uphill slope conditions: 0%, 2% and 7%. Modifications were significant in almost all variables studied, and were more pronounced with increasing incline. Step frequency/length and ground reaction force are adjusted to cope with both the task of uphill progression and the available (limited) metabolic power. From 0% to 7% slope, step frequency and ground reaction force and metabolic cost increased concurrently by 4%, 12% and 53%, respectively (with a 4% step length decrease as well). It is hypothesised that this biomechanical management is allowed by an environment-body communication performed by means of specific muscular activity.     

Primedin situ labeling (PRINS)

PRimedIn Situ labeling (PRINS) is a fast and sensitive alternative to fluorescencein situ hybridization (FISH) for identification of chromosome aberrations. In this article, we present the detailed protocols for detection of repeat sequences using oligonucleotides or fragments of cloned probes as primers for PRINS. We describe a multicolor PRINS procedure for simultaneous visualization of more probes in different colors on a metaphase preparation, and a PRINS-painting procedure, which combines PRINS and chromosome painting. Finally, a protocol for detection of single-copy genes is presented.     

Modelling of the baroreflex-feedback mechanism with time-delay

 The cardiovascular system is considered. A direct modelling of the non-linear baroreflex-feedback mechanism, including time-delay, is developed based on physiological theory and empirical facts. The feedback model is then evaluated on an expanded, but simple, non-pulsatile Windkessel model of the cardiovascular system. The stability of the entire model is analyzed and the effect of the value of the time-delay is investigated and discussed. The time-delay may cause oscillations. A finite number of stability switches may occur dependent on the value of the time-delay. The location of these stability switches turns out to be sensitive to the value of the parameters in the model. We suggest a simple experiment to determine whether or not the time-delay is responsible for the 10 second Mayer waves. Data from an ergometer bicycle test is used for evaluation of the model. Received 1 June 1996; received in revised form 20 November 1996     

Use of conformationally restricted pyridinium alpha-D-N-acetylneuraminides to probe specificity in bacterial and viral sialidases.

Investigations into subtle changes in the catalytic activity of sialidases have been performed using enzymes from several different origins, and their results have been compared. This work highlights the potential pitfalls encountered when extending conclusions derived from mechanistic studies on a single enzyme even to those with high-sequence homology. Specifically, a panel of 5 pyridinium N-acetylneuraminides were used as substrates in a study that revealed subtle differences in the catalytic mechanisms used by 4 different sialidase enzymes. The lowest reactivity towards the artificial (pyridinium) substrates was displayed by the Newcastle disease virus hemagglutinin-neuraminidase. Moreover, in reactions involving aryl N-acetylneuraminides, the activity of the Newcastle enzyme was competitively inhibited by the 3,4-dihydro-2H-pyrano[3,2-c]pyridinium compound with a Ki = 58 micromol/L. Alternatively, the 3 bacterial enzymes tested, from Salmonella typhimurium, Clostridium perfringens, and Vibrio cholerae, were catalytically active against all members of the panel of substrates. Based on the observed effect of leaving-group ability, it is proposed that the rate-determining step for kcat (and likely for kcat/Km as well) with each bacterial enzyme is as follows: sialylation, which is concerted with conformational change for V. cholerae; and conformational change for S. typhimurium and C. perfringens.     

Synthesis and evaluation of 5,6-disubstituted thiopyrimidine aryl aminothiazoles as inhibitors of the calcium-activated chloride channel TMEM16A/Ano1

Transmembrane protein 16A (TMEM16A), also called Ano1, is a Ca²⁺ activated Cl^? channel expressed widely in mammalian epithelia, as well as in vascular smooth muscle and some tumors and electrically excitable cells. TMEM16A inhibitors have potential utility for treatment of disorders of epithelial fluid and mucus secretion, hypertension, some cancers and other diseases. 4-Aryl-2-amino thiazole T16A_inh-01 was previously identified by high-throughput screening. Here, a library of 47 compounds were prepared that explored the 5,6-disubstituted pyrimidine scaffold found in T16A_inh-01. TMEM16A inhibition activity was measured using fluorescence plate reader and short-circuit current assays. We found that very little structural variation of T16A_inh-01 was tolerated, with most compounds showing no activity at 10?μM. The most potent compound in the series, 9bo, which substitutes 4-methoxyphenyl in T16A_inh-01 with 2-thiophene, had IC₅₀ ～1?μM for inhibition of TMEM16A chloride conductance.     

High cell density fed batch and perfusion processes for stable non-viral expression of secreted alkaline phosphatase (SEAP) using insect cells: comparison to a batch Sf-9-BEV system

The development of insect cells expressing recombinant proteins in a stable continuous manner is an attractive alternative to the BEV system for recombinant protein production. High cell density fed batch and continuous perfusion processes can be designed to maximize the productivity of stably transformed cells. A cell line (Sf-9SEAP) expressing high levels of the reporter protein SEAP stably was obtained by lipid-mediated transfection of Sf-9 insect cells and further selection and screening. The expression of the Sf-9SEAP cells was compared with the BEVS system. It was observed that, the yield obtained in BEVS was similar to the batch Sf-9SEAP at 8 and 7 IU/mL, respectively. The productivity of this foreign gene product with the stable cells was enhanced by bioprocess intensification employing the fed-batch and perfusion modes of culture to increase the cell density in culture. The fed batch process yielded a maximum cell density of 28 x 10(6) cells/mL and 12 IU/mL of SEAP. Further improvements in the productivity could be made using the perfusion process, which demonstrated a stable production rate for extended periods of time. The process was maintained for 43 days, with a steady-state cell density of 17-20 x 10(6) cells/mL and 7 IU/mL SEAP. The total yield obtained in the perfusion process (394 IU) was approximately 22 and 8 times higher than that obtained in a batch (17.6 IU) and fed batch (46.1 IU) process, respectively.     

The Pmr1 protein, the major yeast Ca2+-ATPase in the Golgi, regulates intracellular levels of the cadmium ion

Cadmium is a nonessential, highly toxic heavy metal that shows ionic properties similar to calcium. These ionic similarities imply that the cadmium ion, Cd²⁺, is a calcium ion, Ca²⁺, receptor-agonist, affecting the same biochemical pathways involved in Ca²⁺ homeostasis. In the yeast Saccharomyces cerevisiae , the PMC1 and PMR1 genes encode vacuolar and Golgi Ca²⁺-ATPases, respectively. The PMR1 protein product Pmr1p is involved in both Ca²⁺ and Mn²⁺ homeostasis. This study investigated the importance of Pmc1p and Pmr1p for Cd²⁺ cellular detoxification. Using the standard techniques of yeast molecular research and a multielemental procedure named particle-induced X-ray emission, Pmr1p was identified as a protein that directly participates in the detoxification of Cd²⁺, possibly through the secretory pathway. The results allow us to posit a model of Cd²⁺ detoxification where Pmr1p has a central role in cell survival in a Cd²⁺-rich environment.