An 8-month-old, exclusively breastfed girl presented with a five-month history of vomiting with subsequent failure to thrive and lethargy. Family history was notable for the maternal history of gastroschisis. Mother had no dietary restrictions and had successfully breastfed multiple children for >12 months without issue. Initial evaluation was notable for macrocytic anemia. Subsequent serum B
12
levels were undetectable. Upon further questioning, the mother had significant bowel resection as an infant due to complications of gastroschisis. Maternal serum B
12
levels were also undetectable. The infant’s symptoms resolved with supplementation.
Steady state myoplasmic ion concentration and slow ion transport across the sarcolemma can be quantitatively studied by means of ion sensing electrodes (ISE). ISE's readout represents the sum of the electrochemical potential of the ion of interest and the resting membrane potential (Vm). Vm is measured either by using an independent standard microelectrode or a double-barreled capillary. These approaches have known limitations. Ion transport and membrane potential are intricately connected, the resting potential may be unstable or be far from the desired value, ion transport can be electrically silent, and membrane currents may be carried by more than one ion. Thus, an electrophysiological method able to simultaneously measure the concentration of more than one ion, and to measure and control Vm and membrane current (Im) is desirable for physiological and pathophysiological studies. For skeletal fibers, a triple-barreled electrode has been used in the past allowing for current injection to modify Vm under current clamp conditions. Here we describe a novel four-microelectrode system composed of a two-microelectrode voltage-clamp amplifier (TEV-200A, Dagan) and a two channel high impedance electrometer (FD223a, WPI). This approach allows for potentiometric measurements of the concentration and transport of up to two ions in short murine skeletal muscle fibers subjected to either current-or voltage-clamp conditions. Ion translocation by primary and secondary active transport mechanisms, ion exchangers or passive ion diffusion can be studied. The system performance is exemplified by using Na, K and H selective electrodes made with commercially available ionophores and fibers enzymatically dissociated from the flexor digitorum brevis muscles.
The antibody epitope analysis method is a highly adaptable technique of protein conformation elucidation, which can be easily applied without the need for specialized equipment or technical expertise. When applied in a systematic and strategic manner, this method has the potential to reveal novel and biomedically meaningful information for structure-function relationship and evolutionary lineage of proteins.
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