Separation of antihemophilic factor VII from human plasma by column chromatography.

TMAE-Fractogel 650 (M) (Trimethylaminoethyl) is an ion exchange medium can be used to capture factor VIII (F VIII) directly from plasma. Previous reports have focused on the use of DEAE-Fractogel 650 (M) (Dimethylaminoethyl) ion exchange medium to capture F VIII from cryoprecipitate and plasma. Our main objectives were (I) to standardise the purification of FVIII from human plasma by column chromatographic technique. (II) to study the recovery of FVIII activity in purified fraction at 18-20°C process condition. (III) to study the effect of virucidal step on recovery of FVIII activity and (IV) to study the effect of lyophilisation on FVIII activity. In this report, Citrate Phosphate Dextrose (CPD) plasma was batch stirred with dry DEAE-Sephadex A50, filtered, diluted, loaded on to a column packed with TMAE-Fractogel and chromatographed. Most of the unwanted proteins flowed through the gel unadsorbed. Bound F VIII was eluted by increasing the ionic strength of the buffer. This purification step gave an overall 80% recovery from the plasma with a specific activity of 0.97 IU FVIII/mg protein. The purified F VIII fraction was made virus safe by employing the virucidal technique developed by New York Blood Centre (NYBC). There was 48.43% loss of FVIII activity in Virus inactivation treatment and the loss of FVIII activity in lyophilisation was 8.45% which is acceptable. This method of purification gave a higher yield of FVIII than cryoprecipitation, and is a promising alternative method to cryoprecipitation of F VIII.

Target cells promote the development and functional maturation of neurons derived from a sympathetic precursor cell line.

The role of target interactions in the development and functional maturation of peripheral neurons was investigated using an immortalized sympathetic precursor cell line. bMAH cells underwent neuronal differentiation in response to neurotrophic factors, but maintained an immature neuronal phenotype characterized by small cell bodies and continued cell division. Co-culture with cardiac myocytes, a target of sympathetic innervation, promoted the appearance of large-diameter postmitotic bMAH neurons. Analysis of bMAH maturation in the presence and absence of co-cultured myocytes indicated that myocyte-derived factors promoted the survival of maturing bMAH neurons prior to their acquisition of nerve growth factor dependence. Myocyte interactions also promoted the functional maturation of bMAH neurons, leading to an increase in the localization of synaptic vesicle proteins into neuritic varicosities and the acquisition of sympathetic-like intrinsic electrical properties. Like primary sympathetic neurons, mature bMAH neurons formed functional connections to cardiac myocytes as measured by evoked postsynaptic responses in connected myocytes. The effects of myocyte co-culture on developing bMAH neurons could be mimicked by myocyte conditioned medium, indicating that cardiac myocytes produce soluble factors that promote the appearance of mature neurons. These experiments indicate that targets of innervation play a role in directing the development and final maturation of peripheral neurons.