OBJECTIVE: To study the phosphorylation state of neurofilament (NF) proteins and activity of KSPXK kinase in the sciatic nerves of Swiss white (S/W) mice inoculated in the hind foot pads with M. leprae. DESIGN: Test group includes S/W mice inoculated in the foot pads with freshly harvested human derived (viable) M. leprae. Control groups were constituted by (1) Age matched un-inoculated mice, (2) Mice similarly inoculated with M. smegmatis and (3) heat killed M. leprae. Phosphorylation state of NF was studied using Western blot analysis and phosphor-specific NF antibody (SMI 31; Sternberger Monoclonals, Inc.). The KSPXK kinase activity was assayed by using KSPXK fusion protein in a radiometric method using gamma(22)P ATP. RESULTS: Several fold increase in M. leprae numbers was seen in viable M. leprae group while M. smegmatis failed to show any fold increase in the foot pads of S/W mice. Western immunoblot analysis of cytoskeletal preparation from sciatic nerves of uninoculated mice and mice inoculated with M. smegmatis showed immunoreactivity to SMI 31 antibody and protein bands corresponding to both NF-H and NF-M at all the time points from 4-20 months post inoculation. In case of viable M. leprae; SMI 31 reactive protein bands were seen at 4 months but not at any of the later intervals, i.e., between 6-20 months. With heat killed M. leprae transient loss of immunoreactivity to SMI 31 was seen. Decrease in KSPXK kinase activity was recorded in sets inoculated with viable and heat killed M. leprae, and corroborated with loss of immunoreactivity seen in WBs reacted with SMI 31 antibody. CONCLUSIONS: Alterations in the sciatic nerve NF cytoskeleton was seen following inoculation in the hind foot pad with both viable and heat killed M. leprae. The hypophosphorylation of NF observed in this study corroborates with the earlier observations in human leprous nerves.
OBJECTIVE: To study the phosphorylation state of neurofilament (NF) proteins and activity of KSPXK kinase in the sciatic nerves of Swiss white (S/W) mice inoculated in the hind foot pads with M. leprae. DESIGN: Test group includes S/W mice inoculated in the foot pads with freshly harvested human derived (viable) M. leprae. Control groups were constituted by (1) Age matched un-inoculated mice, (2) Mice similarly inoculated with M. smegmatis and (3) heat killed M. leprae. Phosphorylation state of NF was studied using Western blot analysis and phosphor-specific NF antibody (SMI 31; Sternberger Monoclonals, Inc.). The KSPXK kinase activity was assayed by using KSPXK fusion protein in a radiometric method using gamma(22)PATP. RESULTS: Several fold increase in M. leprae numbers was seen in viable M. leprae group while M. smegmatis failed to show any fold increase in the foot pads of S/W mice. Western immunoblot analysis of cytoskeletal preparation from sciatic nerves of uninoculated mice and mice inoculated with M. smegmatis showed immunoreactivity to SMI 31 antibody and protein bands corresponding to both NF-H and NF-M at all the time points from 4-20 months post inoculation. In case of viable M. leprae; SMI 31 reactive protein bands were seen at 4 months but not at any of the later intervals, i.e., between 6-20 months. With heat killed M. leprae transient loss of immunoreactivity to SMI 31 was seen. Decrease in KSPXK kinase activity was recorded in sets inoculated with viable and heat killed M. leprae, and corroborated with loss of immunoreactivity seen in WBs reacted with SMI 31 antibody. CONCLUSIONS: Alterations in the sciatic nerve NF cytoskeleton was seen following inoculation in the hind foot pad with both viable and heat killed M. leprae. The hypophosphorylation of NF observed in this study corroborates with the earlier observations in human leprous nerves.