Multisite phosphorylation of the 80 kDa (MARCKS) protein kinase C substrate in C3H/10T1/2 fibroblasts. Quantitative analysis of individual sites by solid-phase microsequencing.

A synthetic peptide, KKKKRFSFKKSFKLSGFSFKK, containing the phosphorylation sites of the acidic 80-87 kDa protein kinase C substrate was used to identify phosphopeptides in enzyme digests of this protein from mouse fibroblast C3H/10T1/2 cells. Stimulation of phosphorylation occurred, in vivo, with TPA at Ser7, Ser11 and Ser18, and, with two less potent phorbol esters, at Ser7 and Ser18. Okadaic acid effected a net phosphorylation of Ser7 and/or Ser11. Solid-phase sequencing showed that, in vitro, the order of initial rate of phosphorylation was Ser11 greater than Ser7 greater than Ser18, while Ser18 was preferentially phosphorylated when either Ser7 or Ser11 was occupied. No significant phosphorylation of Ser15 was detected.     

Genetic characterization and transmission cycles of Cryptosporidium species isolated from humans in New Zealand

Little is known about the genetic characteristics, distribution, and transmission cycles of Cryptosporidium species that cause human disease in New Zealand. To address these questions, 423 fecal specimens containing Cryptosporidium oocysts and obtained from different regions were examined by the PCR-restriction fragment length polymorphism technique. Indeterminant results were resolved by DNA sequence analysis. Two regions supplied the majority of isolates: one rural and one urban. Overall, Cryptosporidium hominis accounted for 47% of the isolates, with the remaining 53% being the C. parvum bovine genotype. A difference, however, was observed between the Cryptosporidium species from rural and urban isolates, with C. hominis dominant in the urban region, whereas the C. parvum bovine genotype was prevalent in rural New Zealand. A shift in transmission cycles was detected between seasons, with an anthroponotic cycle in autumn and a zoonotic cycle in spring. A novel Cryptosporidium sp., which on DNA sequence analysis showed a close relationship with C. canis, was detected in two unrelated children from different regions, illustrating the genetic diversity within this genus.