ABSTRACT
This article describes a creative maternal and infant home visiting program for first-time parents. Two-year outcomes indicate the program improved parents' infant safety knowledge, positively affected the mother's decision to breastfeed, and promoted infant primary care visits in a cost-effective way.
Subject(s)
Community Health Nursing/organization & administration , Home Care Services/organization & administration , House Calls , Maternal-Child Nursing/organization & administration , Adolescent , Adult , Breast Feeding , California , Humans , Medically Underserved Area , Models, Nursing , Mothers/education , Mothers/psychology , Nursing Evaluation Research , Organizational Objectives , Outcome Assessment, Health Care , Program Evaluation , Prospective Studies , Referral and ConsultationABSTRACT
In an attempt to compare the regulation of chick connexin43 channels to those of mammalian connexin43, we found that the nucleotide sequence reported for chick connexin43 differs from that of the chick connexin gene by two codons that had been entered as histidine49 (H49) and valine50 (V50) (accession no. M29003), but are in fact glutamine49 (Q49) and serine50 (S50). Neuro2A cells were transfected with corrected wild-type (Q49/S50) chick connexin43 (accession no. AF233738), the double-replacement Q49H/S50V connexin43, or the single replacement of Q49H or S50V. All clones had gap junctions in membrane based on immunocytochemistry and immunoblots of the triton-resistant membrane fraction. Wild-type transfectants had three conductance states with a predominant channel conductance of 85 +/- 5 pS. Cells producing the Q49H-Cx43 or the double-replacement Q49H/S50V-Cx43 protein had no detectable connexin43 channels. In contrast, cells expressing S50V-Cx43 gap junctions had channels with reduced conductances (75 +/- 8 pS) compared to wild-type controls. Low or high pH of the bathing solution had no effect on the Q49H-Cx43 channels. We conclude that glutamine49 is important for channel function, and replacement of this residue with histidine most likely distorts secondary structure of the first extracellular loop, possibly by changing the orientation of conserved cysteines, and this inhibits channel function. The S50V substitution may also cause similar but less severe structural changes.
Subject(s)
Cell Communication/physiology , Connexin 43/metabolism , Eukaryotic Cells/metabolism , Gap Junctions/metabolism , Glutamine/metabolism , Serine/metabolism , Amino Acid Sequence/genetics , Animals , Base Sequence/genetics , Chick Embryo , Connexin 43/genetics , Gap Junctions/genetics , Glutamine/genetics , Histidine/genetics , Histidine/metabolism , Hydrogen-Ion Concentration , Immunohistochemistry , Membrane Potentials/genetics , Mice , Molecular Sequence Data , Mutation/genetics , Protein Structure, Tertiary/genetics , Sequence Homology, Amino Acid , Serine/genetics , Transfection , Tumor Cells, CulturedABSTRACT
There is general agreement that the connexin43 gap junction protein is a substrate for phosphorylation by protein kinase C but there is no similar consensus regarding the action of protein kinase A. Our previous studies demonstrated that channels formed by connexin43 were reversibly gated in response to microinjected protein kinase A and protein kinase C, but we did not determine whether these effects involved direct action on the connexin43 protein. Using a combination of in vivo metabolic labeling and in vitro phosphorylation of recombinant protein and synthetic peptides, we now find that connexin43 is a relatively poor substrate for purified protein kinase A compared to protein kinase C, but that phosphorylation can be accelerated by 8-Br-cAMP (8-bromoadenosine 3',5'-cyclic monophosphate) which also enhances connexin43 synthesis but at a much slower rate than phosphorylation. Phosphorylation of a critical amino acid, Ser364, by protein kinase A, appears to be necessary for subsequent multiple phosphorylations by protein kinase C. However, protein kinase C can phosphorylate connexin43 at a reduced level in the absence of prior phosphorylation. The results suggest that the correct regulation of channels formed by connexin43 may require sequential phosphorylations of this protein by protein kinase A and protein kinase C.
