ABSTRACT
Parathyroid hormone-related protein (PTHrP) is produced by the lactating mammary gland and secreted into the milk; however, the function of PTHrP during lactation is unknown. Since messenger RNA for both PTHrP and the PTH/PTHrP receptor have been demonstrated within mammary tissue, a paracrine or autocrine function for PTHrP has been proposed. To investigate this hypothesis in lactating tissue, the expression of PTHrP and the PTH/PTHrP receptor was examined in purified subpopulations of cells derived from lactating rat mammary glands. Subpopulations of stromal, myoepithelial, and alveolar epithelial cells were isolated from mammary tissue using enzymatic digestion and immunomagnetic purification. Isolated cells were phenotypically characterized by immunohistochemistry and ultrastructural morphology. The purity of the separated alveolar and myoepithelial cells was assessed ultrastructurally and ranged from 91 to 96%. Messenger RNA and protein expression of PTHrP and the PTH/PTHrP receptor were examined using reverse transcription polymerase chain reaction, immunohistochemistry, and Western blot analysis, respectively. PTHrP mRNA and protein were expressed in alveolar epithelial cells and stromal fibroblasts, whereas PTH/PTHrP receptor mRNA and protein were expressed in all three cell types. The expression patterns for PTHrP and the PTH/PTHrP receptor support an autocrine or paracrine function for PTHrP in alveolar epithelial cells and stromal fibroblasts and a paracrine function for PTHrP in myoepithelial cells in the rat mammary gland during lactation.
Subject(s)
Epithelial Cells/chemistry , Fibroblasts/chemistry , Lactation , Mammary Glands, Animal/chemistry , Mammary Glands, Animal/cytology , Proteins/isolation & purification , Receptors, Parathyroid Hormone/isolation & purification , Animals , Autocrine Communication , Epithelial Cells/cytology , Female , Fibroblasts/cytology , Immunohistochemistry , Immunomagnetic Separation , Paracrine Communication , Parathyroid Hormone-Related Protein , Proteins/genetics , RNA, Messenger/analysis , Rats , Receptor, Parathyroid Hormone, Type 1 , Receptors, Parathyroid Hormone/genetics , Reverse Transcriptase Polymerase Chain Reaction , Stromal Cells/chemistry , Stromal Cells/cytology , Tissue DistributionABSTRACT
Parathyroid hormone-related protein (PTHrP) produced by the mammary gland has been postulated to have multiple functions in both the mother and neonate. In humans, alternative 3'-mRNA splicing and endoproteolytic processing result in multiple bioactive PTHrP peptides. Multiple PTHrP peptides also have been reported in bovine milk. To investigate the source of molecular heterogeneity of PTHrP in bovine milk, bovine PTHrP was cloned from a bovine brain cDNA library, sequenced and used to characterize the mammary PTHrP transcript. A 1065 bp clone (bP1) for bovine PTHrP was isolated from a brain cDNA library. The bP1 clone contained the entire coding sequence of PTHrP and 61 and 473 nucleotides of the 5'- and 3'-untranslated regions (UTRs) respectively. The predicted amino acid sequence of bovine PTHrP was 72-92% homologous to the sequences of chicken, rat, mouse, human, and canine PTHrP with the highest sequence divergence present in the C-terminal region of the peptide. The 5'- and 3'-UTRs of bovine brain PTHrP have a high degree of homology to exons 4 and 9 of human PTHrP respectively. PTHrP was expressed as a single 1200 nucleotide mRNA transcript in lactating bovine mammary tissue. RT-PCR using region-specific oligonucleotide primers derived from bP1 demonstrated that PTHrP mRNA transcripts in bovine brain and lactating mammary gland utilize the same 5'- and 3'-UTRs. Expression of PTHrP mRNA was localized to secretory and ductular epithelial cells within the lactating mammary gland, as detected using in situ hybridization. Expression of PTHrP mRNA was demonstrated in the mammary gland during late pregnancy and throughout lactation in cows.
Subject(s)
Mammary Glands, Animal/metabolism , Parathyroid Hormone/genetics , Proteins/genetics , RNA, Messenger/genetics , Amino Acid Sequence , Animals , Base Sequence , Cattle , Cloning, Molecular , DNA, Complementary , Female , Humans , Lactation , Molecular Sequence Data , Parathyroid Hormone-Related Protein , Pregnancy , Sequence Homology, Amino AcidABSTRACT
Efficient and reliable protoplasting, regeneration, and fusion techniques were established for the prototrophic strain Bacillus stearothermophilus NUB36. Auxotrophic mutants were isolated, and protoplast fusion was used to construct isogenic mutant strains and for chromosomal mapping. Markers were mapped using two-, three-, and four-factor crosses. The order of the markers was hom-1-thr-1-his-1-(gly-1 or gly-2)-pur-1-pur-2. These markers may be analogous to hom, thrA, hisA, glyC, and purA markers on the Bacillus subtilis chromosome. No analogous pur-1 marker has been reported in B. subtilis. The relative order of three of the markers (hom-1-thr-1-gly-1) was independently confirmed by transduction.
