Developmental and androgenic regulation of the immunocytochemical distribution of mK1, a true tissue kallikrein, in the granular convoluted tubule of the mouse submandibular gland.

The action of androgens on the immunocytochemical distribution of mK1, a true tissue kallikrein, was examined in the submandibular gland (SMG) of developing and adult mice by indirect enzyme-labeled and immunogold-labeled antibody methods for light and electron microscopy, respectively. In both sexes at 3 weeks of age, essentially all of the immature granular convoluted tubule (GCT) cells were uniformly immunostained. At 4 weeks of age (the onset of puberty), morphological differences between the two sexes appeared in the GCTs, in which some cells became immunonegative. Thereafter, the immunonegative GCT cells became more abundant in the SMG of males than of females and considerable intercellular variation in staining intensity for mK1 was seen, especially in males. A few slender GCT cells with strong immunoreactivity appeared in GCT segments only in males. Castration of males resulted in an increase in the number of immunopositive GCT cells, whereas administration of dihydrotestosterone (DHT) decreased the number of immunopositive GCT cells in the SMGs of both sexes. Slender GCT cells immunoreactive for mK1 were seen in females treated with DHT for 6 days. However, there were no immunostained slender GCT cells in female SMGs after injection of DHT for 2 weeks. Immunoelectron microscopy disclosed this type of cell in male SMGs, which closely resembles immature GCT cells of prepubertal mice, with a few small secretory granules uniformly labeled with gold particles, a sparse Golgi apparatus and RER, and basal infoldings. In mature male SMGs and in SMGs of DHT-treated females and castrated males, typical GCT cells had a well-developed Golgi apparatus and a net-like RER but few to no basal infoldings, whereas in the female gland equivalent cells had moderately developed RER and some basal infoldings. These results suggest that mK1 is one of the enzymes characteristically present in immature GCT cells and that its synthesis is inhibited in part by androgens, resulting in decreased numbers of immunopositive cells.     

Plasmin-sensitive dibasic sequences in the third fibronectin-like domain of L1-cell adhesion molecule (CAM) facilitate homomultimerization and concomitant integrin recruitment

L1 is a multidomain transmembrane neural recognition molecule essential for neurohistogenesis. While moieties in the immunoglobulin-like domains of L1 have been implicated in both heterophilic and homophilic binding, the function of the fibronectin (FN)-like repeats remains largely unresolved. Here, we demonstrate that the third FN-like repeat of L1 (FN3) spontaneously homomultimerizes to form trimeric and higher order complexes. Remarkably, these complexes support direct RGD-independent interactions with several integrins, including alpha(v)beta(3) and alpha(5)beta(1). A pep- tide derived from the putative C-C' loop of FN3 (GSQRKHSKRHIHKDHV(852)) also forms trimeric complexes and supports alpha(v)beta(3) and alpha(5)beta(1) binding. Substitution of the dibasic RK(841) and KR(845) sequences within this peptide or the FN3 domain limited multimerization and abrogated integrin binding. Evidence is presented that the multimerization of, and integrin binding to, the FN3 domain is regulated both by conformational constraints imposed by other domains and by plasmin- mediated cleavage within the sequence RK( downward arrow)HSK( downward arrow)RH(846). The integrin alpha(9)beta(1), which also recognizes the FN3 domain, colocalizes with L1 in a manner restricted to sites of cell-cell contact. We propose that distal receptor ligation events at the cell-cell interface may induce a conformational change within the L1 ectodomain that culminates in receptor multimerization and integrin recruitment via interaction with the FN3 domain.     

C9orf10 protein, a novel protein component of Puralpha-containing mRNA-protein particles (Puralpha-mRNPs): characterization of developmental and regional expressions in the mouse brain.

Puralpha has been implicated in mRNA transport and translation in neurons. We previously reported that Puralpha is a component of mRNA/protein complexes (Puralpha-mRNPs) with several other proteins. Among them, we found the C9orf10 (Homo sapiens chromosome 9 open reading frame 10) protein, which was recently characterized as a component of RNA-containing structures. However, C9orf10 itself remains poorly understood. To characterize C9orf10 expression at the protein level, we raised an antibody against C9orf10 and compared the spatial and developmental expressions of this protein and Puralpha in the mouse brain. C9orf10 was expressed as early as embryo stage 12, whereas Puralpha was expressed from 5 days after birth. In adults, C9orf10 expression was most prominent in the hippocampus, caudate putamen, cerebral cortex, and cerebellum, unlike the uniform distribution of Puralpha. C9orf10-positive cells also showed immunoreactivity to Puralpha. C9orf10 expression was restricted to neurons, judging by the immunoreactivity to neuron-specific nuclear protein or CaM kinase II. These observations suggest an accessory role of C9orf10 for Puralpha in a limited brain region in addition to other possible functions that have not yet been determined.     

The Ca/H antiporter TMEM165 expression,localization in the developing,lactating and involuting mammary gland parallels the secretory pathway Ca ATPase (SPCA1)

Plasma membrane Ca²⁺-ATPase 2 (PMCA2) knockout mice showed that ∼60% of calcium in milk is transported across the mammary cells apical membrane by PMCA2. The remaining milk calcium is thought to arrive via the secretory pathway through the actions of secretory pathway Ca²⁺-ATPase’s 1 and/or 2 (SPCA1 and 2). However, another secretory pathway calcium transporter was recently described. The question becomes whether this Golgi Ca²⁺/H⁺ antiporter (TMEM165) is expressed sufficiently in the Golgi of lactating mammary tissue to be a relevant contributor to secretory pathway mammary calcium transport. TMEM165 shows marked expression on day one of lactation when compared to timepoints prepartum. At peak lactation TMEM165 expression was 25 times greater than that of early pregnancy. Forced cessation of lactation resulted in a rapid ∼50% decline in TMEM165 expression at 24 h of involution and TMEM165 expression declined 95% at 96 h involution. It is clear that the timing, magnitude of TMEM165 expression and its Golgi location supports a role for this Golgi Ca2⁺/H⁺ antiporter as a contributor to mammary Golgi calcium transport needs, in addition to the better-characterized roles of SPCA1&2.