Evaluation of the 5'-flanking regions of murine glutathione peroxidase five and cysteine-rich secretory protein-1 genes for directing transgene expression in mouse epididymis.
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Based on strong epididymal expression of the mouse glutathione peroxidase 5 (GPX5) and cysteine-rich secretory protein-1 (CRISP-1) genes, we evaluated whether the 5.0-kilobase (kb)-long GPX5 and 3.8-kb-long CRISP-1 gene 5'-flanking regions could be used to target expression of genes of interest into the epididymis in transgenic mice. Of the two candidate promoters investigated, the CRISP-1 promoter-driven enhanced green fluorescent protein (EGFP) reporter gene was highly expressed in the tubular compartment of the testis in all stages of the seminiferous epithelial cycle between pachytene spermatocytes at stage VII to elongated spermatids at step 16. In contrast to CRISP-1, the 5.0-kb 5' region of the mouse GPX5 gene directed EGFP expression to the epididymis. In the various GPX5-EGFP mouse lines, strongest expression of EGFP mRNA was found in the epididymis, but low levels of reporter gene mRNA were detected in several other tissues. Strong EGFP fluorescence was found in the principal cells of the distal caput region of epididymis, and few fluorescent cells were also detected in the cauda region. No EGFP fluorescence was detected in the corpus region or in the other tissues analyzed. Hence, it is evident that the 5.0-kb 5'-flanking region of GPX5 promoter is suitable for directing the expression of structural genes of interest into the caput epididymidis in transgenic mice. (+info)
Gln-Gly cleavage: correlation between collision-induced dissociation and biological degradation.
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Tryptic digestion of the 150-residue human acidic salivary proline-rich protein 1 (PRP-1) generated eight peptides, two of which corresponded to the N-terminal 30-residue segment. In each of the other six tryptic peptides, a consensus repeat with the structure PQGPPQQGG was present. A facile Gln-Gly cleavage between the second and the third residues of the repeat was observed during collision-induced dissociation experiments. We postulate possible mechanisms to account for this reactivity, involving attack on the peptidyl carbonyl group by the Gln sidechain. Significantly, the Gln-Gly cleavage has been shown to be biologically important in the bacterial degradation of PRPs in saliva, generating bacteria-binding Pro-Gln C-termini. We suggest a link between the gas-phase chemistry and the biochemical degradation of these molecules. (+info)
Resting (basal) secretion of proteins is provided by the minor regulated and constitutive-like pathways and not granule exocytosis in parotid acinar cells.
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Resting secretion of salivary proteins by the parotid gland is sustained in situ between periods of eating by parasympathetic stimulation and has been assumed to involve low level granule exocytosis. By using parotid lobules from ad libitum fed rats stimulated with low doses of carbachol as an in vitro analog of resting secretion, we deduce from the composition of discharged proteins that secretion does not involve granule exocytosis. Rather, it derives from two other acinar export routes, the constitutive-like (stimulus-independent) pathway and the minor regulated pathway, which responds to low doses of cholinergic or beta-adrenergic agonists (Castle, J. D., and Castle, A. M. (1996) J. Cell Sci. 109, 2591-2599). The protein composition collected in vitro mimics that collected from cannulated ducts of glands given low level stimulation in situ. Analysis of secretory trafficking along the two pathways of resting secretion has indicated that the constitutive-like pathway may pass through endosomes after diverging from the minor regulated pathway at a brefeldin A-sensitive branch point. The branch point is deduced to be distal to a common vesicular budding event by which both pathways originate from immature granules. Detectable perturbation of neither pathway in lobules was observed by wortmannin addition, and neither serves as a significant export route for lysosomal procathepsin B. These findings show that parotid acinar cells use low capacity, high sensitivity secretory pathways for resting secretion and reserve granule exocytosis, a high capacity, low sensitivity pathway, for massive salivary protein export during meals. An analogous strategy may be employed in other secretory cell types. (+info)
Anticandida activity is retained in P-113, a 12-amino-acid fragment of histatin 5.
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Through the analysis of a series of 25 peptides composed of various portions of the histatin 5 sequence, we have identified P-113, a 12-amino-acid fragment of histatin 5, as the smallest fragment that retains anticandidal activity comparable to that of the parent compound. Amidation of the P-113 C terminus increased the anticandidal activity of P-113 approximately twofold. The three histidine residues could be exchanged for three hydrophobic residues, with the fragment retaining anticandidal activity. However, the change of two or more of the five basic (lysine and arginine) residues to uncharged residues resulted in a substantial loss of anticandidal activity. A synthetic D-amino-acid analogue, P-113D, was as active against Candida albicans as the L-amino-acid form. In vitro MIC tests in low-ionic-strength medium showed that P-113 has potent activity against Candida albicans, Candida glabrata, Candida parapsilosis, and Candida tropicalis. These results identify P-113 as a potential antimicrobial agent in the treatment of oral candidiasis. (+info)
Human immune response to sand fly salivary gland antigens: a useful epidemiological marker?
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Antibody (IgG) responses to salivary gland homogenate and to a recombinant salivary protein from the sand fly Lutzomyia longipalpis were investigated using sera from children living in an endemic area of visceral leishmaniasis in Brazil. We classified children into four groups according to their responses to Leishmania antigen: (Group I) positive serology and positive delayed type hypersensitivity (DTH), (Group II) positive serology and negative DTH, (Group III) negative serology and positive DTH, and (Group IV) negative serology and negative DTH. A highly significant correlation was found between anti-salivary gland IgG levels and DTH responses. An L. longipalpis salivary recombinant protein used as an antigen in an enzyme-linked immuno sorbent assay (ELISA) gave a significant but different result. A positive correlation was found between anti-Leishmania IgG and anti-recombinant protein IgG titers. The results indicate that sand fly salivary proteins may be of relevance to the study the epidemiology of leishmaniasis. (+info)
Is salivary histatin 5 a metallopeptide?
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Histatins are small histidine-rich salivary polypeptides which exhibit antimicrobial activity against Candida albicans. This antimicrobial activity has been ascribed in part to a high content of basic amino acids. However, unlike most other antimicrobial proteins histatins have a high content of histidine, tyrosine and acidic amino acids known to participate in metal ion coordination. This study was conducted to test whether histatin 5 could bind zinc and copper which are metals present in salivary secretions and whole saliva. Physical binding parameters and spectral properties of zinc- and copper-histatin complexes were investigated in order to obtain direct evidence of these interactions. A spectrophotometric competition assay using the metallochromic indicator murexide showed that histatin 5 dissociates metal indicator complexes containing zinc or copper ions. Absorption spectra of histatin 5 at increasing copper chloride concentrations resulted in higher absorbance in the 230-280 nm wavelength range and this spectral change was saturated at a peptide:metal molar ratio of approx. 1:1. A corresponding band was observed in the visible range of the spectrum with a maximum and molar extinction coefficient corresponding to that of copper binding to an ATCUN motif. Quantitative assessment of zinc and copper binding to histatin 5 using isothermal titration calorimetry revealed at least one high affinity site for each metal, with binding constants of 1.2x10(5) and 2.6x10(7) M(-1), respectively. These results indicate that histatin 5 exhibits metallopeptide-like properties. The precise biological significance of this has not yet been established but histatins may contribute significantly to salivary metal binding capacity. (+info)
In situ transcription and splicing in the Balbiani ring 3 gene.
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The Balbiani ring 3 (BR3) gene contains 38 introns, and more than half of them are co-transcriptionally excised. We have determined the in situ structure of the active BR3 gene by electron tomography. Each of the 20-25 nascent transcripts on the gene is present together with splicing factors and the RNA polymerase II in a nascent transcript and splicing complex, here called the NTS complex. The results indicate that extensive changes in overall shape, substructure and molecular mass take place repeatedly within an NTS complex as it moves along the gene. The volume and calculated mass of the NTS complexes show that, maximally, one complete spliceosome is assembled on the multi-intron transcript at any given time point. The structural data show that the spliceosome is not a structurally well-defined unit in situ and that the C-terminal domain of the elongating RNA polymerase II cannot carry spliceosomal components for all introns in the BR3 transcript. Our data indicate that spliceosomal factors are continuously added to and released from the NTS complexes during transcription elongation. (+info)
Contributions of intercalated duct cells to the normal parenchyma of submandibular glands of adult rats.
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The parenchyma of the submandibular gland in the adult male rat is self-renewing, with most newly formed acinar and granular duct cells believed to differentiate from the rapidly proliferating intercalated duct (ID) compartment. Since the ID cells are phenotypically diverse, based on their different expression of perinatal secretory proteins, we systemically injected tritiated thymidine for 24 hours, and followed the pattern of thymidine distribution in cells by autoradiography and immunocytochemistry of defined cellular phenotypes over a 1-month chase period. Proliferating cells were found within all parenchymal cell compartments; they were most numerous in ID, and primarily in those cells lacking immunoreactivity for the perinatal proteins SMG-B1, -C, and -D. The labeling index (LI) of the ID cells reached a peak at 7 days postinjection, and then decreased over the next 3 weeks. Concurrently, the LI increased significantly in those cells at the junctions of ID with both acini and granular ducts, and also within these larger parenchymal elements. We conclude that the ID cells not reactive for perinatal proteins proliferate to expand the ID compartment, and that ID cells at the ends of the ducts differentiate into both acinar and granular duct cells. Our data provide no evidence for the differentiation of ID cells into cells of striated ducts (SD); however, the small number of excretory duct (ED) profiles seen in our preparations showed extremely high LI (>25%), suggesting that more extensive data might reveal a precursor role for the ED in replacement of SD cells. In addition to the stepwise passage of cells from ID to other parenchymal elements at their junctions, the reported occurrence of occasional clusters of B1-positive acini (BAC) among the typical B1-negative acini had suggested an alternate pathway, in which entire segments of newly expanded ID might develop directly into a recapitulated perinatal stage of B1-reactive cell, pursuant to becoming mature acinar cells. Consistent with this suggestion, the BAC had a fourfold greater LI than typical adult acini; moreover, when analyzed by electron microscopic immunocytochemistry, they appeared similar to the novel perinatal Type III cells both ultrastructurally and in their pattern of B1-immunogold labeling. In contrast, the less common acini showing a sublingual gland phenotype had no significant difference in LI from typical acinar cells. Overall, our results emphasize the importance of the nonimmunoreactive ID cells in normal cellular replacement, and the possibility that ID can undergo en bloc differentiation into replacement acini as well as incremental addition of single cells at the boundaries of ID with acini and with granular ducts. (+info)