Type 2 cystatins comprise a class of cysteine peptidase inhibitor presumed to mediate protective functions at various locations, including the oral cavity. Seven cystatin genes are clustered within a 300-kb region of human 20p11.2. "Salivary" cystatins, encoded by CST1, 2, 4, and 5, are present in saliva at significant levels but have also been reported in other secretions, such as tears, suggesting that during their evolution, these genes have acquired mechanisms directing differential tissue-specific expression. However, their patterns of expression, which might also provide additional clues to their individual functions, have not been determined. Gene-specific RNase protection assays were used to examine the qualitative and quantitative distribution of expression of these seven genes within a collection of 23 adult human tissues. The CST3 gene, encoding cystatin C, was expressed at modest levels in all tissues examined. The presumptive pseudogenes CSTP1 and CSTP2 were not expressed at detectable levels in any tissue. The CST1, 2, 4, and 5 genes were expressed in differential, tissue-specific patterns. Expression of CST2 and CST5 was restricted to the submandibular and parotid glands, while CST1 and CST4 were expressed in these tissues and in the lacrimal gland. Immunohistochemistry studies localized expression to the serous-type secretory end pieces. Coexpression of CST1 and CST4 was also observed in the epithelial lining of the gallbladder and seminal vesicle. The CST1 product was detected in the tracheal glands and CST4 in the kidney and prostate. Despite their different adult patterns of expression, analysis of CST1, 2, 4, and 5 mRNA levels in infant submandibular glands demonstrated a coordinate upregulation of expression of between 3.5 and 9 months of age. The patterns of cystatin gene expression are consistent with several proposed oral functions of the salivary cystatins but also suggest they are important in other locations and that, despite their close sequence similarity, they are individually specialized.
An abundant 1.05 kb human lacrimal gland mRNA has been characterized by cDNA cloning. It encodes a predicted 180 residue, 20546 Da secreted protein, with a charge of +11 at ph 7 and 24.5% proline, designated as Basic Proline-rich Lacrimal Protein (BPLP), Southern blot analysis is consistent with a single BPLP gene. BPLP lacks any distinct repetitive structure, and is unrelated to the salivary proline-rich protein super-family. The pre-proprotein shows modest overall similarity to a superfamily comprising human PRPb, the mouse MSG proteins, and rat VCS-alpha 1, VCS-beta 1 and submandibular apomucin. BPLP also contains a domain with similarity to the Zp2 protein domain found in several otherwise unrelated proteins. Northern blot analysis indicated that the BPLP gene is also expressed at modest levels in the human submandibular gland, and in situ hybridization demonstrated expression of BPLP in the secretory endpieces of the human lacrimal gland. The BPLP cDNA clone defines a new human tear protein, and should provide a useful phenotypic marker of differentiation in in vitro studies of lacrimal gland function.
The family of type 2 cystatin proteins are a class of cysteine proteinase inhibitors found in a variety of human fluids and secretions, where they appear to provide protective functions. To establish the size of the human gene family encoding these proteins, we isolated cosmid and lambda genomic clones. Restriction mapping, partial sequence analysis, and hybridization studies identified a total of seven distinct genes, six of which correspond to known genes and proteins. Sequence analysis showed that the seventh gene, CSTP2, is an apparent pseudogene carrying a nonsense mutation in exon 1 distinct from that in CSTP1. Southern blots of genomic DNA probed with gene-specific probes accounted for all but one or two sets of fragments containing exon 1, and one or two sets of fragments containing exon 3, indicating that the human type 2 cystatin gene family consists of eight or nine members. Southern blot analysis of large restriction fragments using these gene-specific probes indicates that all seven of the cloned type 2 cystatin genes are clustered at a single locus on human chromosome 20. This locus is no larger than about 910 kb, and possibly as small as 365 kb. We designate this as the CST locus and suggest a numbering system for the cystatin genes.
This study quantifies myocardial transfection following percutaneous retrograde coronary venous delivery (RCVD) of a plasmid encoding human Del-1. RCVD of Del-1, GFP plasmid, or marker dye was conducted in 14 pigs. After selective cannulation of a coronary vein, a delivery site was confirmed by contrast injection and myocardial blush. Ten milliliters of plasmid hDel-1 or GFP was administered. Animals were euthanized 3 and 7 days post-RCVD. hDel-1 gene expression was evaluated by quantitative RT-PCR. An average myocardial expression of 4.5 x 10(5) copies hDel-1/microg total RNA was observed within the approximately 5 x 5 cm(2) target tissue of the left ventricle. GFP expression was detected by fluorescent microscopy. hDel-1 protein expression was confirmed by immunohistochemistry. Regionalized myocardial expression was found in all pigs. hDel-1 RNA was not found in distant tissues except in the three pigs with prominent venovenous washout (PVW). These levels were 3 to 4 log unites lower than those found in myocardium. Single retrograde coronary venous administration resulted in efficient regional myocyte transfection of hDel-1 and GFP. This method may be useful and clinically feasible for myocardial angiogenesis therapy.
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