Expression of plasma prekallikrein mRNA in human nonhepatic tissues and cell lineages suggests special local functions of the enzyme.

At present it is generally accepted that plasma prekallikrein (PPK) is synthesized in the liver and secreted into the bloodstream. Surprisingly, it has recently been shown that PPK mRNA is present also in RNA from the kidney, adrenal gland and placenta. In spite of its novelty and possible important physiological implications this finding has been neglected. Here we report that PPK mRNA is expressed also in the human brain, heart, lung, trachea, endothelial cells and leukocytes as well as in a variety of fibroblast and epithelial cell lines. Expression of PPK mRNA in fibroblasts, endothelial cells and leukocytes suggests that PPK mRNA detected in RNA preparations from whole tissue may originate solely from these ubiquitously occurring cells. However, PPK mRNA expression in various epithelial cell lines demonstrates that tissue-specific cells also transcribe the PPK gene. The presence of PPK mRNA in nonhepatic tissues and cells indicates that they have the capacity to synthesize the protein. The physiological role of PPK synthesized in extrahepatic tissue is unknown. It may participate in local actions within tissues as well as contributing to the PPK pool in blood plasma. Cultured cells will provide a valuable model for exploring the physiological significance of extrahepatic PPK expression.     

Cell-specific subcellular localization of soluble epoxide hydrolase in human tissues.

Soluble epoxide hydrolase (sEH) is a phase-I xenobiotic metabolizing enzyme having both an N-terminal phosphatase activity and a C-terminal epoxide hydrolase activity. Endogenous hydrolase substrates include arachidonic acid epoxides, which have been involved in regulating blood pressure and inflammation. The subcellular localization of sEH has been controversial. Earlier studies using mouse and rat liver suggested that sEH may be cytosolic and/or peroxisomal. In this study we applied immunofluorescence and confocal microscopy using markers for different subcellular compartments to evaluate sEH colocalization in an array of human tissues. Results showed that sEH is both cytosolic and peroxisomal in human hepatocytes and renal proximal tubules and exclusively cytosolic in other sEH-containing tissues such as pancreatic islet cells, intestinal epithelium, anterior pituitary cells, adrenal gland, endometrium, lymphoid follicles, prostate ductal epithelium, alveolar wall, and blood vessels. sEH was not exclusively peroxisomal in any of the tissues evaluated. Our data suggest that human sEH subcellular localization is tissue dependent, and that sEH may have tissue- or cell-type-specific functionality. To our knowledge, this is the first report showing the subcellular localization of sEH in a wide array of human tissues.     

Expression of multi-drug resistance 1 mRNA in human and rodent tissues: reduced levels in Parkinson patients

The membrane transporter multi-drug resistance 1 (MDR1, P-gp) regulates the bioavailability of endogenous and exogenous compounds and has been implicated in disorders such as Parkinson's disease, cancer, epilepsy, human immunodeficiency virus disease, and inflammatory bowel disease. To promote further understanding of the role of MDR1 in disease, we have characterized cellular MDR1 mRNA expression in post-mortem human and fresh-frozen Sprague-Dawley rat tissues by using radioactive oligonucleotide probe in situ hybridization. We report MDR1 mRNA in human and rat endothelial cells of small vessels in the brain and pia mater. Mdr1 mRNA is also expressed in the blood vessel walls of rat sensory dorsal root and sympathetic ganglia. In peripheral tissues, we have observed MDR1 mRNA in human and rat liver and renal tubules and in human adrenal cortex and the epithelial lining of rat intestine. In female and male reproductive tissues of rat, strong gene activity has been found in steroid-hormone-synthesizing cells. Quantification of MDR1 mRNA in human striatum has revealed reduced levels in Parkinson patients compared with control individuals. The high expression of MDR1 mRNA in blood vessels of the nervous system, in tissues involved in absorption and excretion, and in tissues forming barriers to the environment support the physiological role of MDR1 as a regulator of intracellular levels of endogenous and exogenous compounds.     

Identification of major cellular proteins synthesized in response to interleukin-1 and interleukin-6 in human hepatoma HepG2 cells

Interleukin-1 (IL-1) and interleukin-6 (IL-6) are principal proinflammatory cytokines inducing the acute phase response of various tissues, including liver. Cultured human hepatoma HepG2 cells were stimulated with IL-1 (10 ng/ml) and IL-6 (10 ng/ml). After 24 h the cells were collected and disrupted by sonication in a lysis buffer containing 8M urea. The extracted cellular proteins were separated by 2D polyacrylamide gel electrophoresis. The gels were stained with Coomassie Brilliant Blue R-250 and the protein spots showing different intensities in comparison to control (unstimulated) cells were excised and subjected to analysis by LC-MS/MS. Alternatively, proteins were stained with SYPRO Ruby. These differentially expressed proteins include seven up-regulated and two down-regulated intracellular proteins of various functions. The identification of three cytokine-responsive proteins was confirmed by biosynthetic labeling with [35S]methionine after incubation of HepG2 cells, and by western blot with specific antisera.     

Immunolocalization of cysteine proteinases (cathepsins) and cysteine proteinase inhibitors (salarin and salmon kininogen) in Atlantic salmon,Salmo salar

Tissue localization of cysteine proteinases (cathepsins) and their inhibitors (salarin, salmon kininogen) was performed in tissues of the Atlantic salmon. In skin, both epidermis and dermis were strongly stained by antisera against salarin and salmon kininogen. In epidermis the intercellular space seemed to be heavily stained (salarin). In kidney, the inhibitors were mainly localized to the interstitial capillaries. Also, some epithelial cells of the tubules (salarin) and some cells of the interstitium were stained. Mostly, the staining had a diffuse cytoplasmic localization. In the liver some hepatocytes were strongly positive for salarin and salmon kininogen. Purified fish cysteine proteinase inhibitors were not found to inhibit the growth of fish pathogenic bacteria and viruses. In the trunk kidney cathepsins B and L were localized in epithelial cells of the tubules (proximal part) and in cells of the interstitium. Mostly, the staining showed a prominent lysosomal localization. In head kidney large macrophage-like cells were positively stained for cathepsin B. The staining was localized to granula/vacuoles in the cytoplasm. In the liver, some hepatocytes were strongly stained and some were less strongly positive for cathepsin B and L. Mostly, the hepatocytes showed lysosomal staining. Cathepsin L was found in some big macrophage-like cells in the spleen. Mucosal epithelial cells of the esophagus and intestine seemed to be strongly stained for cathepsin B and L. The results show that cathepsins and their inhibitors are specifically and widely distributed in the Atlantic salmon skin indicating that they perform some biologically important and specific but so far unknown functions.     

Immunocytochemical demonstration of carbonic anhydrase in human epithelial cells

Human tissues obtained early postmortem were immunostained to demonstrate carbonic anhydrase (CA) and, in some instances, to differentiate CA I and CA II, employing an immunoglobulin-peroxidase bridge method. Optimal immunostaining was obtained in tissues fixed a few hours in Carnoy's fluid or a buffered HgCl2 solution. Specimens fixed 1/2 to 2 hr with buffered formalin or Bouin's fluid stained less well but better than those fixed 24 hr with formalin. In tracheobronchial glands, serous acini and demilunes exhibited intense immunoreactivity demonstrative of the isozyme CA II. In kidney, all cells of the distal convoluted tubules were strongly positive for CA and cortical collecting tubule cells stained strongly but with some variability among individual cells. Cells in medullary collecting tubules ranged from intensely to negligibly reactive. Proximal convoluted tubules and thick ascending limbs showed moderate to light, uniform staining, but the thin limbs of the loop of Henle were negative. Renal cell immunoreactivity occurred only with antiserum to CA II. Seromucous acini in submandibular glands stained strongly and selectively for CA. Ducts in liver and pancreas showed strong selective immunostaining. The most superficial columnar cells lining the main lumen of the colon and appendix displayed strong reactivity, as did columnar cells lining the gall bladder.     

Hsp90 Inhibitor 17-DMAG Decreases Expression of Conserved Herpesvirus Protein Kinases and Reduces Virus Production in Epstein-Barr Virus-Infected Cells

All eight human herpesviruses have a conserved herpesvirus protein kinase (CHPK) that is important for the lytic phase of the viral life cycle. In this study, we show that heat shock protein 90 (Hsp90) interacts directly with each of the eight CHPKs, and we demonstrate that an Hsp90 inhibitor drug, 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG), decreases expression of all eight CHPKs in transfected HeLa cells. 17-DMAG also decreases expression the of the endogenous Epstein-Barr virus protein kinase (EBV PK, encoded by the BGLF4 gene) in lytically infected EBV-positive cells and inhibits phosphorylation of several different known EBV PK target proteins. Furthermore, 17-DMAG treatment abrogates expression of the human cytomegalovirus (HCMV) kinase UL97 in HCMV-infected human fibroblasts. Importantly, 17-DMAG treatment decreased the EBV titer approximately 100-fold in lytically infected AGS-Akata cells without causing significant cellular toxicity during the same time frame. Increased EBV PK expression in 17-DMAG-treated AGS-Akata cells did not restore EBV titers, suggesting that 17-DMAG simultaneously targets multiple viral and/or cellular proteins required for efficient viral replication. These results suggest that Hsp90 inhibitors, including 17-DMAG, may be a promising group of drugs that could have profound antiviral effects on herpesviruses.     

Localisation of immunoreactive kininogen and tissue kallikrein in the human nephron

Summary The cellular localisation of kininogen and its relationships with tissue kallikrein containing cells was studied in the human kidney by the peroxidase-antiperoxidase method using antisera to human LMW kininogen and to human tissue kallikrein. Immunoreactive kininogen was localised in the principal cells of collecting ducts. Immunoreactive tissue kallikrein was detected in the connecting tubule cells segment of the nephron preceeding the cortical collecting ducts. The co-existence of tissue kallikrein and kininogen in the same transitional tubule, but in different cells, was established by the use of serial sections and double immunostaining. This anatomical relationship is in accordance with known studies that describe intermingling of principal cells and connecting tubule cells where connecting tubules merge into cortical collecting ducts in the human nephron. the close relationship between cells that contain tissue kallikrein and its substrate, kininogen, suggests that kinins could be generated in the lumen of distal cortical segments of the human nephron.     

Localisation of immunoreactive kininogen and tissue kallikrein in the human nephron

The cellular localisation of kininogen and its relationships with tissue kallikrein containing cells was studied in the human kidney by the peroxidase-antiperoxidase method using antisera to human LMW kininogen and to human tissue kallikrein. Immunoreactive kininogen was localised in the principal cells of collecting ducts. Immunoreactive tissue kallikrein was detected in the connecting tubule cells, segment of the nephron preceding the cortical collecting ducts. The co-existence of tissue kallikrein and kininogen in the same transitional tubule, but in different cells, was established by the use of serial sections and double immunostaining. This anatomical relationship is in accordance with known studies that describe intermingling of principal cells and connecting tubule cells where connecting tubules merge into cortical collecting ducts in the human nephron. The close relationship between cells that contain tissue kallikrein and its substrate, kininogen, suggests that kinins could be generated in the lumen of distal cortical segments of the human nephron.     

Immunohistochemical localization of D-aspartate oxidase in porcine peripheral tissues

d-Aspartate (d-Asp) is an endogenous substance in mammals. Degradation of d-Asp is carried out only by d-aspartate oxidase (DDO). We measured DDO activity in porcine tissues, and produced an anti-porcine DDO antibody to examine the cellular localization of DDO. All the tissues examined showed DDO activities, whereas the substrate d-Asp was not detected in kidney cortex, liver, heart, and gastric mucosa. In the kidney, intensive immunohistochemical staining for DDO was found in the epithelial cells of the proximal tubules. In the liver, the epithelial cells of interlobular bile ducts, liver sinusoid-lining cells with cytoplasmic processes, and the smooth muscle cells of arterioles were strongly stained for DDO. In the heart, cardiomyocytes and the smooth muscle cells of arterioles showed DDO-immunoreactivity. In the gastric mucosa, only the chief cells were DDO-positive. These newly identified DDO-positive cells seem to actively degrade d-Asp to prevent an excess of d-Asp from exerting harmful effects on the respective functions of porcine tissues.     

Heterogenous staining of D-amino acid oxidase in peroxisomes of rat liver and kidney. A light and electron microscopic study

The localization of D-amino acid oxidase (D-AAOX) in rat liver and kidney has been investigated using the cerium technique for electron microscopy and a recent modification of it for light microscopy. In the liver a mosaic pattern with strongly and weakly stained cells together with some completely negative hepatocytes is observed. The staining is stronger and more uniform in periportal than in perivenous regions of the liver lobule. In the kidney the reaction is confined to the proximal tubules of the renal cortex with the rest of the nephron being negative. At the ultrastructural level in both liver and kidney a marked heterogeneity is observed in the intensity of reaction in peroxisomes of some neighbouring cells. Moreover, in some cells heavily and weakly stained peroxisomes are seen side by side. When Pipes buffer is used in the incubation medium the D-AAOX reaction in kidney peroxiosomes is aggregated in the central region of the matrix with weaker staining of the periphery. A similar result is obtained when the enzyme is localized by immunocytochemistry confirming a recent report by Usuda et al. (1986). The heterogeneous staining of peroxisomes for D-AAOX suggests that subpopulations of this organelle with specialized functions may exist not only in different tissues and cells but even within the same cell.     

Immunohistological analysis of glutathione transferase A4 distribution in several human tissues using a specific polyclonal antibody.

We examined the cellular distribution of glutathione transferase A4 (GSTA4) in various human tissues by indirect immunoperoxidase using a specific polyclonal antibody raised in rabbit. This enzyme was localized in hepatocytes, bile duct cells, and vascular endothelial cells in liver, upper layers of keratinocytes and sebaceous and sweat glands in skin, proximal convoluted tubules in kidney, epithelial cells of mucosa and muscle cells in colon, muscle cells in heart, and neurons in brain. Staining was increased in pathological situations such as cirrhosis, UV-irradiated skin, and myocardial infarction and was strongly decreased in hepatocellular carcinoma. These results strongly support the view of a close correlation between cellular GSTA4 localization and the formation of reactive oxygen species in the tissues investigated.     

Formation of human hepatocyte-like cells with different cellular phenotypes by human umbilical cord blood-derived cells in the human-rat chimeras

We took advantage of the proliferative and permissive environment of the developing pre-immune fetus to develop a noninjury human-rat xenograft small animal model, in which the in utero transplantation of low-density mononuclear cells (MNCs) from human umbilical cord blood (hUCB) into fetal rats at 9-11 days of gestation led to the formation of human hepatocyte-like cells (hHLCs) with different cellular phenotypes, as revealed by positive immunostaining for human-specific alpha-fetoprotein (AFP), cytokeratin 19 (CK19), cytokeratin 8 (CK8), cytokeratin 18 (CK18), and albumin (Alb), and with some animals exhibiting levels as high as 10.7% of donor-derived human cells in the recipient liver. More interestingly, donor-derived human cells stained positively for CD34 and CD45 in the liver of 2-month-old rat. Human hepatic differentiation appeared to partially follow the process of hepatic ontogeny, as evidenced by the expression of AFP gene at an early stage and albumin gene at a later stage. Human hepatocytes generated in this model retained functional properties of normal hepatocytes. In this xenogeneic system, the engrafted donor-derived human cells persisted in the recipient liver for at least 6 months after birth. Taken together, these findings suggest that the donor-derived human cells with different cellular phenotypes are found in the recipient liver and hHLCs hold biological activity. This humanized small animal model, which offers an in vivo environment more closely resembling the situations in human, provides an invaluable approach for in vivo investigating human stem cell behaviors, and further in vivo examining fundamental mechanisms controlling human stem cell fates in the future.     

Distribution and characterization of diazepam binding inhibitor (DBI) in peripheral tissues of rat

We studied the expression and distribution of the polypeptide diazepam binding inhibitor (DBI) in rat peripheral organs by immunocytochemistry, radioimmunoassay, Northern blot analysis and binding assay. Variable amounts of the DBI peptide and DBI mRNA were found in all the tissues examined (liver, duodenum, testis, kidney, adrenal gland, heart, ovary, lung, skeletal muscle and spleen), with the highest level of expression in liver (220 pmol of DBI/mg protein) and the lowest in spleen (11 pmol of DBI/mg protein). A good correlation between DBI-like immunoreactivity (DBI-LI) and mRNA content was found in all tissues except the heart. The immunohistochemical analysis revealed discrete localization of DBI-LI in cell types with specialized functions: for example, the highest DBI-LI content was found in steroid-producing cells (glomerulosa and fasciculata cells of adrenal cortex, Leydig cells of testis); lower DBI-LI immunostaining was found in epithelial cells specialized for water and electrolyte transport (intestinal mucosa, distal convoluted tubules of kidney). Hepatic cells contained moderate immunoreactivity however the total content of DBI in liver is relatively high and is due to the diffuse presence of DBI in every hepatocyte. Cells with high expression of DBI have been shown to contain a high density of mitochondrial benzodiazepine (BZ) binding sites. This observation led us to perform a competitive binding assay between DBI and [3H]PK11195 (a ligand for the mitochondrial BZ binding sites) on mitochondrial membranes of adrenal cortical cells. In this experiment, DBI yielded an apparent competitive inhibition of the binding of PK11195 to the BZ binding sites. Our data support a possible role for DBI as endogenous regulator of intracellular metabolic functions, such as steroidogenesis, via the mitochondrial BZ receptors.     

Expression of liver type pyruvate kinase in insulinoma cells: involvement of LF-B1 (HNF1).

The messages for LF-B1, which interacts with the cis-acting element of PKL-I to play an essential role in expression of L-type pyruvate kinase (PK) in the liver, and L-type PK were found to be present in RIN-m5F insulinoma cells as well as the liver, kidney and small intestine, although the levels of the two mRNAs in these tissues were not correlated. Gel retardation assay suggested that similar nuclear proteins bound to two other cis-acting elements, PKL-II and PKL-III, were expressed in both liver and insulinoma cells, and that additional PKL-III-binding proteins were present only in RIN-m5F cells. Thus, we suggest that the mechanism of L-type PK expression in pancreatic B cells is similar to that in the liver.     

Distribution of the serine protease HtrA1 in normal human tissues.

The human HtrA family of proteases consists of three members: HtrA1, HtrA2, and HtrA3. In bacteria, the chief role of HtrA is recognition and degradation of misfolded proteins in the periplasm, combining a dual activity of chaperone and protease. In humans, the three HtrA homologues appear to be involved in diverse functions such as cell growth, apoptosis, allergic reactions, fertilization, control of blood pressure, and blood clotting. Previous studies using RNA blot hybridization have shown that the expression of HtrA1 is ubiquitous in normal human tissues. Here we show by immunohistochemistry (IHC) that HtrA1 is widely expressed, although different tissue distributions and/or levels of expression were detected in the different tissues examined. In particular, high to medium HtrA1 expression was detected in mature layers of epidermis, in secretory breast epithelium, in liver, and in kidney tubules of cortex, in concordance with its secretory properties. Furthermore, we show a higher protein expression level in the epithelium of proliferative endometrium, in contrast to epithelium of secretory endometrium, which is almost completely negative for this protein. This suggests a possible role for HtrA1 in the modulation of tissue activity in this organ. The various expression levels in human tissues indicate several possible roles for HtrA1 in different cell types.     

Expression of L- and M-type pyruvate kinase in human tissues

Pyruvate kinase (PK) has four isozymes (L, R, M1, M2) that are encoded by two different genes of PK L and M. Differential splicing produces L- and R-type PK mRNA and M1- and M2-type PK mRNA from the PK L gene and the PK M gene, respectively. The nucleotide sequences of the 3'-noncoding region are the same between the L- and the R-type PK and between the M1- and the M2-type PK. We isolated 3'-noncoding sequences of human L- and M2-type PK cDNA to construct L-type and M-type PK specific probes. With these probes, we performed Northern blot analysis of the RNA samples extracted from human tissues. Northern blot analysis showed that both kidney and liver had mRNAs that hybridized with both the L and M probes. Small intestine, skeletal muscle, brain, testis, and lung mRNAs hybridized only with the M probe. Our probes are considered useful for the detection of the types of PK isozymes expressed in small amounts, which are very difficult to detect using the conventional PK polyacrylamide gel electrophoretic method.     

Application of Phase-Contrast Microscopy to Schiff-Positive Material

It was observed that ground substance between the smooth muscle fibers in cerebral arteries stained by periodic acid-Schiff (PAS) was red as seen by ordinary bright-field microscopy (BF), but blue as observed by phase-contrast microscopy (PC). The basement membranes in the small intestine and around the kidney tubules, as well as the striated borders of the intestinal epithelium and the brush borders of the kidney tubules, were seen in blue when stained by PAS and observed by PC. The cytoplasm of PAS stained liver cells, when observed by PC, had irregular shaped areas of blue interspersed between the red material. This blue color was seen by PC after PAS, ninhydrin-Schiff and the Feulgen procedures. Our evidence suggests that this phenomena is characteristic of Schiff-positive material. Digestion by various enzymes: malt diastase, testicular hyaluronidase, collagenase, pepsin, pectinase, trypsin and DNase showed different effects on ground substance, liver cells, basement membranes, and brush and striated borders.