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The genetically hypertensive fawn-hooded (FHH/Eur) rat is characterized by the early presence of systolic and glomerular hypertension, progressive proteinuria (UPV), and albuminuria (UAV), and focal glomerulosclerosis, resulting in premature death from renal failure. Previous studies showed that at least five genetic loci ( Rf-1 to Rf-5) were linked to the development of renal impairment. Of these five, Rf-1 appears to play a major role. To study the impact of Rf-1 in the absence of the other loci, we transferred the Rf-1 region of chromosome 1, between the markers D1Mit34 and D1Rat156, Rf-1B for short, onto the genomic background of the normotensive August × Copenhagen Irish (ACI) rat. In this congenic strain, named ACI.FHH- D1Mit34/ Rat156 or ACI.FHH- Rf1B, we challenged the renal hemodynamic function of these animals by studying the effects of unilateral nephrectomy (UNX) alone, or combined with NG-nitro-l-arginine methyl ester (l-NAME)-induced hypertension. Following UNX, the congenic strain developed significantly more UPV and UAV than the ACI progenitor. The differences were even more pronounced when UNX was combined with an l-NAME-induced rise in systolic blood pressure to about 150 mmHg, i.e., the level of hypertension present in the parental FHH strain. These findings indicate that the Rf-1B region of the FHH rat contains at least one gene affecting the susceptibility to progressive renal failure, especially in the presence of an increase in blood pressure.

Pharmacogenomics offers the potential to define metabolic pathways and to provide increased knowledge of drug actions. We studied relative levels of gene expression in the rat using a microarray with 8448 rat UniGenes (1928 known genes, 6520 unknown ESTs) in the liver and kidney as a function of time of day and then of feeding regime, which are common variables in preclinical pharmacogenomic studies. We identified 597 genes, including several key metabolic pathways, whose relative expression levels are significantly affected by time of day: expression of some was further modified by feeding state. These would have sparked interest in a pharmacogenomic study. Our study demonstrates that two common variables in pharmacogenomic studies can have dramatic effects on gene expression. This study provides investigators with baseline information for both kidney and liver with respect to 'normal' changes in gene expression influenced by time of day and feeding state. It also identifies 18 new genes that should be investigated for a role in circadian rhythms in peripheral tissues.

Over the past 200 years, animal models have been selected and used primarily as surrogates for humans. The primary selection criteria for the animal models have been disease-based phenotypic characteristic(s) similar to those of humans. Indeed, many rat and mouse models share pathobiological characteristics similar to a human condition (Desnick et al. 1982). The idea that genomic organization also tends to be evolutionarily conserved between species was postulated in the early 1900s (Castle and Wachter 1924; Haldane 1927). Studies involving banding conservation and chromosome painting (ZOO-FISH) have since shown that large stretches of DNA are conserved in mammalian species as divergent as humans and fin whales (Nash and O'Brien 1982; Sawyer and Hozier 1986; Scherthan et al. 1994; Weinberg and Stanyon 1995). Although these studies showed genome conservation, they could not show the explicit conserved gene order at high resolution; such detail can only be accomplished at the genetic/physical mapping or sequence level. Several studies evaluating genome conservation at the genetic and physical mapping level have determined that gene order does tend to be conserved between mammals (Oakey et al. 1992; Sellar et al. 1994; Stubbs et al. 1994), opening up the prospect of constructing comparative maps between multiple species based on genetic sequence and map information (Nadeau 1989; Anderson et al. 1996; DeBry and Seldin 1996; Lyons 1997). As genetic and physical maps of human and model organisms developed with the advent of the Human Genome Project in the 1990s and as the number of identified genes increased, the number of possible integration points dramatically enhanced the potential quality and density of comparative maps (O'Brien et al. 1999). The increased number of mapped genes and expressed sequence tag (EST) sites has led to sequence comparisons to identify orthologous genes (homologous genes in different species evolving from the same common ancestral gene; Clark 1999; Fitch 2000). When mapped in both species, these orthologs serve as anchors that are useful in identifying conserved segments between species. However, until absolute phylogeny of the genes is truly known, the ortholog assignments between these species must be considered preliminary; thus, it is prudent to assign gene-based anchors using the more conservative homolog relationships. The Mouse Genome Informatics (MGI) group at The Jackson Laboratories (http://www.informatics.jax.org/; Blake et al. 2000) has curated and assigned 2105 rat-mouse (R-M), 1950 rat-human (R-H), and 5603 mouse-human (M-H) orthologs. However, fewer of these genes have been mapped across all three species, limiting the number of anchors for building comparative maps. Several lower-resolution comparative maps have been generated between rat, mouse, and human using fluorescence in situ hybridization (Levan et al. 1991; Scalzi and Hozier 1998; Grutzner et al. 1999) and combined genetic/radiation hybrid (RH) maps (Watanabe et al. 1999), the later identifying 522 anchor points between rat and human and/or mouse. The combined genetic/RH maps identified 41 conserved segments (identified by containing at least two homologous genes) between rat and mouse and 89 between rat and human (Watanabe et al. 1999). Using the analytical methodology developed by Nadeau and Taylor (1984), Watanabe et al. (1999) predicted the number of evolutionarily conserved segments between rat and human to be 152+21 and between rat and mouse to be 49+7. The emergence of the RH maps in human, rat, and mouse (Gyapay et al. 1996; Steen et al. 1999; VanEtten et al. 1999), coupled with the development of large numbers of UniGenes and ESTs for all three species, has revolutionized the way comparative maps can be built and maintained, before the complete genome sequencing of all three species. Indeed, the mapping approach described here can easily be extended to other mammals with significant EST libraries and RH maps and with entire genome sequences that will not likely be determined. There are many advantages of using the RH maps over curated or integrated genetic maps. First, RH mapping facilitates the integration of genetic markers, genes, and ESTs onto a single backbone map. Second, anchor (homology and map) assignments (based on sequence alignment, UniGene assemblies of ESTs, and map information) between species provide large numbers of hooks on and between the RH maps of rat, mouse, and human, which are useful for further sequence-based annotation of finished sequence from any source and, in particular, annotation of gene function based on results in animal models. Finally, the backbone of the maps has been developed and constructed using sequence-based comparison assignments coupled to a sophisticated scoring algorithm to choose the most likely homologies, thus providing an algorithm for de novo construction of comparative maps as the fundamental EST, gene assembly (UniGene or other), and RH map data sets mature. As the genomic sequence for human and mouse are in finishing and the sequencing of the rat is underway (Marshall 2000; Pennisi 2000a,b), such an RH-based scaffold becomes a powerful tool for early rat physical mapping, sequencing, and annotation of function. Comparative maps as described here provide a powerful platform for the integration of physiological and pharmacological information in the rat with genetic information in the mouse and clinical information in the human.

Smooth muscle cells (SMC) express a battery of lineage-restricted genes whose encoded proteins impart the unique contractile phenotype that characterizes this muscle type. While the encoded function of many SMC-restricted genes has been extensively analyzed, less is known about their position within the genome and the regulatory factors governing their transcription. In this report, we define the gene structure, 5' promoter analysis, and chromosomal mapping of the rat smooth muscle calponin (CnnI) gene. The rat CnnI gene is comprised of seven exons spanning approximately 8 kb of genomic sequence. The intron-exon boundaries of the rat CnnI gene match precisely those in human and mouse. Primer extension and RNase protection assays indicate two major transcription start positions (tsp). Comparative sequence analysis of the 5' promoter region reveals several conserved cis regulatory elements, including a TA-rich element within 30 nt of the tsp that could be a recognition site for TATA-binding protein and two CCAAT boxes. Transient and stable transfection studies support the hypothesis that distal regulatory elements confer SMC-restricted expression of CnnI. Finally, using an F2 intercross, we have mapped the rat CnnI gene to the telomeric end of Chromosome (Chr) 8. These studies provide additional information relating to the control of CnnI gene expression and provide a platform to begin assessing the potential linkage of CnnI to spontaneous and experimental disease phenotypes in rats.

In this report, we describe molecular cloning and characterization of cDNAs encoding a novel rat prolactin-like protein. The rat cDNAs were isolated from the decidua and the gene was named PLP-I. cDNAs for the mouse equivalent were also cloned by the cross-hybridization technique. Pregnancy-specific expression of the rat PLP-I gene was observed in the rat placenta by Northern analysis. Location of signal peptide cleavage sites in rat and mouse pre-PLP-I proteins was predicted using a theoretical method. A molecular phylogenetic tree for the growth hormone-prolactin superfamily including the novel member, PLP-I, constructed using the neighbor-joining method, places rat/mouse PLP-I closest to rat/mouse placental lactogen I and II.

Gamma-glutamyl transpeptidase (gamma-GTP) is a membrane-bound enzyme which is known to play a crucial role in active transport of amino acids across membrane barriers. We prepared a monoclonal antibody recognizing specifically rat gamma-GTP and investigated localization of the enzyme in the rat brain by immunohistochemistry with this antibody. The antigen was localized on the ependyma, epithelia of the choroid plexus and microvessels. More precise localization of gamma-GTP was examined with immuno-electron microscopy. The antigen was recognized on the microvilli and cilia of the ependymal cells, microvilli of the choroid epithelial cells and luminal membranes of the vascular endothelial cells.

The laboratory rat, Rattus novegicus, is a major model system for physiological and pathophysiological studies, and since 1966 more than 422,000 publications describe biological studies on the rat (NCBI/Medline). The rat is becoming an increasingly important genetic model for the study of specific diseases, as well as retaining its role as a major preclinical model system for pharmaceutical development. The initial genetic linkage map of the rat contained 432 genetic markers (Jacob et al. 1995) out of 1171 developed due to the relatively low polymorphism rate of the mapping cross used (SHR x BN) when compared to the interspecific crosses in the mouse. While the rat genome project continues to localize additional markers on the linkage map, and as of 11/97 more than 3,200 loci have been mapped. Current map construction is using two different crosses (SHRSP x BN and FHH x ACI) rather than the initial mapping cross. Consequently there is a need to provide integration among the different maps. We set out to develop an integrated map, as well as increase the number of markers on the rat genetic map. The crosses available for this analysis included the original mapping cross SHR x BN reciprocal F2 intercross (448 markers), a GH x BN intercross (205 markers), a SS/Mcw x BN intercross (235 markers), and a FHH/Eur x ACI/Hsd intercross (276 markers), which is also one of the new mapping crosses. Forty-six animals from each cross were genotyped with markers polymorphic for that cross. The maps appear to cover the vast majority of the rat genome. The availability of these additional markers should facilitate more complete whole genome scans in a greater number of strains and provide additional markers in specific genomic regions of interest.

SRY -related cDNAs, XLS13A and XLS13B , have been isolated from Xenopus laevis ovary. The cDNAs encode polypeptides of 382 and 375 amino acids, respectively. Nucleotide sequences of the two cDNAs are highly homologous to each other. The type-A and type-B XLS13 proteins, and xSox13 reported previously share an identical high mobility group (HMG) box at the amino acid level, although they contain silent nucleotide alterations. The HMG box exhibits strong similarity (>93% amino acid identity) to those of mouse Sox4/human SOX4 and chicken Sox11/human SOX11. The size of XLS13A/XLS13B mRNA was estimated to be 2.8 knt in Xenopus ovary by Northern analysis. Reverse transcription/polymerase chain reaction (RT/PCR) assay indicated that XLS13A and XLS13B mRNAs are present in various tissues of adult frog. The mRNAs of XLS13A and XLS13B of maternal origin found in unfertilized eggs disappear in the early stages of the Xenopus embryo. DNA-binding properties of the XLS13 HMG domain were examined by electrophoretic mobility shift assay (EMSA). The HMG domain preferentially binds to the canonical target sequence of SOX proteins, AACAAT, in vitro.

We investigated whether interleukin-1 (IL-1) activity in the rat hypothalamus was increased by immobilization stress (IS), and whether pretreatment with an interleukin-1 receptor antagonist (IL-1Ra) is capable of inhibiting IS-induced elevations of hypothalamic norepinephrine (NE), dopamine (DA), and serotonin (5-HT) and the levels of their metabolites as well as of plasma adrenocorticotropic hormone (ACTH). IL-1 activity was estimated with a bioassay using mouse thymocyte proliferation in the presence of concanavalin A. IL-1Ra was administered directly into the anterior hypothalamus, and monoamines were determined using a microdialysis technique and an HPLC system. First, we found that levels of IL-1 activity in the rat hypothalamus reached a maximum at 60 min after starting IS. Second, IL-1Ra (2 micrograms) significantly inhibited IS-induced increases in hypothalamic NE, DA, and 5-HT levels as well as the levels of their metabolites. In addition, IL-1Ra (2 micrograms) also inhibited the IS- induced elevation of plasma ACTH levels. Third, timing effects of IL- 1Ra administration on the IS-induced monoamines or ACTH responses were examined. IL-1Ra (2 micrograms) administered at 5 or 60 min before the start of IS, but not at 5 or 60 min after IS had been started, exerted inhibitory effects on these responses, indicating that the effects of IL-1 occurred within 5 min after the initiation of IS. In summary, these results suggest that IS enhances biologically active IL-1 in the hypothalamus, and that hypothalamic IL-1 plays a role in the regulation of IS-induced responses including elevated monoamine release in the hypothalamus and activation of the hypothalamo-pituitary-adrenal axis. Moreover, since 5 min is too short a time for IS to induce production of IL-1, IS may augment the effects of preexisting IL-1 in the hypothalamus.

The homotypic cell aggregation of a carcinoembryonic antigen (CEA) positive colon cancer cell line (Colo 205) was induced in vitro by interferon-gamma (IFN-gamma) treatment. Divalent cations were required for this aggregation, as it was inhibited by EDTA. The partial inhibition by cytochalasin B and the complete inhibition by a mixture of sodium azide and 2-deoxyglucose suggests that the aggregation requires the integrity of cytoskeleton and active metabolism. The expression of CEA was increased in the cytoplasm and on the membrane of Colo 205 by IFN-gamma treatment. Furthermore, this aggregation was inhibited completely by anti-CEA monoclonal antibody (mAb) and partially by mAb against intercellular adhesion molecule-1. This in vitro study suggests that CEA molecule participates in the IFN-gamma induced homotypic adhesion of some CEA positive cancer cells and that IFN-gamma has an important role in the regulation of cell-cell interaction mediated by CEA molecule.

Microwave (MW) has the advantage of accelerating infiltration of fixatives into tissue blocks, but the disadvantage of causing the temperature of the fixatives and tissue blocks to rise. We tried to minimize the temperature elevation during MW irradiation by applying ultrasound (US) in combination with MW oven furnishing US generator. US was uneffective in reducing the rise of temperature, however, it was able to enhance the rapid tissue fixation. Electron microscopic examination showed that the fine structure of organs, such as liver, pancreas, submandibular gland, small intestine, and kidney, was well preserved. Characteristically, the matrix of mitochondria appeared electron-dense, and the marginal zone of zymogen granules in the pancreas was more electron-opaque than the core of the granule. The immunoreactivity of zymogen granules to anti-amylase antibody was strong. The deformation or disorder of cell structure due to the thermal effect and cavitation that can be caused by US was not seen. US can be useful for histologic and immunohistochemical study.

We studied the distribution of gamma-glutamyl transpeptidase (gamma-GT) by use of a monoclonal antibody (MAb) against human kidney gamma-GT in human sweat glands. In the eccrine sweat gland, the enzyme was localized along the luminal membrane and small apocrine extrusions of the superficial cells of the secretory portion. The intercellular canaliculi between basal cells were occasionally immunoreactive. In the secretory portion of the apocrine gland, luminal membrane and apocrine extrusions of various sizes and stages at the apices of the secretory cells exhibited positive reactions. Immunoreaction was also seen in the Golgi area of the cuboidal secretory cells. No positive reaction was observed in the myoepithelial cells of either gland or in the excretory duct cells.

We have determined expression of human GGT gene encoding gamma-glutamyl transpeptidase (GGT) during fetal development of liver using the Northern-blot analysis with a cloned human GGT cDNA and immunohistochemistry with a monoclonal antibody. GGT mRNA could be detected as early as the 12th week of gestation. It then increased gradually to a peak of approx. threefold the amount at week 12, at week 40, just before birth. The size of the mRNA in the fetal liver was 2.7 kb and mRNA of the same size was detected both in the human fetal kidney and human hepatocellular carcinoma as well as normal adult liver. Immunohistochemical analyses show that GGT increased as the fetal liver developed in parallel with the increase in mRNA. Histochemically, GGT was shown to be located in the wall of bile canaliculi when synthesis was low in early development, but to be distributed, in addition, all over the cell membrane of the fetal hepatocytes when synthesis was high at the later stage of development.

The temperature during microwave irradiation in the core of tissue blocks, either 1cm or 2cm cubes of rat kidney and liver, was measured by plunging a sensor into the tissue blocks. The temperature of the tissue blocks was much higher than the outer media, i.e., chemical fixatives. Thus, the temperature of the outer media did not reflect that in the tissue block to be fixed. The temperature of tissue reached 40°C within 5-10sec of irradiation. The highest temperature of tissuee block at 15-second irradiation, for example, was 92.5°C (1cm cube of kidney) and 98°C (2cm cube of liver), using 4% PFA with CaCl2 as a fixative. Microwave fixation is a practical tool for the preparation of tissue spections for either histology or cytology. However, it must be required that the irradiation is controlled so as not to produce high temperature within specimens.

The lack of an appropriate animal model that spontaneously develops diabetic nephropathy has been a significant limitation in the search for genetic factors underlying this disease and the development of new therapeutic strategies to prevent progressive renal disease in diabetes. We introgressed the mitochondria and some passenger loci from the FHH/EurMcwi rat into the genetic background of diabetic GK rats, creating a new rat strain, T2DN (T2DN/Mcwi). Despite the high degree of genetic similarity between T2DN and GK rats (97% at 681 loci), diabetes ensues earlier and progresses more severely in T2DN rats. T2DN rats exhibit proteinuria by 6 months of age, accompanied by renal histologic abnormalities such as focal glomerulosclerosis, mesangial matrix expansion, and thickening of basement membranes. These characteristics progress over time, and nearly all T2DN rats exhibit diffuse global glomerulosclerosis with nodule formation and arteriolar hyalinosis by 18 months of age. The histologic changes in the kidney of T2DN rats closely mimic the changes seen in the kidney of patients with diabetes. These results indicate that the T2DN rat is a suitable model for investigating diabetic nephropathy. Here we report the initial genetic and physiological characterization of this new rat model of diabetic nephropathy.

The number of patients with hypertension-associated end-stage renal failure (ESRF) continues to increase despite improved antihypertensive management and early detection programs. Variation for the development of renal complications in hypertension may reflect independent genetic susceptibility to ESRF. The genetically hypertensive fawn-hooded rat is characterized by the early presence of systolic hypertension, glomerular hypertension, progressive proteinuria (UPV), and focal glomerulosclerosis (FGS), resulting in premature death as a result of renal failure. In the present study, the genetic basis of hypertension-associated ESRF in an F2 intercross consisting of 337 animals, in which systolic BP, UPV, albuminuria, and FGS, were studied at 8 wk after a unilateral nephrectomy performed at 5 to 6 wk of age. A total genome scan, consisting of 418 markers, was used to identify regions that contribute to the pathogenesis of UPV and FGS. Linkage analysis revealed five loci involved in the development of renal impairment. Of these five, two (Rf-1, Rf-2) had been identified previously. There seems to be strong interactive effects between the various loci and their impact on UPV and the other parameters of renal impairment, as well as an interaction with BP. In particular, Rf-1 seems to play a major role in determining the severity of the disease. This study is the first to report the interaction of more than two loci to produce progressive renal failure, suggesting that the genetic dissection of renal failure in humans will require understanding of how multiple genes interact with each other and BP to produce ESRF.

The ultimate informativeness of the zebrafish mutations described in this issue will rest in part on the ability to clone these genes. However, the genetic infrastructure required for the positional cloning in zebrafish is still in its infancy. Here we report a reference cross panel of DNA, consisting of 520 F2 progeny (1040 meioses) that has been anchored to a zebrafish genetic linkage map by 102 simple sequence length polymorphisms. This reference cross DNA provides: (1) a panel of DNA from the cross that was used to construct the genetic linkage map, upon which polymorphic gene(s) and genetic markers can be mapped; (2) a fine order mapping tool, with a maximum resolution of 0.1 cM; and (3) a foundation for the development of a physical map (an ordered array of clones each containing a known portion of the genome). This reference cross DNA will serve as a resource enabling investigators to relate genes or genetic markers directly to a single genetic linkage map and avoid the problem of integrating different maps with different genetic markers, as must be currently done when using randomly amplified polymorphic DNA markers, or as has occurred with human genetic linkage maps.

The mitochondrial FAD-dependent glycerol-3-phosphate dehydrogenase (mtGPD) plays an important role in the regulation of insulin secretion and has been postulated as a candidate responsible for the pathogenesis of non-insulin-dependent diabetes mellitus (NIDDM) in humans as well as in rodent models of NIDDM. Recent molecular genetic studies of the Goto-Kakizaki (GK) rat model of NIDDM have identified loci linked to NIDDM. To elucidate whether rat mtGPD might play a role in the pathogenesis of NIDDM, the rat mtGPD gene (Gpd2) was cloned, and a genetic marker for Gpd2 was developed. The gene mapped to the region of rat chromosome 3 that contains a region linked to NIDDM in the GK rat. Fluorescence in situ hybridization was also carried out to verify the map position.

A family of SRY -related genes has been termed SOX . We have isolated and sequenced a cDNA encoding a novel Sox protein from Xenopus laevis ovary. This cDNA contains an open reading frame (ORF) coding for 362 amino acids, which encompasses an HMG box and exhibits a strong (90%) identity to that of mouse Sox7; the cDNA was named xSox7 in this study. Northern analysis revealed that the xSox7 mRNA was 2.0 kb in length. Various adult frog tissues were tested by reverse transcription/polymerase chain reaction for xSox7 mRNA, and the results showed that xSox7 is expressed in a wide range of tissues. Furthermore, electrophoretic mobility shift assay indicated that recombinant xSox7 is capable of binding to AACAAT sequence.

Several cell-associated cytokines, such as interleukin-1 (IL-1) and tumor necrosis factor, exist on the cell surface and are biologically active. Although extracellular IL-8, a potent chemotactic factor for primarily neutrophils, has been studied extensively, cell-associated IL-8 has barely been studied. In this study, we analyzed the intracellular and cell-surface IL-8 in human blood monocytes in vitro by using flow cytometry and predicted the biological activity of the cell-associated IL-8 in vivo. After fixation with paraformaldehyde, mononuclear cells were divided into two subgroups. One subgroup was left untreated to study cell-associated antigens, and the other subgroup was permeabilized with saponin to detect intracellular antigens. In lipopolysaccharide (LPS)-stimulated monocytes, IL-8 was detected solely intracellularly, whereas both the intracellular and cell-surface IL-1 beta was detectable. In a time-course study, the intracellular IL-8 increased in response to LPS stimulation, but the cell-surface IL-8 was undetectable throughout the course. In an LPS-stimulated monocytic cell line, both ELISA and flow cytometry detected the quantitative change of the intracellular IL-8. The dissimilar localization between IL-8 and IL-1 beta within cells was confirmed by the immunohistochemical analysis. In summary, LPS stimulation induced a time-dependent increase in intracellular but not cell-surface IL-8 in monocytes. Thus, it is unlikely that the cell-associated IL-8 is functioning physiologically. The semiquantitative flow cytometric procedure may be useful for simultaneous examination for cell-surface and intracellular cytokines.

A family of SRY -related genes has been termed SOX . We have isolatedand sequenced a cDNA encoding xSox12 from Xenopus laevis ovary. The cDNA contained an open reading frame (ORF) coding for 470 amino acids encompassing an HMG box characteristic of the SOX family, a leucine zipper motif and glutamine-rich segments. The size of the xSox12 mRNA was determined to be 3.0 knt by Northern analysis. The ovary was the most prominent in the expression of the Sox mRNA among the various tissues of adult frog as far as examined.

We demonstrated that a common antigen (Golgi-associated antigen, GAA 108) is present in the Golgi apparatus of serous exocrine cells, using an immunohistochemical method with monoclonal antibodies (MAb) 108 (IgG1) and 18 (IgM), raised to the microsomal fractions of rat parotid gland. The MAb reacted with polypeptides of molecular weights in the 58-170 KD range in parotid gland on Western blot analysis. The Golgi apparatus of the following cells was immunostained with these MAb: acinar cells of parotid gland, pancreas, and exorbital lacrimal gland, serous cells of sublingual gland, chief cells of stomach, and epithelial cells of rat prostate. However, positive reaction occurred throughout the entire cytoplasm of submandibular gland acinar cells. Immunoelectron microscopy (IM) revealed antigen (GAA 108) localization in the medial and trans-Golgi cisternae and trans-Golgi network (TGN), including condensing vacuoles, in parotid, exorbital lacrimal, and pancreatic acinar cells, and serous acinar cells of sublingual gland. Lysosomes and apical cell membranes also stained positively in some cells. In the submandibular gland reactions were observed in the medial and trans-Golgi cisternae, condensing vacuoles, secretory granule contents, cell membrane, and in some duct lumens. These results suggest that although GAA 108 is found in the Golgi apparatus of most serous exocrine cells, it is secreted by a regulated pathway in the acinar cells of submandibular gland.

We studied in detail the distribution pattern of gamma-glutamyl transpeptidase (gamma-GT) in human salivary glands using a monoclonal antibody (MAb) to human kidney gamma-GT. In the sublingual gland, a strong reactivity of the enzyme was recognized along the luminal and lateral membranes of serous cells. Weak but positive reactivity was noted on the luminal membrane of mucous cells. The intercellular canaliculi in the demilune and luminal surfaces of excretory duct cells were also immunoreactive. In the submandibular gland, a weak reaction was observed on the luminal membrane of intercalated duct cells and striated duct cells. Faint reactivity was seen on the luminal membrane of striated duct cells of the parotid gland. No reaction was observed in serous cells of the parotid gland. The immunoreaction in the sublingual gland was stronger than that in submandibular or parotid glands.

52 The fawn-hooded hypertensive (FHH) rat is an excellent experimental model to analyse the genetics of hypertension-associated renal damage. Male FHH rats spontaneously develop moderate hypertension and marked proteinuria early in life. Previous linkage analyses with crosses between FHH and ACI rats, revealed that at least five quantitative trait loci (QTLs), named Rf-1 to Rf-5 , were linked to proteinuria. The Rf-1 locus on rat chromosome 1 had by far the highest LOD-score suggesting a major role in determining the severity of renal failure. The next step to identify the importance of the various QTLs is the generation of congenic strains for all five chromosomal regions. Here we report the first results obtained in Rf-1 congenic rats. Using marker-assisted backcrossing, we transferred the Rf-1 region, between the markers D1Mit34 and D1Mit156, from the FHH genome onto the genomic background of the normotensive ACI rat. To determine the impact of Rf-1 o n the development of renal damage, we challenged the renal hemodynamic function of these congenic animals by unilateral nephrectomy (UNX) alone, or in combination with L-NAME-induced hypertension. The parental ACI strain was used as control. The various experimental groups consisted of 10-12 rats. During a 24 week follow-up period after UNX, the congenic strain, officially named ACI.FHH- D1Mit34/Mit156 ( Rf-1B for short), did develop significantly more proteinuria and albuminuria than the ACI rats. The differences between the Rf-1B congenic and ACI rats were even more pronounced when UNX was combined with an L-NAME-induced rise in systolic blood pressure to about 150 mm Hg, i.e. the level of hypertension normally present in the parental FHH strain. Histological analysis of the kidney showed that the incidence of glomerulosclerosis was also higher in the Rf-1B congenic rats. These findings provide the first direct evidence that the Rf-1 region of the FHH rat contains at least one gene affecting the susceptibility to progressive renal failure, especially in the presence of an increased blood pressure.

Glutathione S-transferase (GST) EC 2.5.2.18) catalyzes conjugation of reduced glutathione with hydrophobic substrates, such as S-epoxide active molecules. It participates in glutathione metabolism and the gamma-glutamyl cycle, playing an important role in detoxification and biosynthesis of many compounds. It is also known as a marker of pre-neoplasia in chemical hepatocarcinogenesis. Isoelectric focusing studies have revealed that this enzyme is composed of several isozymes, one of which, an acidic form of GST called GST-pi, has been extracted from human placenta. In this study, we prepared monoclonal antibodies (MAb) against human GST-pi from placenta. Specificity was confirmed by immunoblots of GST-pi after polyacrylamide gel electrophoresis and inhibition testing of enzyme activity by the antibody. The subclass of the antibody was IgG1 and the light chain was kappa. In light microscopic immunohistochemical studies of human placenta using the MAb, GST-pi was localized diffusely in the cytoplasm and along the apical cell membranes of syncytial cells in villi and in the cytoplasm of cytotrophoblastic cells in the basal plate. The MAb we prepared may also be useful for analyzing the enzyme's function in detoxification and biosynthesis of many compounds, as well as for oncological studies, such as diagnosis of malignant disease and localization of oncofetal proteins in malignant tissues.

Taking γ-glutamyl transpeptidase (γ-GT)-monoclonal anti γ-GT system as an example, the effect of microwave fixation for the immunohistochemical study was examined in rat kidney, pancreas, duodenum and liver, using a domestic microwave oven. The results were compared with that of the previous study using conventional fixatives. Best results were obtained in tissues irradiated in 4%-8% paraformaldehyde containing 10% glucose or 5%-10% polyethylene glycol 2000 at 45°-50°C. But, the immunoreaction was not demonstratable along the brush border of the duodenum. Though the microwave fixation is a useful tool for immunohistochemistry, it is necessary to adjust the condition of irradiation and the composition of fixatives to keep the physiological state of each organ or tissue well preserved and for the antigen to stay as it is originally located without loosing its antigenicity.

The localization of gamma-glutamyl transpeptidase (γ-GTP) in the rat kidney was examined by immunoelectron microscopy using monoclonal antibody to rat γ-GTP. The enzyme was distributed along the brush border and in the apical canaliculi and vacuoles of the proximal tubules. The immunoreaction of γ-GTP in the brush borders increased from the S1 to S3 segments in the proximal tubules. Conspicuous γ-GTP immunostaining was observed along the basolateral membranes of the S2 and S3 segments of the proximal tubules. In addition, the apical cell membrane of the upper part of descending thin limbs of Henle's loops exhibited positive γ-GTP immunostaining. A faint reaction was occasionally recognized in the apical cell membranes of the distal convoluted cells, including the macula densa.

Monoclonal antibodies of the IgG1 subclass were prepared against rat kidney γ-glutamyl transpeptidase by the hybridoma technique. Eight hybrid clones produced antibodies against the enzyme. Finally two stable clones which produce specific antibodies were obtained. The antibodies specifically recognized a heavy subunit (Mr=51, 000) of the enzyme. Immunohistochemical examination revealed that the reaction was localized in the brush border as well as along the basolateral membrane of the proximal convolution of rat kidney and also in the epididymis and the pancreas of rats. Human kidney showed positive reaction, whereas no reaction was recognized in the kidneys of other species, such as mice, guinea pigs and rabbits.

An applicable method of cryofixation to immunocytochemistry was examined. Fresh tissue blocks of rat pancreas and parotid gland were quickly frozen by the metal contact method using liquid helium and freeze-substituted with one of the following media kept at -80°C for 36hr; pure acetone, 0.1% glutaraldehyde (anhydrous) in acetone, approximately 0.2% paraformaldehyde in acetone, and 10% acrolein in acetone. After freeze-substitution fixation, tissue blocks were embedded in Araldite mixture. Thin sections mounted on nickel grids were processed for immunocytochemical localization of amylase according to the multiple-step protocol of the protein A-gold immunostaining method by Bendayan and Duhr (4). They were then postfixed with 2.5% glutaraldehyde in PBS and stained with uranyl acetate and lead citrate for electron microscopy. Good results were obtained from the materials substituted with glutaraldehyde or paraformaldehyde in acetone. The ultrastructural features of the cells were preserved well, similar to those in the materials substituted with OsO4 in acetone except for negative images of the membranous structures. Secretory granules, condensing vacuoles, and Golgi cisterns were labeled well with immunogold. Labeling density was much higher in the present materials than in those processed by conventional chemical fixation, and the intensity of labeling increased in proportion to increasing electron density of the materials contained within individual subcellular compartments. These results indicate that an application of the cryofixation method is useful for improving resolution and specificity in immunocytochemical postembedding staining.