Extrarenal effects on the pathogenesis and relapse of idiopathic nephrotic syndrome in Buffalo/Mna rats.

Buffalo/Mna rats spontaneously develop a focal segmental glomerulosclerosis with a histological pattern similar to the human disease. In this study, we investigated the potential of recurrence of the disease by transplantation of normal kidneys into Buffalo/Mna recipients. Kidneys from healthy LEW.1W rats were grafted into proteinuric 6-month-old Buffalo/Mna rats without or with specific tolerance induction following donor-specific transfusion (DST) aimed at controlling host anti-donor immune responses. The inverse combination was carried out to determine whether a proteinuric Buffalo/Mna kidney can recover its permselectivity in a normal environment. As a control, LEW.1W kidneys were grafted into Wistar Furth recipients. After transplantation without DST, recurrence of proteinuria in LEW.1W kidneys appeared at approximately 10 days, possibly associated with rejection of the graft. In the same combination with DST, proteinuria occurred after 20 days, and the attendant glomerular damage suggested that the initial kidney disease had recurred. Transplanted control animals remained free of proteinuria. In the opposite combination, the proteinuria and the lesions of Buffalo/Mna kidneys regressed after transplantation into healthy LEW.1W rats. The recurrence of proteinuria after transplantation in Buffalo/Mna and the remission of lesions in Buffalo/Mna kidneys transplanted into normal hosts suggests that Buffalo/Mna rats express circulating albuminuric factors, which may be relevant to the relapse of idiopathic nephrotic syndrome in humans.

Differential effect of acute and permanent heat shock protein 70 overexpression in tumor cells on lysability by cytotoxic T lymphocytes.

We have shown previously that acute heat shock protein (Hsp) 70 induction in a human melanoma cell line containing a doxycycline-inducible Hsp70 expression construct increases lysability of these tumor cells by cytotoxic T lymphocyte (CTL) without interfering with MHC class I expression and antigen presentation. The same parental melanoma cell line has now been transduced retrovirally to overexpress Hsp70 permanently. Here we demonstrate that MHC class I cell surface expression is again not altered and that these cells, in contrast with acutely Hsp70 overexpressing cells, do not show augmentation of CTL-mediated apoptosis. Also, long-term induction of Hsp70 in cells with the doxycycline-inducible Hsp70 construct leads to abrogation of increased lysability. Because, furthermore, after heat shock the same permanently Hsp70 overexpressing cells show Hsp70 induction and increased lysability, it is hypothesized that acutely available Hsp70 is able to chaperone proteins that are involved in CTL-mediated apoptosis of target cells and to thereby improve their lysability. We also observed that permanent but not acute Hsp70 overexpression resulted in decreased levels of Hsc70, the constitutively expressed member of the Hsp70 family. Down-regulation of Hsc70 occurs at the post-transcriptional level and can be observed also after long-term induction of Hsp70 in cells containing the doxycycline-inducible expression system. Hsc70 down-regulation might reflect a functional integration of the overexpressed Hsp70 on the basis of a chaperone network so that only acute induction will provide Hsp70 that can improve tumor cell lysability. The implications of the differential effect of acute versus permanent Hsp70 overexpression for tumor therapy are discussed.

Identification, characterization and cytogenetic mapping of a yeast Vps54 homolog in rat and mouse.

A novel gene, VPS54-like (Vps54l), is described in the rat that is homologous to the yeast Vps54 gene which is known to be involved in intracellular protein sorting. Furthermore, Vps54-related sequences of human, mouse, Drosophila melanogaster, Caenorhabditis elegans and Arabidopsis thaliana could be identified in the EMBL/GenBank/DDBJ database. Each of the deduced amino acid sequences of the Vps54 genes in these species contain a coiled-coil region and eight to 13 dileucine motifs. The rat Vps54l gene could be mapped to the end of chromosome 14 by radiation hybrid analysis 7 cR(3000) from the D14Rat22 marker and to 14q22 by fluorescence in situ hybridization. Using a rat Vps54l-containing P1-derived artificial chromosome (PAC) clone the respective ortholog was mapped to chromosome 11A3 in the mouse. In addition, the rat genome contains a processed pseudogene of Vps54l on chromosome 7q22. PAC clone analysis shows that the rat Vps54l gene maps close to the UDP-glucose-pyrophosphorylase 2 gene. The two genes are in tail to tail orientation with their polyadenylation sites 497 bp apart. Rat Vps54l appears to be expressed ubiquitously, but at a relatively low level. Alternatively spliced transcripts could be isolated which lack the sequence coding for the coiled-coil region.

Unimpaired allorejection of cells deficient for the mannose 6-phosphate receptors Mpr300 and Mpr46.

Cytotoxic T lymphocytes (CTL) play an important role in the rejection of allogeneic cells and organs. CTL secrete granzymes and perforin as cytotoxic effector molecules. The mannose 6-phosphate receptor (Mpr)300 has been reported to function as receptor for granzyme B on target cells and to be essential for the rejection of allogeneic cells in vivo. Using mouse embryonal fibroblasts from Mpr300 and Mpr46 knockout mice, we show that both Mpr 300 and Mpr46 are dispensable on target cells for lysis and apoptosis mediated by alloreactive CTL in vitro and for allorejection in vivo. In agreement with a postulated function of Mpr300 as a tumor suppressor gene, deficiency of Mpr300 appears to promote cellular proliferation and tumorigenicity but not resistance to allorejection.

The rat expresses two complement factor C4 proteins, but only one isotype is expressed in the liver.

The complement component C4 is well known for its complex genetics in human and mouse where it is part of a tandemly duplicated module. For the rat, no such information had been available until recently. A C4 gene duplication could be identified also in the rat, but the duplicated module maps approximately 200 kb centromerically from the canonical C4-1 gene. In this study, we present the genomic organization of the two C4 gene-containing modules and the expression of the two C4 genes in the rat (Rattus norvegicus). The duplicated module contains an intact C4 gene as well as Cyp21 and Stk19 pseudogenes. Quantitative mRNA expression analyses revealed that both C4 genes are transcribed in various organs and tissues, but displaying ample differences of C4-1 and C4-2 expression. Most notably, C4-2 is not expressed in the liver. At variance to the mouse, the expression of the rat C4 genes does not exhibit any sex dependency. By using two-dimensional gel electrophoresis and mass spectrometry, products of both C4 genes could be identified in rat serum samples. These two rat C4 isotypes are nearly identical, but differ in a functionally important amino acid residue that is known to influence the functional properties of the C4 isotypes in human.

MHC class I genes of the tree shrew Tupaia belangeri.

Two MHC class I cDNA sequences from the tree shrew (Tupaia belangeri), Tube-W01 and Tube-W02, have been isolated which are probably derived from classical class I genes. Expression of the tupaia class I genes was investigated in several organs, in particular the brain, in which slightly different amounts of class I transcripts are detectable in different areas. Gene tree analysis performed with Tube-W01 and Tube-W02, and including class I sequences derived from other orders, indicated that the tupaia sequences cluster differently from Primates, Carnivora, Artiodactyla, Perissodactyla, and Rodentia, but might be related to Lagomorpha class I genes.

Physical mapping of the major histocompatibility complex class II and class III regions of the rat.

A contig of overlapping bacterial and P1-derived artificial chromosome (BAC, PAC) clones derived from the inbred rat strain BN was constructed that encompasses the class II and the class III regions of the rat MHC (RT1 complex). The genomic structure of the rat, human, and mouse class II and class III regions is highly similar. However, different from human and mouse, a copy of the C4, Cyp21, and Stk19 genes is found that maps to the class II region in the rat. Gene trees constructed from human, rat, and mouse C4, Cyp21, and Stk19 sequences show species-specific clustering of the duplicated genes. The class II/III contig reported here links two previously published PAC contigs of the BN rat that contain the centromeric and the telomeric class I regions, RT1-A and RT1-C/E/M, respectively. Thus, the MHC of the rat is now completely mapped in a single contig of BAC/PAC clones derived from a single RT1 haplotype and encompasses about 3.7 Mb.

Characterization and phylogenetic relationship of prosimian MHC class I genes.

MHC class I cDNA sequences from the most divergent primate group of extant primates compared to human, the suborder Strepsirrhini (prosimians), are described. The sequences are derived from the gray mouse lemur (Microcebus murinus) and the ring-tailed lemur (Lemur catta), which are members of the malagasy Lemuriformes, as well as from the pygmy slow loris (Nycticebus pygmaeus), a prosimian from East Asia. The M. murinus sequences have been analyzed in detail. Analysis of the expression level, G/C content, and synonymous vs. nonsynonymous substitution rates in the peptide-binding region codons suggests that these cDNA clones represent classical class I (class Ia) genes. According to Southern blot analysis, the genome of the gray mouse lemur might contain about 10 class I genes. In gene tree analysis, the strepsirrhine class Ia genes described here cluster significantly separately from the known class I genes of Catarrhini (humans, apes, Old World monkeys) and Platyrrhini (New World monkeys) species, suggesting that the class I loci of Simiiformes arose by gene duplications which occurred after the divergence of prosimians.

Comparative and evolutionary analysis of the rhesus macaque extended MHC class II region.

The sequence-based map of a part of the rhesus macaque major histocompatibility complex (MHC) extended class II region is presented. The sequenced region encompasses 67,401 bp and contains the SACM2L, RING1, FABGL and KE4 genes, as well as the HTATSF1-like and ZNF-like pseudogenes. Similar to human, but different from rat and mouse, no class I genes are found in the SACM2L- RING1 interval. The rhesus macaque extended MHC class II region shows a high degree of conservation of exonic as well as intronic and intergenic sequences compared with the respective human region. It is concluded that this particular genomic organization of the extended class II region-i.e., the absence of class I genes and the presence of the HTATSF1-like and ZNF-like pseudogenes-can be traced back to a common ancestor of humans and rhesus macaques about 23 million years ago.

Granzyme-mediated cytotoxicity does not involve the mannose 6-phosphate receptors on target cells.

Cytotoxic T lymphocytes (CTL) and natural killer cells secrete granzymes to kill infected or transformed cells. The mannose 6-phosphate receptor (Mpr) 300 on target cells has been reported to function as receptor for secreted granzyme B. Using lymphoblasts and mouse embryonal fibroblast lines from Mpr300 and Mpr46 knockout mice, we show here that both receptors are not essential for CTL-induced apoptosis. Similarly, cells exposed to either monomeric granzyme B or granzyme B-serglycin complexes readily internalize the granzyme and undergo apoptosis in the absence of Mpr300 and Mpr46. Further, no colocalization of granzyme B and Mpr300 could be observed in target cells after internalization. In conclusion, these results strongly argue against an Mpr300- or Mpr46-dependent pathway of granzyme-mediated killing and provide new insight in the internalization of monomeric and complexed granzyme B.

The genomic sequence and comparative analysis of the rat major histocompatibility complex.

We have determined the sequence of a 4-Mb interval on rat chromosome 20p12 that encompasses the rat major histocompatibility complex (MHC). This is the first report of a finished sequence for a segment of the rat genome and constitutes one of the largest contiguous sequences thus far for rodent genomes in general. The rat MHC is, next to the human MHC, the second mammalian MHC sequenced to completion. Our analysis has resulted in the identification of at least 220 genes located within the sequenced interval. Although gene content and order are well conserved in the class II and class III gene intervals as well as the framework gene regions, profound rat-specific features were encountered within the class I gene regions, in comparison to human and mouse. Class I region-associated differences were found both at the structural level, the number, and organization of class I genes and gene families, and, in a more global context, in the way that evolution worked to shape the present-day rat MHC.