Rodent Origin Research Articles

The DNAzymes Rs6, Dz13, and DzF Have Potent Biologic Effects Independent of Catalytic Activity

DNAzymes are catalytic DNA molecules capable of cleaving RNA substrates and therefore constitute a possible gene-suppression technology. We examined whether the previously reported potency of a DNAzyme targeting c-jun (Dz13) could be improved with judicious use of sequence and chemical modifications. Catalytic activity was measured to establish correlations between catalytic activity and biological potency. Surprisingly, Dz13 had significant cytotoxic activity against cells of rodent origin (IC(50) = 20-50 nM) despite having greatly reduced catalytic activity against a rodent target substrate (<25%), the latter being the result of a mismatch to the rodent c-jun sequence. In contrast, a modified Dz13 matching the rodent c-jun sequence (DT1501b) had no activity at similar concentrations against human or rodent cells despite being able to efficiently cleave the rodent c-jun sequence. Overall, catalytic activity against synthetic substrates did not correlate with cytotoxic activity and catalytically inactive mutants had in some cases equal or superior potency in cell cytotoxicity assays. Further examination of other previously published DNAzymes (Rs6 and DzF) revealed other occurrences of this anomalous behaviour. The active sequences all have G-rich 5 termini, suggesting that G-quadruplex formation might be involved. Consistent with this, deaza-guanosine substitutions abrogated cytotoxicity of Dz13. However, Dz13 did not show evidence of quadruplex formation as determined by circular dichroism studies and native electrophoresis. These data reveal that the biologic activity of several published DNAzymes is not mediated through the catalytic degradation of target mRNA.

Pathogens and Host DNA inIxodes ricinusNymphal Ticks from a German Forest

Unfed nymphs of Ixodes ricinus were collected from vegetation in a forest on the outskirts of Berlin, Germany and were analyzed for host and pathogen DNA. Pathogens were detected in 47% of the ticks. Borrelia afzelii was the commonest pathogen detected, followed by Rickettsia helvetica. Other pathogens included B. valaisiana, B. garinii, B. burgdorferi sensu stricto, Anaplasma phagocytophilum, and a relapsing fever-like Borrelia. Most of the host DNA detected was of rodent origin and was associated with infection by B. afzelii, R. helvetica, and A. phagocytophilum. Bird DNA was associated with B. valaisiana and B. garinii, and ruminant DNA with A. phagocytophilum. B. afzelii was also found in two ticks that contained bird DNA.

Determining and understanding the control of glycolysis in fast‐growth tumor cells

Control analysis of the glycolytic flux was carried out in two fast-growth tumor cell types of human and rodent origin (HeLa and AS-30D, respectively). Determination of the maximal velocity (V(max)) of the 10 glycolytic enzymes from hexokinase to lactate dehydrogenase revealed that hexokinase (153-306 times) and phosphofructokinase-1 (PFK-1) (22-56 times) had higher over-expression in rat AS-30D hepatoma cells than in normal freshly isolated rat hepatocytes. Moreover, the steady-state concentrations of the glycolytic metabolites, particularly those of the products of hexokinase and PFK-1, were increased compared with hepatocytes. In HeLa cells, V(max) values and metabolite concentrations for the 10 glycolytic enzyme were also significantly increased, but to a much lesser extent (6-9 times for both hexokinase and PFK-1). Elasticity-based analysis of the glycolytic flux in AS-30D cells showed that the block of enzymes producing Fru(1,6)P2 (i.e. glucose transporter, hexokinase, hexosephosphate isomerase, PFK-1, and the Glc6P branches) exerted most of the flux control (70-75%), whereas the consuming block (from aldolase to lactate dehydrogenase) exhibited the remaining control. The Glc6P-producing block (glucose transporter and hexokinase) also showed high flux control (70%), which indicated low flux control by PFK-1. Kinetic analysis of PFK-1 showed low sensitivity towards its allosteric inhibitors citrate and ATP, at physiological concentrations of the activator Fru(2,6)P2. On the other hand, hexokinase activity was strongly inhibited by high, but physiological, concentrations of Glc6P. Therefore, the enhanced glycolytic flux in fast-growth tumor cells was still controlled by an over-produced, but Glc6P-inhibited hexokinase.

Beilong virus, a novel paramyxovirus with the largest genome of non-segmented negative-stranded RNA viruses

During a subtraction study on gene expression in human kidney mesangial cells (HMCs), cDNA clones with sequence homology to paramyxovirus P, M and F genes were isolated. Subsequent investigation revealed that this particular HMC line was infected with a previously unknown paramyxovirus. Here, we report the isolation and genome characterization of this new virus, now named Beilong virus (BeV). The genome of BeV is 19,212 nucleotides (nt) in length and is the largest among all known members of the order Mononegavirales. The BeV genome contains eight genes in the order 3′-N-P/V/C-M-F-SH-TM-G-L-5′. The SH and TM genes code for a small hydrophobic protein of 76 aa and a transmembrane protein of 254 aa, respectively. The BeV G gene, at 4527 nt, codes for an attachment protein of 734 aa and contains two additional open reading frames (ORFs) in the 3′ half of the gene, coding for putative proteins of 299 and 394 aa in length. Although the exact origin of BeV is presently unknown, we provide evidence indicating that BeV was present in a rat mesangial cell line used in the same laboratory prior to the acquisition of the HMC line, suggesting a potential rodent origin for BeV.

Phenotypic plasticity in the cercarial indices of human and rodent samples ofSchistosoma mansoni(Digenea: Schistosomatidae)

In the epidemiology of schistosomiasis, the cercarial index (CI) has been used for several purposes, including the identification of the species of vertebrate host involved in transmission in a particular setting. The CI of samples of Schistosoma mansoni of human (BH) and rodent (CMO) origin have now been compared, in an attempt to identify morphological markers on cercariae that would indicate whether the eggs from which the cercariae have developed were produced in humans or in rodents. After silver-nitrate impregnation of the cercariae, the CI (and their reaction norms) for the antero-dorsal (AD), antero-lateral (AL) and tail-stem (U) sensory receptors were investigated. The data analysis was based on linear-regression methods, Levene's test for the equality of variances, pooled-variance independent t-tests, and discriminant analysis. The results indicated that CI varied with vertebrate-host origin, that the AD reaction norms were more consistent and plastic than the other reaction norms investigated, that there appeared to be no simple allometric relationship between any of the CI investigated and cercarial body length, and that the BH sample gave larger standard deviations and a higher incidence of mis-classification than the CMO, indicating some developmental instability in the sample of human origin. The relative instability of the BH sample was, however, possibly the result of the experimental procedures. As the parasites used to produce the BH sample had been isolated from humans fairly recently and then routinely maintained in mice, host-induced selection could have affected the sample, altering its CI and their reaction norms. The results of the discriminant analysis indicated that, of the indices investigated, AD was the best morphological marker for distinguishing human and rodent samples of S. mansoni.

Cell line-dependent differences in uptake and retention of the hypoxia-selective nuclear imaging agent Cu-ATSM

Background Cu-diacetyl-bis( N 4-methylthiosemicarbazone) [Cu-ATSM] is a potential marker for tumor hypoxia that has been under evaluation for clinical use. In this study, we examined the mechanisms underlying the uptake of 64Cu in cells incubated with 64Cu-ATSM. Methods The in vitro uptake of 64Cu was determined as a function of oxygenation conditions and incubation time with 64Cu-ATSM using four and two tumor cell lines of human origin and rodent origin, respectively. Additionally, the rate of 64Cu efflux and Cu-ATSM metabolism was determined. Results 64Cu accumulation is rapid during the first 0.5–1 h of incubation. It is highest in anoxic cells but is also significant in normoxic cells. After this initial period, the level of intracellular 64Cu varies depending on the cell line and the oxygenation conditions and, in some circumstances, may decrease. During the first 0.5–1 h, the ratio of 64Cu levels between anoxic and normoxic cells is ∼2:10 and that between hypoxic (0.5% O 2) and normoxic cells is ∼1:2.5, depending on the cell line. These ratios generally decrease at longer times. The 64Cu-ATSM compound was found to be metabolized during incubation in a manner dependent on oxygenation conditions. Within 2 h under anoxic conditions, 64Cu-ATSM could no longer be detected, although 60–90% of the amount of 64Cu added as 64Cu-ATSM was present in the medium. Non-ATSM 64Cu was taken up by the cells, albeit at a much slower rate. Efflux rates of 64Cu were found to be cell line dependent and appeared to be inversely correlated with the final 64Cu uptake levels under anoxic conditions. Conclusion The uptake and retention of 64Cu and their relation to oxygenation conditions were found to be cell line dependent. Given the complexities in the oxygen dependence and cell line-dependent kinetics of uptake and retention of Cu following exposure to Cu-ATSM, the clinical utility of this compound may be disease site specific.

Permeability studies on in vitro blood-brain barrier models: physiology, pathology, and pharmacology.

(1) The specifically regulated restrictive permeability barrier to cells and molecules is the most important feature of the blood-brain barrier (BBB). The aim of this review was to summarize permeability data obtained on in vitro BBB models by measurement of transendothelial electrical resistance and by calculation of permeability coefficients for paracellular or transendothelial tracers. (2) Results from primary cultures of cerebral microvascular endothelial cells or immortalized cell lines from bovine, human, porcine, and rodent origin are presented. Effects of coculture with astroglia, neurons, mesenchymal cells, blood cells, and conditioned media, as well as physiological influence of serum components, hormones, growth factors, lipids, and lipoproteins on the barrier function are discussed. (3) BBB permeability results gained on in vitro models of pathological conditions including hypoxia and reoxygenation, neurodegenerative diseases, or bacterial and viral infections have been reviewed. Effects of cytokines, vasoactive mediators, and other pathogenic factors on barrier integrity are also detailed. (4) Pharmacological treatments modulating intracellular cyclic nucleotide or calcium levels, and activity of protein kinases, protein tyrosine phosphatases, phospholipases, cyclooxygenases, or lipoxygenases able to change BBB integrity are outlined. Barrier regulation by drugs involved in the metabolism of nitric oxide and reactive oxygen species, as well as influence of miscellaneous treatments are also listed and evaluated. (5) Though recent advances resulted in development of improved in vitro BBB model systems to investigate disease modeling, drug screening, and testing vectors targeting the brain, there is a need for checking validity of permeability models and cautious interpretation of data.

Mammalian interchanges between Africa and Eurasia: an analysis of temporal constraints on plausible anthropoid dispersals during the Paleogene

Paleogene temporal and paleogeographic constraints on mammalian faunal interchange between Africa and Eurasia are reassessed in order to discuss the origin of Primates, Anthropoidea, and Catarrhini. During the late Paleocene to early Eocene interval, the fossil record indicates faunal exchange between Africa and the North Tethyan areas involving several groups of mammals, such as the earliest primates, carnivores, and some archaic ungulates. The polarities of such dispersal events are unresolved to date. Regarding the paleogeography of anthropoid origins, previous cladistic assessments argued for a dispersal from Asia to Africa during the early Eocene. However, faunal exchange during that period has been documented only between Africa and Europe. A speculative basis for an Asian origin of African zegdoumyid rodents is the only body of evidence that suggests that the earliest anthropoids might have migrated to Africa from Asia. Finally, according to the putative occurrence of catarrhines (amphipithecids) in the middle Eocene of Asia, a middle Eocene migration event towards Africa is proposed, although this is supported only by hystricognath and anomaluroid rodents, and by anthracotheriid artiodactyls. In conclusion, the early paleobiogeographic history of anthropoids remains conjectural because of the lack of phylogenetic resolution.

Butyrate response factor 1 is regulated by parathyroid hormone and bone morphogenetic protein-2 in osteoblastic cells

Parathyroid hormone (PTH) exerts potent and diverse effects in bone and cartilage through activation of type 1 PTH receptors (PTH1R) capable of coupling to protein kinase A (PKA) and PKC. We have used macroarrays to identify zinc finger protein butyrate response factor-1 (BRF1) as a novel PTH regulated gene in clonal and normal osteoblasts of human and rodent origin. We further demonstrate that in human osteoblast-like OHS cells, biologically active hPTH(1–84) and hPTH(1–34) stimulate BRF1 mRNA expression in a dose- and time-dependent manner, while the amino-terminally truncated hPTH(3–84) which does not activate PTH1R has no effect. Moreover, using specific stimulators or inhibitors of PKA and PKC activity, the PTH-elicited BRF1 mRNA expression is mediated through the PKA signaling pathway. In mouse calvarial osteoblasts, BRF1 mRNA levels are upregulated by PTH(1–84) and reduced in response to bone morphogenetic protein 2 (BMP-2). Hence, our data showing that BRF1 is expressed in osteoblastic cells and regulated by PTH and BMP-2, suggest an important role for BRF1 in osteoblasts within the molecular network of PTH-dependent bone remodeling.

Chapter 8: Adaptation and the Origin of Rodents

To the extent now possible, I trace out what appear to be the adaptive changes involved in the origin of rodents. This requires, as a preliminary, a critical analysis of the existing evidence that bears on their phylogenetic relationship to other groups. Part of the paper provides such an analysis, from an unusual perspective. The evidence for a phylogenetic association of rodents and lagomorphs is weaker than is usually claimed but may nevertheless reflect reality. In particular, the precursors of rodents are not yet adequately identified. The initial adaptations of rodents were for the most part quite different from those of lagomorphs, despite their similar gnawing. There is evidence that the Myomorpha constitute the earliest diverging branch of extant rodents. Conapomorphy and spermativore are new terms.

Mechanisms of cellular senescence in human and mouse cells.

Telomere erosion is considered to be the main cause of the onset of replicative senescence. However, recent findings suggest that a senescent phenotype can be induced by a variety of other stimuli that act independently of telomeres. Moreover, telomere-dependent replicative senescence depends on the species of cell origin, in particular whether cells are of human or rodent origin. In addition, the tissue of origin may also dictate the pathway by which cells undergo replicative senescence. In this Review article, we categorize cellular senescence into two types, which for simplicity we term intrinsic or extrinsic senescence, focus on the differences between human and mouse cells, and discuss the roles of the p53 and pRb tumor suppressor pathways in cellular senescence.

Utilizing Fcε-Bak chimeric protein for studying IgE–FcεRI interactions

We previously constructed a pro-apoptotic Fcε-Bak chimeric protein, targeted against cells expressing the IgE high affinity receptor (FcεRI). We demonstrated that the chimeric protein is internalized by target mast cells and kills them. These results, which constitute a promising basis for applying this approach to antiallergic therapy, raise some theoretical questions with respect to two major issues: (a) is the monomeric Fcε-Bak–FcεRI complex able to undergo endocytosis, and (b) does the receptor binding domain of human IgE (Fcε) react with rodent FcεRI? In an attempt to answer these questions, we have now thoroughly investigate the interaction of human (h) and mouse (m) Fcε-Bak with FcεRI-positive cells. Using established cultures of rodent and human origin, as well as a primary mouse mast cell culture, we demonstrate that binding of the chimeric protein to the membrane is followed by quick endocytosis, leading to the apoptosis of specific cells. We also confirm that this interaction depends on FcεRI and not on other IgE receptors. We found that the effect of Fcε-Bak on the cells depends on the level of surface FcεRI expression, but not on the origin of the target cells or of the Fcε moiety. We suggest that endocytosis of the monomeric Fcε-Bak–FcεRI complex results from the inability of Fcε-Bak to transduce signals, characteristic of the monomeric IgE–FcεRI complex due to the absence of the variable portion of the IgE molecule. Our results also indicate that at least the Fcε fragment of human IgE is able to interact with both human and rodent FcεRI.

Effects of clofibric acid on mRNA expression profiles in primary cultures of rat, mouse and human hepatocytes

The mRNA expression profile in control and clofibric acid (CLO)-treated mouse, rat, and human hepatocytes was analyzed using species-specific oligonucleotide DNA microarrays (Affymetrix). A statistical empirical Bayes procedure was applied in order to select the significantly differentially expressed genes. Treatment with the peroxisome proliferator CLO induced up-regulation of genes involved in peroxisome proliferation and in cell proliferation as well as down-regulation of genes involved in apoptosis in hepatocytes of rodent but not of human origin. CLO treatment induced up-regulation of microsomal cytochrome P450 4a genes in rodent hepatocytes and in two of six human hepatocyte cultures. In addition, genes encoding phenobarbital-inducible cytochrome P450s were also up-regulated by CLO in rodent and human hepatocyte cultures. Up-regulation of phenobarbital-inducible UDP-glucuronosyl-transferase genes by CLO was observed in both rat and human but not in mouse hepatocytes. CLO treatment induced up-regulation of l-fatty acid binding protein ( l-FABP) gene in hepatocytes of both rodent and human origin. However, while genes of the cytosolic, microsomal, and mitochondrial pathways involved in fatty acid transport and metabolism were up-regulated by CLO in both rodent and human hepatocyte cultures, genes of the peroxisomal pathway of lipid metabolism were up-regulated in rodents only. An up-regulation of hepatocyte nuclear factor 1α (HNF1α) by CLO was observed only in human hepatocyte cultures, suggesting that this trans-activating factor may play a key role in the regulation of fatty acid metabolism in human liver as well as in the nonresponsiveness of human liver to CLO-induced regulation of cell proliferation and apoptosis.

Are chromosomal instabilities induced by exposure of cultured normal human cells to low- or high-LET radiation?

Radiation-induced genomic instability has been proposed as a very early, if not an initiating, step in radiation carcinogenesis. Numerous studies have established the occurrence of radiation-induced chromosomal instability in various cells of both human and rodent origin. In many of these studies, however, the cells were not "normal" initially, and in many cases they involved tumor-derived cell lines. The phenomenon clearly would be of even greater interest if it were shown to occur generally in cells that are normal at the outset, rather than cells that may have been "selected" because of a pre-existing susceptibility to induced instability. As a test of the generality of the phenomenon, we studied low-passage normal diploid human fibroblasts (AG1521A) to determine whether they are susceptible to the induction of chromosomal instability in the progeny of surviving cells after exposure in G(0) to low- and high-LET radiation. Cytogenetic assays for instability were performed on both mixed populations of cells and clones of cells surviving exposure. We found no evidence for the induction of such instability as a result of radiation exposure, though we observed a senescence-related chromosomal instability in the progeny of both irradiated and unirradiated cell populations.

Alpha(4)beta(7)/alpha(4)beta(1) dual integrin antagonists block alpha(4)beta(7)-dependent adhesion under shear flow.

The alpha(4) integrin, alpha(4)beta(7), plays an important role in recruiting circulating lymphocytes to the gastrointestinal tract, where its ligand mucosal addressin cell adhesion molecule-1 (MAdCAM-1) is preferentially expressed on high endothelial venules (HEVs). Dual antagonists of alpha(4)beta(1) and alpha(4)beta(7), N-(2,6-dichlorobenzoyl)-(L)-4-(2',6'-bis-methoxyphenyl)phenylalanine (TR14035) and N-(N-[(3,5-dichlorobenzene)sulfonyl]-2-(R)-methylpropyl)-(D)-phenylalanine (compound 1), were tested for their ability to block the binding of alpha(4)beta(7)-expressing cells to soluble ligand in suspension and under in vitro and in vivo shear flow. Compound 1 and TR14035 blocked the binding of human alpha(4)beta(7) to an (125)I-MAdCAM-Ig fusion protein with IC(50) values of 2.93 and 0.75 nM, respectively. Both compounds inhibited binding of soluble ligands to alpha(4)beta(1) or alpha(4)beta(7) on cells of human or rodent origin with similar potency. Under shear flow in vitro, TR14035 and compound 1 blocked binding of human alpha(4)beta(7)-expressing RPMI-8866 cells or murine mesenteric lymph node lymphocytes to MAdCAM-Ig with IC(50) values of 0.1 and 1 microM, respectively. Intravital microscopy was used to quantitate alpha(4)-dependent adhesion of fluorescent murine lymphocytes in Peyer's patch HEVs. When cells were prestimulated with 2 mM Mn(2+) to activate alpha(4)beta(7) binding to ligand, anti-alpha(4) monoclonal antibody (mAb) [10 mg/kg (mpk) i.v.] blocked adhesion by 95%, and anti-beta(1) mAb did not block adhesion, demonstrating that this interaction was dependent on alpha(4)beta(7). TR14035 blocked adhesion to HEVs [ED(50) of 0.01-0.1 mpk i.v.], and compound 1 blocked adhesion by 47% at 10 mpk i.v. Thus, alpha(4)beta(7)/alpha(4)beta(1) antagonists blocked alpha(4)beta(7)-dependent adhesion of lymphocytes to HEVs under both in vitro and in vivo shear flow.

The role of Asia in the origin and diversification of hystricognathous rodents

In the absence of a comprehensive pre‐Oligocene fossil record, the origin and early evolution of hystricognathous rodents have long been the subject of much uncertainty. Baluchimyinae (Rodentia) were initially interpreted as a subfamily of the ctenodactyloid Chappatimyidae (sciurognathous), a group considered to be endemic to the Indian subcontinent and to be closely related to hystricognathous rodents. A newly discovered early Oligocene hystricognathous rodent, Bugtimys zafarullahi gen. n. et sp. n., described herein, from the Bugti Hills (Balochistan, Pakistan) sheds new light on the higher level taxonomy of the previously described Baluchimyinae. As a contribution to the phylogenetic debates regarding the origin of Hystricognathi, we present a cladistic assessment of the dental evidence for the Palaeogene hystricognathous rodent cladogenesis. Our phylogenetic results consistently support the monophyly of the Hystricognathiformes clade (including Tsaganomyidae plus Hystricognathi) of which baluchimyine rodents are clearly members. There is, however, no support for the monophyly of a baluchimyine clade. Nonetheless, ‘baluchimyines’ are for the moment reinterpreted as Hystricognathi incertae sedis. Hystricognathous rodents appear to be well diversified at least since the early Oligocene, both in Africa and South America (phiomorphs and caviomorphs, respectively), and also now in south Asia. Furthermore, our phylogenetic results support close relationships between early hystricognathous and Asian ‘ctenodactyloid’ rodents, which clearly points to an Asian origin for Hystricognathi. In this phylogenetic framework, ‘baluchimyines’ and tsaganomyids are representatives of an initial phase of diversification of hystricognathous rodents in Asia. Oligocene phiomorphs and caviomorphs (sister groups) seem therefore to share a common ‘Asian’ hystricognathous ancestor. This reinforces the possibility that the early dispersal of hystricognathous rodents to South America was not from Africa but from Asia.

Isolation of Mouse Ig Heavy and Light Chain Genomic DNA Clones, and Construction of Gene Knockout Vector for the Generation of Humanized Xenomouse

Background: Monoclonal antibodies (mAb) of rodent origin are produced with ease by hybridoma fusion technique, and have been successfully used as therapeutic reagents for humans after humanization by genetic engineering. However, utilization of these antibodies for therapeutic purpose has been limited by the fact that they act as immunogens in human body causing undesired side effects. So far, there have been several attempts to produce human mAbs for effective in vivo diagnostic or therapeutic reagents including the use of humanized xenomouse that is generated by mating knockout mice which lost Ig heavy and light chain genes by homologous recombination and transgenic mice having both human Ig heavy and light gene loci in their genome. Methods: Genomic DNA fragments of mouse Ig heavy and light chain were obtained from a mouse brain λ genomic library by PCR screening and cloned into a targeting vector with ultimate goal of generating Ig knockout mouse. Results: Through PCR screening of the genomic library, three heavy chain and three light chain Ig gene fragments were identified, and restriction map of one of the heavy chain gene fragments was determined. Then heavy chain Ig gene fragments were subcloned into a targeting vector. The resulting construct was introduced into embryonic stem cells. Antibiotic selection of transfected cells is under the progress. Conclusion: Generation of xenomouse is particularly important in medical biotechnology. However, this goal is not easily achieved due to the technical difficulties as well as huge financial expenses. Although we are in the early stage of a long-term project, our results, at least, partially contribute the successful generation of humanized xenomouse in Korea. (Immune Network 2002; 2(4):233-241)

The athymic nude rat: an animal experimental model to reveal novel aspects of innate immune responses?

Athymic nude rats resemble nude mice in their lack of a normal thymus and functionally mature T cells. They have been useful in the study of mechanisms of tumor growth or graft rejection in immunocompromised hosts since they can accept major histocompatibility complex (MHC) mismatched organ allografts or xenografts for several months and because a number of tumor cell lines of human and rodent origin grow well in these rats. Injection of a few helper T (Th) cells from euthymic littermate rats partly restores the pool of mature T cells as well as full immunocompetence to reject organ allografts and has helped to reveal some of the cell interactions necessary for rejection to occur In contrast, immunologically naive athymic nude rats of certain strains, acutely reject allografts consisting of lymphocytes or bone marrow cells, which is due to the presence of alloreactive natural killer cells. These cells can recognize and kill MHC incompatible hematopoietic cells through the recognition of both mismatches within the classical (RT1.A) and nonclassical (RT1.C/E) MHC class I regions with a repertoire of inhibitory and activating killer lectin-like receptors (KLR) for MHC-I molecules, encoded by the Ly-49 portion of the rat natural killer cell gene complex (NKC). Some of these receptors have been identified and molecularly cloned and show similarities with NK receptors identified in the mouse. Other leukocytes in nude rats, such as dendritic cells, may also contribute to specific innate immune responses in the absence of mature T cells. Nude rats develop T-like cells expressing CD3 and T-cell receptor (TCR) with increasing age. Though their phenotype in peripheral lymphatic tissues resembles that of normal T cells, consisting mainly of CD4+ or CD8+ cells, they lack alloreactivity in vivo and their TCR repertoire is more of an oligoclonal nature. Their contribution to allograft rejection in T-cell-reconstituted rats is therefore questionable, and their role in innate immune response in these rats still enigmatic.

Feline Pit2 functions as a receptor for subgroup B feline leukemia viruses.

Different subgroups of feline leukemia virus (FeLV) use different host cell receptors for entry. Subgroup A FeLV (FeLV-A) is the virus that is transmitted from cat to cat, suggesting that cells expressing the FeLV-A receptor are important targets at the earliest stages of infection. FeLV-B evolves from FeLV-A in the infected cat through acquisition of cellular sequences that are related to the FeLV envelope gene. FeLV-Bs have been shown to infect cells using the Pit1 receptor, and some variants can infect cells at a lower efficiency using Pit2. Because these observations were made using receptor proteins of human or rodent origin, the role that Pit1 and Pit2 may play in FeLV-B replication in the cat is unclear. In this study, the feline Pit receptors were cloned and tested for their ability to act as receptors for different FeLV-Bs. Some FeLV-Bs infected cells expressing feline Pit2 and feline Pit1 with equal high efficiency. Variable region A (VRA) in the putative receptor-binding domain (RBD) was a critical determinant for both feline Pit1 and feline Pit2 binding, although other domains in the RBD appear to influence how efficiently the FeLV-B surface unit can bind to feline Pit2 and promote entry via this receptor. An arginine residue at position 73 in VRA was found to be important for envelope binding to feline Pit2 but not feline Pit1. Interestingly, this arginine is not found in endogenous FeLV sequences or in recombinant viruses recovered from feline cells infected with FeLV-A. Thus, while FeLV-Bs that are able to use feline Pit2 can evolve by recombination with endogenous sequences, a subsequent point mutation during reverse transcription may be needed to generate a virus that can efficiently enter the cells using the feline Pit2 as its receptor. These studies suggest that cells expressing the feline Pit2 protein are likely to be targets for FeLV-B infection in the cat.

Sox-4 Messenger RNA Is Expressed in the Embryonic Growth Plate and Regulated via the Parathyroid Hormone/Parathyroid Hormone-Related Protein Receptor in Osteoblast-like Cells

Parathyroid hormone (PTH) and PTH-related protein (PTHrP) exert potent and diverse effects in cells of the osteoblastic and chondrocytic lineages. However, downstream mediators of these effects are characterized inadequately. We identified a complementary DNA (cDNA) clone encoding the 5' end of the transcription factor Sox-4, using a subtracted cDNA library enriched in PTH-stimulated genes from the human osteoblast-like cell line OHS. The SOX-4 gene is a member of a gene family (SOX and SRY) comprising transcription factors that bind to DNA through their high mobility group (HMG)-type binding domain, and previous reports have implicated Sox proteins in various developmental processes. In situ hybridization of fetal and neonatal mouse hindlimbs showed that Sox-4 messenger RNA (mRNA) was expressed most intensely in the zone of mineralizing cartilage where chondrocytes undergo hypertrophy, and by embryonic day 17 (ED17), after the primary ossification center was formed, its expression was detected only in the region of hypertrophic chondrocytes. Sox-4 mRNA was detected in osteoblast-like cells of both human and rodent origin. In OHS cells, physiological concentrations (10(-10)-10(-9) M) of human PTH 1-84 [hPTH(1-84)] and hPT(1-34), but not hPTH(3-84), stimulated Sox-4 mRNA expression in a time-dependent manner, indicating involvement of the PTH/PTHrP receptor. Sox-4 transcripts also were detected in various nonosteoblastic human cell lines and tissues, in a pattern similar to that previously reported in mice. The presence of Sox-4 mRNA in hypertrophic chondrocytes within the mouse epiphyseal growth plate at sites that overlap or are adjacent to target cells for PTH and PTHrP, and its strong up-regulation via activated PTH/PTHrP receptors in OHS cells, makes it a promising candidate for mediating downstream effects of PTH and PTHrP in bone.