Thehigh growth(hg) Locus Maps to a Deletion in Mouse Chromosome 10

Thehigh growth(hg) locus in mice produces a 30–50% increase in weight gain of homozygous individuals. Here we report that the microsatellite markerD10Mit69is deleted in high growth mice. The deletion ofD10Mit69was uncovered in a screen of the high growth mouse and its progenitor strains for available markers in thehgregion. We demonstrate thathgandD10Mit69cosegregate in a cross of congenic strains C57BL/6J-hghg× C57BL/6J. These results suggest that deletion of a region aroundD10Mit69is responsible for the high growth effect. MarkerD10Mit69will be utilized as an entry point to physical cloning of thehg-containing segment. A dense map of markers aroundhgconstructed here should allow identification of markers in homologous regions in domestic animals and humans, which may be utilized to assess the role of thehglocus in these other species.     

Dominant Lethality of the Mouse Skeletal Mutation Tail-short (Ts) Is Determined by theTsAllele from Mating Partners

Mice with the Tail-short (Ts) mutation have a short, kinky tail and numerous skeletal abnormalities, including a homeotic anteroposterior patterning problem involving the axial skeleton. The viability ofTsheterozygotes varies dramatically, depending on the mouse strain crossed with the mutant strain. At the extremes, the heterozygotes are viable or lethal prenatally. In this study, we found that laboratory mouse strains could be divided into two groups. A cross with strains from the first group yielded viableTsheterozygotes, whereas a cross with the second group resulted in dominant lethalityin utero.We planned to map the gene(s) that controls strain differences in the viability of theTsheterozygotes. The result clearly indicated that a single chromosomal region, genetically inseparable from theTslocus, is responsible for these differences. This suggests that allelism at theTslocus generates variable manifestation of the mutant phenotype. Morphological and histological analyses indicated that embryos from the lethal cross exhibit severe developmental defects from the gastrulation stage through the early fetal stage. In particular, the umbilical vein does not develop properly. All of these results suggest that the phenotype of theTsmutant is modified by theTsalleles of the mating partners.     

Charge Heterogeneity of Insulin Fusion Proteins Expressed in Escherichia coli Is Not Due to Proteolytic Degradation

Evaluation of the yield of expression of exogeneous protein in transformed Escherichia coli cells by means of one-dimensional SDS-PAGE often leads to overestimation and miscalculation. For example, it is possible that proteins of similar size comigrate and thus mask the overexpressed product band. Therefore, two-dimensional electrophoresis was used to analyze two types of recombinant fusion proteins, i.e., a β-galactosidase insulin fusion protein and a interleukin II insulin fusion protein, directly after fermentation. We found that production scale expression products show charge and size heterogeneity. The heterogeneous protein spots were characterized by subsequent blotting onto Immobilon membrane and by N-terminal sequencing. Some of the separated spots were either N-terminally blocked or already degraded to some extent. The integrity of the actual product component of the fusion protein was examined with a C-terminus-specific antibody and by Western blot analysis of the 2D gels.     

The Mouse Severe Combined Immune Deficiency (scid) Mutation Is Closely Linked to the B-Cell-Specific Developmental Genes VpreB and λ5

The mouse severe combined immune deficiency (scid) phenotype is due to a recessive, autosomal mutation which results in failed development of lymphocytes. An important step during normal lymphocyte development is the germline rearrangement of DNA segments to assemble functional immunoglobulin or T cell receptor genes. scid lymphocytes fail to rearrange these genes properly, resulting in the absence of mature B and T lymphocytes. This mutation was originally mapped to chromosome 16 by linkage to the immunoglobulin λ light chain genes (Igl-1) and the coat color mutation mahoganoid . We have typed 288 progeny from backcrosses between MOLF/Ei or CAST/Ei and C.B-17-scid for the scid phenotype and nine other loci mapped to the centromeric region of MMU16. We have established a refined map of this region which places the scid gene between Prm-2 and Igl-1 . In addition, no recombinations were found between scid and three other loci, VpreB, λ5, and D16Mit31, providing markers useful for isolating the scid gene by positional cloning.     

Granzyme A Is Expressed in Mouse Lungs during Mycobacterium tuberculosis Infection but Does Not Contribute to Protection In Vivo

Granzyme A, a serine protease expressed in the granules of cytotoxic T and Natural Killer cells, is involved in the generation of pro-inflammatory cytokines by macrophages. Granzyme A has been described to induce in macrophages in vitro the activation of pro-inflammatory pathways that impair intracellular mycobacterial replication. In the present study, we explored the physiological relevance of Granzyme A in the control of pulmonary Mycobacterium tuberculosis infection in vivo. Our results show that, even though Granzyme A is expressed by cytotoxic cells from mouse lungs during pulmonary infection, its deficiency in knockout mice does not have an effect in the control of M. tuberculosis infection. In addition our findings indicate that absence of Granzyme A does not affect the protection conferred by the live-attenuated M. tuberculosis vaccine MTBVAC. Altogether, our findings are in apparent contradiction with previously published in vitro results and suggest that Granzyme A does not have a crucial role in vivo in the protective response to tuberculosis.     

The Sodium-Dependent Inorganic Phosphate Transporter SLC34A1 (NaPi-IIa) Is Not Localized in the Mouse Brain: A Case of Tissue-Specific Antigenic Cross-Reactivity

The sodium-dependent inorganic phosphate transporter NaPi-IIa is expressed in the kidney. Here, the authors used a polyclonal antiserum raised against NaPi-IIa- and NaPi-IIa-deficient mice to characterize its expression in nervous tissue. Western blots showed that a NaPi-IIa immunoreactive band (~90 kDa) was only present in wild-type kidney membranes and not in kidney knockout or wild-type brain membranes. In the water-soluble fraction of wild-type and knockout brains, another band (~50 kDa) was observed; this band was not detected in the kidney. Light and electron microscopic immunohistochemistry using the NaPi-IIa antibodies showed immunolabeling of kidney tubules in wild-type but not knockout mice. In the brain, labeling of presynaptic nerve terminals was present also in NaPi-IIa-deficient mice. This labeling pattern was also produced by the NaPi-IIa preimmune serum. The authors conclude that the polyclonal antiserum is specific toward NaPi-IIa in the kidney, but in the brain, immunolabeling is caused by a cross-reaction of the antiserum with an unknown cytosolic protein that is not present in the kidney. This tissue-specific cross-reactivity highlights a potential pitfall when validating antibody specificity using knockout mouse-derived tissue other than the specific tissue of interest and underlines the utility of specificity testing using preimmune sera.     

Insulin Stimulated-Glucose Transporter Glut 4 Is Expressed in the Retina

The vertebrate retina is a very metabolically active tissue whose energy demands are normally met through the uptake of glucose and oxygen. Glucose metabolism in this tissue relies upon adequate glucose delivery from the systemic circulation. Therefore, glucose transport depends on the expression of glucose transporters. Here, we show retinal expression of the Glut 4 glucose transporter in frog and rat retinas. Immunohistochemistry and in situ hybridization studies showed Glut 4 expression in the three nuclear layers of the retina: the photoreceptor, inner nuclear and ganglionar cell layers. In the rat retina immunoprecipitation and Western blot analysis revealed a protein with an apparent molecular mass of 45 kDa. ¹⁴C-glucose accumulation by isolated rat retinas was significantly enhanced by physiological concentrations of insulin, an effect blocked by inhibitors of phosphatidyl-inositol 3-kinase (PI3K), a key enzyme in the insulin-signaling pathway in other tissues. Also, we observed an increase in ³H-cytochalasin binding sites in the presence of insulin, suggesting an increase in transporter recruitment at the cell surface. Besides, insulin induced phosphorylation of Akt, an effect also blocked by PI3K inhibition. Expression of Glut 4 was not modified in retinas of a type 1 diabetic rat model. To our knowledge, our results provide the first evidence of Glut4 expression in the retina, suggesting it as an insulin- responsive tissue.     

ARA,a Novel ABC Transporter, Is Located at 16p13.1, Is Deleted in inv(16) Leukemias, and Is Shown To Be Expressed in Primitive Hematopoietic Precursors

ATP-binding cassette (ABC), ATP-dependent transporters are a large superfamily of proteins that include the multidrug resistance proteins, P-glycoprotein and MRP (multidrug resistance protein). TheARA(anthracycline resistance-associated) gene that codes for a putative member of the ABC transporters has recently been cloned and shown to have high sequence homology to the gene for MRP. We have previously shownMRPto be deleted in a subset of inv(16) leukemic patients. The deletion ofMRPwas associated with an improved patient survival compared with inv(16) patients who did not have such a deletion. In this study, theARAgene is mapped to 16p13.1, in the same physical interval as the inv(16) short-arm breakpoint. It is shown to be situated proximal to bothMYH11,the gene involved in the primary breakpoint on the short arm of the inv(16), andMRP.A YAC clone has been isolated containing bothMRPandARA.FISH analysis of metaphase chromosomes from inv(16) patients has established the gene order as telomere–MYH11–MRP–ARA–centromere and demonstrated that bothARAandMRPare deleted in a subgroup of the inv(16) leukemias.ARAandMRPare both shown to be expressed in normal hematopoietic precursors including CD34⁺cells. The mapping ofARAto this region and its homology toMRPraises questions about its potential role in the biology of the inv(16) leukemias.     

B Lymphocyte Stimulator (BLyS) Is Expressed in Human Adipocytes In Vivo and Is Related to Obesity but Not to Insulin Resistance

Inflammation and metabolism have been shown to be evolutionary linked and increasing evidence exists that pro-inflammatory factors are involved in the pathogenesis of obesity and type 2 diabetes. Until now, most data suggest that within adipose tissue these factors are secreted by cells of the innate immune system, e. g. macrophages. In the present study we demonstrate that B lymphocyte stimulator (BLyS) is increased in human obesity. In contrast to several pro-inflammatory factors, we found the source of BLyS in human adipose tissue to be the adipocytes rather than immune cells. In grade 3 obese human subjects, expression of BLyS in vivo in adipose tissue is significantly increased (p<0.001). Furthermore, BLyS serum levels are elevated in grade 3 human obesity (862.5+222.0 pg/ml vs. 543.7+60.7 pg/ml in lean controls, p<0.001) and are positively correlated to the BMI (r = 0.43, p<0.0002). In the present study, bariatric surgery significantly altered serum BLyS concentrations. In contrast, weight loss due to a very-low-calorie-formula-diet (800 kcal/d) had no such effect. To examine metabolic activity of BLyS, in a translational research approach, insulin sensitivity was measured in human subjects in vivo before and after treatment with the human recombinant anti-BLyS antibody belimumab. Since BLyS is known to promote B-cell proliferation and immunoglobulin secretion, the present data suggest that adipocytes of grade 3 obese human subjects are able to activate the adaptive immune system, suggesting that in metabolic inflammation in humans both, innate and adaptive immunity, are of pathophysiological relevance.     

Cloning and Characterization of a Gene (RFN22) Encoding a Novel Brain Expressed Ring Finger Protein (BERP) That Maps to Human Chromosome 11p15.5

We have recently identified a novel RING finger protein expressed in the rat brain, which associates with myosin V and α-actinin-4. Here we have cloned and characterized the orthologous human BERP cDNA and gene (HGMW-approved symbol RNF22). The human BERP protein is encoded by 11 exons ranging in size from 71 to 733 bp, and fluorescence in situ hybridization shows that the BERP gene maps to chromosome 11p15.5, 3′ to the FE65 gene. The human BERP protein is 98% identical to the rat and mouse proteins, and we have identified a highly conserved potential orthologue in Caenorhabditis elegans. BERP belongs to the RING finger–B-box–coiled coil (RBCC) subgroup of RING finger proteins, and a cluster of these RBCC protein genes is present in chromosome 11p15. Chromosome region 11p15 is thought to harbor tumor suppressor genes, and deletions of this region occur frequently in several types of human cancers. These observations indicate that BERP may be a novel tumor suppressor gene.     

Chemoattractant Receptor Homologous to the T Helper 2 Cell (CRTH2) Is Not Expressed in Human Amniocytes and Myocytes

Background

15-deoxy-Δ 12,14- Prostaglandin J2 (15dPGJ2) inhibits Nuclear factor kappa B (NF-κB) in human myocytes and amniocytes and delays inflammation induced preterm labour in the mouse. 15dPGJ2 is a ligand for the Chemoattractant Receptor Homologous to the T helper 2 cell (CRTH2), a G protein-coupled receptor, present on a subset of T helper 2 (Th2) cells, eosinophils and basophils. It is the second receptor for Prostaglandin D2, whose activation leads to chemotaxis and the production of Th2-type interleukins. The cellular distribution of CRTH2 in non-immune cells has not been extensively researched, and its identification at the protein level has been limited by the lack of specific antibodies. In this study we explored the possibility that CRTH2 plays a role in 15dPGJ2-mediated inhibition of NF-κB and would therefore represent a novel small molecule therapeutic target for the prevention of inflammation induced preterm labour.

Methods

The effect of a small molecule CRTH2 agonist on NF-κB activity in human cultured amniocytes and myocytes was assessed by detection of p65 and phospho-p65 by immunoblot. Endogenous CRTH2 expression in amniocytes, myocytes and peripheral blood mononuclear cells (PBMCs) was examined by PCR, western analysis and flow cytometry, with amniocytes and myocytes transfected with CRTH2 acting as a positive control in flow cytometry studies.

Results

The CRTH2 agonist had no effect on NF-κB activity in amniocytes and myocytes. Although CRTH2 mRNA was detected in amniocytes and myocytes, CRTH2 was not detectable at the protein level, as demonstrated by western analysis and flow cytometry. 15dPGJ2 inhibited phospho-65 in PBMC’S, however the CRTH2 antagonist was not able to attenuate this effect. In conclusion, CRTH2 is not expressed on human amniocytes or myocytes and plays no role in the mechanism of 15dPGJ2-mediated inhibition of NF-κB.     

N-Cadherin Gene Maps to Human Chromosome 18 and Is Not Linked to the E-Cadherin Gene

cDNA clones encoding the human N-cadherin cell adhesion molecule have been isolated from an embryonic muscle library by screening with an oligonucleotide probe complementary to the chick brain sequence and chick brain cDNA probe lambda N2. Comparison of the predicted protein sequences revealed greater than 91% homology between chick brain, mouse brain, and human muscle N-cadherin cDNAs over the 748 amino acids of the mature, processed protein. A single polyadenylation site in the chick clone was also present and duplicated in the human muscle sequence. Immediately 3' of the recognition site in chick a poly(A) tail ensued; however, in human an additional 800 bp of 3' untranslated sequence followed. Northern analysis identified a number of major N-cadherin mRNAs. These were of 5.2, 4.3, and 4.0 kb in C6 glioma, 4.3 and 4.0 kb in human foetal muscle cultures, and 4.3 kb in human embryonic brain and mouse brain with minor bands of 5.2 kb in human muscle and embryonic brain. Southern analysis of a panel of somatic cell hybrids allowed the human N-cadherin gene to be mapped to chromosome 18. This is distinct from the E-cadherin locus on chromosome 16. Therefore, it is likely that the cadherins have evolved from a common precursor gene that has undergone duplication and migration to other chromosomal locations.