Plasma cell membrane glycoprotein PC-1. cDNA cloning of the human molecule, amino acid sequence, and chromosomal location

The murine cell membrane glycoprotein PC-1 is a homodimer with restricted tissue distribution, being first characterized in plasma cells. We now describe the isolation of cDNA clones encoding the human homolog of the murine PC-1 protein, its complete amino acid sequence, and its chromosomal location. Overall, the amino acid sequence of the human protein is about 80% identical to the murine protein, although the extent of homology varies in different domains. It had not been possible to assign a definitive amino terminus to the murine protein. Comparison of the murine and human sequence necessitates reassignment of the amino terminus, resulting in a cytoplasmic tail of 24 amino acids rather than 58 amino acids as previously published for the mouse. The sequence of several independently obtained cDNA clones indicates that the 3' end of the mRNA is subject to alternative splicing. Southern blots suggest a single copy gene. In situ chromosomal hybridization localizes the gene for human PC-1 to chromosome 6q22-q23, a common site for deletions in human lymphoid neoplasia.     

Transformation/neodifferentiation of human skin fibroblasts by acute oncornaviruses and dexamethasone

We demonstrated that the Kirsten murine sarcoma virus (KiMSV) and the Harvey murine sarcoma virus (HaMSV) converted human skin fibroblasts (HSF) into adipocytes. Adipocytic conversion of HSF by KiMSV and HaMSV was dependent on the presence of glucocorticosteroids. The Kirsten murine leukemia virus, the Harvey murine sarcoma [corrected] virus and the amphotropic helper virus (AP292) were ineffective by themselves. Balb murine sarcoma virus and Moloney murine sarcoma virus were, to a lesser degree, able to effect adipocytic conversion of HSF. In contrast, the feline sarcoma virus and the simian sarcoma virus did not cause this conversion. Together, the results suggest a role for certain oncogenes and glucocorticosteroids in the transformation/neodifferentiation of human cells.     

Hot spots in apolipoprotein A-II misfolding and amyloidosis in mice and men

ApoA-II is the second-major protein of high-density lipoproteins. C-terminal extension in human apoA-II or point substitutions in murine apoA-II cause amyloidosis. The molecular mechanism of apolipoprotein misfolding, from the native predominantly α-helical conformation to cross-β-sheet in amyloid, is unknown. We used 12 sequence-based prediction algorithms to identify two ten-residue segments in apoA-II that probably initiate β-aggregation. Previous studies of apoA-II fragments experimentally verify this prediction. Together, experimental and bioinformatics studies explain why the C-terminal extension in human apoA-II causes amyloidosis and why, unlike murine apoA-II, human apoA-II normally does not cause amyloidosis despite its unusually high sequence propensity for β-aggregation.     

Four different interleukin-1 species sensitize to the lethal action of tumour necrosis factor

We studied the induction of lethal shock by Tumour Necrosis Factor (TNF) in mice and observed a remarkable difference between the effect of human and murine TNF, which could be eliminated by co-administration of sensitizing agents. We identified interleukin-1 (IL1) as a natural sensitizer, rendering mice as susceptible to human TNF as to murine TNF. This IL1 activity was found to be exerted to the same extent both by human and murine IL1-alpha or IL1-beta, and was also different from the sensitization obtained with galactosamine, since these agents had an additive effect. Pretreatment of the animals with indomethacin, a cyclooxygenase inhibitor, provided partial protection against TNF lethality in IL1-sensitized but not in galactosamine-sensitized mice.     

Recognition of a site of a Kentucky bluegrass pollen allergen by antibodies in the sera of allergic and non-atopic humans and a murine monoclonal antibody

Allergen 27 was isolated from the aqueous extract of Kentucky Bluegrass pollen (KBG-R) with a reversed immunosorbent prepared by coupling murine monoclonal antibody, Mab 27, to Sepharose 4B. Sera of patients allergic to KBG pollen, as well as serum of nonatopic individuals possessing anti-KBG antibodies, inhibited the binding of Mab 27 to either Ag 27 or KBG-R to the extent of 20 to 35% in ELISA. In contrast, sera devoid of antibodies to KBG-R had no inhibitory capacity. In a radioallergosorbent test, it was demonstrated that Mab 27 could inhibit the binding of human IgE antibodies to Ag 27 to the extent of 52%. From these results, it is concluded that Ag 27 contains a determinant recognized by both human IgE and blocking antibodies and a murine Mab.     

Substitution in the murine nectin1 receptor of a single conserved amino acid at a position distal from the herpes simplex virus gD binding site confers high-affinity binding to gD

By analogy with its human nectin1 counterpart, murine nectin1 serves as a cellular receptor for the entry of herpes simplex virus (HSV) into murine cells. HSV entry mediated by either receptor is dependent on the viral glycoprotein D (gD). Whereas human nectin1 binds gD at high affinity and in a saturable manner, murine nectin1 binds gD in a barely detectable fashion, depending on the sensitivity of the assay. The immunoglobulin type V domain of murine nectin differs from its human counterpart in 11 amino acids. To identify the key residues responsible for the high-affinity binding of gD to human nectin1, we replaced each of the 11 divergent amino acids with the human counterparts singly or in groups in an incremental manner. Replacement in murine nectin1 of six amino acids that lie within the gD binding region of human nectin1 (previously mapped to residues 64 to 94, likely the CC'C" surface) increased the gD binding activity to a limited extent. In contrast, the single P138L substitution, which lies distal from the gD binding site, markedly increased gD binding. This substitution, when coupled with downstream substitutions, exerted the greatest effect. Three-dimensional modeling of the nectin1 V domain suggested that P138 in murine nectin1 might decrease the stability of the V domain by reducing the size of beta-strand G. The results support the notion that the overall structure of V nectin1 plays a pivotal role in its ability to bind HSV gD.     

Recessive mutations in ELOVL4 cause ichthyosis, intellectual disability, and spastic quadriplegia

Very-long-chain fatty acids (VLCFAs) play important roles in membrane structure and cellular signaling, and their contribution to human health is increasingly recognized. Fatty acid elongases catalyze the first and rate-limiting step in VLCFA synthesis. Heterozygous mutations in ELOVL4, the gene encoding one of the elongases, are known to cause macular degeneration in humans and retinal abnormalities in mice. However, biallelic ELOVL4 mutations have not been observed in humans, and murine models with homozygous mutations die within hours of birth as a result of a defective epidermal water barrier. Here, we report on two human individuals with recessive ELOVL4 mutations revealed by a combination of autozygome analysis and exome sequencing. These individuals exhibit clinical features of ichthyosis, seizures, mental retardation, and spasticity—a constellation that resembles Sjögren-Larsson syndrome (SLS) but presents a more severe neurologic phenotype. Our findings identify recessive mutations in ELOVL4 as the cause of a neuro-ichthyotic disease and emphasize the importance of VLCFA synthesis in brain and cutaneous development.     

Activity of a partially purified human BCGF on murine assays for B cell stimulatory factors. II. Synergy between two distinct BCGF II-like factors in promoting B cell differentiation

We have previously identified two BCGF II-like factors which can be distinguished by their differential reactivity in several murine BCGF II assays. Prototype sources of these two factors are a partially purified preparation derived from PHA-P-stimulated human peripheral blood T lymphocytes (designated human BCGF), and supernatant from antigen-stimulated D10.G4.1 murine T cells (designated D10 sup). Extending the characterization of these two factors, we show here that human BCGF and D10 sup both cause tritiated thymidine ([3H]TdR) incorporation and IgM secretion by T cell-depleted, in vivo-activated, large murine B cells. In contrast, only the human BCGF consistently induced proliferation and IgM secretion by T cell-depleted, small murine B cells. When simultaneously added to cultures, D10 sup and human BCGF synergized to produce optimal IgM secretion by large murine B cells and murine BCL1 B lymphoma cells. The same factors were tested in an IgM-specific plaque assay, and a similar synergistic response was observed for the large B cells, but not for the BCL1 cells. The combination of factors also produced maximal [3H]TdR incorporation by large murine B cells. In contrast, the addition of human BCGF totally abrogated D10 sup-induced BCL1 proliferation. Together, these data suggest that the synergies observed in IgM secretion result from an increased production of plaque-forming cells (PFC) in cultures of large B cells and an increase in IgM production per responding cell in BCL1 cells. Kinetic analysis of the time of action of the two BCGF II-like lymphokines in the induction of the PFC response by large B cells indicated that human BCGF was required within the first 24 hr of a 4-day culture period, while D10 sup could be added as late as the final 15 hr without significant diminution of the response. In summary, these data provide further support for the existence of two distinct B cell stimulatory factors which cause growth and differentiation of activated B cells, and indicate that these two factors synergize to produce optimal Ig secretion. For ease of discussion, the activity in the human BCGF preparation is referred to as BCGF IIA, and the activity in D10 sup is referred to as BCGF IIB.     

Human carcinoembryonic antigen and biliary glycoprotein can serve as mouse hepatitis virus receptors.

Receptors for murine coronavirus mouse hepatitis virus (MHV) are members of the murine carcinoembryonic antigen (CEA) gene family. Since MHV can also infect primates and cause central nervous system lesions (G. F. Cabirac et al., Microb. Pathog. 16:349-357, 1994; R. S. Murray et al., Virology 188:274-284, 1992), we examined whether human CEA-related molecules can be used by MHV as potential receptors. Transfection of plasmids expressing human carcinoembryonic antigen (hCEA) and human biliary glycoprotein into COS-7 cells, which lack a functional MHV receptor, conferred susceptibility to two MHV strains, A59 and MHV-2. Domain exchange experiments between human and murine CEA-related molecules identified the immunoglobulin-like loop I of hCEA as the region conferring the virus-binding specificity. This finding expands the potential MHV receptors to primate species.     

Lovastatin enhances clearance of apoptotic cells (efferocytosis) with implications for chronic obstructive pulmonary disease

Statins are potent, cholesterol-lowering agents with newly appreciated, broad anti-inflammatory properties, largely based upon their ability to block the prenylation of Rho GTPases, including RhoA. Because phagocytosis of apoptotic cells (efferocytosis) is a pivotal regulator of inflammation, which is inhibited by RhoA, we sought to determine whether statins enhanced efferocytosis. The effect of lovastatin on efferocytosis was investigated in primary human macrophages, in the murine lung, and in human alveolar macrophages taken from patients with chronic obstructive pulmonary disease. In this study, we show that lovastatin increased efferocytosis in vitro in an 3-hydroxyl-3-methylglutaryl coenzyme A (HMG-CoA) reductase-dependent manner. Lovastatin acted by inhibiting both geranylgeranylation and farnesylation, and not by altering expression of key uptake receptors or by increasing binding of apoptotic cells to phagocytes. Lovastatin appeared to exert its positive effect on efferocytosis by inhibiting RhoA, because it 1) decreased membrane localization of RhoA, to a greater extent than Rac-1, and 2) prevented impaired efferocytosis by lysophosphatidic acid, a potent inducer of RhoA. Finally, lovastatin increased efferocytosis in the naive murine lung and ex vivo in chronic obstructive pulmonary disease alveolar macrophages in an HMG-CoA reductase-dependent manner. These findings indicate that statins enhance efferocytosis in vitro and in vivo, and suggest that they may play an important therapeutic role in diseases where efferocytosis is impaired and inflammation is dysregulated.     

Inhibition of Candida albicans Growth by Murine Peritoneal Neutrophils and Augmentation of the Inhibitory Activity by Bacterial Lipopolysaccharide and Cytokines

Anti-Candida activity of murine neutrophils and its regulation by immunomodulators were studied in vitro. Murine neutrophils which were prepared from peritoneal-exudated cells inhibited the growth of Candida albicans at an effector: target (E/T) ratio of 30/1 or above. This anti-Candida activity of neutrophils was augmented by lipopolysaccharide from Escherichia coli, murine tumor necrosis factor (TNF), murine interferon-γ (IFN-γ) and murine granulocyte macrophage colony-stimulating factor (GM-CSF) but not by granulocyte colony-stimulating factor (G-CSF) added to the incubation medium. Greater extent of augmentation was obtained when TNF plus GM-CSF or INF-γ plus GM-CSF were used in combination. These results indicate that anti-Candida activity of murine neutrophils is regulated similarly to that of the human neutrophils reported previously. Therefore murine peritoneal neutrophils can be used as a favorable substitute for human neutrophils in studies on protective machinery against C. albicans infection.     

The X chromosome deletion in HYP mice extends into the intergenic region but does not include the SAT gene downstream from Phex

The murine Hyp mutation is a model for X-linked hypophosphatemia (XLH), the most prevalent form of inherited rickets in humans. Although mutations in the murine Phex gene and the human PHEX gene have been identified in both murine and human disorders, the extent of the Hyp deletion on the mouse X chromosome has not been delineated. In the present study we demonstrate that the Hyp deletion starts in the middle of Phex intron 15 and includes approximately 48 kb of the 3' region of the Phex gene and approximately 10 kb of intergenic sequence on the mouse X chromosome. In addition, we show that the Hyp deletion does not involve the downstream spermidine/spermine N1-acetyl transferase (Sat; formerly Ssat) gene and thus is not a contiguous gene deletion syndrome. Our data indicate that the Hyp mouse is a true homolog of XLH in humans and underscore the validity of this murine model in studies of XLH pathophysiology and for testing novel treatment modalities.     

Antigen-specific activity of murine leukocyte dialysates containing transfer factor on human leukocytes in the leukocyte migration inhibition (LMI) assay

We report on the extension of the direct leukocyte migration inhibition (LMI) test as an assay for antigen-specific activity in human leukocyte dialysates (DLE) containing transfer factor to an evaluation of antigen-specific activity in DLE prepared from inbred mice. Murine DLE was observed to cause antigen-dependent and antigen-specific effects on the inhibition of migration of nonimmune human leukocyte populations. Pulsing of nonimmune human leukocyte with DLE preparations from BALB/c and SJL mice immunized with Candida, diphtheria toxoid, and SK-SD resulted in their inhibition of migration in the presence of the respective antigens. The antigen-specific activity in murine DLE was found to be present in lymph node cell preparations and to be absent from spleen cell preparations of the same donors. The activity of DLE in lymph node cells was found to be present in the theta-cell enriched subpopulation of nonadherent lymphocytes after passage through nylon wool columns. The antigen-specific activity of murine DLE, as we have reported for human DLE, was found to reside in the < 3500 dalton dialysis fraction and not in the < 3500 dalton fraction. We conclude that nonimmune human leukocytes in the LMI test provide a suitable assay for the detection of antigen-specific activity in murine DLE as well as that in human DLE. Additionally, murine DLE is active across species barriers and appears to share properties with human DLE.     

Oxidative responses of human and murine macrophages during phagocytosis of Leishmania chagasi.

Leishmania chagasi, the cause of South American visceral leishmaniasis, must survive antimicrobial responses of host macrophages to establish infection. Macrophage oxidative responses have been shown to diminish in the presence of intracellular leishmania. However, using electron spin resonance we demonstrated that murine and human macrophages produce O2-during phagocytosis of opsonized promastigotes. Addition of the O2- scavenger 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl to cultures resulted in increased infection, suggesting that O2- enhances macrophage leishmanicidal activity. The importance of NO. produced by inducible NO synthase (iNOS) in controlling murine leishmaniasis is established, but its role in human macrophages has been debated. We detected NO. in supernatants from murine, but not human, macrophages infected with L. chagasi. Nonetheless, the iNOS inhibitor N(G)-monomethyl-L-arginine inhibited IFN-gamma-mediated intracellular killing by both murine and human macrophages. According to RNase protection assay and immunohistochemistry, iNOS mRNA and protein were expressed at higher levels in bone marrow of patients with visceral leishmaniasis than in controls. The iNOS protein also increased upon infection of human macrophages with L. chagasi promastigotes in vitro in the presence of IFN-gamma. These data suggest that O2- and NO. each contribute to intracellular killing of L. chagasi in human and murine macrophages.     

Complete murine cDNA sequence, genomic structure, and tissue expression of the high mobility group protein HMG-I(Y)

A cDNA coding for the non-histone chromosomal protein HMG-I, or its isoform HMG-Y, was isolated from a murine Friend cell library using synthetic oligonucleotide hybridization probes. Sequence analysis showed that the 1670-base pair full length cDNA insert consists of a 201-base pair, G/C-rich (74%), 5'-untranslated region, a 288-base pair amino acid coding sequence, and an unusually long 1182-base pair 3'-untranslated region. The deduced 96-residue amino acid coding sequence of the murine HMG-I(Y) cDNA is very similar to the reported amino acid sequence of human HMG-I, except that it lacks 11 internal amino acids reported in the human protein. Based on Southern blot hybridization analysis of genomic DNA, there appear to be fewer than five copies of HMG-I(Y) genes in the haploid murine genome. These murine HMG-I(Y) genes contain a large (at least 890 base pairs) exon that includes most, or all, of the 3'-untranslated region; whereas the much shorter 5'-untranslated region and amino acid coding sequences are interrupted by at least one intron. A single size class (approximately 1700 nucleotides in murine cells and 2000 nucleotides in human cells) of HMG-I(Y) mRNAs was detected at high levels in total RNA extracts from rapidly dividing, transformed cells, but to a lesser extent, or not at all, in extracts from slowly or non-dividing cells.     

Ligand-induced phosphorylation of the human interferon-gamma receptor. Dependence on the presence of a functionally active receptor

Phosphorylation of the human interferon-gamma (IFN gamma) receptor was studied in three cell lines of distinct lineages using radiophosphate labeling techniques. Receptors from unstimulated Colo-205 displayed a low level of constitutive phosphorylation which was enhanced 5.3-fold following exposure of the cells to either purified recombinant human IFN gamma or phorbol myristate acetate. Enhanced receptor phosphorylation was specific, dose- and time-dependent, reversible, and affected only serine and threonine residues. Increased phosphorylation was observed only when cells were treated with human IFN gamma or phorbol myristate acetate and not with murine IFN gamma, human IFN alpha, human tumor necrosis factor-alpha, or epidermal growth factor. The biologic relevance of IFN gamma receptor phosphorylation was suggested by three additional observations; 1) there was a close correlation between the extent of receptor phosphorylation and the magnitude of the cellular response induced, 2) TNF alpha concomitantly enhanced both IFN gamma-dependent HLA-DR expression and IFN gamma-dependent receptor phosphorylation on Colo-205, and 3) phosphorylation of functionally inactive recombinant murine IFN gamma receptors expressed on transfected human 293 or Colo-205 cells was not induced by murine IFN gamma but was induced by the homologous human ligand. These results suggest that phosphorylation of the IFN gamma receptor is an important step in the development of IFN gamma-dependent cellular responses and indicates that phosphorylation requires a functionally active receptor.     

Controlled formaldehyde fixation of fibronectin layers for expansion of mesenchymal stem cells

Extracellular cell matrices deposited by cells stimulate cell proliferation. However, their generation is cumbersome and time consuming. We show here that controlled fixation of fibronectin layers after coating culture vessels significantly enhances expansion of murine and human mesenchymal stem cells (MSCs) and, to a lesser extent, primary fibroblasts. In contrast, fibronection fixation did not stimulate proliferation of established cancer cell lines. Fixed vitronectin or collagen IV layers also enhanced proliferation of murine MSCs. Thus, controlled formaldehyde fixation of layers formed by fibronectin or some other extracellular matrix components represents a simple and reproducible way to enhance proliferation of primary cells.     

Ultrastructural morphology and domain structure of a unique collagenous component of basement membranes

A disulfide cross-linked collagenous fragment (7 S) has been isolated by pepsin solubilization from several tissues rich in basement membranes including bovine lung, human placenta, and the murine EHS tumor. Examination of this material by the rotary shadowing technique indicates that these fragments are similar to but not identical with the 7S collagen described recently [Risteli, J., B?chinger, H.P., Engel, J., Furthmayr, H., & Timpl, R. (1980) Eur. J. Biochem. 108, 239-250]. The central rodlike portion of the particles was found to be similar in length; however, the peripheral four arms of 7S particles from bovine and murine sources are 10 nm longer in comparison to those from human sources. In addition, about 5-7% of all the particles contain a fifth arm. Specific antibodies to bovine 7 S cross-react with murine 7 S but only to a rather limited extent with human 7 S. These antibodies react with antigenic sites located at the ends of the peripheral arms of the fragment as visualized directly with rotary shadowing techniques. The data are consistent with a structural difference in type IV collagens from bovine, human, and mouse which leads to pepsin cleavage at different sites in a particular noncollagenous region adjacent to 7 S. However, since bovine 7S antibodies cross-react with human and murine tissues by immunofluorescence despite the lack of complete serological cross-reactivity, it is suggested that type IV collagens from all three species have some degree of homology in this region.     

Synthesis of platelet-activating factor (PAF) in transformed cell lines of a different origin

Interest in the possible involvement of the platelet-activating factor (PAF) in tumor growth and invasiveness has been stimulated by the recognition that PAF influences various biological responses relevant to metastatic diffusion, such as angiogenesis, adhesiveness to endothelia and cellular motility. In the present study, we investigated the extent to which PAF is synthesized by a series of human and murine transformed cell lines of a different histotype. Synthesis of PAF was studied by combining the 14C-acetate incorporation into PAF with the quantitative analysis of PAF performed by a procedure based on gas chromatography-mass spectrometry with a negative ion chemical ionization. In the presence of the Ca2+ ionophore A23187, cultures of human melanoma (Hs294T), fibrosarcoma (HT1080) and colon carcinoma (LS180) cell lines synthesized conspicuous amounts of PAF, comparable to those produced by resident peritoneal macrophages. Substantial quantities of PAF were also synthesized by the murine melanoma (F10-M3 cells). PAF synthesis was rather limited in RSV-transformed Balb/c3T3 (B77-3T3) cells and in one of their high metastatic variants (B77-AA6 cells), although it was more abundant in the latter. We also investigated whether certain cytokines, such as TNFalpha and IFNgamma might induce PAF synthesis in our systems of cell lines which we found to express mRNAs encoding receptors for these cytokines. We observed that PAF synthesis was enhanced in human melanoma and colon carcinoma cell lines and in the murine B77-AA6 cells to levels comparable to those obtained with the Ca2+ ionophore. Synthesis of PAF was not inducible by TNFalpha in murine F10-M3 melanoma cells. IFNgamma also stimulated PAF synthesis in human and murine melanoma lines, and in human LS180 colon carcinoma line, but not in the B77-AA6 cells. PAF synthesis was also inducible by exogenous PAF in the human and murine melanoma lines, and in the human LS180 colon carcinoma line, all of which expressed cell surface PAF receptors. PAF synthesis was not inducible by exogenous PAF in the B77-AA6 cells, which do not express PAF receptors.