Correction of murine galactosialidosis by bone marrow-derived macrophages overexpressing human protective protein/cathepsin A under control of the colony-stimulating factor-1 receptor promoter

Galactosialidosis (GS) is a human neurodegenerative disease caused by a deficiency of lysosomal protective protein/cathepsin A (PPCA). The GS mouse model resembles the severe human condition, resulting in nephropathy, ataxia, and premature death. To rescue the disease phenotype, GS mice were transplanted with bone marrow from transgenic mice overexpressing human PPCA specifically in monocytes/macrophages under the control of the colony stimulating factor-1 receptor promoter. Transgenic macrophages infiltrated and resided in all organs and expressed PPCA at high levels. Correction occurred in hematopoietic tissues and nonhematopoietic organs, including the central nervous system. PPCA-expressing perivascular and leptomeningeal macrophages were detected throughout the brain of recipient mice, although some neuronal cells, such as Purkinje cells, continued to show storage and died. GS mice crossed into the transgenic background reflected the outcome of bone marrow-transplanted mice, but the course of neuronal degeneration was delayed in this model. These studies present definite evidence that macrophages alone can provide a source of corrective enzyme for visceral organs and may be beneficial for neuronal correction if expression levels are sufficient.

The inhibition of antigen-presenting activity of dendritic cells resulting from UV irradiation of murine skin is restored by in vitro photorepair of cyclobutane pyrimidine dimers

Exposing skin to UVB (280–320 nm) radiation suppresses contact hypersensitivity by a mechanism that involves an alteration in the activity of cutaneous antigen-presenting cells (APC). UV-induced DNA damage appears to be an important molecular trigger for this effect. The specific target cells in the skin that sustain DNA damage relevant to the immunosuppressive effect have yet to be identified. We tested the hypothesis that UV-induced DNA damage in the cutaneous APC was responsible for their impaired ability to present antigen after in vivo UV irradiation. Cutaneous APC were collected from the draining lymph nodes of UVB-irradiated, hapten-sensitized mice and incubated in vitro with liposomes containing a photolyase (Photosomes; Applied Genetics, Freeport, NY), which, upon absorption of photoreactivating light, splits UV-induced cyclobutane pyrimidine dimers. Photosome treatment followed by photoreactivating light reduced the number of dimer-containing APC, restored the in vivo antigen-presenting activity of the draining lymph node cells, and blocked the induction of suppressor T cells. Neither Photosomes nor photoreactivating light alone, nor photoreactivating light given before Photosomes, restored APC activity, and Photosome treatment did not reverse the impairment of APC function when isopsoralen plus UVA (320–400 nm) radiation was used instead of UVB. These controls indicate that the restoration of APC function matched the requirements of Photosome-mediated DNA repair for dimers and post-treatment photoreactivating light. These results provide compelling evidence that it is UV-induced DNA damage in cutaneous APC that leads to reduced immune function.

Selective increase in specific alternative splice variants of tyrosinase in murine melanomas: A projected basis for immunotherapy

Melanomas tend to become less pigmented in the course of malignant progression. Thus, as proliferation increases, the tumors are decreasingly characterized by the tissue-specific phenotype of normally differentiated melanocytes. To learn whether the decline in melanization is associated with a shift from constitutive to alternative splicing of some pigment gene pre-mRNAs, melanomas were collected from Tyr-SV40E transgenic mice of the standard C57BL/6 strain. The mRNAs of the tyrosinase gene, which has a key role in melanogenesis, were analyzed by quantitative reverse transcriptase–PCR in 34 samples from 16 cutaneous tumors and 9 metastases. The cutaneous tumors included some cases with distinct melanotic and amelanotic zones, which were separately analyzed. All tyrosinase transcripts found in the melanomas were also found in normal skin melanocytes. However, the Δ1b and Δ1d alternatively spliced transcripts, due to deletions within the first exon, were specifically augmented in most of the tumors over their very low levels in skin; the exceptions were some all-amelanotic tumors in which no tyrosinase transcripts were detected. The level of Δ1b rose as high as 11.3% of total tyrosinase mRNAs as compared with 0.6% in skin; Δ1d reached 4.0% as compared with 0.8% in skin. Expression of these splice variants was highest in the melanotic components of zonal primary tumors, relatively lower in their amelanotic components, and still lower in all-amelanotic primary tumors and amelanotic metastases. The increase in Δ1b and Δ1d transcripts may be predicted to increase the levels of unusual peptides, which could have antigenic potential in the tumors, especially in the relatively early phases of malignancy. Analyses of the alternative transcripts of other pigment genes may identify additional candidate antigens, ultimately enabling melanoma cells in all phases of the disease to be represented as a basis for immune intervention.

Electrostatic interaction between helical macromolecules in dense aggregates: An impetus for DNA poly- and meso-morphism

DNA exhibits a surprising multiplicity of structures when it is packed into dense aggregates. It undergoes various polymorphous transitions (e.g., from the B to A form) and mesomorphous transformations (from hexagonal to orthorhombic or monoclinic packing, changes in the mutual alignment of nearest neighbors, etc). In this report we show that such phenomena may have their origin in the specific helical symmetry of the charge distribution on DNA surface. Electrostatic interaction between neighboring DNA molecules exhibits strong dependence on the patterns of molecular surface groups and adsorbed counter-ions. As a result, it is affected by such structural parameters as the helical pitch, groove width, the number of base pairs per helical turn, etc. We derive expressions which relate the energy of electrostatic interaction with these parameters and with the packing variables characterizing the axial and azimuthal alignment between neighboring macromolecules. We show, in particular, that the structural changes upon the B-to-A transition reduce the electrostatic energy by ≈kcal/mol per base pair, at a random adsorption of counter ions. Ion binding into the narrow groove weakens or inverts this effect, stabilizing B-DNA, as it is presumably the case in Li+-DNA assemblies. The packing symmetry and molecular alignment in DNA aggregates are shown to be affected by the patterns of ion binding.

Targeted disruption of the murine dihydrolipoamide dehydrogenase gene (Dld) results in perigastrulation lethality

The Dld gene product, known as dihydrolipoamide dehydrogenase or the E3 component, catalyzes the oxidation of dihydrolipoyl moieties of four mitochondrial multienzyme complexes: pyruvate dehydrogenase, α-ketoglutarate dehydrogenase, branched-chain α-ketoacid dehydrogenase, and the glycine cleavage system. Deficiency of E3 activity in humans results in various degrees of neurological dysfunction and organic acidosis caused by accumulation of branched-chain amino acids and lactic acid. In this study, we have introduced a null mutation into the murine Dld gene (Dldtm1mjp). The heterozygous animals are shown to have approximately half of wild-type activity levels for E3 and all affected multienzyme complexes but are phenotypically normal. In contrast, the Dld−/− class dies prenatally with apparent developmental delay at 7.5 days postcoitum followed by resorption by 9.5 days postcoitum. The Dld−/− embryos cease to develop at a time shortly after implantation into the uterine wall when most of the embryos have begun to gastrulate. This null phenotype provides in vivo evidence for the requirement of a mitochondrial oxidative pathway during the perigastrulation period. Furthermore, the early prenatal lethal condition of the complete deficiency state may explain the low incidence of detectable cases of E3 deficiency in humans.

A targeted mutation in the murine gene encoding the high density lipoprotein (HDL) receptor scavenger receptor class B type I reveals its key role in HDL metabolism

Plasma high density lipoprotein (HDL), which protects against atherosclerosis, is thought to remove cholesterol from peripheral tissues and to deliver cholesteryl esters via a selective uptake pathway to the liver (reverse cholesterol transport) and steroidogenic tissues (e.g., adrenal gland for storage and hormone synthesis). Despite its physiologic and pathophysiologic importance, the cellular metabolism of HDL has not been well defined. The class B, type I scavenger receptor (SR-BI) has been proposed to play an important role in HDL metabolism because (i) it is a cell surface HDL receptor which mediates selective cholesterol uptake in cultured cells, (ii) its physiologically regulated expression is most abundant in the liver and steroidogenic tissues, and (iii) hepatic overexpression dramatically lowers plasma HDL. To test directly the normal role of SR-BI in HDL metabolism, we generated mice with a targeted null mutation in the SR-BI gene. In heterozygous and homozygous mutants relative to wild-type controls, plasma cholesterol concentrations were increased by ≈31% and 125%, respectively, because of the formation of large, apolipoprotein A-I (apoA-I)-containing particles, and adrenal gland cholesterol content decreased by 42% and 72%, respectively. The plasma concentration of apoA-I, the major protein in HDL, was unchanged in the mutants. This, in conjunction with the increased lipoprotein size, suggests that the increased plasma cholesterol in the mutants was due to decreased selective cholesterol uptake. These results provide strong support for the proposal that in mice the gene encoding SR-BI plays a key role in determining the levels of plasma lipoprotein cholesterol (primarily HDL) and the accumulation of cholesterol stores in the adrenal gland. If it has a similar role in controlling plasma HDL in humans, SR-BI may influence the development and progression of atherosclerosis and may be an attractive candidate for therapeutic intervention in this disease.

Degeneration of neurons, synapses, and neuropil and glial activation in a murine Atm knockout model of ataxia–telangiectasia

Neural degeneration is one of the clinical manifestations of ataxia–telangiectasia, a disorder caused by mutations in the Atm protein kinase gene. However, neural degeneration was not detected with general purpose light microscopic methods in previous studies using several different lines of mice with disrupted Atm genes. Here, we show electron microscopic evidence of degeneration of several different types of neurons in the cerebellar cortex of 2-month-old Atm knockout mice, which is accompanied by glial activation, deterioration of neuropil structure, and both pre- and postsynaptic degeneration. These findings are similar to those in patients with ataxia–telangiectasia, indicating that Atm knockout mice are a useful model to elucidate the mechanisms underlying neurodegeneration in this condition and to develop and test strategies to palliate and prevent the disease.

Targeting of a Germ Cell-specific Type 1 Hexokinase Lacking a Porin-binding Domain to the Mitochondria as Well as to the Head and Fibrous Sheath of Murine Spermatozoa

Multiple isoforms of type 1 hexokinase (HK1) are transcribed during spermatogenesis in the mouse, including at least three that are presumably germ cell specific: HK1-sa, HK1-sb, and HK1-sc. Each of these predicted proteins contains a common, germ cell-specific sequence that replaces the porin-binding domain found in somatic HK1. Although HK1 protein is present in mature sperm and is tyrosine phosphorylated, it is not known whether the various potential isoforms are differentially translated and localized within the developing germ cells and mature sperm. Using antipeptide antisera against unique regions of HK1-sa and HK1-sb, it was demonstrated that these isoforms were not found in pachytene spermatocytes, round spermatids, condensing spermatids, or sperm, suggesting that HK1-sa and HK1-sb are not translated during spermatogenesis. Immunoreactivity was detected in protein from round spermatids, condensing spermatids, and mature sperm using an antipeptide antiserum against the common, germ cell-specific region, suggesting that HK1-sc was the only germ cell-specific isoform present in these cells. Two-dimensional SDS-PAGE suggested that all of the sperm HK1-sc was tyrosine phosphorylated, and that the somatic HK1 isoform was not present. Immunoelectron microscopy revealed that HK1-sc was associated with the mitochondria and with the fibrous sheath of the flagellum and was found in discrete clusters in the region of the membranes of the sperm head. The unusual distribution of HK1-sc in sperm suggests novel functions, such as extramitochondrial energy production, and also demonstrates that a hexokinase without a classical porin-binding domain can localize to mitochondria.

Proteolytic Processing and Ca2+-binding Activity of Dense-Core Vesicle Polypeptides in Tetrahymena

Formation and discharge of dense-core secretory vesicles depend on controlled rearrangement of the core proteins during their assembly and dispersal. The ciliate Tetrahymena thermophila offers a simple system in which the mechanisms may be studied. Here we show that most of the core consists of a set of polypeptides derived proteolytically from five precursors. These share little overall amino acid identity but are nonetheless predicted to have structural similarity. In addition, sites of proteolytic processing are notably conserved and suggest that specific endoproteases as well as carboxypeptidase are involved in core maturation. In vitro binding studies and sequence analysis suggest that the polypeptides bind calcium in vivo. Core assembly and postexocytic dispersal are compartment-specific events. Two likely regulatory factors are proteolytic processing and exposure to calcium. We asked whether these might directly influence the conformations of core proteins. Results using an in vitro chymotrypsin accessibility assay suggest that these factors can induce sequential structural rearrangements. Such progressive changes in polypeptide folding may underlie the mechanisms of assembly and of rapid postexocytic release. The parallels between dense-core vesicles in different systems suggest that similar mechanisms are widespread in this class of organelles.

Endogenous tumor necrosis factor protects the adult cardiac myocyte against ischemic-induced apoptosis in a murine model of acute myocardial infarction

Previous studies have shown that proinflammatory cytokines, such as tumor necrosis factor (TNF), are expressed after acute hemodynamic overloading and myocardial ischemia/infarction. To define the role of TNF in the setting of ischemia/infarction, we performed a series of acute coronary artery occlusions in mice lacking one or both TNF receptors. Left ventricular infarct size was assessed at 24 h after acute coronary occlusion by triphenyltetrazolium chloride (TTC) staining in wild-type (both TNF receptors present) and mice lacking either the type 1 (TNFR1), type 2 (TNFR2), or both TNF receptors (TNFR1/TNFR2). Left ventricular infarct size as assessed by TTC staining was significantly greater (P < 0.005) in the TNFR1/TNFR2-deficient mice (77.2% ± 15.3%) when compared with either wild-type mice (46.8% ± 19.4%) or TNFR1-deficient (47.9% ± 10.6%) or TNFR2-deficient (41.6% ± 16.5%) mice. Examination of the extent of necrosis in wild-type and TNFR1/TNFR2-deficient mice by anti-myosin Ab staining demonstrated no significant difference between groups; however, the peak frequency and extent of apoptosis were accelerated in the TNFR1/TNFR2-deficient mice when compared with the wild-type mice. The increase in apoptosis in the TNFR1/TNFR2-deficient mice did not appear to be secondary to a selective up-regulation of the Fas ligand/receptor system in these mice. These data suggest that TNF signaling gives rise to one or more cytoprotective signals that prevent and/or delay the development of cardiac myocyte apoptosis after acute ischemic injury.

An autologous oral DNA vaccine protects against murine melanoma

We demonstrated that peripheral T cell tolerance toward murine melanoma self-antigens gp100 and TRP-2 can be broken by an autologous oral DNA vaccine containing the murine ubiquitin gene fused to minigenes encoding peptide epitopes gp10025–33 and TRP-2181–188. These epitopes contain dominant anchor residues for MHC class I antigen alleles H-2Db and H-2Kb, respectively. The DNA vaccine was delivered by oral gavage by using an attenuated strain of Salmonella typhimurium as carrier. Tumor-protective immunity was mediated by MHC class I antigen-restricted CD8+ T cells that secreted TH1 cytokine IFN-γ and induced tumor rejection and growth suppression after a lethal challenge with B16G3.26 murine melanoma cells. Importantly, the protective immunity induced by this autologous DNA vaccine against murine melanoma cells was at least equal to that achieved through xenoimmunization with the human gp10025–33 peptide, which differs in its three NH2-terminal amino acid residues from its murine counterpart and was previously reported to be clearly superior to an autologous vaccine in inducing protective immunity. The presence of ubiquitin upstream of the minigene proved to be essential for achieving this tumor-protective immunity, suggesting that effective antigen processing and presentation may make it possible to break peripheral T cell tolerance to a self-antigen. This vaccine design might prove useful for future rational designs of other recombinant DNA vaccines targeting tissue differentiation antigens expressed by tumors.

Rapid regulated dense-core vesicle exocytosis requires the CAPS protein

Although many proteins essential for regulated neurotransmitter and peptide hormone secretion have been identified, little is understood about their precise roles at specific stages of the multistep pathway of exocytosis. To study the function of CAPS (Ca2+-dependent activator protein for secretion), a protein required for Ca2+-dependent exocytosis of dense-core vesicles, secretory responses in single rat melanotrophs were monitored by patch-clamp membrane capacitance measurements. Flash photolysis of caged Ca2+ elicited biphasic capacitance increases consisting of rapid and slow components with distinct Ca2+ dependencies. A threshold of ≈10 μM Ca2+ was required to trigger the slow component, while the rapid capacitance increase was recorded already at a intracellular Ca2+ activity < 10 μM. Both kinetic membrane capacitance components were abolished by botulinum neurotoxin B or E treatment, suggesting involvement of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor)-dependent vesicle fusion. The rapid but not the slow component was inhibited by CAPS antibody. These results were further clarified by immunocytochemical studies that revealed that CAPS was present on only a subset of dense-core vesicles. Overall, the results indicate that dense-core vesicle exocytosis in melanotrophs occurs by two parallel pathways. The faster pathway exhibits high sensitivity to Ca2+ and requires the presence of CAPS, which appears to act at a late stage in the secretory pathway.

Expression of αv and β3 integrin chains on murine lymphocytes

The vitronectin receptor is a member of the integrin family of adhesion protein receptors and binds a broad spectrum of ligands, including fibronectin and fibrinogen in addition to vitronectin. We have generated four mAbs that recognize the murine αvβ3 vitronectin receptor. Biochemical and expression analyses showed that two of the mAbs are specific for the αv chain, and two are specific for the β3 chain. The mAbs are effective blocking reagents and inhibited cell adhesion to vitronectin, fibrinogen, and fibronectin. Staining analysis revealed expression of αv and β3 on certain populations of murine thymocytes, splenocytes, and bone marrow cells. The expression of αv and β3 appeared to be modulated at specific stages of thymocyte development, suggesting a possible function for the αvβ3 vitronectin receptor in T cell development.

Molecular cloning and characterization of a murine pre-B-cell growth-stimulating factor/stromal cell-derived factor 1 receptor, a murine homolog of the human immunodeficiency virus 1 entry coreceptor fusin

Pre-B-cell growth-stimulating factor/stromal cell-derived factor 1 (PBSF/SDF-1) is a member of the CXC group of chemokines that is initially identified as a bone marrow stromal cell-derived factor and as a pre-B-cell stimulatory factor. Although most chemokines are thought to be inducible inflammatory mediators, PBSF/SDF-1 is essential for perinatal viability, B lymphopoiesis, bone marrow myelopoiesis, and cardiac ventricular septal formation, and it has chemotactic activities on resting lymphocytes and monocytes. In this paper, we have isolated a cDNA that encodes a seven transmembrane-spanning-domain receptor, designated pre-B-cell-derived chemokine receptor (PB-CKR) from a murine pre-B-cell clone, DW34. The deduced amino acid sequence has 90% identity with that of a HUMSTSR/fusin, a human immunodeficiency virus 1 (HIV-1) entry coreceptor. However, the second extracellular region has lower identity (67%) compared with HUMSTSR/fusin. PB-CKR is expressed during embryo genesis and in many organs and T cells of adult mice. Murine PBSF/SDF-1 induced an increase in intracellular free Ca2+ in DW34 cells and PB-CKR-transfected Chinese hamster ovary (CHO) cells, suggesting that PB-CKR is a functional receptor for murine PBSF/SDF-1. Murine PBSF/SDF-1 also induced Ca2+ influx in fusin-transfected CHO cells. On the other hand, considering previous results that HIV-1 does not enter murine T cells that expressed human CD4, PB-CKR may not support HIV-1 infection. Thus, PB-CKR will be an important tool for functional mapping of HIV-1 entry coreceptor fusin and for understanding the function of PBSF/SDF-1 further.

Human cytomegalovirus harbors its own unique IL-10 homolog (cmvIL-10)

We identified a viral IL-10 homolog encoded by an ORF (UL111a) within the human cytomegalovirus (CMV) genome, which we designated cmvIL-10. cmvIL-10 can bind to the human IL-10 receptor and can compete with human IL-10 for binding sites, despite the fact that these two proteins are only 27% identical. cmvIL-10 requires both subunits of the IL-10 receptor complex to induce signal transduction events and biological activities. The structure of the cmvIL-10 gene is unique by itself. The gene retained two of four introns of the IL-10 gene, but the length of the introns was reduced. We demonstrated that cmvIL-10 is expressed in CMV-infected cells. Thus, expression of cmvIL-10 extends the range of counter measures developed by CMV to circumvent detection and destruction by the host immune system.