Mice lacking histidine decarboxylase exhibit abnormal mast cells

Histidine decarboxylase (HDC) synthesizes histamine from histidine in mammals. To evaluate the role of histamine, we generated HDC-deficient mice using a gene targeting method. The mice showed a histamine deficiency and lacked histamine-synthesizing activity from histidine. These HDC-deficient mice are viable and fertile but exhibit a decrease in the numbers of mast cells while the remaining mast cells show an altered morphology and reduced granular content. The amounts of mast cell granular proteases were tremendously reduced. The HDC-deficient mice provide a unique and promising model for studying the role of histamine in a broad range of normal and disease processes.     

Anti-cathepsin L monoclonal antibodies that distinguish cathepsin L from cathepsin V

Cathepsin L is a lysosomal cysteine protease involved in intracellular protein degradation. Recently, several new cysteine proteases have been identified. Human cathepsin V, a thymus- and testis-specific human cysteine protease, shares 78% sequence identity with human cathepsin L. Due to the strong sequence similarity, highly selective reagents are needed to elucidate the physiological functions of the two enzymes. Monoclonal antibodies (mAbs) have been prepared against recombinant human cathepsin L. Antibodies produced by five clones reacted with procathepsin L and mature cathepsin L. They also reacted with cathepsin L in complex with a peptide fragment, which is identical to the alternatively spliced segment of the p41 form of MHC Class II associated invariant chain. Two mAbs, (M105 and H102) were specific for cathepsin L, while three (N135, B145 and D24) cross-reacted with cathepsin V. None of the mAbs cross-reacted with cathepsins B, H and S. We have developed a sandwich enzyme-linked immunosorbent assay (ELISA) for quantifying cathepsin L. This sandwich ELISA uses a combination of two monoclonal antibodies which recognize different, non-overlapping epitopes on the cathepsin L molecule. The lower detection limit of the sandwich ELISA was 5 ng of cathepsin L per ml.     

Mice lacking Homer 1 exhibit a skeletal myopathy characterized by abnormal transient receptor potential channel activity

Transient receptor potential (TRP) channels are nonselective cation channels, several of which are expressed in striated muscle. Because the scaffolding protein Homer 1 has been implicated in TRP channel regulation, we hypothesized that Homer proteins play a significant role in skeletal muscle function. Mice lacking Homer 1 exhibited a myopathy characterized by decreased muscle fiber cross-sectional area and decreased skeletal muscle force generation. Homer 1 knockout myotubes displayed increased basal current density and spontaneous cation influx. This spontaneous cation influx in Homer 1 knockout myotubes was blocked by reexpression of Homer 1b, but not Homer 1a, and by gene silencing of TRPC1. Moreover, diminished Homer 1 expression in mouse models of Duchenne's muscular dystrophy suggests that loss of Homer 1 scaffolding of TRP channels may contribute to the increased stretch-activated channel activity observed in mdx myofibers. These findings provide direct evidence that Homer 1 functions as an important scaffold for TRP channels and regulates mechanotransduction in skeletal muscle.     

Expression of mouse cathepsin L cDNA in Saccharomyces cerevisiae: evidence that cathepsin L is sorted for targeting to yeast vacuole.

To investigate the intracellular transport mechanism of lysosomal cathepsin L in yeast cells, we attempted to produce mouse cathepsin L in Saccharomyces cerevisiae by placing the coding region under the control of the promoter of the yeast glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene. Immunoblotting analysis by the use of an antibody specific for rat cathepsin L revealed that the yeast cells carrying the cathepsin L coding sequence produced 39- and 30-kDa products, which correspond to the rat procathepsin L and the single-chain form of mature cathepsin L, respectively. The precursor polypeptide showed sensitivity toward endoglycosidase H treatment. Cell fractionation experiments demonstrated that the processed form of 30-kDa cathepsin L was found to be colocalized to the yeast vacuole with the marker enzyme carboxypeptidase Y in a Ficoll step gradient. In the prepared vacuolar fraction, a considerable amount of cathepsin L was revealed to be cofractionated with the vacuolar membranes. Furthermore, the phase separation experiments with Triton X-114 provide the first evidence showing that the mature form of cathepsin L polypeptide is strongly associated with the vacuolar membranes. Therefore, the present results suggest that the mouse cathepsin L precursor polypeptide is initially synthesized as the proenzyme in the yeast cells and then correctly delivered to the vacuole. During the intracellular sorting pathway, the procathepsin L would undergo the post-translational proteolytic processing step to generate the mature enzyme. Based on these lines of evidence, we propose that cathepsin L is recognized by mechanisms similar to those for the intracellular sorting and processing of vacuolar proteins in the yeast cells.     

Transgenic regal pelargoniums that express the rolC gene from Agrobacterium rhizogenes exhibit a dwarf floral and vegetative phenotype

Summary The regal pelargonium, ev. Dubonnet, was transformed using the disarmed Agrobacterium tumefaciens strains LBA4404 or EHA105 containing the binary vector pLN70. This plasmid carries on its T-DNA the rolC gene from Agrobacterium rhizogenes under control of the CaMV 35S promoter and the npt II selectable marker gene under a NOS promoter. Six independent transformants were produced and grouped according to their phenotypic characteristics. Two transformants showed the same phenotype as the untransformed control plants. Three transformants exhibited a dwarf phenotype and one displayed a super-dwarf phenotype. Southern hybridization analyses of the T-DNA left border region using a npt II probe showed that the six transformants all arose from independent transformation events. Northern hybridization analyses showed that the rolC gene was expressed only in the four transformants that exhibited a dwarf phenotype. Our data show that the phenotypic effects of rolC expression in regal pelargoniums include reductions in plant height, leaf area, petal area, and corolla length. Earlier flowering of the rolC transgenics by up to 22d was also observed.     

Derivation of cytotoxic T cell lines that express T cell receptor but exhibit reversible loss of stable antigen binding

Stable cell lines lacking cytotoxic activity against specific target cells were derived from highly active murine CTL clones by the omission of antigen from the culture for several weeks. Several independent CTL clones cultured in the absence of antigen showed a gradual decline in cytotoxic activity, resulting in complete loss by 5 to 10 wk. Such noncytotoxic (NC) cells lacked the ability to form stable conjugates with specific target cells, but were able to kill all target cells tested in the presence of Con A. It was shown by subcloning at limiting dilution that all cells in the starting population were cytolytically active, and that all cells in the NC population derived from such a clone were cytolytically inactive against target cells bearing an appropriate antigen under normal assay conditions. By using the monoclonal antibody F23.1, which reacted with the antigen receptors of two of the CTL clones, it was shown that the NC cells derived from these clones continued to express the receptor at normal levels. Levels of expression of Thy-1.2, Lyt-2.2, and LFA-1 were also similar in all cytotoxic cell lines and their noncytotoxic derivatives. The F23.1 antibody induced an increase in cytoplasmic free Ca2+ in both CTL and NC cells, and NC cells lysed F23.1 hybridoma cells in the absence of Con A. When cells expressing appropriate target cell antigen were added back to cultures of NC cells, cytotoxic activity of appropriate specificity was fully recovered in 2 wk. These results indicate that expression of an apparently functional antigen receptor alone is insufficient for stable binding of CTL to specific target cells, and that other factors dependent upon antigen stimulation may be involved in the recognition process. A difference in affinity for antigen between CTL and NC cells is suggested as a possible explanation for these observations.     

Mice deficient in endothelin-converting enzyme-2 exhibit abnormal responses to morphine and altered peptide levels in the spinal cord

An increasing body of evidence suggests that endothelin-converting enzyme-2 (ECE-2) is a non-classical neuropeptide processing enzyme. Similar to other neuropeptide processing enzymes, ECE-2 exhibits restricted neuroendocrine distribution, intracellular localization, and an acidic pH optimum. However, unlike classical neuropeptide processing enzymes, ECE-2 exhibits a non-classical cleavage site preference for aliphatic and aromatic residues. We previously reported that ECE-2 cleaves a number of neuropeptides at non-classical sites in vitro; however its role in peptide processing in vivo is poorly understood. Given the recognized roles of neuropeptides in pain and opiate responses, we hypothesized that ECE-2 knockout (KO) mice might show altered pain and morphine responses compared with wild-type mice. We find that ECE-2 KO mice show decreased response to a single injection of morphine in hot-plate and tail-flick tests. ECE-2 KO mice also show more rapid development of tolerance with prolonged morphine treatment and fewer signs of naloxone-precipitated withdrawal. Peptidomic analyses revealed changes in the levels of a number of spinal cord peptides in ECE-2 KO as compared to wild-type mice. Taken together, our findings suggest a role for ECE-2 in the non-classical processing of spinal cord peptides and morphine responses; however, the precise mechanisms through which ECE-2 influences morphine tolerance and withdrawal remain unclear.     

Mice deficient for the lysosomal proteinase cathepsin D exhibit progressive atrophy of the intestinal mucosa and profound destruction of lymphoid cells.

Mice deficient for the major lysosomal aspartic proteinase cathepsin D, generated by gene targeting, develop normally during the first 2 weeks, stop thriving in the third week and die in a state of anorexia at day 26 +/- 1. An atrophy of the ileal mucosa first observed in the third week progresses towards widespread intestinal necroses accompanied by thromboemboli. Thymus and spleen undergo massive destruction with fulminant loss of T and B cells. Lysosomal bulk proteolysis is maintained. These results suggest, that vital functions of cathepsin D are exerted by limited proteolysis of proteins regulating cell growth and/or tissue homeostasis, while its contribution to bulk proteolysis in lysosomes appears to be non-critical.     

Mice deficient in heparan sulfate 6-O-sulfotransferase-1 exhibit defective heparan sulfate biosynthesis, abnormal placentation, and late embryonic lethality

Heparan sulfate (HS) plays critical roles in a variety of developmental, physiological, and pathogenic processes due to its ability to interact in a structure-dependent manner with numerous growth factors that participate in cellular signaling. The divergent structures of HS glycosaminoglycans are the result of the coordinate actions of several N- and O-sulfotransferases, C5-epimerase, and 6-O-endosulfatases. We have shown that 6-O-sulfation of the glucosamine residues in HS are catalyzed by the sulfotransferases HS6ST-1, -2, and -3. To determine the biological and physiological importance of HS6ST-1, we now describe the creation of transgenic mice that lack this sulfotransferase. Most of our HS6ST-1-null mice died between embryonic day 15.5 and the perinatal stage, and those mice that survived were considerably smaller than their wild-type littermates. Some of these HS6ST-1-null mice exhibited development abnormalities, and histochemical and molecular analyses of these mice revealed an approximately 50% reduction in the number of fetal microvessels in the labyrinthine zone of the placenta relative to that in the wild-type mice. Because we observed a modest reduction in VEGF-A mRNA and protein in the tissues of HS6ST-1-null mice, an HS-dependent defect in cytokine signaling probably contributes to increased embryonic lethality and decreased growth. Biochemical studies of the HS chains isolated from various organs of our HS6ST-1-null mice revealed a marked reduction of GlcNAc(6SO(4)) and HexA-GlcNSO(3)(6SO(4)) levels and a reduced ability to bind Wnt2. Thus, despite the presence of three closely related 6-O-sulfotransferase genes in the mouse genome, HS6ST-1 is the primary one used in HS biosynthesis in most tissues.     

Mice lacking Dad1, the defender against apoptotic death-1, express abnormal N-linked glycoproteins and undergo increased embryonic apoptosis

Dad1 has been shown to play a role in preventing apoptotic cell death and in regulating levels of N-linked glycosylation in Saccharomyces cerevisiae and the BHK hamster cell line. To address the in vivo role of Dad1 in these processes during multicellular development, we have analyzed mice carrying a null allele for Dad1. Embryos homozygous for this mutation express abnormal N-glycosylated proteins and are developmentally delayed by embryonic day 7.5. Such mutants exhibit aberrant morphology, impaired mesodermal development, and increased levels of apoptosis in specific tissues. These defects culminate in homozygous embryos failing to turn the posterior axis and subsequent lethality by embryonic day 10.5. Thus, Dad1 is required for proper processing of N-linked glycoproteins and for certain cell survival in the mouse.     

Exosomes from human lymphoblastoid B cells express enzymatically active CD38 that is associated with signaling complexes containing CD81, Hsc-70 and Lyn

Exosome vesicles of endocytic origin are involved in communication between tumor and immune cells. In addition, membrane rafts (MR) may support the sorting of proteins associated with exosomes. CD38 is found at the plasma membrane and in recycling endosomes, which are both redistributed toward the immunological synapse (IS) upon T cell antigen receptor (TCR) engagement. The data of this study provide evidence that CD38 is expressed on the surface of secreted exosomes derived from lymphoblastoid B cells. Exosomic CD38 is associated with the signaling molecules CD81, Hsc-70 and Lyn. Likewise, in MR, CD38 is associated with CD81, CD19, Lyn, Gαi-2, Hsc-70 and actin. Therefore, a high degree of overlap in the pattern of signaling proteins associated with CD38 in exosomes and MR exists. Exosomic and MR CD38, by virtue of these interactions, have signaling potential. Indeed, CD38 is enzymatically active in both exosomes and MR, and CD38 ligation induces Akt/PKB and Erk activation, which is accompanied by increased translocation of CD38 into MR. In conclusion, the present study indicates that CD38 localizes to MR, where it promotes cell signaling, and it is exported out of the cells through the exosome-mediated exocytic pathway, where it may act as an intercellular messenger.     

Trypanosoma brucei: chemical evidence that cathepsin L is essential for survival and a relevant drug target

The protozoan parasite causing human African trypanosomiasis, Trypanosoma brucei, displays cysteine peptidase activity, the chemical inhibition of which is lethal to the parasite. This activity comprises a cathepsin B (TbCATB) and a cathepsin L (TbCATL). Previous RNA interference (RNAi) data suggest that TbCATB rather than TbCATL is essential to survival even though silencing of the latter was incomplete. Also, chemical evidence supporting the essentiality of either enzyme which would facilitate a target-based drug development programme is lacking. Using specific peptidyl inhibitors and substrates, we quantified the contributions of TbCATB and TbCATL to the survival of T. brucei. At 100 μM, the minimal inhibitory concentration that kills all parasites in culture, the non-specific cathepsin inhibitors, benzyloxycarbonyl-phenylalanyl-arginyl-diazomethyl ketone (Z-FA-diazomethyl ketone) and (l-3-trans-propylcarbamoyloxirane-2-carbonyl)-l-isoleucyl-l-proline methyl ester (CA-074Me) inhibited TbCATL and TbCATB by >99%. The cathepsin L (CATL)-specific inhibitor, ((2S,3S)-oxirane-2,3-dicarboxylic acid 2-[((S)-1-benzylcarbamoyl-2-phenyl-ethyl)-amide] 3-{[2-(4-hydroxy-phenyl)-ethyl]-amide}) (CAA0225), killed parasites with >99% inhibition of TbCATL but only 70% inhibition of TbCATB. Conversely, the cathepsin B (CATB)-specific inhibitor, (l-3-trans-propylcarbamoyloxirane-2-carbonyl)-l-isoleucyl-l-proline (CA-074), did not affect survival even though TbCATB inhibition at >95% was statistically indistinguishable from the complete inhibition by Z-FA-diazomethyl ketone and CA-074Me. The observed inhibition of TbCATL by CA-074 and CA-074Me was shown to be facilitated by the reducing intracellular environment. All inhibitors, except the CATB-specific inhibitor, CA-074, blockaded lysosomal hydrolysis prior to death. The results suggest that TbCATL, rather than TbCATB, is essential to the survival of T. brucei and an appropriate drug target.     

Identification of 9 uterine genes that are regulated during mouse pregnancy and exhibit abnormal levels in the cyclooxygenase-1 knockout mouse

Background  

Preterm birth is the leading cause of all infant mortality. In 2004, 12.5% of all births were preterm. In order to understand preterm labor, we must first understand normal labor. Since many of the myometrial changes that occur during pregnancy are similar in mice and humans and mouse gestation is short, we have studied the uterine genes that change in the mouse during pregnancy. Here, we used microarray analysis to identify uterine genes in the gravid mouse that are differentially regulated in the cyclooxygenase-1 knockout mouse model of delayed parturition.     

Inhibitor studies indicate that active cathepsin L is probably essential to its own processing in cultured fibroblasts.

Somatostatin (somatotropin release inhibiting factor; SRIF) has widespread functions as a modulator of neural activity as well as of endocrine and exocrine secretion. In the present paper, the binding characteristics of somatostatin receptors have been investigated in rat long bones using the stable analogue, 125I-SDZ 204-090, as a ligand. Binding studies revealed the presence of a single class of high-affinity binding sites for 125I-SDZ 204-090 on cells prepared from neonatal rat long bones with an equilibrium dissociation constant (KD) of 70.1 +/- 8.2 pM (n = 3). An excellent correlation was found between the ability of various somatostatin analogues to inhibit growth hormone in pituitary cells and to displace the binding of 125I-SDZ 204-090 to the bone cell preparation, indicating that the receptors are very similar, if not identical. The localization of the somatostatin-binding sites was examined by autoradiography after labelling in vitro and in vivo. The binding sites were shown by both procedures to be selectively localized to the metaphysis of rat long bones. The labelling experiments in vivo indicate that these receptors can be reached in the living animal by circulating somatostatin analogues. In addition, the analogue SMS 201-995 inhibited the forskolin-stimulated adenylate cyclase activity in bone cell suspensions. These results suggest that somatostatin could be an important regulatory factor in bone metabolism.     

A new pathway that regulates 53BP1 stability implicates cathepsin L and vitamin D in DNA repair

Genomic instability due to telomere dysfunction and defective repair of DNA double-strand breaks (DSBs) is an underlying cause of ageing-related diseases. 53BP1 is a key factor in DNA DSBs repair and its deficiency is associated with genomic instability and cancer progression. Here, we uncover a novel pathway regulating the stability of 53BP1. We demonstrate an unprecedented role for the cysteine protease Cathepsin L (CTSL) in the degradation of 53BP1. Overexpression of CTSL in wild-type fibroblasts leads to decreased 53BP1 protein levels and changes in its cellular distribution, resulting in defective repair of DNA DSBs. Importantly, we show that the defects in DNA repair associated with 53BP1 deficiency upon loss of A-type lamins are due to upregulation of CTSL. Furthermore, we demonstrate that treatment with vitamin D stabilizes 53BP1 and promotes DNA DSBs repair via inhibition of CTSL, providing an as yet unsuspected link between vitamin D action and DNA repair. Given that CTSL upregulation is a hallmark of cancer and progeria, regulation of this pathway could be of great therapeutic significance for these diseases.     

L3T4 (CD4+) cells that mediate contact sensitivity to trinitrochlorobenzene express I-A determinants

The present study has further characterized the T cell-mediated inflammatory response of contact sensitivity (CS) to the hapten trinitrochlorobenzene (TNCB) in mice. A discernible CS response was found to be induced as early as 2 days after epicutaneous application of TNCB. The response peaked on Days 4 to 5 and it then declined to a nearly undetectable level by Days 10 to 11. Examination of the draining lymph nodes demonstrated that development of CS coincided with an increase in cellular proliferation and in the total number of cells present. Despite a severalfold increase in the cellular contents of the draining lymph nodes of sensitized mice, the relative percentages of most subsets of T cells remained unchanged. Flow cytometric studies revealed that the subpopulation of T cells characterized as Thy 1.2+ L3T4+ I-A+ increased substantially in comparison to its presence in unsensitized mice. Whether the Thy 1.2+ L3T4+ I-A+ cells that increased following sensitization represented the effector population that mediates CS was then examined. Four-day immune lymph node T cells or L3T4 cells positively selected from them were capable of adoptively transferring CS to normal mice. However, these cells, after treatment with anti-Ia antibody or anti-I-A monoclonal antibody and complement, were unable to transfer CS. These findings imply that expression of I-A determinants may indicate antigen-induced T cell activation in vivo and that L3T4 cells that mediate CS are I-A positive.     

Spontaneous germ cell death in the testis of the adult rat takes the form of apoptosis: re-evaluation of cell types that exhibit the ability to die during spermatogenesis

Spontaneous germ cell degeneration occurs in the testis of the adult rat. Accumulating data supports the idea that this degeneration takes place via apoptosis. We have determined that morphology, acridine orange staining and ultrastructural features of these cell deaths clearly take the form of apoptosis. Furthermore, with acridine orange staining it was possible to detect a cell population showing early signs of death. The characterization of the main morphological features of these cells allowed us to identify several steps of maturing germ cells undergoing degeneration that have not previously been described. We have reevaluated in toluidine blue stained semithin sections the germ cell types that undergo cell death at every stage of the spermatogenic cycle in the adult rat and concluded that, spermatogonia undergo cell death coinciding with their mitotic peaks, spermatocytes during preleptotene, leptotene, zygotene, pachytene and during metaphase I and spermatids during all their maturation steps. The biological significance of these cell deaths, at these steps of germ cell development, in relation to apoptosis, is discussed.     

A novel bud mutation that confers abnormal patterns of lycopene accumulation in sweet orange fruit (Citrus sinensis L. Osbeck)

A novel, pleiotropic sweet orange (Citrus sinensis L. Osbeck) mutant, 'Hong Anliu', is described. This mutation causes carotenoid accumulation, high sugar, and low acid in the fruits. Gas chromatographic analysis revealed that high sugar and low acid in the fruit were caused by the accumulation of sucrose and the deficiency of citric acid. The dominant carotenoid accumulated in albedo, segment membranes, and juice sacs is lycopene, which can reach levels that are a 1000-fold higher than those in comparable wild-type fruits. This mutation does not affect the carotenoid composition of leaves. Carotenoid concentration and biosynthetic gene expression of albedo, segment membranes, and juice sacs were dramatically altered by the mutation. Lycopene accumulation in the juice sacs was regulated by co-ordinate expression of carotenoid biosynthetic genes. However, in albedo and segment membranes, the expression of downstream carotenogenic genes seems to be feedback induced by lycopene accumulation. This implies that there must be at least two modes regulating lycopene accumulation in 'Hong Anliu' fruit. Taken together, these results suggest that massive amounts of lycopene might be synthesized in the juice sacs and then transported to the segment membrane and the albedo, which leads to lycopene accumulation there.