Mutations in a gene encoding an ABC transporter cause pseudoxanthoma elasticum.

Pseudoxanthoma elasticum (PXE) is a heritable disorder characterized by calcification of elastic fibres in skin, arteries and retina that results in dermal lesions with associated laxity and loss of elasticity, arterial insufficiency and retinal haemorrhages leading to macular degeneration. PXE is usually found as a sporadic disorder, but examples of both autosomal recessive and autosomal dominant forms of PXE have been observed. Partial manifestations of the PXE phenotype have also been described in presumed carriers in PXE families. Linkage of both dominant and recessive forms of PXE to a 5-cM domain on chromosome 16p13.1 has been reported (refs 8,9). We have refined this locus to an 820-kb region containing 6 candidate genes. Here we report the exclusion of five of these genes and the identification of the first mutations responsible for the development of PXE in a gene encoding a protein associated with multidrug resistance (ABCC6).

A novel fibrotic disorder associated with increased dermal fibroblast proliferation and downregulation of genes of the microfibrillar network.

Clinical evaluation of a young woman with subcutaneous fibrotic nodules, progressive distal joint contractures and marfanoid stature revealed a previously unrecognized fibrotic disorder characterized by several unique phenotypic features and some features overlapping with known disorders. Mutational analysis of the FBN1 and FBN2 genes excluded Marfan syndrome and congenital contractural arachnodactyly. MMP2 gene sequence analysis excluded multicentric osteolysis, nodulosis and arthropathy. The lack of mutations within the MAGP2 gene also excluded an MAGP2-associated disorder. In order to establish the mechanistic basis for the severe skin pathology noted in this patient, we performed transcriptional profiling of dermal fibroblasts, and candidate gene expression studies in conjunction with immunocytochemistry and cell-based and functional assays. Data from these experiments have further excluded any previously recognized fibrotic disorder and identified a unique pattern of gene expression in this patient consistent with progressive fibrosis. The pathogenic mechanisms included persistent proliferation of dermal fibroblasts in coexistence with a disarray of the microfibrillar network. Collagen accumulation, moreover, could be linked to extensive crosslinking resulting from increased activities of lysyl oxidases (LOX and LOXL), and lack of remodelling due to deficiencies in collagenolytic matrix metalloproteinases. The disorder may represent a novel syndrome in which transforming growth factor-ß1-independent dermal fibrosis, unlike known microfibrillar disorders caused by single gene deficiencies, associates with a disarray of the microfibrillar network.

Lysyl oxidases: a novel multifunctional amine oxidase family.

Lysyl oxidase (LOX), a copper-containing amine oxidase, belongs to a heterogeneous family of enzymes that oxidize primary amine substrates to reactive aldehydes. LOX has been traditionally known for one function, the extracellular catalysis of lysine-derived cross-links in fibrillar collagens and elastin. More recently, diverse roles have been attributed to lysyl oxidase and these novel activities cover a spectrum of diverse biological functions such as developmental regulation, tumor suppression, cell motility, and cellular senescence. Lysyl oxidase has also been shown to have both intracellular and intranuclear locations. The multifunctional properties of lysyl oxidase (LOX) and our recent discovery of three novel members of this amine oxidase family, LOX-like (LOXL), LOXL2, and LOXL3, indicate the possibility that these varied functions are performed in both intracellular and extracellular environments by individual novel members of the LOX amine-oxidase family. Structural similarities of the highly conserved copper-binding and lysyl-tyrosylquinone cofactor sites among the LOX and LOX-like proteins may result in similar amine oxidase activities. However, specific novel functions, such as a potential role in cell adhesion and cell growth control, will be determined by other, conserved domains such as the cytokine receptor-like domain that is shared by all LOXs and by multiple scavenger receptor cysteine-rich (SRCR) domains present in LOXL2 and LOXL3. Furthermore, these functions may be carried out in a temporally and spatially regulated fashion.

Regulatory elements downstream of the promoter of an rRNA gene of E. coli.

Previously we have shown that plasmid constructs carrying a reporter gene fused to the P2 promoter of the E. coli rrnB gene exhibited a strange two-phase kinetics of expression depending on the physiological conditions of the cell if a short DNA region downstream of the promoter was present between the promoter and the reporter gene (Lukacsovich et al. (1987) J. Bacteriol. 169, 272-277). Insertion of a synthetic oligonucleotide corresponding to the first half of this region into constructs where the reporter directly follows the promoter, leads to a complete block of expression in vivo, while in vitro--in a purified system--transcription is not inhibited. Band-shift experiments indicate that the putative regulatory region downstream of the promoter specifically binds protein(s) present in total bacterial extracts.

Structural and functional diversity of lysyl oxidase and the LOX-like proteins.

Lysyl oxidase (LOX) and four lysyl oxidase-like proteins, LOXL, LOXL2, LOXL3 and LOXL4, each contain a copper binding site, conserved lysyl and tyrosyl residues that may contribute to quinone co-factor formation, and a cytokine receptor-like domain. Each protein differs mainly in their N-terminal sequence, which may confer individual functions. Processing of the LOX proteins by BMP-1 and possibly other mechanisms may result in multiple functional forms. Splicing, reported for LOXL3, may also generate additional variants with unique functions. Each LOX, with its individual, developmentally regulated tissue and cell-specific expression and localization, results in a complex structural and functional variation for the LOX amine oxidases. The presence of only two LOX-like proteins in Drosophila, each with distinct spatial and temporal expression, allows for the assignment of individual function to one of these amine oxidases. Comparative expression analysis of each LOX protein is presented to help determine their functional significance.

Lysyl oxidase-like 2 (LOXL2) and E47 EMT factor: novel partners in E-cadherin repression and early metastasis colonization.

Epithelial-mesenchymal transition (EMT) has been associated with increased aggressiveness and acquisition of migratory properties providing tumor cells with the ability to invade into adjacent tissues. Downregulation of E-cadherin, a hallmark of EMT, is mediated by several transcription factors (EMT-TFs) that act also as EMT inducers, among them, Snail1 and the bHLH transcription factor E47. We previously described lysyl oxidase-like 2 (LOXL2), a member of the lysyl oxidase family, as a Snail1 regulator and EMT inducer. Here we show that LOXL2 is also an E47-interacting partner and functionally collaborates in the repression of E-cadherin promoter. Loss and gain of function analyses combined with in vivo studies in syngeneic breast cancer models demonstrate the participation of LOXL2 and E47 in tumor growth and their requirement for lung metastasis. Furthermore, LOXL2 and E47 contribute to early steps of metastatic colonization by cell and noncell autonomous functions regulating the recruitment of bone marrow progenitor cells to the lungs and by direct transcriptional regulation of fibronectin and cytokines TNFα, ANG-1 and GM-CSF. Moreover, fibronectin and GM-CSF proved to be necessary for LOXL2/E47-mediated modulation of tumor growth and lung metastasis.

A novel human lysyl oxidase-like gene (LOXL4) on chromosome 10q24 has an altered scavenger receptor cysteine rich domain.

We have identified a novel 14-exon human lysyl oxidase-like gene, LOXL4, on chromosome 10q24. The cDNA and derived amino acid sequence of LOXL4 demonstrates a conserved C-terminal region including the characteristic copper-binding site, lysyl and tyrosyl residues and a cytokine receptor-like domain. One of the four N-terminal SRCR domains contains a 13 amino acid insertion encoded by a short exon not present within the closely homologous LOXL2 and LOXL3 genes. The 3.5-kb LOXL4 mRNA is present in pancreas and testis and at lower levels in several other tissues. Fibroblasts, smooth muscle and osteosarcoma (HOS) cells express LOXL4. No expression was detected in HCT-116 and DLD-1 colon, MCF-7 breast and DU-145 prostate cancer cell lines.

The mouse lysyl oxidase-like 2 gene (mLOXL2) maps to chromosome 14 and is highly expressed in skin, lung and thymus.

The predicted amino acid sequence derived from a mouse expressed sequence tag (EST) contig contained two domains that are highly conserved among members of the lysyl oxidase gene family: a copper binding-site with four histidines and a catalytic domain that includes a tryptophan residue. This new cDNA sequence showed the highest level of sequence homology with the human loxl2 cDNA and suggested that it encoded the mouse equivalent of hLOXL2. The mLOXL2 gene was mapped to chromosome 14 by radiation hybrid analysis. The mLOXL2 locus was tightly linked with a LOD score over 9 to the marker D14Mit32. The mLOXL2 gene is expressed as a 4-kb mRNA in almost all tissues analyzed, with highest levels of mRNA in skin, lung and thymus.

Hyperglycemia enhances DNA fragmentation after transient cerebral ischemia.

Previous histopathologic results have suggested that one mechanism whereby hyperglycemia (HG) leads to exaggerated ischemic damage involves fragmentation of DNA. DNA fragmentation in normoglycemia (NG) and HG rats subjected to 30 minutes of forebrain ischemia was studied by terminal deoxynucleotidyl transferase mediated DNA nick-labeling (TUNEL) staining, by pulse-field gel electrophoresis (PFGE), and by ligation-mediated polymerase chain reaction (LM-PCR). High molecular weight DNA fragments were detected by PFGE, whereas low molecular weight DNA fragments were detected using LM-PCR techniques. The LM-PCR procedure was performed on DNA from test samples with blunt (without Klenow polymerase) and 3'-recessed ends (with Klenow polymerase). In addition, cytochrome c release and caspase-3 activation were studied by immunocytochemistry. Results show that HG causes cytochrome c release, activates caspase-3, and exacerbates DNA fragments induced by ischemia. Thus, in HG rats, but not in control or NGs, TUNEL-stained cells were found in the cingulate cortex, neocortex, thalamus, and dorsolateral crest of the striatum, where neuronal death was observed by conventional histopathology, and where both cytosolic cytochrome c and active caspase-3 were detected by confocal microscopy. In the neocortex, both blunt-ended and stagger-ended fragments were detected in HG, but not in NG rats. Electron microscopy (EM) analysis was performed in the cingulate cortex, where numerous TUNEL-positive neurons were observed. Although DNA fragmentation was detected by TUNEL staining and electrophoresis techniques, EM analysis failed to indicate apoptotic cell death. It is concluded that HG triggers a cell death pathway and exacerbates DNA fragmentation induced by ischemia.

Transcriptional analysis of the cyanobacterial gvpABC operon in differentiated cells: occurrence of an antisense RNA complementary to three overlapping transcripts.

Cyanobacteria are photosynthetic prokaryotes able to colonize almost all kinds of ecosystems. Some of them exhibit differentiation processes and/or may establish tight symbiotic associations. Upon changes in the environmental conditions, the cyanobacterium Calothrix 7601 differentiates hormogonia which are short filaments of small cells resulting from cellular division and fragmentation of the long filaments of vegetative cells. In Calothrix, hormogonia are characterized by their gliding motility and by a massive production of gas vesicles which confer buoyancy. At least four genes are involved in the formation of gas vesicles, three of which are organized in one operon (gvpABC). Four different RNA species, only present after induction of hormogonia differentiation, result from transcription of this operon. Mapping of the 5' and 3' ends of these transcripts demonstrates the presence of gvpA, gvpAB and gvpABC transcripts, all three having the same 5' end. Each of the three transcripts terminates a few bases downstream from stem-and-loop structures. Most interestingly, the fourth transcript is an antisense RNA starting from the 3' end of the gvpB gene and ending within the gvpA gene. This antisense RNA can thus form an homologous duplex with the three other transcripts, thereby being able to impair translation and/or modify mRNA stability.

A developmentally regulated gvpABC operon is involved in the formation of gas vesicles in the cyanobacterium Calothrix 7601.

In the filamentous cyanobacterium Calothrix PCC7601, gas-vesicle (GV) formation is restricted to specialized filaments, called hormogonia. The differentiation of these cells is controlled by environmental factors, such as light intensity and/or wavelength. The structural gene (gvpA) encoding a GV protein in this cyanobacterium has been previously cloned and sequenced. Two other genes, gvpB and gvpC have been found in the sequence downstream from gvpA. The gvpB gene corresponds to a second copy of gvpA, encoding an identical protein. Unlike the GV protein, the product of the gvpC gene is predominantly hydrophilic, as deduced from nucleotide sequence. Interestingly, the internal part of the gvpC gene is composed of four contiguous repeats, each containing 99 bp, forming highly homologous repeats in the deduced amino acid sequence. Another kind of periodicity has been detected inside the 99-bp repeats, suggesting that the gvpC gene might have evolved by amplification of a 33-bp-long primordial building block. The function of this gene remains to be elucidated. Finally, we have shown that the three genes, gvpA, gvpB, and gvpC, are organized in an operon that is exclusively expressed during GV formation in hormogonia.

Free radical spin trap alpha-phenyl-N-tert-butyl-nitron inhibits caspase-3 activation and reduces brain damage following a severe forebrain ischemic injury.

It has been documented that alpha-phenyl-N-tert-butyl-nitron (PBN) possesses a potent neuroprotective effect when administered after transient focal cerebral ischemia. However, contradicting results were reported regarding its effect in transient global ischemia. To further elucidate the mechanism of PBN action, we have studied the effect of PBN on animal survival, histopathological outcome, and activation of caspase-3 following 30 min of global ischemia in vehicle- and PBN-treated rats. The results showed that 30 min of global ischemia was such a severe insult that no animal could survive beyond 2 d of reperfusion. Histopathological evaluation showed severe tissue edema and microinfarct foci in the neocortex and thalamus. Close to 100% damage was observed in the stratum and hippocampal CA1, CA3, and dentate gyrus subregions. Postischemic PBN treatment significantly enhanced animal survival and reduced damage in the neocortex, thalamus, and hippocampus. Immunohistochemistry demonstrated that caspase-3 was activated following ischemia in the striatum and the neocortex. PBN suppressed the activation of caspase-3 in both structures. It is concluded that PBN is a potent neuroprotectant against both focal and global ischemia; besides its function as a free radical scavenger, PBN may reduce ischemic brain damage by blocking cell death pathways that involve caspase-3 activation.

Analysis of the 5'-upstream regions of the human relaxin H1 and H2 genes and their chromosomal localization on chromosome 9p24.1 by radiation hybrid and breakpoint mapping.

Relaxins are known endocrine and autocrine/paracrine hormones that play a major role in reproduction. In the human there are two relaxin genes, H1 and H2 which share 90% sequence homology within their coding region. The biological and evolutionary significance of two highly homologous and biologically active human relaxins is unknown. In order to achieve a better understanding of the regulatory mechanisms involved in the differential expression of these two genes and to gain insight into their role(s) in the preterm premature rupture of the membranes, we have investigated the properties of their 5'-upstream regions and mapped them both by radiation hybrid and breakpoint mapping into the same chromosome 9p24.1 locus. The 5' ends of these relaxin genes could be divided into a proximal highly homologous segment and a distal non-homologous region. Within the proximal region are contained several putative regulatory elements common to both genes, suggesting a similar regulatory mechanism. The clustering of the relaxin genes within the same chromosomal locus suggests that these genes may be under a common regulation. On the other hand, a distinct gene-specific regulation may also exist for the individual relaxin genes since cis elements specific to each gene were identified at their 5' ends. Moreover, the observed divergence at the distal region of their 5'-upstream sequences may provide the structural features that act as gene-specific transcription regulators. Since the two genes are highly homologous in both their coding and flanking regions, the divergence at the distal region of their 5' ends may be important in the regulation of these genes and in their involvement in the pathology of preterm birth.

Lysyl oxidases: expression in the fetal membranes and placenta.

The cross-linking of the connective tissues in the fetal membranes and placenta is important for their tensile strength and elasticity. We have studied the expression of lysyl oxidase (LOX) because it is the classical enzyme responsible for the cross-linking of collagen and elastin. We have also studied the two recently described, genetically distinct lysyl oxidase-like genes and proteins, lysyl oxidase-like (LOXL) and lysyl oxidase-like 2 (LOXL2), of unknown functions. Specific antisera have been used for immunolocalization in fetal membranes and placentae from early pregnancy terminations and after caesarean section at both preterm and term, prior to labour. In addition, the steady state mRNA levels of the three genes has been quantitated in separated amnion, chorion, decidua and placentae collected at term before labour. The immunocytochemistry shows that the spatial expression of the three lysyl oxidases is similar in early pregnancy in both the fetal membranes and placentae. However, by preterm this pattern had diverged and becomes greatest at term. The expression of the genes found at term was similar to the results of protein expression obtained by immunocytochemistry, with the exception of LOXL which had high placental gene expression, but low levels of immunolocalized protein. Thus by term, LOX was expressed predominantly in the amniotic epithelium, with little expression in the placenta, while LOXL showed highest gene expression in the placenta and lowest expression in the amnion. LOXL2 expression was again different and was expressed predominantly in the chorionic cytotrophoblast of the membranes with low expression in both the amnion and placentae. These results suggest that these three members of the lysyl oxidase family may have similar roles in early pregnancy during the development of the placenta and fetal membranes, but their divergence as pregnancy advances to term, may reflect changes in substrate specificity and connective tissue composition.

Detection of elastin in the human fetal membranes: proposed molecular basis for elasticity.

The human fetal membranes provide a sterile biomechanical container which adjust by growth to mid-pregnancy to the increase in fetal size, and by elasticity to the forceful movements of the fetus. The molecular basis for this elasticity is not known, yet reduced elasticity may lead to their premature rupture and preterm birth, a major problem in perinatal medicine. Classically, elastin confers the property of elastic recoil to elastic fibres which are assembled from a family of tropoelastin precursors. These are covalently cross-linked to form insoluble elastin by formation of desmosine and isodesmosine, catalysed by the enzyme lysyl oxidase. The amnion, chorion and decidua were shown by Northern analysis and RT-PCR to contain detectable levels of tropoelastin mRNA and the mRNA encoding lysyl oxidase. The proteins encoded by these mRNAs were also identified by Western blotting and immunolocalization. Further, insoluble elastin was extracted from the human fetal membranes and shown by comparison to elastin preparations from other elastic tissues to have a reasonable desmosine content. Finally, scanning electron microscopy confirmed the presence of multiple layers of an apparently very thin elastic system in this tissue. This biochemical and histopathologic study has demonstrated therefore that the human fetal membranes synthesize and deposit a novel elastic fibre. The presence of such an elastic system in these tissues provides, for the first time, a probable molecular basis for the elastic properties of this tissue.

Does long-term glucose infusion reduce brain damage after transient cerebral ischemia?

A recent study reported that hyperglycemia of a brief duration worsens, and of long duration reduces, ischemic brain damage. To test whether this is a valid conception, we induced 10 min of transient forebrain ischemia, recorded postischemic seizures, and evaluated brain morphology. The results showed that administration of glucose 2 h before ischemia aggravated brain damage, induced seizures, and caused animal death in the same manner as was previously observed when glucose was given 30 min before ischemia. Thus, the conclusion that the influence of glucose on an ischemic transient is dependent upon the duration of hyperglycemia is unsubstantiated.

Intrathecal cyclosporin prolongs survival of late-stage ALS mice.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by upper and lower motor neuron death with ascending paralysis leading to death. In a transgenic mouse model of ALS (SOD1-G93A) weakness appears at 3 months of age, and because of progressive paralysis leads to death by 5 months. Cyclosporin A (CsA) is well known, for its extracerebral effect, as an immunosuppressant in organ transplantation. When able to access the brain, CsA is an effective neuroprotective agent mainly due to its protection of mitochondria through inhibition of the mitochondrial permeability transition. CsA does not cross the intact blood-brain barrier and was in the present study delivered to the brain through an infusion into the lateral cerebral ventricle. Injections started at the onset of late disease when weakness of the hindlimbs was apparent. CsA treatment prolonged the survival of ALS transgenic mice as compared to vehicle-treated controls. This finding implicates mitochondrial function in ALS and may have significance for human disease.

Mechanism of action of tricyclic drugs on Escherichia coli and Yersinia enterocolitica plasmid maintenance and replication.

Tricyclic medical compounds like many other non-antibiotics exhibit antimicrobial activities. Two chemically representative groups were tested in plasmid DNA transformation and replication to assign intracellular target sites responsible for the multiple effects in Escherichia coli and Yersinia enterocolitica cells. To analyse the mechanism of action at the molecular level, the effects of chlorpromazine, 7,8 dioxochlorpromazine, promethazine, methylene blue, imipramine, cannabidiolic acid and tetrahydrocannabidiolic acid were examined at several points in the course of transformation, in plasmid replication and on the topological state of plasmid DNA. Two possible target sites were identified, one of them involving membrane binding sites which participate in plasmid DNA replication. Drug binding at these sites interfered with the replicating plasmid DNA and membrane protein complex, preventing the proper processing of the replication that resulted in plasmid loss. The other in vivo and in vitro effect was observed on the topological state of plasmid DNA. Tricyclic drugs intefered with energy dependent gyrase activity and promoted the relaxation of plasmid DNA, causing disturbances in gene expression and in plasmid replication. The results give insight into the chemical structures connected with significant specific antimicrobial effects.

Novel trisaccharide fatty acid ester identified from the fruits of Morinda citrifolia (Noni).

Two known glycosides and a novel trisaccharide fatty acid ester were isolated from the n-butanol-soluble fraction of the fruits of Morinda citrifolia (noni). Structure determination was carried out by spectral techniques such as MS, IR, NMR, and 2D-NMR. The novel trisaccharide fatty acid ester was elucidated as 2, 6-di-O-(beta-D-glucopyranosyl)-1-O-octanoyl-beta-D-glucopyranose. The known compounds were identified as rutin and asperulosidic acid.

Characterization of the region encompassing the human lysyl oxidase locus.

A 46,823 bp region of human chromosome 5q23.1 encompassing the seven-exon lysyl oxidase gene was characterized at the primary sequence level. Approximately 17.4% of this region is comprised of repetitive elements. The gene colocalizes with microsatellite marker D5S467. It is flanked by two candidate nuclear matrix association regions (MARs). The 5' MAR centered at position 12,500 is of the AT-rich and curved DNA class. This is followed by a large CpG island containing fifty-seven putative regulatory elements which extend from just upstream of exon 1 to intron 2. The larger 3' MAR, spans position 35,050-39,750 and is characterized by a TG-rich kinked structure that also contains a topoisomerase II binding site. Based on these results model of the transcriptional regulation of the lysy/oxidase gene is presented.