ABSTRACT
Effective data management and sharing have become increasingly crucial in biomedical research; however, many laboratory researchers lack the necessary tools and knowledge to address this challenge. This article provides an introductory guide into research data management (RDM), and the importance of FAIR (Findable, Accessible, Interoperable, and Reusable) data-sharing principles for laboratory researchers produced by practicing scientists. We explore the advantages of implementing organized data management strategies and introduce key concepts such as data standards, data documentation, and the distinction between machine and human-readable data formats. Furthermore, we offer practical guidance for creating a data management plan and establishing efficient data workflows within the laboratory setting, suitable for labs of all sizes. This includes an examination of requirements analysis, the development of a data dictionary for routine data elements, the implementation of unique subject identifiers, and the formulation of standard operating procedures (SOPs) for seamless data flow. To aid researchers in implementing these practices, we present a simple organizational system as an illustrative example, which can be tailored to suit individual needs and research requirements. By presenting a user-friendly approach, this guide serves as an introduction to the field of RDM and offers practical tips to help researchers effortlessly meet the common data management and sharing mandates rapidly becoming prevalent in biomedical research.
Subject(s)
Biomedical Research , Data Management , Information Dissemination , Humans , Biomedical Research/methods , Biomedical Research/standards , Data Management/methods , Information Dissemination/methods , Research PersonnelABSTRACT
Hyperargininemia is a rare autosomal recessive disorder of the last step of the urea cycle characterized by a deficiency in liver arginase1. Clinically, it differs from other urea cycle defects by a progressive paraparesis of the lower limbs (spasticity and contractures) with hyperreflexia, neurodevelopmental delay and regression in early childhood. Growth is affected as well. Hyperammonemia is episodic, if present at all. The disease is caused by mutations in the ARG1 gene; there are approximately 20 different known ARG1 mutations with considerable genetic heterogeneity. We describe two Arab siblings with a late diagnosis of hyperargininemia and present the genetic findings in their family. As ARG1 sequencing was unrevealing despite suggestive clinical and laboratory findings, molecular cDNA analysis was performed. The ARG1 expression pattern identified a 125-bp out-of-frame insertion between exons 3 and 4, leading to the addition of 41 amino acids and a premature termination codon TGA at the sixth codon downstream. The insertion originated at intron 3 and was attributable to a novel c.305 + 1323 t > c intronic base change that enabled an enhancement phenomenon. This is the first reported exon-splicing-enhancer mutation in patients with hyperargininemia. The clinical course and genetic findings emphasize the possibility that hyperargininemia causes neurological deterioration in children and the importance of analyzing the expression pattern of the candidate gene when sequencing at the DNA level is unrevealing.
ABSTRACT
BACKGROUND: Patients with Xp22.3 interstitial and terminal deletions have been shown to be affected by intellectual disability (ID) or autism. Previously, VCX-A (variably charged protein X-A), located at Xp22.3, was introduced as a gene for ID and its presence was suggested to be sufficient to maintain normal mental development. Recent reports suggest that mutations in NLGN4 (neuroligin 4), located at that same region, are involved in autistic disorders and ID. METHODS: In the current case study, we clinically and molecularly describe a pedigree of three generations affected by contiguous gene syndrome that includes features of X-linked ichthyosis and Kallmann syndrome. RESULTS: Molecular analysis revealed the presence of an interstitial deletion spanning approximately 4.5Mb at Xp22.3. The centromeric breakpoint was localized between markers DXS1467 and DXS8051, proximal to KAL-1. The telomeric breakpoint was localized between markers DXS89 and DXS1060, distal to NLGN4. The deletion of VCX-A and NLGN4 in this family prompted us to examine the cognitive functions of our two adult patients using comprehensive intellectual and neurocognitive assessment. Normal intellectual function was found in one patient and mild ID was revealed in the other. Neither patient met any Diagnostic and Statistical Manual of Mental Disorder, Fourth Edition criteria for a pervasive developmental disorder such as autism. CONCLUSIONS: These findings suggest that deletion of VCX-A and NLGN4 can result in variable phenotypic features and that normal mental development can be achieved despite this deletion, emphasizing the importance of environmental factors and possible modifier genes.
Subject(s)
Carrier Proteins/genetics , Gene Deletion , Intellectual Disability/genetics , Intelligence , Membrane Proteins/genetics , Nuclear Proteins/genetics , Phenotype , Adult , Cell Adhesion Molecules, Neuronal , Chromosomes, Human, X/genetics , Cognition Disorders/diagnosis , Cognition Disorders/epidemiology , DNA Mutational Analysis , Female , Genetic Markers , Humans , Intellectual Disability/epidemiology , Male , Middle Aged , Pedigree , Point Mutation/genetics , Surveys and QuestionnairesABSTRACT
Deficiency of the hepatic cytosolic enzyme tyrosine aminotransferase (TAT) causes marked hypertyrosinaemia leading to painful palmoplantar hyperkeratoses, pseudodendritic keratitis and variable mental retardation (oculocutaneous tyrosinaemia type II or Richner-Hanhart syndrome). Parents may therefore seek prenatal diagnosis, but this is not possible by biochemical assays as tyrosine does not accumulate in amniotic fluid and TAT is not expressed in chorionic villi or amniocytes. Molecular analysis is therefore the only possible approach for prenatal diagnosis and carrier detection. To this end, we sought TAT gene mutations in 9 tyrosinaemia II patients from three consanguineous Palestinian kindreds. In two kindreds (7 patients), the only potential abnormality identified after sequencing all 12 exons and exon-intron boundaries was homozygosity for a silent, single-nucleotide transversion c.1224G > T (p.T408T) at the last base of exon 11. This was predicted to disrupt the 5' donor splice site of exon 11 and result in missplicing. However, as TAT is expressed exclusively in liver, patient mRNA could not be obtained for splicing analysis. A minigene approach was therefore used to assess the effect of c.1224G > T on exon 11 splicing. Transfection experiments with wild-type and c.1224G > T mutant minigene constructs demonstrated that c.1224G > T results in complete exon 11 skipping, illustrating the utility of this approach for confirming a putative splicing defect when cDNA is unavailable. Homozygosity for a c.1249C > T (R417X) exon 12 nonsense mutation (previously reported in a French patient) was identified in both patients from the third kindred, enabling successful prenatal diagnosis of an unaffected fetus using chorionic villous tissue.
Subject(s)
Eye Diseases/genetics , Mutation , Skin Diseases/genetics , Tyrosine Transaminase/genetics , Tyrosinemias/genetics , Adult , Alternative Splicing , Child , Child, Preschool , DNA Mutational Analysis/methods , Exons , Female , Humans , Infant , Infant, Newborn , Israel , Male , Molecular Sequence Data , PedigreeABSTRACT
Four members of an extended consanguineous Bedouin family presented with different phenotypic variants of an autosomal recessive lysosomal free sialic acid storage disease. One affected individual had congenital ascites followed by rapid clinical deterioration and death, a presentation concordant with the clinical course of infantile free sialic acid storage disorder. His three first cousins had a more slowly progressive neurodegenerative disease, in line with the clinical phenotype of the milder form (Salla type) of this lysosomal disorder. Diagnosis of free sialic acid storage disease was based on clinical findings, histology, and biochemical assays of sialic acid. Molecular studies showed that all four affected individuals were homozygous for the same novel 983G > A mutation in exon 8 of the SLC17A5 gene, replacing glycine with glutamic acid at position 328 of the sialin protein. This family demonstrates the significant phenotypic variability of the disease in affected members of a single inbred kindred with precisely the same mutation, suggesting a role for modifier genes or environmental factors. It also highlights the need to consider this rare disorder in the differential diagnosis of congenital ascites and of unexplained psychomotor retardation, ataxia, and hypomyelination in infancy.
Subject(s)
Arabs/genetics , Consanguinity , Mutation/genetics , Organic Anion Transporters/genetics , Sialic Acid Storage Disease/genetics , Sialic Acid Storage Disease/physiopathology , Symporters/genetics , Adult , Base Sequence , Child, Preschool , Female , Genetic Markers/genetics , Humans , Infant , Male , N-Acetylneuraminic Acid/analysis , Phenotype , Polymorphism, Genetic/genetics , Sialic Acid Storage Disease/diagnosisSubject(s)
Mucolipidoses/genetics , Mutation , Transferases (Other Substituted Phosphate Groups)/genetics , Adolescent , Adult , Child , Child, Preschool , DNA Mutational Analysis , Exons , Genome, Human , Humans , Introns , Mucolipidoses/diagnosis , Mucolipidoses/pathology , Protein Subunits/geneticsABSTRACT
OBJECTIVES: Mucolipidosis IIIC (MLIIIC) is a rare autosomal recessive lysosomal storage disease resulting from defective mannose 6-phosphate-dependent lysosomal enzyme trafficking; mutations of the gamma subunit of N-acetylglucosamine-1 phosphotransferase (GINAcPT) were recently found to cause its pathogenesis. We report here for the first time prenatal diagnosis (PND) for MLIIIC by means of chorionic villous sampling (CVS). METHODS AND RESULTS: A fetus in a large Bedouin-Moslem family was found to be homozygous for the founder haplotype and the mutational SSCP pattern of MLIIIC. The diagnosis was confirmed by markedly reduced lysosomal enzyme activities in cultured chorionic villi. The molecular identification of the disease-causing mutation in this large Bedouin-Moslem kindred permitted, for the first time, identification of carriers and couples at risk. CONCLUSIONS: The feasibility of early PND for a progressive disabling disease is important for its prevention. Nevertheless, the feasibility of PND raises a serious dilemma since affected individuals might have a variable phenotype and the disease is progressive and non-lethal. In addition, religious and social constraints are important factors to be taken into consideration in the genetic counseling of couples at risk.
Subject(s)
Mucolipidoses/diagnosis , Prenatal Diagnosis , Arabs , DNA Mutational Analysis , Female , Genetic Counseling , Genotype , Homozygote , Humans , Islam , Lysosomes/enzymology , Male , Mucolipidoses/enzymology , Mucolipidoses/genetics , Pedigree , Polymorphism, Single-Stranded ConformationalABSTRACT
PURPOSE: Niemann-Pick disease type C (NP-C) is an autosomal recessive lipid storage disease manifested by an impairment in cellular cholesterol homeostasis. The clinical phenotype of NP-C is extremely variable, ranging from an acute neonatal form to an adult late-onset presentation. To facilitate phenotype-genotype studies, we have analyzed multiple Israeli NP-C families. METHODS: The severity of the disease was assessed by the age at onset, hepatic involvement, neurological deterioration, and cholesterol esterification studies. Screening of the entire NPC1 coding sequence allowed for molecular characterization and identification of disease causing mutations. RESULTS: A total of nine NP-C index cases with mainly neurovisceral involvement were characterized. We demonstrated a possible link between the severity of the clinical phenotype and the cholesterol esterification levels in fibroblast cultures following 24 hours of in vitro cholesterol loading. In addition, we identified eight novel mutations in the NPC1 gene. CONCLUSIONS: Our results further support the clinical and allelic heterogeneity of NP-C and point to possible association between the clinical and the biochemical phenotype in distinct affected Israeli families.
Subject(s)
Carrier Proteins/genetics , Membrane Glycoproteins/genetics , Mutation/genetics , Niemann-Pick Diseases/genetics , Niemann-Pick Diseases/physiopathology , Age of Onset , Cell Line , Child, Preschool , Cholesterol/metabolism , Consanguinity , Esterification , Fibroblasts , Gene Frequency/genetics , Genotype , Humans , Intracellular Signaling Peptides and Proteins , Israel , Niemann-Pick C1 Protein , Niemann-Pick Diseases/epidemiology , Niemann-Pick Diseases/metabolism , PhenotypeABSTRACT
The present study assesses the applicability of human skin-SCID (severe combined immunodeficiency) mouse chimeras in testing antipsoriatic therapeutics. Three agents were examined: (1) a monoclonal antibody to the alpha subunit of leukocyte function associated antigen-1 integrin (CD11a); (2) Cyclosporin A; and (3) clobetasol propionate (Temovate), a potent topical corticosteroid used clinically in the treatment of psoriasis. Skin transplanted to SCID mice from normal human volunteers or from psoriatic lesional skin was allowed to heal for 3 to 5 weeks before application of test reagents. During this period, psoriatic skin, which was 3.8-fold thicker than the corresponding normal skin before transplantation, maintained its phenotype (ie, increased epidermal thickness, rete ridges with blunted ends, and intralesional presence of T lymphocytes). Transplanted normal human skin, however, underwent a hyperplastic response during this period, resulting in a 2.4-fold increase in epidermal thickness. After the healing period, animals transplanted with normal or psoriatic skin were treated for 14 days by daily intraperitoneal injection of either Cyclosporin A or a monoclonal antibody to human CD11a, or by topical application of clobetasol propionate. At the end of the treatment period, the mice were killed and the tissue evaluated morphometrically for changes in epidermal thickness and immunohistologically for the presence of T lymphocytes. Both Cyclosporin A and anti-CD11a reduced the epidermal thickness of transplanted psoriatic skin, whereas neither reagent significantly reduced the thickness of transplanted normal skin. T lymphocytes were detected in the skin from treated animals; there did not seem to be any reduction in the number of T lymphocytes. Clobetasol propionate reduced the epidermal thickness of both normal and psoriatic skin. These data indicate that, in this model, therapies directed against pathophysiologic mechanisms that contribute to psoriasis can be distinguished from treatments that block epidermal hyperplasia occurring as a consequence of xenografting. Our observations provide evidence for the activity of anti-CD11a in an animal model of human psoriasis.
Subject(s)
Antibodies, Monoclonal/therapeutic use , Lymphocyte Function-Associated Antigen-1/immunology , Psoriasis/drug therapy , Skin Transplantation , Transplantation, Heterologous , Administration, Topical , Animals , Anti-Inflammatory Agents/therapeutic use , Clobetasol/analogs & derivatives , Clobetasol/therapeutic use , Cyclosporine/therapeutic use , Dermatologic Agents/therapeutic use , Glucocorticoids , Humans , Mice , Mice, SCID , Psoriasis/pathology , Reference Values , Skin/drug effects , Skin/pathologyABSTRACT
Wolman disease results from an inherited deficiency of lysosomal acid lipase (LAL; EC 3.1.1.13). This enzyme is essential for the hydrolysis of cholesteryl esters and triacylglycerols derived from endocytosed lipoproteins. Because of a complete absence of LAL activity, Wolman patients accumulate progressive amounts of cholesteryl esters and triacylglycerols in affected tissues. To investigate the nature of the genetic defects causing this disease, mutations in the LAL gene from three subjects of Moslem-Arab and Russian descent living in Israel were determined. Two homozygotes for a novel 1-bp deletion introducing a premature in-frame termination codon at amino acid position 106 (S106X) were identified. A third subject was a homozygote for a G-5R signal peptide substitution and a G60V missense mutation. The functional significance of these mutations was tested by in vitro expression of single and double mutants in Spodoptera frugiperda cells. Single mutants G60V and S106X and double mutant G-5R/G60V displayed a virtual absence of lipase activity in cell extracts and culture medium. Signal peptide mutant G-5R retained lipase activity in cell extracts and showed a drastically reduced enzyme activity in culture supernatant, indicating that the mutation may affect secretion of active enzyme from cells. These results support the notion that Wolman disease is a genetically heterogeneous disorder of lipid metabolism.
Subject(s)
Cholesterol Esters/metabolism , Fetal Diseases/genetics , Lipase/deficiency , Lipase/genetics , Lysosomes/metabolism , Triglycerides/metabolism , Wolman Disease/genetics , Animals , Fetal Diseases/metabolism , Fibroblasts/metabolism , Humans , Infant , Lysosomes/ultrastructure , Mutation/genetics , Peptides/genetics , Protein Sorting Signals/genetics , Sequence Deletion/genetics , Spodoptera/genetics , Wolman Disease/metabolismABSTRACT
The gene MCOLN1 is mutated in Mucolipidosis type IV (MLIV), a neurodegenerative, recessive, lysosomal storage disorder. The disease is found in relatively high frequency among Ashkenazi Jews due to two founder mutations that comprise 95% of the MLIV alleles in this population [Bargal et al., 2000]. In this report we complete the mutation analysis of Jewish and non-Jewish MLIV patients whose DNA were available to us. Four novel mutations were identified in the MCOLN1 gene of severely affected patients: two missense, T232P and F465L; a nonsense, R322X; and an 11-bp insertion in exon 12. The nonsense mutation (R322X) was identified in two unrelated patients with different haplotypes in the MCOLN1 chromosomal region, indicating a mutation hotspot in this CpG site. An in-frame deletion (F408del) was identified in a patient with unusual mild psychomotor retardation. The frequency of MLIV in the general Jewish Ashkenazi population was estimated in a sample of 2,000 anonymous, unrelated individuals assayed for the two founder mutations. This analysis indicated a heterozygotes frequency of about 1/100. A preferred nucleotide numbering system for MCOLN1 mutations is presented and the issue of a screening program for the detection of high-risk families in the Jewish Ashkenazi population is discussed.
Subject(s)
Jews/genetics , Membrane Proteins/genetics , Mucolipidoses/epidemiology , Mucolipidoses/genetics , Mutation/genetics , White People/genetics , Codon, Nonsense/genetics , CpG Islands/genetics , DNA Mutational Analysis , DNA Primers/genetics , Exons/genetics , Founder Effect , Gene Frequency/genetics , Genetic Predisposition to Disease/genetics , Genetic Testing , Haplotypes/genetics , Heterozygote , Humans , Molecular Sequence Data , Mucolipidoses/classification , Mutagenesis, Insertional/genetics , Mutation, Missense/genetics , Polymerase Chain Reaction , TRPM Cation Channels , Transient Receptor Potential ChannelsABSTRACT
Sun-protected human skin was maintained in organ culture and treated with all-trans retinoic acid in the presence or absence of reversible or irreversible pharmacologic antagonists of c-erbB receptor tyrosine kinase activity. In the absence of these inhibitors, all-trans retinoic acid induced epidermal hyperplasia comparable to that induced in intact skin by all-trans retinol or all-trans retinoic acid itself. There was a strong correlation between inhibition of epidermal hyperplasia in organ culture and inhibition of epidermal-growth-factor-dependent keratinocyte growth in monolayer culture. In additional studies it was shown that all-trans retinoic acid could overcome the known inhibitory effects of calcium on expression of HB-EGF-like growth factor mRNA in organ-cultured skin. Further, it was shown that an antibody to HB-EGF-like growth factor inhibited retinoid-stimulated epidermal hyperplasia in organ culture and reduced proliferation in cultured keratinocytes. In contrast, the c-erbB receptor tyrosine kinase antagonists and the neutralizing HB-EGF-like growth factor antibody were ineffective in inhibiting all-trans-retinoic-acid-dependent survival and proliferation of human dermal fibroblasts. Taken together, these data indicate (i) that retinoid-induced epidermal hyperplasia in human skin proceeds through c-erbB, and (ii) that HB-EGF-like growth factor is one of the c-erbB ligands mediating this effect.
Subject(s)
Epidermal Growth Factor/metabolism , Epidermis/pathology , ErbB Receptors/metabolism , Keratinocytes/metabolism , Keratolytic Agents/pharmacology , Tretinoin/pharmacology , Adult , Antibodies/pharmacology , Calcium/pharmacology , Cell Division/drug effects , Cell Survival/drug effects , Epidermal Growth Factor/genetics , Epidermal Growth Factor/immunology , Epidermis/drug effects , Epidermis/metabolism , ErbB Receptors/antagonists & inhibitors , Fibroblasts/cytology , Fibroblasts/drug effects , Gene Expression/drug effects , Heparin-binding EGF-like Growth Factor , Humans , Hyperplasia , Intercellular Signaling Peptides and Proteins , Keratinocytes/cytology , Keratinocytes/drug effects , Organ Culture Techniques , RNA, Messenger/metabolism , Receptors, Cell SurfaceABSTRACT
Mucolipidosis type IV (MLIV) is an autosomal recessive, neurodegenerative, lysosomal storage disorder characterized by psychomotor retardation and ophthalmological abnormalities including corneal opacities, retinal degeneration and strabismus. Most patients reach a maximal developmental level of 12?15 months. The disease was classified as a mucolipidosis following observations by electron microscopy indicating the lysosomal storage of lipids together with water-soluble, granulated substances. Over 80% of the MLIV patients diagnosed are Ashkenazi Jews, including severely affected and mildly affected patients. The gene causing MLIV was previously mapped to human chromosome 19p13.2-13.3 in a region of approximately 1 cM (ref. 7). Haplotype analysis in the MLIV gene region of over 70 MLIV Ashkenazi chromosomes indicated the existence of two founder chromosomes among 95% of the Ashkenazi MLIV families: a major haplotype in 72% and a minor haplotype in 23% of the MLIV chromosomes (ref. 7, and G.B., unpublished data). The remaining 5% are distinct haplotypes found only in single patients. The basic metabolic defect causing the lysosomal storage in MLIV has not yet been identified. Thus, positional cloning was an alternative to identify the MLIV gene. We report here the identification of a new gene in this human chromosomal region in which MLIV-specific mutations were identified.
Subject(s)
Membrane Proteins/genetics , Mucolipidoses/genetics , Mutation , Amino Acid Sequence , Base Sequence , Blotting, Northern , Chromosome Mapping , Chromosomes, Human, Pair 19 , Cloning, Molecular , CpG Islands , DNA Mutational Analysis , Exons , Expressed Sequence Tags , Female , Gene Deletion , Genes, Recessive , Genetic Markers , Haplotypes , Humans , Male , Models, Genetic , Molecular Sequence Data , Pedigree , Polymorphism, Genetic , RNA Splicing , RNA, Messenger/metabolism , Restriction Mapping , Reverse Transcriptase Polymerase Chain Reaction , Sequence Homology, Amino Acid , TRPM Cation Channels , Transient Receptor Potential ChannelsABSTRACT
BACKGROUND: Psoriasis is often treated with agents that activate nuclear hormone receptors for glucocorticoids, retinoids, and vitamin D. The peroxisome proliferator-activated receptor-gamma (PPARgamma) is a related nuclear hormone receptor that can be activated by its ligands, including the thiazolidinediones. OBJECTIVE: To assess whether treatment with troglitazone, a currently available thiazolidinedione used to treat diabetes mellitus, has an effect on psoriasis in normoglycemic patients and whether ligands for PPARgamma have an effect on models of psoriasis. DESIGN: Open-label administration of troglitazone in patients with psoriasis and evaluation of drug actions in cellular, organ, and transplant models of psoriasis. SETTING: University and community hospital outpatient departments and university laboratories. PATIENTS: Patients with chronic, stable plaque psoriasis and control subjects. Five patients with psoriasis received troglitazone (none withdrew); 10 different untreated patients and 10 controls provided tissue samples. INTERVENTIONS: Oral troglitazone therapy at various dosages in patients with psoriasis; also, use of troglitazone, ciglitazone, and 15-deoxy-delta-12,14-prostaglandinJ2 in psoriasis models. MAIN OUTCOME MEASURES: Investigator-determined clinical results in patients and cell counts and histological evidence in models. RESULTS: All patients' psoriasis improved substantially during troglitazone therapy. Peroxisome proliferator-activated receptor-gamma was expressed in human keratinocytes; ligands for PPARgamma inhibited the proliferation of normal and psoriatic human keratinocytes in culture. Troglitazone treatment normalized the histological features of psoriatic skin in organ culture and reduced the epidermal hyperplasia of psoriasis in the severe combined immunodeficient mouse and human skin transplant model of psoriasis (P<.05 compared with untreated controls). CONCLUSIONS: Peroxisome proliferator-activated receptor-gamma might be a useful intracellular target for the treatment of psoriasis; further study is needed to assess the clinical value of ligands for PPARgamma, including troglitazone.
Subject(s)
Antineoplastic Agents/therapeutic use , Chromans/therapeutic use , Psoriasis/drug therapy , Receptors, Cytoplasmic and Nuclear/metabolism , Skin Diseases/drug therapy , Thiazoles/therapeutic use , Thiazolidinediones , Transcription Factors/metabolism , Adult , Animals , Antineoplastic Agents/metabolism , Cell Differentiation , Chromans/metabolism , DNA Primers , Female , Humans , Keratinocytes/cytology , Ligands , Male , Mice , Mice, SCID , Psoriasis/metabolism , RNA, Messenger/metabolism , Receptors, Cytoplasmic and Nuclear/genetics , Reverse Transcriptase Polymerase Chain Reaction , Skin Diseases/metabolism , Thiazoles/metabolism , Transcription Factors/genetics , TroglitazoneABSTRACT
Mucolipidosis IIIC, or variant pseudo-Hurler polydystrophy, is an autosomal recessive disease of lysosomal hydrolase trafficking. Unlike the related diseases, mucolipidosis II and IIIA, the enzyme affected in mucolipidosis IIIC (N-Acetylglucosamine-1-phosphotransferase [GlcNAc-phosphotransferase]) retains full transferase activity on synthetic substrates but lacks activity on lysosomal hydrolases. Bovine GlcNAc-phosphotransferase has recently been isolated as a multisubunit enzyme with the subunit structure alpha(2)beta(2)gamma(2). We cloned the cDNA for the human gamma-subunit and localized its gene to chromosome 16p. We also showed, in a large multiplex Druze family that exhibits this disorder, that MLIIIC also maps to this chromosomal region. Sequence analysis of the gamma-subunit cDNA in patients from 3 families identified a frameshift mutation, in codon 167 of the gamma subunit, that segregated with the disease, indicating MLIIIC results from mutations in the phosphotransferase gamma-subunit gene. This is to our knowledge the first description of the molecular basis for a human mucolipidosis and suggests that the gamma subunit functions in lysosomal hydrolase recognition.
Subject(s)
Lysosomes/metabolism , Mucolipidoses/genetics , Transferases (Other Substituted Phosphate Groups)/genetics , Amino Acid Sequence , Base Sequence , Chromosomes, Human, Pair 16 , Cloning, Molecular , Female , Fibroblasts , Frameshift Mutation , Genetic Linkage , Humans , Lod Score , Lysosomes/enzymology , Male , Molecular Sequence Data , Mucolipidoses/etiology , Pedigree , RNA, Messenger/metabolism , Sequence Analysis , Transferases (Other Substituted Phosphate Groups)/metabolismABSTRACT
Tissue from 54 histologically-identified basal cell carcinomas of the skin was obtained at surgery and assayed using a combination of functional and immunochemical procedures for matrix metalloproteinases (MMPs) with collagenolytic activity and for MMPs with gelatinolytic activity. Collagenolytic enzymes included MMP-1 (interstitial collagenase), MMP-8 (neutrophil collagenase) and MMP-13 (collagenase-3). Gelatinolytic enzymes included MMP-2 (72-kDa gelatinase A/type IV collagenase) and MMP-9 (92-kDa gelatinase B/type IV collagenase). Inhibitors of MMP activity including tissue inhibitor of metalloproteinases-1 and -2 (TIMP-1 and TIMP-2) were also assessed. All three collagenases and both gelatinases were detected immunochemically. MMP-1 appeared to be responsible for most of the functional collagenolytic activity while gelatinolytic activity reflected both MMP-2 and MMP-9. MMP inhibitor activity was also present, and appeared, based on immunochemical procedures, to reflect the presence of TIMP-1 but not TIMP-2. As a group, tumours identified as having aggressive-growth histologic patterns were not distinguishable from basal cell carcinomas with less aggressive-growth histologic patterns. In normal skin, the same MMPs were detected by immunochemical means. However, only low to undetectable levels of collagenolytic and gelatinolytic activities were present. In contrast, MMP inhibitor activity was comparable to that seen in tumour tissue. In previous studies we have shown that exposure of normal skin to epidermal growth factor in organ culture induces MMP up-regulation and activation. This treatment concomitantly induces stromal invasion by the epithelium (Varani et al (1995) Am J Pathol 146: 210-217; Zeigler et al (1996b) Invasion Metastasis 16: 11-18). Taken together with these previous data, the present findings allow us to conclude that the same profile of MMP/MMP inhibitors that is associated with stromal invasion in the organ culture model is expressed endogenously in basal cell carcinomas of skin.
Subject(s)
Carcinoma, Basal Cell/metabolism , Collagen/metabolism , Enzyme Inhibitors/metabolism , Gelatin/metabolism , Matrix Metalloproteinases/metabolism , Skin Neoplasms/metabolism , Skin/metabolism , Blotting, Western , Carcinoma, Basal Cell/enzymology , Caseins/metabolism , Humans , Hydrolysis , Immunohistochemistry , Matrix Metalloproteinase Inhibitors , Skin/enzymology , Skin Neoplasms/enzymologyABSTRACT
Invasion is an essential cellular response that plays an important role in a number of physiological and pathological processes. Matrix metalloproteinase (MMP) production and cell movement are diverse cellular responses integral to the process of invasion. The complexity of the invasive process suggests the necessity of coordinate activation of more than one signaling pathway in order to activate specific factors responsible for regulating these cellular responses. In this report, we demonstrate that cell movement and MMP-9 production are both directly dependent on the activation of endogenous ERK signaling in hepatocyte growth factor (HGF)-or epidermal growth factor (EGF)-stimulated human epidermal keratinocytes. The kinetic profiles of endogenous MEK and ERK activity suggest that prolonged activation of these signal transducers is an underlying mechanism involved in stimulating cell motility and MMP-9 production. In support of this finding, a transient MEK/ERK signal elicited by keratinocyte growth factor (KGF) or insulin-like growth factor-1 (IGF-1) fails to stimulate these invasion-related responses. Specific inhibition of MEK leads to suppression of ERK activation, marked reduction in steady-state levels of c-Fos, and inhibition of cell movement and MMP-9 production. This occurs despite continued activation of JNK and c-Jun signaling in the presence of MEK-specific inhibition. In contrast, when JNK activity is specifically inhibited in HGF-stimulated cells, AP-1 activity is suppressed but cell motility is not affected. This evidence suggests that while ERK and JNK activity are necessary for AP-1 activation, ERK but not JNK is sufficient in stimulating cell motility.
Subject(s)
Calcium-Calmodulin-Dependent Protein Kinases/metabolism , Collagenases/metabolism , Fibroblast Growth Factors , Hepatocyte Growth Factor/pharmacology , JNK Mitogen-Activated Protein Kinases , Keratinocytes/enzymology , MAP Kinase Kinase 4 , Mitogen-Activated Protein Kinase Kinases , Mitogen-Activated Protein Kinases , Protein Kinases/metabolism , Antigen-Antibody Complex , Calcium-Calmodulin-Dependent Protein Kinases/analysis , Calcium-Calmodulin-Dependent Protein Kinases/immunology , Cell Division/drug effects , Cell Division/physiology , Cell Movement/drug effects , Cell Movement/physiology , Cells, Cultured , Epidermal Cells , Epidermal Growth Factor/pharmacology , Fibroblast Growth Factor 10 , Fibroblast Growth Factor 7 , Gene Expression Regulation, Enzymologic , Growth Substances/pharmacology , Humans , Immunoblotting , Insulin-Like Growth Factor I/pharmacology , Keratinocytes/cytology , Keratinocytes/drug effects , Kinetics , Luciferases , MAP Kinase Kinase 1 , MAP Kinase Kinase 2 , Matrix Metalloproteinase 9 , Mitogen-Activated Protein Kinase 1 , Mitogen-Activated Protein Kinase 3 , Protein Kinases/analysis , Protein Kinases/immunology , Protein Serine-Threonine Kinases/analysis , Protein Serine-Threonine Kinases/immunology , Protein Serine-Threonine Kinases/metabolism , Protein-Tyrosine Kinases/analysis , Protein-Tyrosine Kinases/immunology , Protein-Tyrosine Kinases/metabolism , Signal Transduction/physiology , Transcription Factor AP-1/genetics , Transcription Factor AP-1/metabolismABSTRACT
Subunit immunogens containing tandemly repeated copies of T- and B-cell epitopes have been shown to be more immunogenic than the respective immunogen containing only a single copy of the sequence. It has been unclear, however, whether the increased immunogenicity of a tandemly repeated B-cell epitope necessarily results from increased helper T-cell responses to intrinsic T-cell epitopes in the tandemly repeated sequences, or to neodeterminant T-cell epitopes created at the junction of adjacent repeat sequences. We examined this question by comparing the immunogenicity in mice of two immunogens containing one or eight tandemly repeated copies of an HIV-1 V3 loop haptenic sequence. Our results show that the tandemly repeated haptenic sequence potentiates the immunogenicity of the protein construct, likely through the facilitation of enhanced B-cell interaction with the tandem repeat construct and the consequent elicitation of increased carrier protein-specific helper T-cell responses.
Subject(s)
Carrier Proteins/immunology , HIV Envelope Protein gp160/immunology , Haptens/immunology , ISCOMs/immunology , Peptides/immunology , T-Lymphocytes, Helper-Inducer/immunology , Amino Acid Sequence , Animals , B-Lymphocytes/immunology , H-2 Antigens/immunology , H-2 Antigens/metabolism , HIV Antibodies/biosynthesis , HIV Infections/immunology , HIV Infections/prevention & control , HIV-1/chemistry , HIV-1/immunology , Male , Mice , Mice, Inbred BALB C , Mice, Nude , Molecular Sequence Data , Repetitive Sequences, Amino AcidABSTRACT
Mucolipidosis type IV (MLIV) is a neurodegenerative lysosomal storage disease in which most of the patients diagnosed hitherto are Ashkenazi Jews. The basic metabolic defect causing this disease is still unknown and the relevant gene has not yet been mapped or cloned. Seventeen Israel Ashkenazi families with MLIV patients had been interviewed to study their family origin. Although the families immigrated to Israel from various European countries they all could trace their roots three to four generations back to northern Poland or the immediate neighbouring country, Lithuania. Furthermore, there are only one or two ultraorthodox families among the 70-80 Ashkenazi families with MLIV patients worldwide, a marked under-representation of this group which constitutes at least 10% of the Ashkenazi population. This data indicate that MLIV mutation occurred only around the 18th and 19th centuries, after the major expansion of this population, in a founder in this defined European region belonging to a more modern, secular family.
Subject(s)
Jews/genetics , Mucolipidoses/genetics , Emigration and Immigration , Founder Effect , Humans , Israel , Lithuania/ethnology , Mucolipidoses/ethnology , Poland/ethnologyABSTRACT
Functional and immunochemical approaches were used to assess matrix metalloproteinase (MMP) inhibitors, e.g., tissue inhibitor of metalloproteinases 1 and 2 (TIMP-1 and TIMP-2), in organ cultures of normal human skin maintained under growth factor free conditions or in medium supplemented with a combination of growth factors including epidermal growth factor, insulin, and pituitary extract. It has previously been shown that under growth factor free conditions, normal skin structure and function are maintained for several days, while in the presence of these exogenous growth factors, the epithelial cells invade the stroma [Invasion and Metastasis 1993;13:225-233]. TIMP-1 was detected in equivalent amounts in organ culture fluids under both conditions. TIMP-2 was not detected under either condition. Normal epidermal keratinocytes, normal dermal fibroblasts, and three different epithelial tumor cell lines were also examined for MMP inhibitor expression. Keratinocytes and fibroblasts produced high levels of both TIMP-1 and TIMP-2, but in neither cell type was there a significant difference between growth factor free and growth factor containing conditions. In contrast, the three epithelial tumor cell lines produced low to undetectable levels of both TIMP-1 and TIMP-2. These data suggest that acquisition of local invasive capacity is not dependent on a reduction in MMP inhibitor expression. A reduction in MMP inhibitors may accompany the transition from invasive to metastatic tumors.