PubMed Health⌕ Search

Biomedical subjects

Leena Pulkkinen

Publications and source records attributed to Leena Pulkkinen.

14 recordsLinked to original sources
PubMed2006

Lung development in laminin gamma2 deficiency: abnormal tracheal hemidesmosomes with normal branching morphogenesis and epithelial differentiation.

BACKGROUND: Laminin gamma2 (Lamc2), one of the polypeptides in laminin-332 (laminin-5), is prominent in the basement membrane of alveolar walls and airways of developing and adult lung. Laminins are important for lung morphogenesis and based on its localization, a function for laminin gamma2 in lung development has been hypothesized. Targeted deletion of the laminin gamma2 gene in mice results in skin blistering and neonatal death at 3-5 days after birth due to failure to thrive. METHODS: Examination of lung development in Lamc2-/- mice through 1-2 days postnatal was accomplished by morphometric analysis, lung bud culture, electron microscopy, immunohistochemical and immunofluorescence staining. RESULTS: Compared to littermate controls, Lamc2-/- lungs were similar in morphology during embryonic life. At post-natal day 1-2, distal saccules were mildly dilated by chord length measurements. Epithelial differentiation as evaluated by immunohistochemical staining for markers of ciliated cells, Clara cells, alveolar type I cells and alveolar type II cells did not reveal a difference between Lamc2-/- and littermate control lungs. Likewise, vascular development, smooth muscle cell differentiation, and elastic fiber formation looked similar, as did airway basement membrane ultrastructure. Branching morphogenesis by lung bud culture was similar in Lamc2-/- and littermate control lungs. Since laminin-332 is important for hemidesmosome formation, we examined the structure of tracheal hemidesmosomes by transmission electron microscopy. Compared to littermate controls, Lamc2-/- tracheal hemidesmosomes were less organized and lacked the increased electron density associated with the basement membrane abutting the hemidesmosome. CONCLUSION: These findings indicate that laminin gamma2 and laminin-332, despite their prominence in the lung, have a minimal role in lung development through the saccular stage.

Animals↗
PubMed2006

Association between ghrelin gene variations and blood pressure in subjects with impaired glucose tolerance.

BACKGROUND: Ghrelin is a gut-brain hormone, which stimulates food intake and controls energy balance. Recently, it has been shown that ghrelin may also play a role in the regulation of blood pressure (BP) by acting at the sympathetic nervous system. In the present study we genotyped six variants of the ghrelin gene and its promoter, and tested whether these single nucleotide polymorphisms (SNPs) were associated with BP levels in participants of the Finnish Diabetes Prevention Study. METHODS: The Finnish Diabetes Prevention Study was a longitudinal study where 522 subjects with impaired glucose tolerance were randomized into either an intervention or control group. DNA was available from 507 subjects (mean body mass index [BMI] 31.2+/-4.5 kg/m2, age 55+/-7 years). All six SNPs were screened by the restriction fragment length polymorphism method. RESULTS: Subjects with the most common genotype combination of the following four SNPs, -604G/A, -501A/C, Leu72Met, and Gln90Leu, had the lowest systolic (131+/-11 v 137+/-13 mm Hg, P=.003) and diastolic BP levels (79+/-7 v 83+/-7 mm Hg, P=.004) at the baseline of the study and during 3 years of follow-up compared to all other genotypes. Adjustments for age, gender, antihypertensive medication, BMI, waist circumference, and alcohol intake did not change this association. CONCLUSIONS: Several ghrelin gene variations were associated with BP levels in subjects with impaired glucose tolerance.

Adult↗
PubMed2005

Tissue-specific expression of the ABCC6 gene.

The ABCC6 gene encodes MRP6, a member of the multidrug resistance-associated protein (MRP) family. Interest in ABCC6/MRP6 derives, in part, from the fact that mutations in this gene/protein system have been identified in families with pseudoxanthoma elasticum (PXE). Early studies indicated that ABCC6 is expressed primarily in the liver and to a lesser extent in the kidney, but more recently a widespread distribution has been suggested. To explore the tissue-specific expression of ABCC6, we first examined various mouse tissues by RT-PCR. The results indicated high levels of mRNA in the liver, whereas low level of expression was noted in the kidney and small intestine. To explore other tissues in which initial RT-PCR was essentially negative, a second-round nested PCR was performed, which revealed expression also in the brain, tongue, stomach, and eye. Unexpectedly, however, distinct PCR products of smaller molecular weight were noted in these tissues. Subcloning and sequencing of these PCR products indicated that they reflected aberrant splicing in the 3' end of the ABCC6 mRNA, resulting in each case in a premature termination codon. Similar results were noted with RT-PCR analysis using RNA isolated from cultured human epidermal keratinocytes and dermal fibroblasts. Collectively, our results confirm high level of expression of ABCC6 in the liver and the kidney, whereas very low level of expression in a variety of other tissues was noted. The results have implications for mutation detection strategies in PXE by RT-PCR, and they further support the notion that PXE is a primary metabolic disorder.

ATP-Binding Cassette Transporters↗
PubMed2005

Targeted ablation of the abcc6 gene results in ectopic mineralization of connective tissues.

Pseudoxanthoma elasticum (PXE), characterized by connective tissue mineralization of the skin, eyes, and cardiovascular system, is caused by mutations in the ABCC6 gene. ABCC6 encodes multidrug resistance-associated protein 6 (MRP6), which is expressed primarily in the liver and kidneys. Mechanisms producing ectopic mineralization as a result of these mutations remain unclear. To elucidate this complex disease, a transgenic mouse was generated by targeted ablation of the mouse Abcc6 gene. Abcc6 null mice were negative for Mrp6 expression in the liver, and complete necropsies revealed profound mineralization of several tissues, including skin, arterial blood vessels, and retina, while heterozygous animals were indistinguishable from the wild-type mice. Particularly striking was the mineralization of vibrissae, as confirmed by von Kossa and alizarin red stains. Electron microscopy revealed mineralization affecting both elastic structures and collagen fibers. Mineralization of vibrissae was noted as early as 5 weeks of age and was progressive with age in Abcc6(-/-) mice but was not observed in Abcc6(+/-) or Abcc6(+/+) mice up to 2 years of age. A total body computerized tomography scan of Abcc6(-/-) mice revealed mineralization in skin and subcutaneous tissue as well as in the kidneys. These data demonstrate aberrant mineralization of soft tissues in PXE-affected organs, and, consequently, these mice recapitulate features of this complex disease.

ATP-Binding Cassette Transporters↗
PubMed2004

Differential structural properties and expression patterns suggest functional significance for multiple mouse desmoglein 1 isoforms.

The four isoforms of desmosomal cadherin desmogleins (Dsg1-4) are expressed in epithelial tissues in a differentiation-specific manner. Extensive sequencing of the human genome has revealed only one copy of the Dsg1 gene. However, we recently cloned two novel additional mouse Dsg1 genes, Dsg1-beta and -gamma, which flank the original Dsg1-alpha on chromosome 18. Sequence conservation between the Dsg1 isoforms diverged significantly at exon 11, particularly in the region that encodes for the extracellular anchoring (EA) domains. Computational analysis revealed very low hydrophilic potential of the Dsg1-gamma EA compared with the corresponding sequences of Dsg1-alpha and -beta, suggesting that the Dsg1-gamma EA domain may have a stronger affinity to the cell membrane. We generated antibodies using synthetic peptides or recombinant proteins localized within the EA domains. These antibodies were tested for their specificity and were then used to demonstrate expression of Dsg1 isoforms in various tissues. In the epidermis, all Dsg1 isoforms were differentially expressed in the differentiating cell layers. In the hair follicle, all Dsg1 isoforms were present throughout the entire process of its development and cycling but the expression of Dsg1 isoforms is subject to significant hair cycle-dependent changes. Dsg1-beta and -gamma, but not Dsg1-alpha, were detected in the sebaceous gland epithelium and the stratified epithelium of the stomach. Finally, Dsg1-alpha and Dsg1-beta, but not Dsg1-gamma, are proteolytically cleaved by exfoliative toxin A. These results suggest that the developmental complexity of mouse tissues, including skin and hair, may play a significant role in the evolutionary driving force to maintain multiple Dsg1 genes in mouse.

Amino Acid Sequence↗
PubMed2003

Prenatal diagnosis for epidermolysis bullosa: a study of 144 consecutive pregnancies at risk.

Epidermolysis bullosa (EB) is a group of inherited disorders characterized by increased skin fragility, resulting in blisters and erosions after minor trauma. Mutations in 10 structural genes expressed in the cutaneous basement membrane zone have been reported. The DebRA Molecular Diagnostics Laboratory at Jefferson Medical College has performed 144 DNA-based prenatal diagnoses since 1993 in families at risk for recurrence of the most severe forms of EB, including the recessive dystrophic EB (RDEB), junctional EB (JEB), EB with pyloric atresia (EB-PA), and EB simplex (EBS). A mutation-detection strategy using either conformation-sensitive gel electrophoresis (CSGE) or denaturing high-performance liquid chromatography (dHPLC) scanning analysis, followed by nucleotide sequencing, was applied to most cases with DEB and to all JEB, EB-PA, and EBS families. For some RDEB families, linkage analysis was performed, either alone when the inheritance pattern was clear or in combination with one mutation. Among the 144 prenatal diagnoses, 63 were for RDEB, 69 for JEB, 6 for EB-PA, and 6 for EBS. Twenty-eight normal, 73 heterozygous carrier, and 28 affected RDEB, JEB, and EB-PA pregnancies were reported in these recessively inherited diseases. Two affected and four normal pregnancies were predicted in dominantly inherited EBS. Among the 144 pregnancies, 9 were terminated without confirmation, 13 cases were lost to follow-up, and 6 pregnancies are ongoing. There were 6 families with inconclusive results due either to recombination events between flanking markers, absence of informative markers for one allele, or lack of sample from the previously affected child. There were three discordant results, one that was explained by maternal contamination of the chorionic villus sample and two that were unresolved. Overall, the availability, relative ease, and over 98% success rate make molecular DNA-based prenatal diagnosis a viable option for EB families at risk.

Adult↗
PubMed2003

Targeted inactivation of murine laminin gamma2-chain gene recapitulates human junctional epidermolysis bullosa.

Junctional forms of epidermolysis bullosa (JEB) are associated with mutations in six distinct genes expressed in the cutaneous basement membrane zone; these include LAMA3, LAMB3, and LAMC2, which encode laminin 5 subunit polypeptides, the alpha3-, beta3-, and gamma2-chains, respectively. Here we generated a mouse model for JEB by inactivating the laminin gamma2-chain gene by targeted frameshift deletion of exon 8 in Lamc2. Heterozygous mice were phenotypically normal, whereas the majority of Lamc2-/- mice showed blistering phenotype on days 1 to 2 and died within 5 days of birth. The Lamc2-/- mice demonstrated absent expression of laminin gamma2-chain on the basement membrane zone as well as attenuated expression of alpha3- and beta3-chains of laminin. Transmission electron microscopy revealed rudimentary, poorly developed hemidesmosomes. The epidermis of the Lamc2-/- mice revealed induced apoptosis in the basal cells of the blistered skin, suggesting that cell-matrix adhesion provided by laminin 5 plays a role in cell survival in vivo. Cultured Lamc2-/- keratinocytes demonstrated slightly positive staining with gamma2-chain-specific antibodies, which could be explained by the presence of a transcript with partial restoration of the reading frame owing to alternative splicing in vitro. These cells proliferated in different matrices and attached to type IV collagen and Matrigel as efficiently as the wild-type keratinocytes, whereas their attachment on plastic and laminin was significantly weaker. In summary, Lamc2-/- mouse recapitulates human JEB and provides novel insight into the role of laminin 5 in keratinocyte biology.

Animals↗
PubMed2002

Interspecies conservation and differential expression of mouse desmoglein gene family.

Epithelial cell adhesion is mediated by intercellular junctions, called desmosomes. Desmogleins (Dsg; Dsg1, Dsg2 and Dsg3) are calcium-dependent transmembrane adhesion components of the desmosomes. While Dsg1 and Dsg3 are mainly restricted to stratified squamous epithelia, Dsg2 is expressed in essentially all desmosome-containing epithelia. In the epidermis, Dsg2 and Dsg3 are expressed in the basal keratinocytes while Dsg1 is expressed throughout the upper differentiating cell layers. To date, in mouse, only Dsg3 has been characterized by molecular cloning. In this study, we have cloned and characterized the mouse Dsg1 and Dsg2 genes. The full-length mouse Dsg1 cDNA (5.5 kb) contains an open reading frame (ORF) of 3171 bp encoding a precursor protein of 1057 amino acids. The Dsg2 cDNA (6.3 kb) has an ORF of 3366 bp coding for a precursor protein of 1122 amino acids. Mouse Dsg2 protein shares 76% identity with human DSG2 but only 26% and 33% identity with mouse Dsg1 and Dsg3, respectively. Analysis of intron/exon organization of the desmoglein genes revealed significant conservation. However, the mRNA expression patterns of these desmogleins during mouse embryonic development and in various adult tissues are variable. While Dsg2 and Dsg3 are expressed in all developmental stages, Dsg1 expression is delayed until day 15 of mouse embryos. In adult mouse tissues, Dsg2 is widely expressed while the expression of Dsg1 and Dsg3 is restricted to select tissues. In summary, while desmogleins share high homology at both the gene and protein level, their expression is spatially and temporally regulated, potentially contributing to their significant role in cell-cell adhesion during development.

Amino Acid Sequence↗
PubMed2002

Compound heterozygosity for non-sense and mis-sense mutations in desmoplakin underlies skin fragility/woolly hair syndrome.

The constitutive desmosomal plaque protein desmoplakin plays a vital part in keratinocyte adhesion in linking the transmembranous desmosomal cadherins to the cytoplasmic keratin filament network. Recently, mutations in desmoplakin have been shown to underlie some cases of the autosomal dominant disorder, striate palmoplantar keratoderma, as well as an autosomal recessive condition characterized by dilated cardiomyopathy, woolly hair, and keratoderma. Here, we describe two unrelated individuals with a new autosomal recessive genodermatosis characterized by focal and diffuse palmoplantar keratoderma, hyperkeratotic plaques on the trunk and limbs, varying degrees of alopecia, but no apparent cardiac anomalies. Mutation screening of desmoplakin demonstrated compound heterozygosity for a non-sense/mis-sense combination of mutations in both cases, C809X/N287K and Q664X/R2366C, respectively. Heterozygous carriers of any of these mutations displayed no phenotypic abnormalities. Immunohistochemistry of skin biopsies from both affected individuals revealed that desmoplakin was not just located at the cell periphery but there was also cytoplasmic staining. In addition, electron microscopy demonstrated acantholysis throughout all layers of the skin, focal detachment of desmosomes into the intercellular spaces, and perinuclear condensation of the suprabasal keratin intermediate filament network. Clinicopathologic and mutational analyses therefore demonstrate that desmoplakin haploinsufficiency can be tolerated in some cases, but that in combination with a mis-sense mutation on the other allele, the consequences are a severe genodermatosis with specific clinical manifestations.

Amino Acid Sequence↗
PubMed2002

Progress in molecular genetics of heritable skin diseases: the paradigms of epidermolysis bullosa and pseudoxanthoma elasticum.

The 42nd Annual Symposium on the Biology of the Skin, entitled "The Genetics of Skin Disease", was held in Snowmass Village, Colorado, in July 1993. That meeting presented the opportunity to discuss how modern approaches to molecular genetics and molecular biology could be applied to understanding the mechanisms of skin diseases. The published proceedings of this meeting stated that "It is an opportune time to examine the genetics of skin disease" (Norris et al, 1994). Indeed, this meeting just caught the wave of early pioneering studies that have helped us to understand the molecular basis of a large number of genodermatoses. This overview presented in the 50th Annual Symposium on the biology of the skin, highlights the progress made in the molecular genetics of heritable skin diseases over the past decade.

Epidermolysis Bullosa↗
PubMed2002

Loss of cell adhesion in Dsg3bal-Pas mice with homozygous deletion mutation (2079del14) in the desmoglein 3 gene.

Pemphigus encompasses a group of autoimmune blistering diseases with circulating pathogenic autoantibodies recognizing several proteins, including the desmosomal cadherin, desmoglein 3. Targeted disruption of the Dsg3 gene by homologous recombination (Dsg3tm1stan) in mouse results in fragility of the skin and oral mucous membranes, analogous to the human disease. In addition, the Dsg3tm1stan mice develop phenotypic runting and hair loss, identical to that of the mouse mutant, Dsg3bal-2J. The Dsg3bal-2J mice are homozygous for a 1 bp insertion (2275insT) in the Dsg3 gene resulting in a nonfunctional Dsg3 mRNA. In this study, we characterized an allelic mutation, Dsg3bal-Pas, with clinical features similar to those in Dsg3bal-2J. We have identified a 14 bp deletion in exon 13 of the Dsg3 gene resulting in a frameshift and premature termination codon 7 bp downstream from the site of the deletion and causing a truncation of the desmoglein 3 polypeptide by 199 amino acids, eliminating virtually all of the intracellular domain. We demonstrate that, although a Dsg3 mRNA transcript was detectable in Dsg3bal-Pas skin, the corresponding protein for desmoglein 3 was completely absent in the oral mucosal epithelium of homozygous Dsg3bal-Pas compared with that of +/Dsg3bal-Pas mice. No significant changes in the expression of desmogleins 1 and 2 were detected. To elucidate a potential mechanism causing loss of cell adhesion in the Dsg3bal-Pas mice, we generated a myc-tagged truncated Dsg3bal-Pas desmoglein 3 protein and expressed it in keratinocytes. The myc-tagged truncated Dsg3bal-Pas desmoglein 3 protein was found predominantly in the cytoplasm possibly due to increased proteolytic degradation. Cell surface staining was also detected but was jagged, not linear along the cell-cell border like that observed for the full-length desmoglein 3. The expression of the myc-tagged truncated Dsg3bal-Pas desmoglein 3 protein resulted in a reduction in staining of other desmosomal proteins, including desmoglein 1 and 2, plakophilin 2, and plakoglobin. In addition, the cells expressing myc-tagged truncated Dsg3bal-Pas desmoglein 3 protein underwent dramatic changes in cell morphology and exhibited striking extensive filopodia. Collectively, these data showed that the perturbation of desmoglein 3 found in the Dsg3bal-Pas mice resulted in disadhesion of keratinocytes manifested with blistering phenotype.

Alopecia↗
PubMed2002

Junctional epidermolysis bullosa in the Middle East: clinical and genetic studies in a series of consanguineous families.

BACKGROUND: Junctional epidermolysis bullosa (JEB) is a group of inherited blistering diseases characterized by epidermal-dermal separation resulting from mutations that affect the function of critical components of the basement membrane zone. This group of autosomal recessive diseases is especially prevalent in regions where consanguinity is common, such as the Middle East. However, the clinical and genetic epidemiology of JEB in this region remains largely unexplored. OBJECTIVE: The aim of the present study was to assess a series of consanguineous JEB families originating from the Middle East. METHODS: We identified 7 families referred to us between 1998 and 1999 and originating from the United Arab Emirates, Saudi Arabia, Sudan, Yemen, and Israel. Histologic, immunofluorescence, and electron microscopy studies were performed to direct the subsequent molecular analysis. DNA obtained from all family members was amplified by means of polymerase chain reaction and analyzed by conformation-sensitive gel electrophoresis with subsequent direct sequencing. RESULTS: In 6 families presenting with the clinical and histologic features distinctive for JEB, mutations in genes encoding 1 of the 3 subunit polypeptides of laminin-5 were identified. Two families each had mutations in LAMB3, 2 in LAMA3, and 2 in LAMC2. Out of 7 distinct mutations, 5 were novel and 2 were recurrent. No relationship was found between the presence of nonsense/frameshift mutations in laminin-5 genes and perinatal mortality, contradicting a major genotype-phenotype correlation previously reported in the European and US literature. Similarly, none of the recurrent LAMB3 hot spot mutations previously described in other populations was found in our series. Finally, in a family with the clinical diagnosis of generalized atrophic benign epidermolysis bullosa, a homozygous non-sense mutation in Col17A1 gene (encoding the BPAG2 antigen) was identified. CONCLUSION: The present report suggests (1) the existence of a unique spectrum of mutations in the Middle East populations and (2) the need for the implementation of a diagnostic strategy tailored to the genetic features of JEB in this region.

Autoantigens↗
PubMed2001

Laminin 5 mutations in junctional epidermolysis bullosa: molecular basis of Herlitz vs. non-Herlitz phenotypes.

Junctional epidermolysis bullosa (JEB) is a group of heritable blistering diseases in which tissue separation occurs within the lamina lucida of the cutaneous basement membrane zone. Clinically, two broad subcategories have been recognized: The Herlitz variant (H-JEB; OMIM 226700) is characterized by early demise of the affected individuals, usually within the first year of life, while non-Herlitz (nH-JEB; OMIM 226650) patients show a milder phenotype with life-long blistering, yet with normal lifespan. In this study, we have examined a cohort of 27 families, 15 with Herlitz and 12 with non-Herlitz JEB, for mutations in the candidate genes, LAMA3, LAMB3, and LAMC2, encoding the subunit polypeptides of laminin 5. The mutation detection strategy consisted of PCR amplification of all exons in these genes, followed by heteroduplex scanning and nucleotide sequencing. We were able to identify pathogenic mutations in both alleles of each proband, the majority of the mutations being in the LAMB3 gene. Examination of the mutation database revealed that most cases with Herlitz JEB harbored premature termination codon (PTC) mutations in both alleles. In non-Herlitz cases, the PTC mutation was frequently associated with a missense mutation or a putative splicing mutation in trans. In three cases with putative splicing mutations, RT-PCR analysis revealed a repertoire of splice variants in-frame, predicting the synthesis of either shortened or lengthened, yet partly functional, polypeptides. These observations would explain the relatively mild phenotype in cases with splicing mutations. Collectively, these findings, together with the global laminin 5 mutation database, contribute to our understanding of the genotype/phenotype correlations explaining the Herlitz vs non-Herlitz phenotypes.

Adolescent↗