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Biomedical subjects

M J Fazio

Publications and source records attributed to M J Fazio.

16 recordsLinked to original sources
PubMed1995

Full-thickness skin grafts. Clinical observations on the impact of using epinephrine in local anesthesia of the donor site.

BACKGROUND AND DESIGN: A recent animal study indicated that epinephrine in local anesthesia adversely affects the survival of full-thickness skin grafts. Because animal models do not always correlate to humans, we performed a prospective observational study to elucidate the clinical outcome of using epinephrine in local anesthesia of the donor site. Seventy-two patients had cutaneous tumors excised by the Mohs micrographic technique. The resultant surgical defects were repaired using full-thickness skin grafts. Patients were randomly divided into two groups based on the local anesthetic used at the donor site: (1) 1% lidocaine (Xylocaine) (n = 33) or (2) 1% lidocaine with 1:100,000 epinephrine (n = 39). RESULTS: Assessment of the skin grafts at 1 week revealed a significantly increased risk of developing graft complications in the lidocaine with epinephrine group compared with the plain lidocaine group. The overall cosmetic outcome of the grafted site at 6 weeks revealed no significant difference between the two groups. CONCLUSIONS: Because there was only a minimal clinical effect of epinephrine on graft survival observed at 1 week and there was no effect on the 6-week cosmetic outcome, we do not recommend harvesting all full-thickness skin grafts with plain lidocaine. In certain clinical circumstances with compromised vascular supply or poor oxygenation, the use of plain lidocaine may be advantageous.

Aged↗
PubMed1992

De Barsy syndrome: report of a case, literature review, and elastin gene expression studies of the skin.

Several "progeroid" syndromes have now been identified. The De Barsy syndrome is an autosomal recessive syndrome of dwarfism, mental deficiency, an "aged" appearance at birth, abnormal elastic fibers on skin biopsy, and lax skin, large helices, eye abnormalities, lax joints, hypotonia, and athetoid posturing. We report one case and review 11 cases from the literature. To understand the abnormal appearance of the elastic fibers on biopsy, we performed elastin gene expression studies on fibroblasts cultured from our patient's skin. Molecular hybridization studies revealed reduced elastin mRNA steady-state levels as compared with age matched control individuals. Assuming normal rates of mRNA translation, reduced elastin synthesis would occur. Diminished dermal elastin content could explain the altered cutaneous elasticity, decreased elastic fibers in the skin, and many clinical manifestations of individuals with this condition.

Child, Preschool↗
PubMed1991

Human nidogen gene: structural and functional characterization of the 5'-flanking region.

Nidogen is a sulfated multifunctional glycoprotein present in basement membranes. In this study, we have cloned the 5'-flanking region of the human nidogen gene. Initially, an approximately 35-kb DNA clone (NCos4) was isolated from a human cosmid genomic library. Southern hybridization of EcoRI-digested NCos4 allowed isolation of a 3.7-kb fragment, which was shown to contain a portion of intron 1, the entire exon 1, and approximately 0.9 kb of 5'-flanking sequences of the nidogen gene. Nucleotide sequencing of the 5'-flanking DNA revealed the presence of two canonic CCAAT consensus sequences in the antisense strand and a potential variant of the TATA motif, TATTT, in the sense strand. One putative AP-2 and six putative SP1 binding sites were also present. To test the functional promoter activity of the 5'-flanking genomic DNA, two nidogen promoter/CAT reporter gene constructs, with the promoter segment spanning from -864 to -1 and from -534 to -1, respectively, were developed and analyzed in transient transfections of human and mouse cell cultures. Both constructs showed clearly detectable promoter activity, and the activity of the larger construct could be up-regulated by 12-O-tetradecanoyl phorbol 13-acetate up to 2.5 times. The results indicate that the nidogen promoter/CAT gene constructs developed in this study provide a means to examine the transcriptional regulation of nidogen gene expression in human diseases of the basement membrane zone.

Amino Acid Sequence↗
PubMed1991

Reconstruction of the nose with local flaps.

The author presents the results of a study of 200 patients with surgical defects of the nose following excision of skin malignancies. The location, size, depth, and quality of the adjacent skin, the reconstruction choice, and the cosmetic result were recorded. Healing by second intention was most useful for wounds in concave areas. Full-thickness skin grafts were used for defects too large for local flaps, or for defects on the nasal tip or alar surface. Local flaps were the most useful choice for nasal reconstruction. Transposition flaps, in particular, were most useful for each cosmetic sub unit of the nose.

Humans↗
PubMed1991

Human elastin gene: new evidence for localization to the long arm of chromosome 7.

In this study we have utilized human elastin cDNAs in molecular hybridizations to establish the chromosomal location of the human elastin gene. First, in situ hybridizations were performed with metaphase chromosomes from phytohemagglutinin-stimulated human peripheral blood lymphocytes. In three separate experiments using two different regions of human elastin cDNAs, the distribution of grains was found to be concentrated on the long arm of chromosome 7 within the [q11.1-21.1] region, and the peak number of grains coincided with the locus 7q11.2. Second, hybridizations with a panel of human-rodent cell hybrids showed concordance with human chromosome 7. Third, PCR analyses with elastin-specific primers of DNA from a hybrid cell line containing chromosome 7 as the only human chromosome yielded a product of the expected size, while DNA containing human chromosome 2, but not chromosome 7, did not result in a product. The results indicate that the human elastin gene is located in the proximal region of the long arm of chromosome 7. The precise localization of the elastin gene in the human genome is useful in establishing genetic linkage between inheritance of an allele with a mutated elastin gene and a heritable disorder.

Alleles↗
PubMed1990

Deletion analyses of 5'-flanking region of the human elastin gene. Delineation of functional promoter and regulatory cis-elements.

To delineate cis-acting regulatory elements of the human elastin gene, several elastin promoter region/chloramphenicol acetyltransferase reporter gene constructs were developed. The spectrum of inserts, spanning from -2260 to +2, was shown to contain several SP-1 and AP2 binding sites, as well as putative glucocorticoid, cAMP, and 12-O-tetradecanoylphorbol-13-acetate responsive elements. Assay of promoter activity in transient transfections of rat aortic smooth muscle cells, human skin fibroblasts, HT-1080 human fibrosarcoma cells, HeLa cells, or mouse NIH-3T3 cells allowed delineation of several functional subregions within 2.26 kilobases of the 5'-flanking DNA. The results suggest that the basic promoter element resides within the region -128 to -1, and the 5'-flanking DNA contains several functional regulatory subregions. Also, the regulatory function of three putative SP-1 binding sites was demonstrated by transfections with a plasmid devoid of such sequences. These findings attest to the complexity of transcriptional regulation of the elastin gene.

Animals↗
PubMed1990

Regulation of elastin gene expression: evidence for functional promoter activity in the 5'-flanking region of the human gene.

Analysis of nucleotide sequences in the 5'-flanking region of the human elastin gene has revealed several unusual features, suggesting that regulation of elastin gene expression is complex. To identify any cis-acting regulatory promoter elements, a 35-kb fragment of DNA (CosE) was isolated from a human genomic cosmid library by hybridizations with a human elastin cDNA. Southern blots of EcoRI digests of CosE DNA, utilizing a 5'-end labeled 21-mer oligonucleotide corresponding to the signal sequence of elastin, revealed the presence of a single 7.8-kb genomic fragment. Partial dideoxynucleotide sequencing of this EcoRI genomic subclone revealed that it extended approximately 2.5 kb 3' of the translation initiation site (ATG), encompassing exon 1 and a portion of the first intron, while the remaining DNA encompassed the 5'-flanking region. Exonuclease III digestion (3'----5') was performed to remove sequences of the first intron and first exon, including the ATG site. One clone, approximately 5 kb in size, had the 3' end located 14 bp upstream of the ATG site. A 462-bp 3' portion of this 5-kb fragment was subcloned into a Bluescript/CAT chimeric plasmid (pBS0CAT) to generate an elastin gene promoter/CAT reporter gene construct (pEP6CAT). Transient transfection experiments with pEP6CAT using human skin fibroblasts, human HT-1080, mouse NIH-3T3, or freshly isolated neonatal rat aortic smooth muscle cells revealed significant CAT activity in each cell line. These results suggest that the 5'-flanking region of the elastin gene contains the cis-acting regulatory elements necessary for transcription. The chimeric plasmid pEP6CAT provides a means to study the transcriptional control of elastin gene expression by exogenous affector molecules, as well as in human dermatologic diseases.

Base Sequence↗
PubMed1989

Human nidogen: complete amino acid sequence and structural domains deduced from cDNAs, and evidence for polymorphism of the gene.

Nidogen, a multifunctional glycoprotein, is an integral part of all basement membranes. In this study, human nidogen cDNAs were isolated and characterized from human placental and skin fibroblast cDNA libraries by hybridization with a mouse nidogen cDNA probe. Six overlapping clones covering 4.9 kb were characterized. The composite cDNA contained a 3,741-nucleotide open reading frame which coded for a 1,247-amino-acid peptide that included a hydrophobic signal sequence. The deduced amino acid sequence contains seven epidermal growth factor-like cysteine-rich repeats, one possible tyrosine O-sulfation site, and a possible N-glycosylation site. The tripeptide sequence -Arg-Gly-Asp- (RGD), a potential cell attachment site, was also present. Human and mouse nidogen sequences were 84% homologous at the nucleotide level and 85% homologous at the deduced amino acid level. Southern blotting of human leukocyte DNA from 23 individuals indicated that nidogen probably is a single-copy gene and shows multiple restriction fragment length polymorphisms when cleaved with Eco RI, Pvu II, Taq I, and Msp I. In particular, digestions with Pvu II revealed polymorphism in four discrete DNA fragments, which could be discriminated by hybridizations with nidogen subclones. One of the polymorphisms revealed an allelic frequency of 0.52/0.48. Thus, human nidogen gene displays RFLPs which provide analytical tools to establish genetic linkage between the nidogen gene and a clinical phenotype.

Amino Acid Sequence↗
PubMed1989

Extracellular matrix of the skin: 50 years of progress.

The extracellular connective tissue matrix of the skin is a complex aggregate of distinct collagenous and non-collagenous components. Optimal quantities and delicate interactions of these components are necessary to maintain normal physiologic properties of skin. This overview summarizes the progress made in understanding the normal biology and biochemistry of the extracellular matrix, and will highlight cutaneous diseases with underlying molecular defects in the structure and expression of extracellular matrix components.

Animals↗
PubMed1989

Molecular mechanisms of cutaneous aging. Age-associated connective tissue alterations in the dermis.

Clinically detectable, age-associated cutaneous changes result from two independent processes: chronologic aging and actinic irradiation. Several lines of evidence suggest that these two processes have different biologic, biochemical, and molecular mechanisms. This review summarizes the current understanding of age-associated alterations in the biochemistry and molecular biology of the extracellular matrix.

Aged↗
PubMed1989

Skin aging: lessons from cutis laxa and elastoderma.

Several lines of evidence suggest that elastic fibers provide resilience to normal human skin, and that abnormalities in elastin may be the primary event leading to the clinical appearance of aged skin. Innately aged skin exhibits a paucity and fragmentation of elastic fibers, while actinically damaged skin consists of an abnormal accumulation of elastotic material within the dermis. Cutis laxa and elastoderma are two cutaneous diseases that manifest as selective alterations in elastic fibers. Clinical and histopathologic findings in these diseases are similar to those found in cutaneous aging. Thus, it appears that alterations in the quantity and/or quality of the elastic fibers may contribute to age-associated cutaneous changes.

Adolescent↗
PubMed1988

Cutis laxa: reduced elastin gene expression in skin fibroblast cultures as determined by hybridizations with a homologous cDNA and an exon 1-specific oligonucleotide.

Fibroblast cultures were established from six patients with cutis laxa, and elastin gene expression was analyzed by RNA hybridizations with a 2.5-kilobase human elastin cDNA or an exon 1-specific 35-base oligomer. Northern analyses using either probe detected mRNA transcripts of approximately 3.5 kilobases, and no qualitative difference between the control and cutis laxa mRNAs was detected. However, quantitation of the elastin mRNA abundance by slot blot hybridizations revealed markedly reduced levels in all cutis laxa cell strains. Assuming equal translational activity of the control and cutis laxa mRNAs, the reduced mRNA levels could result in diminished elastin production, providing an explanation for the paucity of elastic fibers in the skin and other tissues in cutis laxa.

Base Sequence↗
PubMed1988

Cloning of full-length elastin cDNAs from a human skin fibroblast recombinant cDNA library: further elucidation of alternative splicing utilizing exon-specific oligonucleotides.

A human cDNA library was constructed utilizing RNA isolated from cultured skin fibroblasts. Recombinant clones containing elastin sequences were identified by plaque hybridizations with previously characterized human placental elastin cDNAs. Seven positive recombinant clones with inserts of approximately 3.2-2.2 kb were isolated. Characterization of the clones by restriction endonuclease analysis and dot-blot hybridizations with exon-specific synthetic oligonucleotides demonstrated considerable variability in the primary nucleotide sequence. Dideoxy nucleotide sequencing confirmed this finding. The variability is most likely a result of alternative splicing of exons from the primary elastin transcripts. The two largest clones contained approximately 1 kb of 3' untranslated sequence and approximately 2.2 kb of translated sequence encoding 730 amino acids. Six amino acids, encoded by exon 12A, have not been previously noted in human elastin cDNAs. In addition, these human skin fibroblast clones contained a 49 bp 5' untranslated sequence. These results demonstrate that there is considerable variability in the processed nucleotide sequence of the elastin mRNAs. These transcripts may code for isoforms of tropoelastin with different biologic properties.

Amino Acid Sequence↗
PubMed1988

Isolation and characterization of human elastin cDNAs, and age-associated variation in elastin gene expression in cultured skin fibroblasts.

In attempts to isolate human elastin cDNAs, a human placental lambda gt11 cDNA library was screened with a 1.3 kilobase sheep genomic DNA subclone, corresponding to the 3'-end of the elastin mRNA. The four largest clones, the largest being approximately 3 kilobase, were characterized by Northern transfer analyses, restriction endonuclease digestions and dideoxy nucleotide sequencing. Northern transfer analyses of poly(A)+RNA revealed hybridization to mRNA transcripts in the region of 3.5 kilobase. Restriction endonuclease mapping and nucleotide sequencing demonstrated distinct domains characteristic of elastin, and identified areas of variability which apparently reflects alternative splicing of the primary elastin transcripts. To demonstrate the utilization of these cDNAs for studies on elastin gene expression in human cells, elastin mRNA was examined in fibroblast cultures established from the skin of several individuals of varying ages. Northern transfer analyses and slot blot hybridizations demonstrated that elastin gene expression is initiated early during fetal development, and continues at a relatively constant level through several decades. The lowest abundance of elastin mRNA was noted in the cell cultures established for the oldest individual studied (61-year-old female). Demonstration of elastin gene expression in cultured fibroblasts provides a system to study diseases affecting the elastic fibers.

Aging↗
PubMedDate not supplied

Membrane skeletal proteins and their integral membrane protein anchors are targets for tyrosine and threonine kinases in Euglena.

Proteins of the membrane skeleton of Euglena gracilis were extensively phosphorylated in vivo and in vitro after incubation with [32P]-orthophosphate or gamma-[32P] ATP. Endogenous protein threonine/serine activity phosphorylated the major membrane skeletal proteins (articulins) and the putative integral membrane protein (IP39) anchor for articulins. The latter was also the major target for endogenous protein tyrosine kinase activity. A cytoplasmic domain of IP39 was specifically phosphorylated, and removal of this domain with papain eliminated the radiolabeled phosphoamino acids and eliminated or radically shifted the PI of the multiple isoforms of IP39. In gel kinase assays IP39 autophosphorylated and a 25 kDa protein which does not autophosphorylate was identified as a threonine/serine (casein) kinase. Plasma membranes from the membrane skeletal protein complex contained threonine/serine (casein) kinase activity, and cross-linking experiments suggested that IP39 was the likely source for this membrane activity. pH optima, cation requirements and heparin sensitivity of the detergent solubilized membrane activity were determined. Together these results suggest that protein kinases may be important modulators of protein assembly and function of the membrane skeleton of these protistan cells.

Adenosine Triphosphate↗