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Biomedical subjects

M K Duncan

Publications and source records attributed to M K Duncan.

At least 19 recordsLinked to original sources
PubMed2002

Abdominal pressure during laparoscopy: effects of fentanyl.

BACKGROUND: In patients breathing spontaneously during anaesthesia, expiration is active and opioids enhance this effect. The mechanical consequences are not well characterized. METHODS: We studied 14 patients undergoing laparoscopy for minor gynaecological procedures, anaesthetized with isoflurane in nitrous oxide, and breathing spontaneously through a laryngeal mask airway. We made direct recordings of intra-abdominal pressure and respiratory flow before and after giving fentanyl 25 microg i.v. RESULTS: Satisfactory records were obtained in 11 patients. Before fentanyl, the abdominal pressure changes were small and had an inconsistent pattern, increasing in inspiration in seven patients and during expiration in five. After fentanyl, an increase in pressure during inspiration was seen in only two patients, and the intra-abdominal pressure during expiration was increased. The mean value of maximum abdominal pressure (which always occurred during expiration) increased from 17 (SD 5) cm H2O before to 25 (9) cm H2O after fentanyl (P<0.01). CONCLUSIONS: Direct measurements support previous findings that opioids stimulate active phasic expiratory activity and can cause large increases in abdominal pressure.

Abdominal Muscles↗
PubMed2001

An immunohistochemical method for the detection of proteins in the vertebrate lens.

Fluorescence immunohistochemistry has traditionally been difficult or impossible to perform on the vertebrate lens because of its extremely high protein content. Described here is a robust and rapid method for preparing and labeling vertebrate eyes for confocal microscopy. This technique has successfully been applied to localize proteins in the lens epithelium and capsule, as well as the primary and secondary fibers. This technique preserves tissue morphology and coupled with double and triple labeling, has allowed localization of proteins bound to plasma membrane, basement membrane, nucleus, endoplasmic reticulum as well as sub-nuclear compartments. In addition, the present technique has proven useful for fluorescent immunohistochemical analysis of diverse tissues including whole embryos, adult muscle, pancreas, and liver. This procedure allowed us to successfully localize a wide variety of antigens on diverse vertebrate tissues including the more challenging vertebrate lens.

Animals↗
PubMed2001

The mouse beta B1-crystallin promoter: strict regulation of lens fiber cell specificity.

Previous studies have shown that the chicken beta B1-crystallin promoter (-434/+30) contains all of the signals necessary to specifically direct high level expression of heterologous genes to the lens fiber cells of mice. In the present study, the mouse beta B1-crystallin gene was cloned, and its regulation was investigated to further elucidate the mechanisms controlling lens fiber cell-specific gene expression. Phylogenetic footprinting analysis of the 5' flanking sequence from the mouse, rat, human and chicken beta B1-crystallin genes identified several known and putative functional cis elements including the PL2 element which is required for lens-specific expression of the chicken beta B1 promoter. Surprisingly, however, all six mouse beta B1-crystallin/CAT constructs tested (-1493/+44, -1493/+30, -870/+30, -250/+30, -135/+30 and -98/+30) were inactive in three different mammalian lens-derived cell lines while only the -870/+30 and -98/+30 constructs were active in chicken primary patched lens epithelial cells. In contrast, the chicken beta B1-crystallin promoter (-434/+30) was transcriptionally active in all lens-derived cells tested. Transgenic mice harboring a mouse beta B1-crystallin -1493/+44 CAT construct did express the transgene specifically in lens fiber cells, however, at lower levels than that previously reported for a chicken -434/+30 CAT construct. These data suggest that, as in other crystallin genes, the regulatory signals controlling lens fiber cell-specific expression are conserved between chicken and mouse. However, the inability of the mouse beta B1-crystallin promoter to function in mammalian lens-derived cultured cells implies that this gene has acquired additional cis-regulatory elements to ensure lens fiber cell specificity.

Amino Acid Sequence↗
PubMed2001

Characterization and expression of calpain 10. A novel ubiquitous calpain with nuclear localization.

Calpains are calcium-dependent intracellular nonlysosomal proteases that are believed to hydrolyze specific substrates important in calcium-regulated signaling pathways. Recently, an atypical member of the calpain family, calpain 10, was described, and genetic variation in this gene was associated with an increased risk of type II diabetes mellitus in humans. In the present report, a polyclonal antibody directed against rat calpain 10 was developed. This antibody was used to monitor the expression of calpain 10 protein in tissues from rats, mice, and humans. Calpain 10 protein was found to be present in all tissues examined by Western blotting including the lens, retina, brain, heart, and skeletal muscle. Although some calpain 10 was detectable in the water-soluble protein fraction of these tissues, it was preferentially found in the water-insoluble fraction. In the lens, immunohistochemistry revealed that calpain 10 was predominately located in the cytoplasm of epithelial and newly differentiating lens fibers at the transition zone. However, calpain 10 was found to be associated with the plasma membrane of differentiated lens fiber cells and the sarcolemma of skeletal muscle. In the lens epithelium-derived cell line, alphaTN4-1, the calpain 10 protein was found in a punctate distribution in the cell nucleus as well as the cytoplasm. After the elevation of intracellular calcium levels with ionomycin, calpain 10 protein levels in the nucleus of alphaTN4-1 cells increased markedly, whereas those in the cytoplasm decreased. In the lens, the elevation of intracellular calcium levels after selenite administration resulted in increased levels of calpain 10 RNA within 1 day and a loss of calpain 10 protein from the lens nucleus coincident with the onset of selenite cataract. In conclusion, calpain 10 seems to be a ubiquitous calpain, the expression level and subcellular distribution of which are dynamically influenced by calcium.

Adult↗
PubMed2001

Production of monoclonal antibodies against chicken Pop1 (BVES).

Pop1 (BVES) is a member of the Popeye gene family which was named for its high-level expression in the heart and other muscle lineages. However, these proteins have no sequence similarity to any other gene family and their function is unclear. Here we report the production of recombinant chicken Pop1/BVES protein and the generation of two Pop1/BVES specific monoclonal antibodies (MAbs) 3F11-D9-E8 and 1B3-G11-A8. These antibodies detect recombinant Pop1/BVES in ELISAs and endogenous chicken Pop1/BVES in chicken heart extracts by Western blotting. Further, both 3F11-D9-E8 and 1B3-G11-A8 detect Pop1/BVES specifically in the cardiomyocytes of 8-day-old embryonic chicken hearts by immunofluorescence. These MAbs will be useful in immunolocalization and immunoblotting experiments of different tissue types to determine the location and levels of Pop1/BVES expression throughout development, as well as further analysis of the biochemistry of this protein.

Animals↗
PubMed2000

Overexpression of PAX6(5a) in lens fiber cells results in cataract and upregulation of (alpha)5(beta)1 integrin expression.

The PAX6 gene, a key regulator of eye development, produces two major proteins that differ in paired domain structure: PAX6 and PAX6(5a). It is known that an increase in the PAX6(5a) to PAX6 ratio leads to multiple ocular defects in humans. Here, transgenic mice were created that overexpress human PAX6(5a) in the lens. These mice develop cataracts with abnormalities in fiber cell shape as well as fiber cell/lens capsule and fiber cell/fiber cell interactions. While the structure of the actin cytoskeleton appeared relatively normal, the cataractous lens expresses increased amounts of paxillin and p120(ctn) as well as large aggregates of (alpha)5(beta)1 integrin in the dysgenic fiber cells. The elevated amounts of these proteins in the transgenic lens correlated well with elevated levels of their respective mRNAs. To investigate the role of Pax-6(5a) in the upregulation of these genes, a series of gel shift experiments using truncated proteins and consensus oligonucleotides demonstrated the complexity of Pax-6 and Pax-6(5a) binding to DNA, aiding our identification of potential binding sites in the human (&agr;)5- and (beta)1-integrin promoters. Consequent gel shift analysis demonstrated that these putative regulatory sequences bind Pax-6 and/or Pax-6(5a) in lens nuclear extracts, suggesting that the human (alpha)5 and (beta)1 integrin promoters contain PAX6/PAX6(5a) binding sites and maybe directly regulated by this transcription factor in the transgenic lens. We conclude that these transgenic mice are good models to study a type of human cataract and for identifying batteries of genes that are directly or indirectly regulated by both forms of Pax-6.

Animals↗
PubMed2000

Truncated forms of Pax-6 disrupt lens morphology in transgenic mice.

PURPOSE: Extensive literature shows that Pax-6 is critical for lens development and that Paxb mutations can result in aniridia in humans. In addition, it has been reported that truncated Pax-6 molecules can act as dominant-negative repressors of wild-type Pax-6 activity in cultured cells. This study was designed to determine whether Pax-6 molecules without either the activation domain (AD) or the homeodomain (HD) and the AD can function as dominant-negative repressors in vivo and alter the phenotype of the lens. METHODS: Transgenic mice were created harboring the alphaA-crystallin promoter linked to a cDNA encoding either a truncated Pax-6 without the C terminus (paired domain [PD] + homeodomain) or Pax-6 consisting of only the PD. The phenotype of the resultant animals was investigated by light and electron microscopy as well as atomic absorption spectroscopy. RESULTS: Two lines of PD + HD mice and three lines of PD mice were generated, all of which exhibit posterior nuclear and/or cortical cataracts of variable severity. The lenses from mice transgenic for either Pax-6 truncation are smaller and more hydrated than normal. Morphologically, the mice expressing the PD + HD of Pax-6 have swollen lens fibers with attenuated ball-and-socket junctions. In contrast, the lenses from mice overexpressing the PD of Pax-6 have posterior nuclear cataracts composed of cell debris, whereas the remaining fiber cells appear generally normal. CONCLUSIONS: The presence of truncated Pax-6 protein in the lens is sufficient to induce cataract in a wild-type genetic background. The simplest explanation for this phenomenon is a dominant-negative effect; however, a number of other possible mechanisms are presented.

Animals↗
PubMed2000

Influence of specific regions in Lp82 calpain on protein stability, activity, and localization within lens.

PURPOSE: To determine the influence of specific regions within Lp82 calpain on protein stability, enzymatic activity, and localization within lens and to test the influence of an Lp82 knockout mouse on normal maturational proteolysis in lens. METHODS: DNA constructs for Lp82 and Lp82-related proteins were subcloned into the pcDNA 3.1 vector. The constructs contained a substitution of the novel sequence (NS) region from p94 for the AX1 N-terminal region of Lp82 and insertions of the p94 IS1 and IS2 regions into Lp82. Transient expression of these Lp82-related proteins was performed in COS-7 mammalian cells. Immunoblotting and casein zymography were used to measure protein stability and enzymatic activity of the expressed proteins. Homologous recombination was used to knock out p94 gene expression and p94 splice variants such as Lp82 and Lp85 in the lenses of 10-day-old mice. Confocal microscopy revealed the immunohistochemical localization Lp82 and Lp85 within lens. RESULTS: Insertion of IS1 into Lp82 resulted in a lack of stable protein and loss of enzymatic activity. In contrast, substitution of the NS region for AX1 and insertion of IS2 into Lp82 had no effect on the stability of the Lp82-related proteins. p94 knockout mice at 10 days of age exhibited a total absence of Lp82 activity in the lens but normal activity for the separate mu- and m-calpain gene products. Calcium-induced in vitro proteolysis was retarded in these Lp82/p94 knockout lenses. Lp82 and Lp85 immunostaining was intense throughout the cytoplasm of the cortical and nuclear fibers of newborn mouse lenses with little staining in the epithelium. In contrast, immunostaining for the ubiquitous m-calpain was highest in the epithelium and bow region, with much lower levels in the nucleus. The naturally occurring IS3 insert in Lp85 also promoted the association of Lp85 with the perinuclear region of the nucleated lens fibers. CONCLUSIONS: The lack of the IS1 region in Lp82 accounts for the stability and abundance of enzymatically active Lp82 protein in rodent lenses. Conversely, the presence of the IS1 region is responsible for the lability of p94 and Rt88 calpains in muscle and retina, respectively. The insert in Lp85 may promote membrane association. A consequence of the specific loss of Lp82 in the lens may be to retard normal maturational proteolysis.

Animals↗
PubMed1998

Dual roles for Pax-6: a transcriptional repressor of lens fiber cell-specific beta-crystallin genes.

It has been demonstrated previously that Pax-6, a paired domain (PD)/homeodomain (HD) transcription factor critical for eye development, contributes to the activation of the alphaB-, alphaA-, delta1-, and zeta-crystallin genes in the lens. Here we have examined the possibility that the inverse relationship between the expression of Pax-6 and beta-crystallin genes within the developing chicken lens reflects a negative regulatory role of Pax-6. Cotransfection of a plasmid containing the betaB1-crystallin promoter fused to the chloramphenicol acetyltransferase reporter gene and a plasmid containing the full-length mouse Pax-6 coding sequences into primary embryonic chicken lens epithelial cells or fibroblasts repressed the activity of this promoter by as much as 90%. Pax-6 constructs lacking the C-terminal activation domain repressed betaB1-crystallin promoter activity as effectively as the full-length protein, but the PD alone or Pax-6 (5a), a splice variant with an altered PD affecting its DNA binding specificity, did not. DNase footprinting analysis revealed that truncated Pax-6 (PD+HD) binds to three regions (-183 to -152, -120 to -48, and -30 to +1) of the betaB1-crystallin promoter. Earlier experiments showed that the betaB1-crystallin promoter sequence from -120 to -48 contains a cis element (PL2 at -90 to -76) that stimulates the activity of a heterologous promoter in lens cells but not in fibroblasts. In the present study, we show by electrophoretic mobility shift assay and cotransfection that Pax-6 binds to PL2 and represses its ability to activate promoter activity; moreover, mutation of PL2 eliminated binding by Pax-6. Taken together, our data indicate that Pax-6 (via its PD and HD) represses the betaB1-crystallin promoter by direct interaction with the PL2 element. We thus suggest that the relatively high concentration of Pax-6 contributes to the absence of betaB1-crystallin gene expression in lens epithelial cells and that diminishing amounts of Pax-6 in lens fiber cells during development allow activation of this gene.

Animals↗
PubMed1997

Expression of the helix-loop-helix genes Id-1 and NSCL-1 during cerebellar development.

Neurons throughout the central nervous system (CNS) undergo proliferation, migration, and differentiation during their histogenesis. Although numerous regulatory molecules are expressed in developing neurons, it is unknown whether most of these molecules have the same function throughout the CNS or play different roles in different neuronal populations. Previous studies have shown that Id-1 and NSCL-1 are expressed at high levels in the ventricular and subependymal zones, respectively, of the embryonic brain. In the present study, the expression of Id-1 and NSCL-1 was further investigated during postnatal development of the cerebellum. By Northern blot hybridization analysis, the expression levels of Id-1 and NSCL-1 mRNA were developmentally regulated in the cerebellum, with the highest mRNA levels coinciding with the time of maximal granule cell histogenesis. By in situ hybridization, NSCL-1 mRNA was found in the premigratory zone of the external granule layer (EGL), a structure developmentally analogous to the subependymal zone of the embryonic brain. In normal mice, Id-1 mRNA was found to be transiently expressed in the upper internal granule layer (IGL), a population of cells that recently completed their migration from the EGL. In the mouse mutant weaver, Id mRNA was only seen in granule cells that have reached their normal positions in the IGL. No Id-1 hybridization signal was observed in the large numbers of granule cells remaining in the EGL of weaver mice, indicating that Id-1 expression is controlled by spatial cues. The lack of Id-1 expression in ectopic weaver granule cells is compatible with previous suggestions of arrested differentiation. These results support the idea that transcriptional regulators of the helix-loop-helix gene family play important roles in neuronal development, exhibiting region-specific expression and function.

Animals↗
PubMed1997

Eyes absent: a gene family found in several metazoan phyla.

Genes related to the Drosophila eyes absent gene were identified in vertebrates (mouse and human), mollusks (squid), and nematodes (C. elegans). Proteins encoded by these genes consist of conserved C-terminal and variable N-terminal domains. In the conserved 271-amino acid C-terminal region, Drosophila and vertebrate proteins are 65-67% identical. A vertebrate homolog of eyes absent, designated Eya2, was mapped to Chromosome (Chr) 2 in the mouse and to Chr 20q13.1 in human. Eya2 shows a dynamic pattern of expression during development. In the mouse, expression of Eya2 was first detected in 8.5-day embryos in the region of head ectoderm fated to become the forebrain. At later stages of development, Eya2 is expressed in the olfactory placode and in a variety of neural crest derivatives. In the eye, expression of Eya2 was first detected after formation of the lens vesicle. At day 17.5, the highest level of Eya2 mRNA was observed in primary lens fibers. Low levels of Eya2 expression was detected in retina, sclera, and cornea. By postnatal day 10, Eya2 was expressed in secondary lens fibers, cornea, and retina. Although Eya2 is expressed relatively late in eye development, it belongs to the growing list of factors that may be essential for eye development across metazoan phyla. Like members of the Pax-6 gene family, eyes absent gene family members were probably first involved in functions not related to vision, with recruitment for visual system formation and function occurring later.

Age Factors↗
PubMed1996

Structure and chromosomal localization of the human homeobox gene Prox 1.

The genomic organization and nucleotide sequence of the human homeobox gene Prox 1 as well as its chromosomal localization have been determined. This gene spans more than 40 kb, consists of at least 5 exons, and encodes an 83-kDa protein. It shows 89% identity with the chicken sequence at the nucleotide level in the coding region, while the human and chicken proteins are 94% identical. Among the embryonic tissues analyzed (lens, brain, lung, liver, and kidney), the human Prox 1 gene is most actively expressed in the developing lens, similar to the expression pattern of the chicken Prox 1 gene. The Prox 1 gene was mapped to human chromosome 1q32.2-q32.3.

Adult↗
PubMed1996

Chicken homeobox gene Prox 1 related to Drosophila prospero is expressed in the developing lens and retina.

Prox 1 is the vertebrate homolog of Drosophila prospero, a gene known to be expressed in the lens-secreting cone cells of fly ommatidia. Chicken Prox 1 cDNAs were isolated from 14 day embryonic chicken lenses, and a complete open reading frame encoding an 83 kDa protein was elucidated. The homeodomains of chicken and mouse Prox 1 are identical at the amino acid level and are 65-67% similar to the homeodomains of Drosophila and C. elegans prospero. The homology between these proteins extends beyond the homeodomain. There is 56% identity between chicken Prox 1 and Drosophila prospero in the C-terminal region downstream of the homeodomain, whereas there is little similarity upstream of the homeodomain. Prox 1 is expressed most actively in the developing lens and midgut and at lower levels in the developing brain, heart, muscle, and retina. cDNA sequencing has established that there are alternatively spliced forms of the single Prox 1 gene, which probably account for the two abundant RNAs of about 2 and 8 kb and two less abundant RNAs close to 3.5 kb in length in the lens. In the lens fibers, only the shortest mRNA was present, whereas, in the epithelial cells, both short and long mRNAs were detected. By using in situ hybridization, expression of the Prox 1 gene was first detected at stage 14 in the early lens placode and slightly preceded the expression of delta 1-crystallin, the first crystallin gene expressed in the developing chicken lens. At later stages of development, Prox 1 mRNA was observed throughout the lens, but it appeared more abundant around the bow region of the equator than in the anterior epithelium or the fibers. In the retina, expression of the Prox 1 gene was detected mainly in the inner nuclear layer during later stages of histogenesis. The conserved pattern of Prox 1/prospero gene expression in vertebrates and Drosophila suggests that Prox 1, like Pax-6, may be essential for eye development in different systematic groups.

Amino Acid Sequence↗
PubMed1996

Spatial and temporal activity of the alpha B-crystallin/small heat shock protein gene promoter in transgenic mice.

In order to study the spatial and temporal activity of the mouse alpha B-crystallin/small heat shock gene promoter during embryogenesis, we generated mice harboring a transgene consisting of approximately 4 kbp of alpha B-crystallin promoter sequence fused to the Escherichia coli lacZ reporter gene. beta-galactosidase activity was first observed in the heart rudiment of 8.5 days post coitum (d.p.c.) embryos. An identical expression pattern was obtained for the endogenous alpha B-crystallin gene by whole mount in situ hybridization. At 9.5 d.p.c., beta-galactosidase activity was detected in the lens placode, in the myotome of the somites, in Rathke's pouch (future anterior pituitary), and in some regions of oral ectoderm. We also examined the stress inducibility of the alpha B-crystallin promoter in vivo. Injection of sodium arsenite into mice resulted in increased endogenous alpha B-crystallin expression in the adrenal gland and possibly the liver. Our results indicate that visualization of beta-galactosidase activity provides an accurate reflection of endogenous alpha B-crystallin expression and demonstrate that the complex developmental pattern of mouse alpha B-crystallin gene expression is regulated at the transcriptional level. This expression pattern, coupled with the present literature which addresses functions of the protein, suggests a role for the alpha B-crystallin/small heat shock protein in intermediate filament turnover and cellular remodeling which occur during normal development and differentiation.

Animals↗
PubMed1996

Sequence and expression of chicken beta A2- and beta B3-crystallins.

Crystallins are a diverse group of proteins that contribute to the transparency and refractive properties of the eye lens. Previously, the chicken orthologs of four out of the six known bovine beta-crystallin genes have been cloned and sequenced. In the present study, cDNAs corresponding to the chicken orthologs of beta A2- and beta B3-crystallin, the two previously unidentified chicken beta-crystallins, have been isolated. In addition, sequence analysis of three independent chicken beta B2-crystallin cDNAs yielded a deduced connecting peptide sequence which is considerably shorter than that reported previously. Thus, direct homologs of all of the known bovine beta-crystallins are expressed in the chicken lens. This demonstrates that the duplications giving rise to the known vertebrate beta-crystallins occurred over 300 million years ago. beta B2- and beta B3/A1-crystallin are the most highly conserved of the beta-crystallins suggesting that these genes may be important for other functions besides their refractive role in the lens. By Northern blot hybridization analysis, both beta A2- and beta B3-crystallin were shown to be lens-specific in the chicken embryo. The relative levels of beta A2-crystallin remained stable from five days of embryogenesis until adulthood, while the relative amounts of beta B3-crystallin increased until hatching and were appreciably lower in the adult lens. Approximately equal relative amounts of beta A2-crystallin mRNA were found in the lens epithelia and fibers of 5 day embryonic chicken embryos; by contrast, beta B3-crystallin mRNA was detected preferentially in the lens fibers. These data in combination with previous studies suggest that beta-crystallin genes are regulated independently from each other in the developing chicken lens. The elucidation of the primary structures for all seven chicken beta-crystallin polypeptides will facilitate future studies on the structure/function relationships responsible for lens transparency and on the molecular basis for beta-crystallin gene expression during development.

Amino Acid Sequence↗
PubMed1996

Developmental regulation of the chicken beta B1-crystallin promoter in transgenic mice.

The cis-elements responsible for the high-level, lens-specific expression of the chicken beta B1-crystallin gene were investigated by generating mice harboring beta B1-crystallin promoter/chloramphenicol acetyl transferase (CAT) transgenes. Deletion of promoter sequences -434/-153 and -152/-127 as well as site-directed mutagenesis of the PL1 (-116/-102) and Pl2 (-90/-76) elements significantly decreased CAT gene expression in the lenses of adult transgenic mice. Transfection studies using multimerized PL1 and PL2 elements fused to the chicken beta-actin basal promoter indicated that PL1 is a general activating element while PL2 is involved in the lens-specificity of the chicken beta B1-crystallin promoter. CAT histochemistry demonstrated that the chicken beta B1-crystallin promoter (-434/+30) was active in both primary and secondary lens fiber cells from 12.5 days post coitum (dpc) until adulthood. Activity of the -152/+30/CAT transgene was relatively low and confined to the primary lens fiber cells of 16.5 dpc mice. Together, these data suggest that the reduced activity of this promoter in the adult lens is due both to this developmentally restricted expression pattern and a reduction in promoter activity. RNA hybridization studies demonstrated that the chicken beta B1-crystallin/CAT (-434/+30) transgene was expressed at similar levels in the same cells as the endogenous mouse beta B1-crystallin gene in 16.5 dpc transgenic mouse embryos. These data show a strict conservation of the lens-specific spatial and temporal regulation of the chicken and mouse beta B1-crystallin genes.

Age Factors↗