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Biomedical subjects

K Guan

Publications and source records attributed to K Guan.

36 records · Page 2Linked to original sources
PubMed1999

Induction of cellular differentiation by retinoic acid in vitro.

Cellular differentiation by the vitamin A derivative retinoic acid (RA) has been studied with undifferentiated pluripotent embryonic carcinoma (EC) and embryonic stem (ES) cells in vitro. Both cellular systems are suitable to study differentiation of various cell types, because they recapitulate early stages of mouse embryogenesis. In vivo, RA was identified as a morphogenic and teratogenic compound and furthermore as a signalling molecule influencing gene expression in a complex manner via a family of RA receptors. Here, we summarize in vitro studies with ES and EC cells in comparison to in vivo studies that have contributed to our understanding how RA influences differentiation and regulates gene expression. We demonstrate that modulation of ES cell differentiation in vitro by RA depends on the concentration and developmental stage of application which is comparable to its stage-dependent influence on embryonic development in vivo.

Animals↗
PubMed1999

Embryonic Stem Cells in vitro - Prospects for Cell and Developmental Biology, Embryotoxicology and Cell Therapy.

Embryonic stem (ES) cells are able to differentiate in vitro via embryo-like aggregates, so-called "embryoid bodies", into derivatives of the endodermal, ectodermal and mesodermal lineage. We established standardised protocols for cardiogenesis, myogenesis, neurogenesis and vascular smooth muscle cell differentiation in vitro. The developmentally controlled expression of tissue-specific genes, proteins, ion channels and receptors during ES cell differentiation is the basis of several in vitro approaches: (1) "Loss of function" assays with ES cells containing homozygous mutations of specific genes, (2) "Gain of function" assays with ES cells overexpressing exogenous genes, (3) Developmental analysis of teratogenic/embryotoxic compounds in vitro, (4) Pharmacological assays and the establishment of model systems for pathological cell functions, and (5) The application of differentiation and growth factors for induction of selectively differentiated cells which, in the future, may be used as a source for tissue grafts. We propose the ES cell technology as valuable in vitro system to substitute and reduce the use of animals in basic and applied research.

Journal Article↗
PubMed1998

Growth hormone stimulates phosphorylation and activation of elk-1 and expression of c-fos, egr-1, and junB through activation of extracellular signal-regulated kinases 1 and 2.

Growth hormone (GH), a major regulator of normal body growth and metabolism, regulates cellular gene expression. The transcription factors Elk-1 and Serum Response Factor are necessary for GH-stimulated transcription of c-fos through the Serum Response Element (SRE). GH stimulates the serine phosphorylation of Elk-1, thereby enabling Elk-1 to mediate transcriptional activation. The contribution of the Ras/mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway to Elk-1-mediated transcriptional activation of the c-fos SRE in response to GH was examined. The MEK inhibitor PD098059 attenuated GH-induced expression of the endogenous SRE-regulated genes c-fos, egr-1, and junB as well as transcriptional activation mediated by the c-fos promoter. The MEK inhibitor blocked GH-stimulated activation of MEK, phosphorylation of ERK1/ERK2, and MAP kinase activity in 3T3-F442A cells. Blocking MEK activation prevented GH-induced phosphorylation of Elk-1, as well as the ability of Elk-1 to mediate transcriptional activation in response to GH. Overexpression of dominant-negative Ras or the ERK-specific phosphatase, mitogen-activated protein kinase phosphatase-1, blocked the Ras/MEK/ERK pathway and abrogated GH-induced phosphorylation of Elk-1. GH failed to stimulate phosphorylation or activation of Jun N-terminal kinase under the conditions used. GH slightly increased p38-mediated mitogen-activated protein kinase-activated protein (MAPKAP) kinase-2 activity, but the p38 inhibitor SB203580 did not attenuate GH-promoted Elk-1 phosphorylation. Wortmannin, which inhibited GH-induced ERK phosphorylation, also attenuated transcriptional activation of c-fos by GH. Taken together, these data suggest that GH-dependent activation of the Ras/MEK/ERK pathway and subsequent serine phosphorylation of Elk-1 contribute to GH-stimulated c-fos expression through the SRE.

3T3 Cells↗
PubMed1998

Cloning and characterization of GETS-1, a goldfish Ets family member that functions as a transcriptional repressor in muscle.

An Ets transcription factor family member, GETS-1, was cloned from a goldfish retina cDNA library. GETS-1 contains a conserved Ets DNA-binding domain at its N-terminus and is most similar to ternary complex factor (TCF) serum-response-factor protein-1a (SAP-1a). GETS-1 is expressed in many tissues, but is enriched in retina and brain. As with the TCFs SAP-1a and ets-related protein (ERP), overexpression of the GETS-1 promoter suppresses nicotinic acetylcholine receptor epsilon-subunit gene expression in cultured muscle cells. A consensus Ets binding site sequence in the promoter of the epsilon-subunit gene is required for GETS-1-mediated repression. GETS-1 repressor activity is abrogated by overexpression of an activated Ras/mitogen-activated protein kinase (MAP kinase) or by mutation of Ser-405, a MAP kinase phosphorylation site in GETS-1. Fusion proteins created between GETS-1 and the Gal4 DNA-binding domain show that, like other TCFs, GETS-1 contains a C-terminal activation domain that is activated by a Ras/MAP kinase signalling cascade. Interestingly, mutation of Ser-405 located within this activation domain abrogated transcriptional activation of the fusion protein.

Amino Acid Sequence↗
PubMed1998

Loss of beta1 integrin function results in a retardation of myogenic, but an acceleration of neuronal, differentiation of embryonic stem cells in vitro.

Integrin cell surface receptors play an important role for cell adhesion, migration, and differentiation during embryonic development by mediating cell-cell and cell-matrix interactions. Less is known about the function of integrins during commitment and lineage determination of early embryogenesis. Homozygous inactivation of the beta1 integrin gene results in embryonal death in mice around the time of implantation. In vitro, differentiation of embryonic stem (ES) cells which lack beta1 integrin (beta1-/-) into the cardiogenic lineage is delayed and results in a disordered cellular specification (Fässler et al., J. Cell Sci. 109, 2989-2999, 1996). To analyze beta1 integrin function during myogenesis and neurogenesis we studied differentiation of beta1-/- ES cells via embryoid bodies into skeletal muscle and neuronal cells in vitro. beta1-/- cells showed delayed and reduced myogenic differentiation compared to wildtype and heterozygous (beta1+/-) ES cells. RT-PCR analysis demonstrated delayed expression of skeletal muscle-specific genes in the absence of beta1 integrin. Immunofluorescence studies with antibodies against the sarcomeric proteins myosin heavy chain, titin, nebulin, and slow C-protein showed that myotubes formed, but their number was reduced and the assembly of sarcomeric structures was retarded. In contrast, neuronal cells differentiating from beta1-/- ES cells appeared earlier than wildtype and heterozygous (beta1+/-) ES cells. This was shown by the accelerated expression of neuron-specific genes and an increased number of neuronal cells in beta1-/- embryoid bodies. However, neuronal outgrowth was retarded in the absence of beta1 integrin. No functional difference between wildtype and beta1-/- cells was found with respect to secretion of gamma-aminobutyric acid, the main neurotransmitter of ES cell-derived neuronal cells. The lineage-specific effects of loss of beta1 integrin function, that is the inhibition of mesodermal and acceleration of neuroectodermal differentiation, were supported by differential expression of genes encoding lineage-specific transcription factors (Brachyury, Pax-6, Mash1) and signaling molecules (BMP-4 and Wnt-1). Because of the reduced and delayed expression of the BMP-4 encoding gene in beta1-/- cells, we analyzed in wildtype and beta1-/- cells the regulatory role of exogenously added BMP-4 on the expression of the mesodermal and neuronal marker genes, Brachyury and wnt-1, respectively. The data suggest that BMP-4 plays a regulatory role during differentiation of wildtype and beta1-/- cells by modifying mesodermal and neuronal pathways. The reduced expression of BMP-4 in beta1-/- cells may account for the accelerated neuronal differentiation in beta1-/- ES cells.

Animals↗
PubMed1998

Formation of postsynaptic-like membranes during differentiation of embryonic stem cells in vitro.

To analyze the formation of neuromuscular junctions, mouse pluripotent embryonic stem (ES) cells were differentiated via embryoid bodies into skeletal muscle and neuronal cells. The developmentally controlled expression of skeletal muscle-specific genes coding for myf5, myogenin, myoD and myf6, alpha 1 subunit of the L-type calcium channel, cell adhesion molecule M-cadherin, and neuron-specific genes encoding the 68-, 160-, and 200-kDa neurofilament proteins, synaptic vesicle protein synaptophysin, brain-specific proteoglycan neurocan, and microtubule-associated protein tau was demonstrated by RT-PCR analysis. In addition, genes specifically expressed at neuromuscular junctions, the gamma- and epsilon-subunits of the nicotinic acetylcholine receptor (AChR) and the extracellular matrix protein S-laminin, were found. At the terminal differentiation stage characterized by the formation of multinucleated spontaneously contracting myotubes, the myogenic regulatory gene myf6 and the AChR epsilon-subunit gene, both specifically expressed in mature adult skeletal muscle, were found to be coexpressed. Only the terminally differentiated myotubes showed a clustering of nicotinic acetylcholine receptors (AChR) and a colocalization with agrin and synaptophysin. The formation of AChRs was also demonstrated on a functional level by using the patch clamp technique. Taken together, our results showed that during ES cell differentiation in vitro neuron- and muscle-specific genes are expressed in a developmentally controlled manner, resulting in the formation of postsynaptic-like membranes. Thus, the embryonic stem cell differentiation model will be helpful for studying cellular interactions at neuromuscular junctions by "loss of function" analysis in vitro.

Agrin↗
PubMed1998

[The indication and mode of surgery for the treatment of patients with severe acute pancreatitis].

OBJECTIVE: To study the indication and mode of surgical management in patients with severe acute pancreatitis (SAP). METHOD: 41 patients with SAP were grouped for conservative treatment or surgical. The main procedures of surgical management were pancreatic debridement, peripancreatic and coeliac drainage, combined with peripancreatic instillation and suction alternatively. RESULT: Conservative treatment was adopted in 22 patients (16 patients in grade I, 6 in grade II), among them 19 recovered, and 3 of grade II died. 19 patients underwent surgical treatment including 7 in grade I, 12 in grade II). 15 patients were cured, 4 patients of grade II died. CONCLUSION: Timely operation is necessary for the patients with infection and complications. Surgical intervention is recommended for patients older than 60 years old. Surgery should be less traumatic, simple and prompt. Surgical or conservative treatment are chosen according to the patients' conditions. The cure rate of SAP can be improved.

Adult↗
PubMed1997

Structure-function studies of p38 mitogen-activated protein kinase. Loop 12 influences substrate specificity and autophosphorylation, but not upstream kinase selection.

Several mitogen-activated protein kinase (MAPK) cascades have been identified in eukaryotic cells. The activation of MAPKs is carried out by distinct MAPK kinases (MEKs or MKKs), and individual MAPKs have different substrate preferences. Here we have examined how amino acid sequences encompassing the dual phosphorylation motif located in the loop 12 linker (L12) between kinase subdomains VII and VIII and the length and amino acid sequence of L12 influence autophosphorylation, substrate specificity, and upstream kinase selectivity for the MAPK p38. Conversion of L12 of p38 to an "ERK-like" structure was accomplished in several ways: (i) by replacing glycine with glutamate in the dual phosphorylation site, (ii) by placing a six-amino acid sequence present in L12 of ERK (but absent in p38) into p38, and (iii) by mutations of amino acid residues in loop 12. Two predominant effects were noted: (i) the Xaa residue in the dual phosphorylation motif Thr-Xaa-Tyr as well as the length of L12 influence p38 substrate specificity, and (ii) the length of L12 plays a major role in controlling autophosphorylation. In contrast, these modifications do not result in any change in the selection of p38 by individual MAPK kinases.

Amino Acid Sequence↗
PubMed1996

Differentiation and integrity of cardiac muscle cells are impaired in the absence of beta 1 integrin.

Cellular interactions with substrata of the microenvironment are one of the major mechanisms for differentiation and morphogenesis. Many of these interactions are mediated via the beta 1 integrin subfamily of cell surface receptors, which are believed to transduce signals upon cell adhesion. We have used beta 1 integrin-deficient embryonic stem cells to test their ability to differentiate into cardiac muscle cells. We show here by several approaches that beta 1 integrin is important for normal cardiogenesis. First, the in vitro differentiation of beta 1 integrin-deficient embryonic stem cells into cardiac muscle cells is retarded. This is demonstrated by the delayed expression of cardiac muscle-specific genes and action potentials. Second, the specification of cardiac precursor cells into pacemaker-, atrial- and ventricular-like cells is significantly impaired in beta 1 integrin-deficient cells. The occurrence of atrial- and ventricular-like cells is reduced and transient. Only cells exhibiting peacemaker-like action potentials of high frequency and arrhythmias survive. Third, the sarcomeric architecture is incomplete and disarranged in the absence of beta 1 integrin. Fourth, beta 1-deficient embryonic stem cells can contribute to the developing heart in chimaeric mice but many areas with beta 1-null cells contain cell debris. The number of beta 1-null cells decrease from prenatal to postnatal stages and is lost completely in 6-month-old hearts. Thus, we conclude that interactions with the extracellular matrix via beta 1 integrin is necessary for differentiation and the maintenance of a specialized phenotype of cardiac muscle cells.

Animals↗
PubMed1994

Simian virus 40 small t antigen cooperates with mitogen-activated kinases to stimulate AP-1 activity.

The simian virus 40 small tumor antigen (small t) specifically interacts with protein phosphatase type 2A (PP2A) in vivo and alters its catalytic activity in vitro. Among the substrates for PP2A in vitro are the activated forms of MEK and ERK kinases. Dephosphorylation of the activating phosphorylation sites on MEK and ERKs by PP2A in vitro results in a decrease in their respective kinase activities. Recently, it has been shown that overexpression of small t in CV-1 cells results in an inhibition of PP2A activity toward MEK and ERK2 and a constitutive upregulation of MEK and ERK2 activity. Previously, we have observed that overexpression of either ERK1, MEK1, or a constitutively active truncated form of c-Raf-1 (BXB) is insufficient to activate AP-1 in REF52 fibroblasts. We therefore examined whether overexpression of small t either alone or in conjunction with ERK1, MEK1, or BXB could activate AP-1. We found that coexpression of small t and either ERK1, MEK1, or BXB resulted in an increase in AP-1 activity, whereas expression of either small t or any of the kinases alone did not have any effect. Similarly, coexpression of small t and ERK1 activated serum response element-regulated promoters. Coexpression of kinase-deficient mutants of ERK1 and ERK2 inhibited the activation of AP-1 caused by expression of small t and either MEK1 or BXB. Coexpression of an interfering MEK, which inhibited AP-1 activation by small t and BXB, did not inhibit the activation of AP-1 caused by small t and ERK1. In contrast to REF52 cells, we observed that overexpression of either small or ERK1 alone in CV-1 cells was sufficient to stimulate AP-1 activity and that this stimulation was not enhanced by expression of small t and ERK1 together. These results show that the effects of small t on immediate-early gene expression depend on the cell type examined and suggest that the mitogen-activated protein kinase activation pathway is distinctly regulated in different cell types.

Animals↗
PubMed1993

"Chitin Leash": a polysaccharide heterobifunctional cross-linking agent which can be cleaved by lysozyme.

6-O-[(2-Hydroxyethyl)poly(2-oxyethyl)]chitosan ("glycolchitosan") was oxidatively cleaved with nitrous acid and then partly acetylated with acetic anhydride, reacted with bromoacetyl-N-hydroxysuccinimide, and reacted further with acetic anhydride. Conditions were selected, including fractionation by size-exclusion chromatography, so that the resulting "Chitin Leash" had an estimated, average molecular weight of 10,000 (dextran standards), corresponding to a length of approximately 40 sugar residues. It possessed 0.9 terminal aldehyde and 2.6 random (presumably) side-chain bromoacetyl reactive groups per chain (average values). As a model system, the Chitin Leash was used to crosslink staphylococcal nuclease (SNase) to ribonuclease A (RNase) with retention of 75 and 78%, respectively, of the starting enzyme activities. For this coupling, the Nase was first converted to a sulfhydryl SNase derivative which retained 74% of the activity of starting enzyme. The yields in this synthesis were: 13% Chitin Leash from glycolchitosan, 24% Chitin Leash-RNase from Chitin Leash and 45% SNase-Chitin Leash-RNase from the latter conjugate. The ratio of SNase to RNase in this conjugate was 1.0:0.94. In a second preparation, in which [14C]acetic anhydride was used, a longer reaction time was employed for the coupling of Chitin Leash to RNase. This gave a 1.0:1.8:0.95 molar ratio of Nase: [14C]Chitin Leash: RNase, revealing multiple attachment of the [14C]Chitin Leash to RNase. The activity of the RNase in the final conjugate was 20%. The latter conjugate was approximately 70% hydrolyzed by diaminooctyl-succinyl-lysozyme, disconnecting the two enzymes while not affecting their activities.

Carbohydrate Sequence↗
PubMed1992

A yeast protein phosphatase related to the vaccinia virus VH1 phosphatase is induced by nitrogen starvation.

A phosphatase related to the vaccinia virus VH1 phosphatase has been cloned from Saccharomyces cerevisiae. The yeast phosphatase is related to the Schizosaccharomyces pombe cdc25 gene product and to a protein encoded by a mammalian open reading frame known as 3CH134, which is an immediate early gene responding to serum stimulation. The phosphatase activity of the yeast gene product appears to be restricted to the hydrolysis of phosphotyrosine-containing substrates, whereas the vaccinia phosphatase hydrolyzes both phosphoserine- and phosphotyrosine-containing substrates. The mRNA encoding the yeast phosphatase is dramatically induced by nitrogen starvation. Inactivation of the yeast phosphatase gene results in a decrease in growth rate.

Amino Acid Sequence↗
PubMed1992

Isolation and characterization of a second protein tyrosine phosphatase gene, PTP2, from Saccharomyces cerevisiae.

A putative protein tyrosine phosphatase (PTPase) gene, PTP2, was cloned from Saccharomyces cerevisiae. The complete yeast PTP2 gene encodes a 750-amino acid residue protein with a predicted mass of 86 kDa. The conserved PTPase domain was localized in the C-terminal half of the protein. Amino acid sequence alignment of the yeast PTPase domain with other phosphatases indicated approximately 20-25% sequence identity with the mammalian PTPase and a similar degree of identity with the PTPase encoded by the yeast PTP1 gene. The PTP2 gene is closely linked to the yeast RET1 and STE4 genes and is localized on the right arm of chromosome 15. Gene disruption experiments demonstrated that neither PTP2 alone nor PTP2 in combination with PTP1 was essential for growth under the conditions tested. The ability of PTP2 to complement the cdc25-22 mutant of Schizosaccharomyces pombe was also examined, and unlike the human T-cell PTPase, which was able to complement the cdc25-22 mutant, the S. cerevisiae PTP2 was unable to complement the cdc25-22 mutant of S. pombe.

Amino Acid Sequence↗
PubMed1991

De novo purine nucleotide biosynthesis: cloning, sequencing and expression of a chicken PurH cDNA encoding 5-aminoimidazole-4-carboxamide-ribonucleotide transformylase-IMP cyclohydrolase.

The purH cDNA, encoding 5-aminoimidazole-4-carboxamide-ribonucleotide (AICAR) transformylase-inosine monophosphate cyclohydrolase (ATIC), was cloned by functional complementation of an Escherichia coli purH mutant using a chicken liver cDNA expression library. This represents the first report of the cloning of any eukaryotic ATIC-encoding cDNA (PurH). The avian ATIC mRNA is 2.3 kb long and encodes a protein with an Mr of 64,422. The deduced amino acid sequence is 36% identical to the bacterial purH-encoded enzymes from Bacillus subtilis and E. coli. The avian cDNA was expressed as a glutathione S-transferase (GST) fusion protein that was purified in a single step by affinity chromatography. A novel vector was employed which permits rapid and highly efficient cleavage of the GST fusion protein yielding 10 mg of purified PurH product per liter of bacterial culture. Km values were determined with the purified fusion protein utilizing AICAR and (6-R)N10-formyl-tetrahydrofolate as substrates. These values compare favorably with the isolated avian enzyme, supporting the idea that kinetic, as well as other physical properties of the recombinant fusion protein are similar to the native avian enzyme. Large quantities of purified enzyme and the ability to generate site-directed mutations should make mechanistic studies possible. The recombinant enzyme also affords a simple and reliable approach to identifying new antifolates.

Acyltransferases↗
PubMedDate not supplied

Normal mitochondrial structure and genome maintenance in yeast requires the dynamin-like product of the MGM1 gene.

The isolation and characterization of MGM1, an yeast gene with homology to members of the dynamin gene family, is described. The MGM1 gene is located on the right arm of chromosome XV between STE4 and PTP2. Sequence analysis revealed a single open reading frame of 902 residues capable of encoding a protein with an approximate molecular mass of 101 kDa. Loss of MGM1 resulted in slow growth on rich medium, failure to grow on non-fermentable carbon sources, and loss of mitochondrial DNA. The mitochondria also appeared abnormal when visualized with an antibody to a mitochondrial-matrix marker. MGM1 encodes a dynamin-like protein involved in the propagation of functional mitochondria in yeast.

Base Sequence↗