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Biomedical subjects

A F Stewart

Publications and source records attributed to A F Stewart.

At least 19 recordsLinked to original sources
PubMed2002

Codon-improved Cre recombinase (iCre) expression in the mouse.

By applying the mammalian codon usage to Cre recombinase, we improved Cre expression, as determined by immunoblot and functional analysis, in three different mammalian cell lines. The improved Cre (iCre) gene was also designed to reduce the high CpG content of the prokaryotic coding sequence, thereby reducing the chances of epigenetic silencing in mammals. Transgenic iCre expressing mice were obtained with good frequency, and in these mice loxP-mediated DNA recombination was observed in all cells expressing iCre. Moreover, iCre fused to two estrogen receptor hormone binding domains for temporal control of Cre activity could also be expressed in transgenic mice. However, Cre induction after administration of tamoxifen yielded only low Cre activity. Thus, whereas efficient activation of Cre fusion proteins in the brain needs further improvements, our studies indicate that iCre should facilitate genetic experiments in the mouse.

Animals↗
PubMed2001

The Saccharomyces cerevisiae Set1 complex includes an Ash2 homologue and methylates histone 3 lysine 4.

The SET domain proteins, SUV39 and G9a have recently been shown to be histone methyltransferases specific for lysines 9 and 27 (G9a only) of histone 3 (H3). The SET domains of the Saccharomyces cerevisiae Set1 and Drosophila trithorax proteins are closely related to each other but distinct from SUV39 and G9a. We characterized the complex associated with Set1 and Set1C and found that it is comprised of eight members, one of which, Bre2, is homologous to the trithorax-group (trxG) protein, Ash2. Set1C requires Set1 for complex integrity and mutation of Set1 and Set1C components shortens telomeres. One Set1C member, Swd2/Cpf10 is also present in cleavage polyadenylation factor (CPF). Set1C methylates lysine 4 of H3, thus adding a new specificity and a new subclass of SET domain proteins known to methyltransferases. Since methylation of H3 lysine 4 is widespread in eukaryotes, we screened the databases and found other Set1 homologues. We propose that eukaryotic Set1Cs are H3 lysine 4 methyltransferases and are related to trxG action through association with Ash2 homologues.

Amino Acid Sequence↗
PubMed2001

The S. cerevisiae SET3 complex includes two histone deacetylases, Hos2 and Hst1, and is a meiotic-specific repressor of the sporulation gene program.

Set3 is one of two proteins in the yeast Saccharomyces cerevisiae that, like Drosophila Trithorax, contains both SET and PHD domains. We found that Set3 forms a single complex, Set3C, with Snt1, YIL112w, Sif2, Cpr1, and two putative histone deacetylases, Hos2 and NAD-dependent Hst1. Set3C includes NAD-dependent and independent deacetylase activities when assayed in vitro. Homology searches suggest that Set3C is the yeast analog of the mammalian HDAC3/SMRT complex. Set3C represses genes in early/middle of the yeast sporulation program, including the key meiotic regulators ime2 and ndt80. Whereas Hos2 is only found in Set3C, Hst1 is also present in a complex with Sum1, supporting previous characterizations of Hst1 and Sum1 as repressors of middle sporulation genes during vegetative growth. However, Hst1 is not required for meiotic repression by Set3C, thus implying that Set3C (-Hst1) and not Hst1-Sum1, is the meiotic-specific repressor of early/middle sporulation genes.

Amino Acid Sequence↗
PubMed2001

Self-deleting suicide vectors (SDSV): selective killing of p53-deficient cancer cells.

A self-deleting retrovirus vector carrying a herpes simplex virus (HSV)-thymidine kinase suicide gene has been developed to selectively kill cancer cells expressing a dysfunctional p53 tumor suppressor protein. When cells containing functional p53 are infected with the virus, the integrated provirus and the HSV-thymidine kinase gene are deleted from the genome by site-specific recombination (Cre/loxP). In contrast, cells without p53 or cells expressing a DNA-binding mutant of p53 retain the provirus and become susceptible to killing by ganciclovir. This strategy provides a new concept for the selective killing of cancer cells that can be adapted to any other dysfunctional transcription factor expressed by different tumors.

Animals↗
PubMed2001

NSD3, a new SET domain-containing gene, maps to 8p12 and is amplified in human breast cancer cell lines.

We describe the isolation and characterization of NSD3, the third member of a gene family including Nsd1 and NSD2. Murine Nsd1 was isolated in a search for proteins that interact with the ligand-binding domain of retinoic acid receptor alpha. NSD2 (also known as WHSC1 and MMSET) is located in the Wolf-Hirschhorn syndrome (WHS) critical region on 4p16.3 and is involved in multiple myeloma with t(4;14) translocations. The proteins Nsd1, NSD2, and NSD3 are highly similar within a block of about 700 amino acids. This block contains several conserved domains, such as the SET domain and the PHD finger, present in proteins involved in development and/or chromatin reorganization. The NSD3 gene consists of an 8.5-kb transcript composed of 23 coding exons and spans >90 kb of genomic DNA. NSD3 maps to chromosome band 8p12 and is amplified in several tumor cell lines and primary breast carcinomas.

Alternative Splicing↗
PubMed2001

Independent regulation of cardiac Kv4.3 potassium channel expression by angiotensin II and phenylephrine.

Hypertrophied cardiac myocytes exhibit prolonged action potentials and decreased transient outward potassium current (I(to)). Because Kv4.3 is a major contributor to I:(to), we studied regulation of its expression in neonatal rat cardiac myocytes in response to the known stimulators of cardiac myocyte hypertrophy, angiotensin II (Ang II) and phenylephrine (PE). RNase protection assays and immunoblots revealed that Ang II and PE each downregulate Kv4.3 mRNA and protein. However, although PE induces a faster and more extensive hypertrophic response than Ang II, the PE effect on Kv4.3 mRNA develops slowly and is sustained, whereas Ang II rapidly and transiently decreases Kv4.3 mRNA expression. Turnover measurements revealed that Kv4.3 mRNA is very stable, with a half-life >20 hours. This suggests that Ang II must destabilize the channel mRNA. In contrast, PE does not affect the rate of Kv4.3 mRNA degradation. To test for transcriptional regulation, the 5' flanking region of the rat Kv4.3 gene was cloned, and Kv4.3 promoter-reporter constructs were expressed in cardiac myocytes. Whereas Ang II was found to have no effect on transcription, PE inhibits Kv4.3 promoter activity. Pharmacological experiments also indicate that PE and Ang II act independently to downregulate Kv4.3 gene expression. Thus, regulation of Kv4.3 gene expression is not a simple secondary response to hypertrophy. Rather, Ang II and PE use different mechanisms to decrease Kv4.3 channel expression in neonatal rat cardiac myocytes.

Angiotensin II↗
PubMed2001

Efficient FLP recombination in mouse ES cells and oocytes.

We report an improved vector, pCAGGS-FLPe, for transient expression of the enhanced FLP recombinase in mouse ES cells and oocytes. In standard transfection experiments, about 6% of total ES colonies showed FLP recombination, albeit with mosaicism within each colony. After microinjection of pCAGGS-FLPe into oocytes, about one-third of heterozygotic mice born showed complete FLP recombination. Thus pCAGGS-FLPe presents two practical options for removal of FRT cassettes in mice.

Amino Acid Sequence↗
PubMed2001

A CamKIIalpha iCre BAC allows brain-specific gene inactivation.

We describe the generation of transgenic mouse lines expressing the Cre recombinase enzyme in brain under control of the CamKIIalpha gene present in a BAC expression vector. The CamKIIalpha BAC transgene gave a faithful expression pattern resembling the pattern of the endogenous CamKIIalpha gene. Specifically, high levels of CamKIIalpha Cre were detected in hippocampus, cortex, and amygdala, and lower levels were detected in striatum, thalamus, and hypothalamus. As expected, no expression was detected in the cerebellum or outside of the brain. The expression level of the BAC CamKIIalpha driven Cre was shown to be copy number dependent. To test the activity of the Cre recombinase, the transgenic mice were crossed with mice harbouring the CREB (cAMP response element binding protein) allele with the 10th exon flanked by two loxP sites, and recombination was monitored by the disappearance of the CREB protein. Finally, evaluation of the developmental postnatal expression of the CamKIIalpha Cre BAC revealed the expression of the Cre recombinase as early as P3.

Animals↗
PubMed2001

Techniques: Recombinogenic engineering--new options for cloning and manipulating DNA.

Driven by the needs of functional genomics, DNA engineering by homologous recombination in Escherichia coli has emerged as a major addition to existing technologies. Two alternative approaches, RecA-dependent engineering and ET recombination, allow a wide variety of DNA modifications, including some which are virtually impossible by conventional methods. These approaches do not rely on the presence of suitable restriction sites and can be used to insert, delete or substitute DNA sequences at any desired position on a target molecule. Furthermore, ET recombination can be used for direct subcloning and cloning of DNA sequences from complex mixtures, including bacterial artificial chromosomes and genomic DNA preparations. The strategies reviewed in this article are applicable to modification of DNA molecules of any size, including very large ones, and present powerful new avenues for DNA manipulation in general.

Cloning, Molecular↗
PubMed2001

Alteration of Cre recombinase site specificity by substrate-linked protein evolution.

Directed molecular evolution was applied to generate Cre recombinase variants that recognize a new DNA target sequence. Cre was adapted in a three-stage strategy to evolve recombinases to specifically recombine the new site. This complex multicycle task was made feasible by an improved directed-evolution procedure that relies on placing the recombination substrate next to the recombinase coding region. Consequently, those DNA molecules carrying the coding region for a successful recombinase are physically marked by the action of that recombinase on the linked substrate and are easily retrieved from a large background of unsuccessful candidates by PCR amplification. We term this procedure substrate-linked protein evolution (SLiPE). The method should facilitate the development of new recombinases and other DNA-modifying enzymes for applications in genetic engineering, functional genomics, and gene therapy.

3T3 Cells↗
PubMed2001

Parathyroid hormone-related protein and its receptors: nuclear functions and roles in the renal and cardiovascular systems, the placental trophoblasts and the pancreatic islets.

The cloning of the so-called 'parathyroid hormone-related protein' (PTHrP) in 1987 was the result of a long quest for the factor which, by mimicking the actions of PTH in bone and kidney, is responsible for the hypercalcemic paraneoplastic syndrome, humoral calcemia of malignancy. PTHrP is distinct from PTH in a number of ways. First, PTHrP is the product of a separate gene. Second, with the exception of a short N-terminal region, the structure of PTHrP is not closely related to that of PTH. Third, in contrast to PTH, PTHrP is a paracrine factor expressed throughout the body. Finally, most of the functions of PTHrP have nothing in common with those of PTH. PTHrP is a poly-hormone which comprises a family of distinct peptide hormones arising from post-translational endoproteolytic cleavage of the initial PTHrP translation products. Mature N-terminal, mid-region and C-terminal secretory forms of PTHrP are thus generated, each of them having their own physiologic functions and probably their own receptors. The type 1 PTHrP receptor, binding both PTH(1-34) and PTHrP(1-36), is the only cloned receptor so far. PTHrP is a PTH-like calciotropic hormone, a myorelaxant, a growth factor and a developmental regulatory molecule. The present review reports recent aspects of PTHrP pharmacology and physiology, including: (a) the identification of new peptides and receptors of the PTH/PTHrP system; (b) the recently discovered nuclear functions of PTHrP and the role of PTHrP as an intracrine regulator of cell growth and cell death; (c) the physiological and developmental actions of PTHrP in the cardiovascular and the renal glomerulo-vascular systems; (d) the role of PTHrP as a regulator of pancreatic beta cell growth and functions, and, (e) the interactions of PTHrP and calcium-sensing receptors for the control of the growth of placental trophoblasts. These new advances have contributed to a better understanding of the pathophysiological role of PTHrP, and will help to identify its therapeutic potential in a number of diseases.

Animals↗
PubMed2001

The C-terminal region of PTHrP, in addition to the nuclear localization signal, is essential for the intracrine stimulation of proliferation in vascular smooth muscle cells.

PTHrP is secreted by most cell types. In addition to a paracrine/autocrine role, PTHrP has "intracrine" actions, entering the nuclear compartment under the direction of a classic bipartite nuclear localization signal. In vascular smooth muscle cells, nuclear entry stimulates mitogenesis. In the current study, we sought to more precisely define the regions of PTHrP required for the activation of mitogenesis in vascular smooth muscle cells. PTHrP deletion mutants missing large regions [i.e. the signal peptide, N terminus (1--36), mid region (38--86), nuclear localization signal, C terminus (108--139), or combinations of the above] were expressed in A-10 vascular smooth muscle cells. The consequences on nuclear localization and proliferation were examined. Deletion of the nuclear localization signal prevented nuclear entry and slowed proliferation. Deletion of the highly conserved N terminus or mid region had no impact on nuclear localization or on proliferation. Deletion of the C terminus had no deleterious effect on nuclear localization but dramatically reduced proliferation. Thus, the nuclear localization signal is both necessary and sufficient for nuclear localization of PTHrP. In contrast, activation of proliferation in vascular smooth muscle cells requires both an intact nuclear localization signal and an intact C terminus. Whereas the nuclear localization signal is required for nuclear entry, the C terminus may serve a trans-activating function to stimulate mitogenesis once inside the nucleus of vascular smooth muscle cells.

Amino Acid Sequence↗
PubMed2001

Using beta-cell growth factors to enhance human pancreatic Islet transplantation.

This is a particularly exciting time in the field of pancreatic islet growth, development, and survival. The recent publication of a study demonstrating that human pancreatic islet transplantation is both technically and immunologically feasible has highlighted the need for large supplies of pancreatic islets or pancreatic beta cells for larger-scale islet transplantation in patients with diabetes. This, together with a rapid expansion in the past several years of the understanding of mechanisms of islet growth, development, and survival, has accelerated and invigorated efforts to therapeutically harness the cellular mechanisms responsible for pancreatic beta-cell proliferation, survival, and development and to take advantage of this new knowledge to enhance the availability, survival, and function of pancreatic beta cells in human islet transplantation for diabetes mellitus. Here, we briefly review the confluence of events that have provided optimism and energy to the islet transplant field, and we focus on peptide growth factors that eventually may be deployed in the effort to augment islet mass and function in patients with diabetes.

Animals↗
PubMed2001

Parathyroid hormone-related protein-(1--36) stimulates renal tubular calcium reabsorption in normal human volunteers: implications for the pathogenesis of humoral hypercalcemia of malignancy.

All would agree that hypercalcemia occurs among patients with humoral hypercalcemia of malignancy (HHM) as a result of osteoclastic bone resorption. Some studies suggest that enhanced renal calcium reabsorption, which plays an important pathophysiological role in the hypercalcemia occurring in primary hyperparathyroidism, is also important pathophysiologically in HHM. Other studies have not agreed. In large part, these differences result from the inability to accurately assess creatinine and calcium clearance in critically ill subjects with HHM. To circumvent these issues, we have developed steady state 48-h PTH-related protein (PTHrP) infusion and 8-h hypercalcemic calcium clamp protocols. These techniques allow assessment of the effects of steady state PTHrP and calcium infusions in normal healthy volunteers in a setting in which renal function is stable and measurable and in which the filtered load of calcium can be matched in PTHrP- and calcium-infused subjects. Normal subjects were infused with saline (placebo), PTHrP, or calcium. Subjects receiving PTHrP, as expected, displayed mild hypercalcemia (10.2 mg/dL), suppression of endogenous PTH-(1--84), and phosphaturia. Subjects receiving the hypercalcemic calcium clamp displayed indistinguishable degrees of hypercalcemia and PTH suppression. Despite their matched degrees of hypercalcemia and PTH suppression, the two groups differed importantly with regard to fractional calcium excretion (FECa). The hypercalcemic calcium clamp group was markedly hypercalciuric (FECa averaged 6.5%), whereas FECa in the PTHrP-infused subjects was approximately 50% lower (between 2.5--3.7%), and no different from that in the normal controls, which ranged from 1.5--3.0%. These studies demonstrate that PTHrP is able to stimulate renal calcium reabsorption in healthy volunteers. These studies suggest that PTHrP-induced renal calcium reabsorption, in concert with the well established acceleration of osteoclastic bone resorption, contributes in a significant way to the hypercalcemia observed in patients with HHM.

Absorption↗
PubMed2001

Midregion parathyroid hormone-related protein inhibits growth and invasion in vitro and tumorigenesis in vivo of human breast cancer cells.

Parathyroid hormone-related protein (PTHrP) is critical for normal mammary development and is overexpressed by breast cancers. PTHrP is a peptide hormone that undergoes extensive post-translational processing, and PTHrP(38-94)-amide is one of the mature secretory forms of the peptide. In this study, we explored the effect of PTHrP(38-94)-amide in a panel of six breast cancer cell lines "in vitro" and in MDA-MB231 cells "in vivo" specifically examining cell viability, proliferation, invasiveness, and growth in nude mice. PTHrP(38-94)-amide markedly inhibited proliferation and also caused striking toxicity and accelerated cell death in breast cancer cells. In addition, direct injection of PTHrP(38-94)-amide into MDA-MB231 breast cancer cells passaged in immunodeficient mice produced a marked reduction in tumor growth. These studies (i) indicate breast cancer cells are one of the few tissues in which specific effects of midregion PTHrP have been established to date, (ii) support a role for midregion secretory forms of PTHrP in modulating not only normal but also pathological mammary growth and differentiation, (iii) add further evidence for the existence of a specific midregion PTHrP receptor, and (iv) provide a novel molecule for modeling of small molecule analogues that may have anti-breast cancer effects.

Animals↗
PubMed2001

Transgenic overexpression of hepatocyte growth factor in the beta-cell markedly improves islet function and islet transplant outcomes in mice.

Recent advances in human islet transplantation have highlighted the need for expanding the pool of beta-cells available for transplantation. We have developed three transgenic models in which growth factors (hepatocyte growth factor [HGF], placental lactogen, or parathyroid hormone-related protein) have been targeted to the beta-cell using rat insulin promoter (RIP). Each displays an increase in islet size and islet number, and each displays insulin-mediated hypoglycemia. Of these three models, the RIP-HGF mouse displays the least impressive phenotype under basal conditions. In this study, we show that this mild basal phenotype is misleading and that RIP-HGF mice have a unique and salutary phenotype. Compared with normal islets, RIP-HGF islets contain more insulin per beta-cell (50 +/- 5 vs. 78 +/- 9 ng/islet equivalent [IE] in normal vs. RIP-HGF islets, P < 0.025), secrete more insulin in response to glucose in vivo (0.66 +/- 0.06 vs. 0.91 +/- 0.10 ng/ml in normal vs. RIP-HGF mice, P < 0.05) and in vitro (at 22.2 mmol/l glucose: 640 +/- 120.1 vs. 1,615 +/- 196.9 pg. microg protein(-1). 30 min(-1) in normal vs. RIP-HGF islets, P < 0.01), have two- to threefold higher GLUT2 and glucokinase steady-state mRNA levels, take up and metabolize glucose more effectively, and most importantly, function at least twice as effectively after transplantation. These findings indicate that HGF has surprisingly positive effects on beta-cell mitogenesis, glucose sensing, beta-cell markers of differentiation, and transplant survival. It appears to have a unique and unanticipated effective profile as an islet mass- and function-enhancing agent in vivo.

Animals↗