Nucleotide excision repair gene (ERCC1) deficiency causes G(2) arrest in hepatocytes and a reduction in liver binucleation: the role of p53 and p21.

A wide range of DNA lesions, both UV and chemically induced, are dealt with by the nucleotide excision repair (NER) pathway. Defects in NER result in human syndromes such as xeroderma pigmentosum (XP), where there is a 1000-fold increased incidence of skin cancer. The ERCC1 protein is essential for NER, but ERCC1 knockout mice are not a model for XP.

Hedgehog signals regulate multiple aspects of gastrointestinal development.

The gastrointestinal tract develops from the embryonic gut, which is composed of an endodermally derived epithelium surrounded by cells of mesodermal origin. Cell signaling between these two tissue layers appears to play a critical role in coordinating patterning and organogenesis of the gut and its derivatives. We have assessed the function of Sonic hedgehog and Indian hedgehog genes, which encode members of the Hedgehog family of cell signals.

Early mouse endoderm is patterned by soluble factors from adjacent germ layers.

Endoderm that forms the respiratory and digestive tracts is a sheet of approximately 500-1000 cells around the distal cup of an E7.5 mouse embryo. Within 2 days, endoderm folds into a primitive gut tube from which numerous organs will bud.

Endoderm development: from patterning to organogenesis.

Although the ectoderm and mesoderm have been the focus of intensive work in the recent era of studies on the molecular control of vertebrate development, the endoderm has received less attention. Because signaling must occur between germ layers in order to achieve a properly organized body, our understanding of the coordinated development of all organs requires a more thorough consideration of the endoderm and its derivatives. This review focuses on present knowledge and perspectives concerning endoderm patterning and organogenesis.

Vertebrate endoderm development.

Endoderm, one of the three principal germ layers, contributes to all organs of the alimentary tract. For simplicity, this review divides formation of endodermal organs into four fundamental steps: (a) formation of endoderm during gastrulation, (b) morphogenesis of a gut tube from a sheet of cells, (c) budding of organ domains from the tube, and (d) differentiation of organ-specific cell types within the growing buds. We discuss possible mechanisms that regulate how undifferentiated endoderm becomes specified into a myriad of cell types that populate the respiratory and gastrointestinal tracts.

A single amino acid alteration (101L) introduced into murine PrP dramatically alters incubation time of transmissible spongiform encephalopathy.

A mutation equivalent to P102L in the human PrP gene, associated with Gerstmann-Straussler syndrome (GSS), has been introduced into the murine PrP gene by gene targeting. Mice homozygous for this mutation (101LL) showed no spontaneous transmissible spongiform encephalopathy (TSE) disease, but had incubation times dramatically different from wild-type mice following inoculation with different TSE sources. Inoculation with GSS produced disease in 101LL mice in 288 days.

Ataxia in prion protein (PrP)-deficient mice is associated with upregulation of the novel PrP-like protein doppel.

The novel locus Prnd is 16 kb downstream of the mouse prion protein (PrP) gene Prnp and encodes a 179 residue PrP-like protein designated doppel (Dpl). Prnd generates major transcripts of 1.7 and 2.

Screening for novel pancreatic genes expressed during embryogenesis.

We have combined suppressive subtractive hybridization with in situ hybridization to identify genes expressed at early stages of pancreas development. By using polymerase chain reaction amplification and subtractive hybridization, this protocol for screening can be applied when the amount of RNA is limited. Seven genes expressed in or adjacent to the pancreas anlage were isolated, three of which show similarity to known genes.

Angiotensin AT1 receptor inhibition, angiotensin-converting enzyme inhibition, and combination therapy with developing heart failure: cellular mechanisms of action.

Background: Past studies have shown that angiotensin-converting enzyme inhibition (ACEI) alone, angiotensin AT1 receptor blockade (AT1 block) alone, and combined treatment have differential effects on left ventricular (LV) function and geometry with developing congestive heart failure (CHF). The purpose of this study was to more carefully examine the cellular basis for these differential effects by using a model of pacing CHF.

Methods And Results: Pigs were randomly assigned to five groups: (1) rapid pacing (240 bpm) for 3 weeks (n = 9), (2) concomitant ACEI (benazeprilat, 0.

Structure-function relations of heparin-mimetic sulfated xylan oligosaccharides: inhibition of human immunodeficiency virus-1 infectivity in vitro.

Heparins/heparan sulfates modulate the function of proteins and cell membranes in numerous biological systems including normal and disease processes in humans. Heparin has been used for many years as an anticoagulant, and anticoagulant heparin-mimetics were developed several decades ago by chemical sulfation of non-mammalian polysaccharides, e.g.

Efficient BLG-Cre mediated gene deletion in the mammary gland.

Using the phage P1-derived Cre/loxP recombination system, we have developed a strategy for efficient mammary tissue specific inactivation of floxed genes. Transgenic mice were generated which express Cre DNA-recombinase under the control of the mammary gland specific promoter of the ovine beta-lactoglobulin (BLG) gene. To test the specificity of Cre mediated recombination, we crossed these mice to animals harbouring a floxed DNA ligase I allele.

Pancreas development is promoted by cyclopamine, a hedgehog signaling inhibitor.

Exposure to cyclopamine, a steroid alkaloid that blocks Sonic hedgehog (Shh) signaling, promotes pancreatic expansion in embryonic chicks. Heterotopic development of pancreatic endocrine and exocrine structures occurs in regions adjacent to the pancreas including stomach and duodenum, and insulin-producing islets in the pancreas are enlarged. The homeodomain transcription factor PDX1, required for pancreas development, is expressed broadly in the posterior foregut but pancreas development normally initiates only in a restricted region of PDX1-expressing posterior foregut where endodermal Shh expression is repressed.

Mixer, a homeobox gene required for endoderm development.

An expression cloning strategy in Xenopus laevis was used to isolate a homeobox-containing gene, Mixer, that can cause embryonic cells to form endoderm. Mixer transcripts are found specifically in the prospective endoderm of gastrula, which coincides with the time and place that endodermal cells become histologically distinct and irreversibly determined. Loss-of-function studies with a dominant inhibitory mutant demonstrate that Mixer activity is required for endoderm development.

Mutant Vg1 ligands disrupt endoderm and mesoderm formation in Xenopus embryos.

The Xenopus Vg1 gene, a TGFbeta superfamily member, is expressed as a maternal mRNA localized to prospective endoderm, and mature Vg1 protein can induce both endodermal and mesodermal markers in embryonic cells. Most previous work on embryonic inducers, including activin, BMPs and Vg1, has relied on ectopic expression to assay for gene function. Here we employ a mutant ligand approach to block Vg1 signaling in developing embryos.

Notochord repression of endodermal Sonic hedgehog permits pancreas development.

Notochord signals to the endoderm are required for development of the chick dorsal pancreas. Sonic hedgehog (SHH) is normally absent from pancreatic endoderm, and we provide evidence that notochord, in contrast to its effects on adjacent neuroectoderm where SHH expression is induced, represses SHH expression in adjacent nascent pancreatic endoderm. We identify activin-betaB and FGF2 as notochord factors that can repress endodermal SHH and thereby permit expression of pancreas genes including Pdx1 and insulin.

Lessons from keratin 18 knockout mice: formation of novel keratin filaments, secondary loss of keratin 7 and accumulation of liver-specific keratin 8-positive aggregates.

Here, we report on the analysis of keratin 18 null mice. Unlike the ablation of K8, which together with K18 is expressed in embryonic and simple adult epithelia, K18 null mice are viable, fertile, and show a normal lifespan. In young K18 null mice, hepatocytes were completely devoid of keratin filaments.

Mice with gene targetted prion protein alterations show that Prnp, Sinc and Prni are congruent.

Classical genetic analysis has identified Sinc/Prni as the major gene controlling mouse scrapie incubation time. Sinc/Prni is linked to Prnp, the gene encoding the prion protein (PrP). Prnp alleles express distinct PrP protein variants, PrP A and PrP B, which arise from codon 108L/F and 189 T/V dimorphisms.

Cells from ERCC1-deficient mice show increased genome instability and a reduced frequency of S-phase-dependent illegitimate chromosome exchange but a normal frequency of homologous recombination.

The ERCC1 protein is essential for nucleotide excision repair in mammalian cells and is also believed to be involved in mitotic recombination. ERCC1-deficient mice, with their extreme runting and polyploid hepatocyte nuclei, have a phenotype that is more reminiscent of a cell cycle arrest/premature ageing disorder than the classic DNA repair deficiency disease, xeroderma pigmentosum. To understand the role of ERCC1 and the link between ERCC1-deficiency and cell cycle arrest, we have studied primary and immortalised embryonic fibroblast cultures from ERCC1-deficient mice and a Chinese hamster ovary ERCC1 mutant cell line.

Modulation of the renin-angiotensin pathway through enzyme inhibition and specific receptor blockade in pacing-induced heart failure: II. Effects on myocyte contractile processes.

Background: The goal of this study was to determine the effects of ACE inhibition alone, AT1 angiotensin (Ang) II receptor blockade alone, and combined ACEI and AT1 Ang II receptor blockade in a model of congestive heart failure (CHF) on isolated LV myocyte function and fundamental components of the excitation-contraction coupling process.

Methods And Results: Pigs were randomly assigned to one of five groups: (1) rapid atrial pacing (240 bpm) for 3 weeks (n=9), (2) concomitant ACEI (benazeprilat, 0.187 mg x kg(-1) x d(-1)) and rapid pacing (n=9), (3) concomitant AT1 Ang II receptor blockade (valsartan, 3 mg/kg/d) and rapid pacing (n=9), (4) concomitant ACEI and AT1 Ang II receptor blockade (benazeprilat/valsartan, 0.

Modulation of the renin-angiotensin pathway through enzyme inhibition and specific receptor blockade in pacing-induced heart failure: I. Effects on left ventricular performance and neurohormonal systems.

Background: The goal of this study was to determine the effects of ACE inhibition (ACEI) alone, AT1 angiotensin (Ang) II receptor blockade alone, and combined ACEI and AT1 Ang II receptor blockade on LV function, systemic hemodynamics, and neurohormonal system activity in a model of congestive heart failure (CHF).

Methods And Results: Pigs were randomly assigned to each of 5 groups: (1) rapid atrial pacing (240 bpm) for 3 weeks (n=9), (2) ACEI (benazeprilat, 0.187 mg x kg(-1) x d(-1)) and rapid pacing (n=9), (3) AT1 Ang II receptor blockade (valsartan, 3 mg x kg(-1) x d(-1)) and rapid pacing (n=9), (4) ACEI and AT1 Ang II receptor blockade (benazeprilat/valsartan, 0.

Notochord to endoderm signaling is required for pancreas development.

The role of the notochord in inducing and patterning adjacent neural and mesodermal tissues is well established. We provide evidence that the notochord is also required for one of the earliest known steps in the development of the pancreas, an endodermally derived organ. At a developmental stage in chick embryos when the notochord touches the endoderm, removal of notochord eliminates subsequent expression of several markers of dorsal pancreas bud development, including insulin, glucagon and carboxypeptidase A.

Stability of HPRT marker gene expression at different gene-targeted loci: observing and overcoming a position effect.

For sophisticated gene targeting procedures requiring two sequential selective steps to operate efficiently it is essential that the marker genes used are not prone to position effects. The double replacement gene targeting procedure, to produce mice with subtle gene alterations, is based on the use of hypoxanthine phosphoribosyltransferase ( HPRT) minigenes in HPRT-deficient embryonic stem cells. Our standard HPRTminigene, under the control of the mouse phosphoglycerate kinase-1 gene promoter, was stably expressed at five of six target loci examined.

Laparoscopy in the evaluation and treatment of patients with AIDS and acute abdominal complaints.

Background: The evaluation of AIDS patients with acute abdominal complaints (AAC) is quite difficult, and surgical intervention is associated with a high complication rate. The intent of this study is to evaluate the application of laparoscopy in the diagnosis and treatment of AIDS patients with AAC.

Methods: This is a retrospective analysis of 10 consecutive AIDS patients who presented with AAC.

Transgenic analysis of prion diseases.

Prion diseases are fatal transmissible neurological disorders afflicting a range of mammalian species. Although still controversial, a large body of data suggests that the causative agent may be composed entirely of a small glycoprotein. The brains of infected animals have accumulations of a pathogenic protease-resistant isoform (PrPsc) of a normal host-encoded glycoprotein, PrPc or prion protein.

Xnr4: a Xenopus nodal-related gene expressed in the Spemann organizer.

We have isolated a novel TGF beta-like gene from Xenopus which is highly related to mouse nodal The gene, Xnr4, is expressed at the gastrula stage in the Spemann organizer and at later stages in the notochord and neural tube. Ectopic expression of Xnr4 can induce and dorsalize mesoderm. These studies suggest that Xnr4, along with other nodal-related genes (Xnr1-3), may participate in mesodermal patterning and possibly neural development.