Clayton A. Buck

Further characterization of endometrial integrins during the menstrual cycle and in pregnancy

OBJECTIVEnTo profile the changes in integrin expression in cycling and pregnant endometrium.nnnDESIGNnImmunohistochemistry was performed on endometrium from proliferative, early, and midsecretory phase and early pregnancy using a blinded panel of monoclonal antibodies directed against integrins. Closer examination of the cycle-dependent integrins was performed in 112 patients throughout the cycle and from the first trimester of pregnancy.nnnSETTINGnAn academic teaching hospital.nnnPATIENTSnOvulatory women or women undergoing pregnancy termination.nnnMAIN OUTCOME MEASUREnStaining intensity of each antibody in epithelial or stromal cells.nnnRESULTSnCertain integrins were present constitutively throughout the menstrual cycle whereas others were expressed only during the luteal phase. Four integrins increased in the decidua of pregnancy. The timing of expression of the two cycle-dependent integrins (alpha 4 beta 1 and alpha v beta 3) framed the putative window of implantation and suggests a role in establishment of uterine receptivity.nnnCONCLUSIONSnOur findings confirm the patterns of endometrial integrins and suggest important roles for integrins in the process of implantation and decidualization.

Adenoviral Gene Transfer of β3 Integrin Subunit Induces Conversion from Radial to Vertical Growth Phase in Primary Human Melanoma

Expression of the beta3 subunit of the alphavbeta3 vitronectin receptor on melanoma cells is associated with tumor thickness and the ability to invade and metastasize. To address the role of alphavbeta3 in the complex process of progression from the nontumorigenic radial to the tumorigenic vertical growth phase of primary melanoma, we examined the biological consequences of overexpressing alphavbeta3 in early-stage melanoma cells using an adenoviral vector for gene transfer. Overexpression of functional alphavbeta3 in radial growth phase primary melanoma cells 1) promotes both anchorage-dependent and -independent growth, 2) initiates invasive growth from the epidermis into the dermis in three-dimensional skin reconstructs, 3) prevents apoptosis of invading cells, and 4) increases tumor growth in vivo. Thus, alphavbeta3 serves diverse biological functions during the progression from the nontumorigenic radial growth phase to the tumorigenic and-invasive vertical growth phase primary melanoma.

Glycopeptides from the Surface of Control and Virus-Transformed Cells

Glycopeptides were removed by trypsin digestion from the surface of control cells and cells transformed by Rous sarcoma virus, murine sarcoma virus, or polyoma virus. After digestion with pronase, the glycopeptides were analyzed by gel filtration. The elution profiles suggest that there are differences in the glycopeptides from the surface of control cells and those from transformed cells.

Studies on mitochondrial tRNA from animal cells: I. A comparison of mitochondrial and cytoplasmic tRNA and aminoacyl-tRNA synthetases

Abstract Mitochondrial and cytoplasmic aminoacyl-tRNAs of rat liver have been compared by chromatography on methylated albumin kieselguhr. This comparison revealed species of leucyl-, tyrosyl-, aspartyl-, valyl- and seryl-tRNA which were found exclusively in mitochondria. These tRNAs did not arise as artifacts during the isolation or acylation procedures. This was shown by the fact that elution profiles could not be altered as a result of denaturing the tRNAs prior to acylation. Furthermore, the relative positions of these tRNAs remained unaltered as a result of interchanging synthetases. It was also found that cytoplasmic synthetases were unable to acylate species of tRNA which were exclusively mitochondrial. These studies add further support to the idea that the protein-synthesizing apparatus of the mitochondria is distinct from that operating in other areas of the cytoplasm.

Transcriptional Regulation of Cardiac Development: Implications for Congenital Heart Disease and DiGeorge Syndrome

In recent years, impressive advances have occurred in our understanding of transcriptional regulation of cardiac development. These insights have begun to elucidate the mystery of congenital heart disease at the molecular level. In addition, the molecular pathways emerging from the study of cardiac development are being applied to the understanding of adult cardiac disease. Preliminary results support the contention that a thorough understanding of molecular programs governing cardiac morphogenesis will provide important insights into the pathogenesis of human cardiac diseases. This review will focus on examples of transcription factors that play critical roles at various phases of cardiac development and their relevance to cardiac disease. This is an exciting and burgeoning area of investigation. It is not possible to be all-inclusive, and the reader will note important efforts in the areas of cardiomyocyte determination, left-right asymmetry, cardiac muscular dystrophies, electrophysiology and vascular disease are not covered. For a more complete discussion, the reader is referred to recent reviews including the excellent compilation of observations assembled by Harvey and Rosenthal (1).

Studies on mitochondrial tRNA from animal cells: II. Hybridization of aminoacyl-tRNA from rat liver mitochondria with heavy and light complementary strands of mitochondrial DNA

Abstract The sequence homology of mitochondrial and cytoplasmic aminoacyl-tRNAs with mitochondrial DNA of rat liver has been studied by performing hybridization at low temperatures in the presence of 50% formamide. The tRNA was acylated with radioactive amino acids so that specific aminoacyl-tRNA-DNA hybridization could be followed in the presence of other species of RNA. With a constant amount of DNA and increasing RNA concentrations, a saturation plateau was reached with all tRNAs tested. Competition experiments showed that mitochondrial aminoacyl-tRNA competed far more efficiently with mitochondrial tyrosyl-, phenylalanyl-, seryl- or leucyl-tRNA for hybridization sites on mitochondrial DNA than did cytoplasmic aminoacyl-tRNA. No hybridization was observed with rat liver nuclear DNA or Escherichia coli DNA. Hybridization of mitochondrial aminoacyl-tRNAs was also performed with isolated complementary heavy (H) and light (L) strands of mitochondrial DNA. The DNA was prepared by equilibrium centrifugation in alkaline cesium chloride. It was shown by electron microscopy that at least 20% of the single-stranded DNA molecules present in H and L fractions consisted of full-length 5 μ linear or circular forms. It was found that mitochondrial leucyl- and phenylalanyl-tRNA hybridized exclusively with the H strand, whereas tyrosyl- and seryl-tRNA hybridized exclusively with the L strand of mitochondrial DNA. These studies show (1) that at least four mitochondrial tRNAs are potentially transcribed from mitochondrial DNA; (2) these mitochondrial tRNAs differ in base sequence from their cytoplasmic counterparts; and (3) some species of mitochondrial tRNA may be transcribed in vivo from one strand and others from the complementary strand of mitochondrial DNA.

p57Kip2 expression is enhanced during mid-cardiac murine development and is restricted to trabecular myocardium.

During embryonic development the heart is required to grow in size and cell number, undergo complex morphologic alterations, and function to circulate the blood. Between embryonic d 10.5 (E10.5) and E11.5, cardiac myocytes undergo rapid cell division, resulting in doubling of cardiac mass, while metabolic requirements are increased and contraction force is enhanced. Accelerated cardiomyocyte differentiation is accompanied by a significant increase in trabeculation of ventricular myocardium. Many single gene mutations in the mouse result in a thinned myocardium and embryonic lethality between E10.5 and E13.5 secondary to heart failure. This is the case in the Splotch mouse in which a mutation of the Pax3 gene results in neural crest and cardiac defects. Nevertheless, the molecular events governing these important developmental steps remain largely unknown. Here, we describe the use of suppression subtractive hybridization to identify mRNA transcripts whose expression is enhanced during this critical period in normal hearts. These genes encode functions related to maturation of the contractile apparatus, cardiomyocyte differentiation, altered cellular metabolism, and transcriptional regulation. One of the genes that we identified, p57Kip2, encodes a cyclin-dependent kinase inhibitor of the p21 family. We show that p57Kip2 is normally expressed in the inner trabecular layer of the developing heart. In Splotch embryos, expression of p57Kip2 is expanded to encompass the entire thickness of the myocardium. This result and further structural analysis suggests that the myocardial defect of Splotch embryos is associated with precocious cardiomyocyte differentiation.

Rnf4, a RING protein expressed in the developing nervous and reproductive systems, interacts with Gscl, a gene within the DiGeorge critical region

A yeast 2‐hybrid screen was performed to identify possible transcriptional modulators interactive with goosecoid‐like (gscl), a transcription factor with suppressive activity, expressed during early brain and gonad development. The screen resulted in the identification of a RING protein known as rnf4 or snrf. Gscl/rnf4 interactions were confirmed by affinity chromatography and by immunoprecipitation. Northern analysis confirmed earlier reports of ubiquitous rnf4 expression in adult tissues. Immunohistochemical analysis of mouse embryos revealed expression primarily in the developing nervous system, with strong expression in the dorsal root ganglia and developing gonads. In contrast to previous reports, both cytoplasmic and nuclear expression of rnf4 was documented. The results reported here confirm and extend earlier reports of rnf4 expression. They suggest for the first time, that in addition to acting as a modulator of transcriptional activity, rnf4 may function, as do other RING proteins, to promote the formation of intracytoplasmic complexes involved in shuttling information between the cytoplasm and the nucleus. Dev Dyn;218:102–111.

Adenoviral‐mediated expression of antisense RNA to fibroblast growth factors disrupts murine vascular development

Fibroblast growth factors (FGFs) are expressed in the developing embryo and are postulated to regulate embryonic and vascular growth. The goal of this study was to elucidate the role of basic fibroblast growth factor (FGF‐2) in early murine embryonic cardiovascular development in the mouse embryo. Gestation day 7.5 embryos were harvested and placed in culture, and 12 hr later replication‐defective adenovirus (0.5 × 106 plaque forming units) encoding either β‐galactosidase or antisense FGF‐2 RNA was injected into the sinus venosus of the cultured embryos. Embryos receiving only replication‐defective adenovirus expressing the β‐galactosidase gene continued to develop normally over the next 12 hr. In contrast, those receiving adenovirus encoding antisense FGF‐2 RNA displayed marked morphogenetic abnormalities, including cessation of growth and abnormal yolk sac vascular development, even though the embryonic hearts continued to beat. Abnormal development of the yolk sac vasculature was confirmed by microangiography and by histologic examination. Coinjection of virus carrying FGF‐2 cDNA in the sense orientation reversed the effects of antisense FGF‐2 RNA expression. These results confirm the efficacy of the replication‐defective adenovirus for targeting gene expression to the developing vasculature and provide evidence for a critical role of FGF in the normal vascular assembly in the early embryo. Cessation of embryonic growth on expression of antisense FGF‐2 RNA was most likely attributable to failure of efficient circulation of the early embryonic blood cells from the yolk sac into the embryo. Dev. Dyn. 1998;213:421–430.

Integrins and other cell adhesion molecules in cardiac development

Cardiac morphogenesis is dependent on the coordinated and programmed expression of cell surface receptors that can mediate cell-cell associations or promote adhesion of the cell to its extracellular environment. This article briefly reviews the unique adhesive characteristics and expression profiles of the integrins and other families of adhesion molecules that have been implicated in critical events of early heart development. Understanding the developmental repertoire of receptor expression, which in most cases cannot be predicted from that seen in the mature heart, is an important component in unraveling the molecular events of normal cardiac development, congenital heart disease, and certain pathologic conditions seen in the adult heart.