Adenoviral delivery of human CDC5 promotes G2/M progression and cell division in neonatal ventricular cardiomyocytes.

Heart failure results from the cumulative death of cardiomyocytes, and the inability of remaining cells to regenerate. Efforts toward transcriptional reprogramming of cardiomyocytes by overexpressing E1A or E2F1 have been limited by the inability of cardiomyocytes to enter and complete mitosis. Human CDC5 (hCDC5), a component of the pre-mRNA splicing complex, has been shown to regulate G2/M transit in asynchronously dividing cells. We now show that co-infection of recombinant adenoviruses expressing E1A/E1B and hCDC5 promotes cell cycle re-entry and G2/M progression in post-mitotic cardiomyocytes. Co-expression of E1A/E1B and hCDC5 induced nuclear localization of cyclin-dependent kinase 1 and cyclin B1, and was sufficient to promote mitotic entry as determined by an increase in mitotic index only in co-infected cells. E1A/E1B and hCDC5 promoted cell division, as evidenced by an increase in the number of cardiomyocytes following co-infection. Thus, overexpression of E1A/E1B and hCDC5 resulted in cell cycle re-entry, DNA synthesis, cell division, and an increase in cardiomyocyte number, suggesting the formation of new cardiomyocytes. These studies suggest that G1/S-phase transcriptional regulators, in combination with pre-mRNA splicing factors, such as CDC5, that regulate rate-limiting G2/M target genes may prove useful in developing therapies to stimulate myocardial regeneration.

Human Cdc5, a regulator of mitotic entry, can act as a site-specific DNA binding protein.

G(2)/M progression requires coordinated expression of many gene products, but little is known about the transcriptional regulators involved. We recently identified human Cdc5, a positive regulator of G(2)/M in mammalian cells. We also demonstrated the presence of a latent activation domain in its carboxyl terminus, suggesting that human Cdc5 regulates G(2)/M through transcriptional activation. Despite the presence of a DNA binding domain, studies by others have failed to identify a preferential binding site for Cdc5 family members. In addition, Cdc5 recently has been associated with the splicesome in several organisms, suggesting that it may not act through DNA binding. We now report the identification of a 12 bp sequence to which human Cdc5 binds specifically and with high affinity through its amino terminus. We show that this DNA-protein interaction is capable of activating transcription. We also used a selection system in yeast to identify human genomic fragments that interact with human Cdc5. Several of these contained sequences similar to the binding site. We demonstrate that these bind human Cdc5 with similar specificity and affinity. These experiments provide the first evidence that Cdc5 family members can act as site-specific DNA binding proteins, and that human Cdc5 may interact with specific, low abundance sequences in the human genome. This raises the possibility that Cdc5 proteins may participate in more than one process necessary for regulated cell division.

A mammalian homolog of fission yeast Cdc5 regulates G2 progression and mitotic entry.

Progression through G2/M of the mammalian cell division cycle requires the coordinated expression of many gene products, but little is known of the transcriptional regulators involved. Schizosaccharomyces pombe Cdc5 is a putative transcription factor implicated in G2/M transit. We recently identified a cDNA encoding a putative human transcription factor, now designated human Cdc5 (hCdc5), with homology to S. pombe Cdc5. Widespread expression of hCdc5 in human tissues and homology with expressed sequences in other eukaryotes suggested an evolutionarily conserved general function. Nuclear import of hCdc5 upon serum stimulation of mammalian cells suggested a possible role in cell proliferation. We now report that overexpression of hCdc5 in mammalian cells shortened G2 and reduced cell size. A dominant negative mutant of hCdc5 lacking the carboxyl-terminal activation domain slowed G2 progression and delayed entry into mitosis. Thus, hCdc5 is the first transcriptional regulator shown to affect G2 progression and mitotic entry in mammalian cells.

Pombe Cdc5-related protein. A putative human transcription factor implicated in mitogen-activated signaling.

The Schizosaccharomyces pombe cdc5 gene product is a cell cycle regulator that exerts its effects at the G2/M transition in fission yeast. We describe the cloning of a putative human transcription factor, pombe Cdc5-related protein (PCDC5RP), which bears significant homology to S. pombe Cdc5 and to expressed sequences in mouse, nematode, and budding yeast. PCDC5RP is expressed widely in normal adult human tissues and thus may have an important general function that has been preserved evolutionarily. PCDC5RP contains two tandem repeats of a helix-turn-helix DNA binding motif, four consensus nuclear localization signals, and a hydrophilic, proline-rich central region similar to the transcriptional activating domain in Myb family members. Remarkably, PCDC5RP moved rapidly from cytoplasm to nucleus upon serum stimulation of cultured cells. This movement correlated temporally with an increase in PCDC5RP phosphorylation. Thus, PCDC5RP is a presumed transcription factor that appears to transduce cytoplasmic signals to the nucleus upon serum stimulation.

Fulminant development of pulmonary arteriovenous fistulas in an infant after total cavopulmonary shunt.

An infant with cyanotic congenital heart disease and polysplenia syndrome developed profound cyanosis within months of undergoing bilateral cavopulmonary anastomoses. Intrapulmonary shunting was diagnosed by contrast echocardiography with peripheral venous and selective pulmonary artery injection. Histopathology revealed abnormal, thin-walled vessels within the interstitium of the lung lobule. These vessels have not been reported previously and are likely to be the anatomic site of arteriovenous shunting. This case demonstrates that pulmonary arteriovenous fistulas (PAVFs) may develop rapidly after cavopulmonary anastomosis in young infants. It also illustrates the use of contrast echocardiography for following PAVF progression in these patients.

Development of pulmonary arteriovenous fistulae in children after cavopulmonary shunt.

BACKGROUND: The cavopulmonary shunt procedure is now used for palliation of complex congenital heart lesions in infants. While pulmonary arteriovenous fistulae (PAVF) are a well-known complication of this surgery in older patients, no study of the prevalence of this condition in children and young infants has been reported. METHODS AND RESULTS: We compared 29 patients with cavopulmonary shunts or total caval exclusion with 53 control subjects evaluated by contrast echocardiography at the University of California, San Francisco. The primary cardiac lesion, age at the time of surgery, type of right heart bypass procedure, provision of auxiliary pulmonary blood flow, and changes in oxygen saturation over time were compared. The prevalence of PAVF in children after cavopulmonary anastomosis is 60%, higher than previously reported. The prevalence is significantly higher in infants < 6 months old and in those with a heterotaxy syndrome. The provision of an additional source of pulsatile, pulmonary blood flow appears to have little effect on the development of PAVF. Patients who developed PAVF had arterial oxygen saturations at the time of discharge from surgery similar to those who did not develop them. Those with PAVF had significantly lower arterial and pulmonary venous oxygen saturations at follow-up as a result of their intrapulmonary shunt. CONCLUSIONS: Contrast echocardiography provides a sensitive method for the detection of PAVF. While the origins, natural history, and ultimate clinical significance of PAVF in children after cavopulmonary anastomosis are unclear, surveillance by contrast echocardiography is indicated for all patients who have had this procedure because PAVF may cause significant intrapulmonary right-to-left shunting in some patients.

The levoatriocardinal vein: morphology and echocardiographic identification of the pulmonary-systemic connection.

OBJECTIVES: This study considers the array of pulmonary-systemic connections made by the levoatriocardinal vein. The primary and associated lesions that play a role in forming this vein are examined, and echocardiography is discussed as a method for its rapid identification. BACKGROUND: The levoatriocardinal vein is a pulmonary-systemic connection that provides an alternative egress for pulmonary venous blood in left-sided obstructive lesions. It is thought to result from the persistence of anastomotic channels that connect the capillary plexus of the embryonic foregut to the cardinal veins. Only 12 cases of levoatriocardinal vein have been reported since its first description in 1926. A comprehensive description of the morphology and echocardiographic identification of this lesion has been unavailable because of its rarity. METHODS: A retrospective study was performed in 13 patients with a levoatriocardinal vein from the University of California, San Francisco. Echocardiographic findings were compared with those obtained by angiography or at necropsy. In addition, the details of 12 previously published case reports were reviewed. Age at presentation, primary obstruction to pulmonary venous return, integrity of the atrial septum and origin and drainage of the levoatriocardinal vein were compared. RESULTS: Patient age at presentation was < 2 years, with most patients presenting before age 6 months. Variations of the hypoplastic left heart syndrome accounted for the majority of primary defects encountered, although multiple but less severe left-sided lesions were seen. The atrial septum was functionally intact in most patients. The levoatriocardinal vein, defined echocardiographically, originated predominantly from the smooth-walled left atrium and drained to the superior vena cava or innominate vein; however, variations of this pattern existed. CONCLUSIONS: As a physiologic entity, the levoatriocardinal vein provides a mechanism for decompression of pulmonary venous return primarily in patients with left ventricular inflow obstruction. A levoatriocardinal vein is thought to form when the atrial septum fails to provide an alternate egress for left atrial blood. However, when a septal defect or alternative shunt occurs in conjunction with a levoatriocardinal vein, the clinical presentation may be postponed. Echocardiography provides a rapid, noninvasive modality for identifying the pulmonary-systemic connection, which may masquerade as the vertical vein in anomalous pulmonary venous connection or act as an occult source of left to right shunting in patients undergoing surgery for hypoplastic left heart syndrome.

Tethered ligand library for discovery of peptide agonists.

We exploited the mechanism underlying thrombin receptor activation to develop a novel screening method to identify peptide agonists. The thrombin receptor is activated by limited proteolysis of its amino-terminal exodomain. Thrombin cleaves this domain to unmask a new amino terminus, which then functions as a tethered peptide agonist, binding intramolecularly to the body of the receptor to trigger signaling. The thrombin receptor's amino-terminal exodomain can also donate the tethered agonist intermolecularly to activate nearby thrombin receptors. We utilized this ability by co-expressing a "tethered ligand library," which displayed the thrombin receptor's amino-terminal exodomain bearing random pentapeptides in place of the native tethered ligand together with target receptors in Xenopus oocytes. Clones that conferred thrombin-dependent signaling by intermolecular ligation of the target receptor were isolated by sib selection. Agonists for the thrombin receptor itself (GFIYF) and for the formyl peptide receptor (MMWLL) were identified. Surprisingly, the latter agonist was quite active at the formyl peptide receptor even without N-formylation, and its formylated form, fMMWLL, was more potent than the classical formyl peptide receptor agonist fMLF. In addition to identifying novel peptide agonists for targets of pharmacological interest, this method might be used to discover agonists for orphan receptors. It also suggests a possible evolutionary path from peptide to protease-activated receptors.

Prognosis of fetuses with a cystic hygroma.

This paper reports our experience with 55 fetuses identified in utero to have a cystic hygroma. The outcome of fetuses with an isolated cystic hygroma, cystic hygroma with non-immune hydrops, and cystic hygroma with multiple anomalies was evaluated. Approximately two-thirds of karyotypes were aneuploid, and a strong association of septation and aneuploidy existed. Only five cases, four of which had isolated hygromas, came to term and resulted in live births. Two of these involved small non-septated lesions which resolved in utero.

Fabry disease: six gene rearrangements and an exonic point mutation in the alpha-galactosidase gene.

Fabry disease, an X-linked recessive disorder of glycosphingolipid catabolism, results from the deficient activity of the lysosomal hydrolase, alpha-galactosidase. Southern hybridization analysis of the alpha-galactosidase gene in affected hemizygous males from 130 unrelated families with Fabry disease revealed six with different gene rearrangements and one with an exonic point mutation resulting in the obliteration of an Msp I restriction site. Five partial gene deletions were detected ranging in size from 0.4 to greater than 5.5 kb. Four of these deletions had breakpoints in intron 2, a region in the gene containing multiple Alu repeat sequences. A sixth genomic rearrangement was identified in which a region of about 8 kb, containing exons 2 through 6, was duplicated by a homologous, but unequal crossover event. The Msp I site obliteration, which mapped to exon 7, was detected in an affected hemizygote who had residual enzyme activity. Genomic amplification by the polymerase chain reaction and sequencing revealed that the obliteration resulted from a C to T transition at nucleotide 1066 in the coding sequence. This point mutation, the first identified in Fabry disease, resulted in an arginine356 to tryptophan356 substitution which altered the enzyme's kinetic and stability properties. The detection of these abnormalities provided for the precise identification of Fabry heterozygotes, thereby permitting molecular pedigree analysis in these families which revealed paternity exclusions and the first documented new mutations in this disease.

Fabry disease: molecular diagnosis of hemizygotes and heterozygotes.

Fabry disease, an X-linked inborn error of glycosphingolipid catabolism, results from the deficient activity of the lysosomal hydrolase, alpha-galactosidase A. Previously, the diagnosis of affected hemizygous males and heterozygous females was based on clinical findings and the levels of alpha-galactosidase A activity in easily obtained sources such as plasma and isolated lymphocytes or granulocytes. Since the gene encoding alpha-galactosidase A undergoes random X-inactivation, the expressed level of enzymatic activity in females heterozygous for the disease gene may vary significantly, thereby making accurate carrier detection difficult. The recent cloning and characterization of the full-length cDNA encoding human alpha-galactosidase A now permits the accurate diagnosis of affected hemizygotes and heterozygous females. In families with gene rearrangements or an altered restriction endonuclease cleavage site, precise diagnosis can be accomplished by Southern hybridization analysis using the alpha-galactosidase A cDNA as probe. In families with normal restriction patterns, two restriction fragment length polymorphisms have been identified in and adjacent to the alpha-galactosidase A gene which also allow precise hemizygote and heterozygote diagnosis. In addition, the recent identification of polymorphic, random DNA sequences (DXS17 and DXS87) located near the alpha-galactosidase A locus permits molecular diagnosis in informative families. Further evaluation of DXS17, DXS87 and other closely linked random DNA probes is required in order to determine their informativeness, proximity to the alpha-galactosidase A locus and, hence, accuracy for molecular diagnosis.

Human alpha-galactosidase A: nucleotide sequence of a cDNA clone encoding the mature enzyme.

The complete nucleotide sequence has been determined for a lambda gt11 cDNA clone (lambda AG18) containing the full-length coding region for the mature lysosomal form of human alpha-galactosidase A (alpha-Gal A; EC 3.2.1.22). The lambda AG18 insert contained a 1226-base-pair sequence with an open reading frame encoding 398 amino acids of the mature polypeptide (predicted Mr = 45,356) and the last 5 amino acids of the propeptide sequence. The poly(A) signals AATACA and ATTAAA occurred 28 and 11 nucleotides prior to the TAA stop codon, respectively. There was no 3' untranslated region as the poly(A) sequence immediately followed the TAA termination codon; a second independently cloned cDNA confirmed this finding. The predicted amino acid sequence was colinear with 86 nonoverlapping residues (22% of the mature subunit) determined by microsequencing amino-terminal, tryptic, and cyanogen bromide peptides of the purified mature enzyme. Four potential N-glycosylation sites were identified, all of which occurred at predicted beta turns in hydrophilic regions of secondary structure. RNA transfer hybridization analysis of HeLa poly(A)+ RNA demonstrated a single 1.45-kilobase band whose signal was decreased by prior immunoabsorption of polysomes with monospecific alpha-Gal A antibodies. Searches of nucleic acid and protein data bases did not reveal significant homology even with the limited sequences available for mammalian lysosomal enzymes.

Fabry disease: isolation of a cDNA clone encoding human alpha-galactosidase A.

Fabry disease is an X-linked inborn error of metabolism resulting from the deficient activity of the lysosomal hydrolase, alpha-galactosidase A (alpha-Gal A; alpha-D-galactoside galactohydrolase, EC 3.2.1.22). To investigate the structure, organization, and expression of alpha-Gal A, as well as the nature of mutations in Fabry disease, a clone encoding human alpha-Gal A was isolated from a lambda gt11 human liver cDNA expression library. To facilitate screening, an improved affinity purification procedure was used to obtain sufficient homogeneous enzyme for production of monospecific antibodies and for amino-terminal and peptide microsequencing. On the basis of an amino-terminal sequence of 24 residues, two sets of oligonucleotide mixtures were synthesized corresponding to adjacent, but not overlapping, amino acid sequences. In addition, an oligonucleotide mixture was synthesized based on a sequence derived from an alpha-Gal A internal tryptic peptide isolated by reversed-phase HPLC. Four positive clones were initially identified by antibody screening of 1.4 X 10(7) plaques. Of these, only one clone (designated lambda AG18) demonstrated both antibody binding specificity by competition studies using homogeneous enzyme and specific hybridization to synthetic oligonucleotide mixtures corresponding to amino-terminal and internal amino acid sequences. Nucleotide sequencing of the 5' end of the 1250-base-pair EcoRI insert of clone lambda AG18 revealed an exact correspondence between the predicted and known amino-terminal amino acid sequence. The insert of clone lambda AG18 appears to contain the full-length coding region of the processed, enzymatically active alpha-Gal A, as well as sequences coding for five amino acids of the amino-terminal propeptide, which is posttranslationally cleaved during enzyme maturation.