Differential expression of the cyclic GMP-stimulated phosphodiesterase PDE2A in human venous and capillary endothelial cells.

We developed selective monoclonal antibodies and used them for Western and immunocytochemical analyses to determine the tissue and cellular distribution of the human cyclic GMP-stimulated phosphodiesterase (PDE2). Western analysis revealed PDE2A expression in a variety of tissue types, including cerebellum, neocortex, heart, kidney, lung, pulmonary artery, and skeletal muscle. Immunocytochemical analysis revealed PDE2A expression in a subset of tissue endothelial cells. PDE2A immunostaining was detected in venous and capillary endothelial cells in cardiac and renal tissue but not in arterial endothelial cells. These results were confirmed by in situ hybridization. PDE2A immunostaining was also absent from luminal endothelial cells of large vessels, such as aorta, pulmonary, and renal arteries, but was present in the endothelial cells of the vasa vasorum. PDE2A immunostaining was detected in the endothelial cells of a variety of microvessels, including those in renal and cardiac interstitial spaces, renal glomerulus, skin, brain, and liver. Although PDE2A was not readily detected in arterial endothelial cells by immunocytochemistry of intact tissue, it was detected at low levels in cultured arterial endothelial cells. These results suggest a possible role for PDE2A in modulating the effects of cyclic nucleotides on fluid and inflammatory cell transit through the endothelial cell barrier.

Isolation and characterization of cDNAs encoding PDE5A, a human cGMP-binding, cGMP-specific 3',5'-cyclic nucleotide phosphodiesterase.

Human cGMP-binding, cGMP-specific 3',5'-cyclic nucleotide phosphodiesterase (PDE5A) cDNAs were isolated. A 3.1-kb composite DNA sequence assembled from overlapping cDNAs encodes an 875-amino-acid protein with a predicted molecular mass of 100012 Da (PDE5A1). Extracts prepared from yeast expressing human PDE5A1 hydrolyzed cGMP. This activity was inhibited by the selective PDE5 inhibitors zaprinast and DMPPO. PDE5A mRNA is expressed in aortic smooth muscle cells, heart, placenta, skeletal muscle and pancreas and, to a much lesser extent, in brain, liver and lung. A 5'-splice variant, PDE5A2, encodes an 833-amino-acid protein with eight unique amino acids at the amino terminus. PDE5A maps to chromosome 4q 25-27.

Identification and characterisation of a human calmodulin-stimulated phosphodiesterase PDE1B1.

A cDNA encoding a calmodulin-stimulated 3',5'-cyclic nucleotide phosphodiesterase (PDE) was isolated from a human brain cDNA library. The cDNA, designated HSPDE1B1, encoded a protein of 536 amino acids that shared 96% sequence identity with the bovine "63 kDa" calmodulin-stimulated PDE. The recombinant protein had cyclic nucleotide phosphodiesterase activity that was stimulated approximately 2-fold by Ca2+/calmodulin and preferred cGMP as substrate. In addition, the enzymatic activity of HSPDE1B1 was inhibited by phosphodiesterase inhibitors with potencies similar to that displayed toward the bovine PDE1 enzymes: IBMX approximately equal to 8-methoxymethyl-IBMX > vinpocetine approximately equal to zaprinast > cilostamide > rolipram. HSPDE1B1 mRNA was found predominantly in the brain. Lower mRNA levels were found in heart and skeletal muscle. In situ hybridisation of brain revealed expression of HSPDE1B1 predominately in neuronal cells of the cerebellum, hippocampus and caudate. The HSPDE1B1 gene was mapped to human chromosome 12. A partial genomic sequence of HSPDE1B1 was isolated and shown to contain two splice junctions that are conserved in the rat PDE4 and the Drosophila dunce genes.

Overlapping expression of Xwnt-3A and Xwnt-1 in neural tissue of Xenopus laevis embryos.

Xwnt-3A is a member of the Xenopus-Wnt gene family, a class of secreted, cysteine-rich proteins implicated in intercellular signaling during early development. Here we describe the full-length coding sequence of Xwnt-3A, as well as the spatial expression pattern of this Xwnt gene as determined by whole-mount in situ hybridization analysis. While Xwnt-3A shares considerable amino acid identity with both Wnt-3 (87%) and Wnt-3A (85%), its spatial expression pattern is most like that of Wnt-3A. Xwnt-3A, which is first detected at the neurula stage of development, is expressed exclusively along the dorsal midline of the developing brain and neural tube and along the dorsal surface of the otic vesicle. While the expression of Xwnt-1 extensively overlaps that of Xwnt-3A, Xwnt-1 is uniquely expressed along the midbrain/hindbrain boundary and is absent from the otic vesicle. The expression of Xwnt-3A in neural ectoderm is dependent upon neural induction as determined by experiments with recombined ectoderm and mesoderm tissue. These results suggest that Xwnt-3A may participate in patterning the central nervous system during early Xenopus development. Last, the ectopic expression of Xwnt-3A induces the formation of a secondary axis at the anterior end of the embryo.

Cloning and developmental expression in Xenopus laevis of seven additional members of the Wnt family.

Degenerate oligonucleotide primers encoding highly conserved regions of Wnt-related proteins were used with the polymerase chain reaction (PCR) to amplify cDNA derived from Xenopus laevis embryos. cDNA sequences partially encoding seven additional members of the Xwnt gene family were isolated using this strategy. These cDNAs have been given the designation Xwnt-2, Xwnt-6, Xwnt-7A, Xwnt-7B, Xwnt-7C, Xwnt-8B and Xwnt-10 based on their amino acid identity with previously described Wnts. With regard to the timing of expression of these Xwnts during embryonic development, Xwnt-2, the least abundant transcript, was first detected during the neurula stage, while Xwnt-8B transcripts were first detected at the gastrula stage, and decreased by the tailbud stage. Multiple transcripts of Xwnt-6 were detected at varied times during development beginning at the gastrula stage. In contrast, Xwnt-7A, -7B and -10 transcripts were not detected until the tailbud stage. With regard to expression in adult tissues, Xwnt-6, -7A, -7B, -8B and -10 were all expressed abundantly in the brain, and to a lesser extent in a variety of other tissues. Whole-mount in situ hybridization was then employed to monitor the spatial expression of selected Xwnts. Xwnt-7A and -10 transcripts were detected in distinct areas of the developing brain of tailbud-stage embryos. The temporal and spatial differences in expression suggest different roles for these new Xwnt family members in Xenopus development.

Molecular genetic analysis of cytoskeletal proteins in development and implications for the membrane skeleton.

The membrane skeleton of nonerythroid cells may be involved in a variety of processes, including the formation and maintenance of specific membrane-cytoskeletal domains. Although much has been learned about the ultrastructure and protein chemistry of the membrane skeleton, there are few direct tests of the in vivo functions of the constituent proteins of the membrane skeleton. Recent advances in molecular genetic analysis provide techniques for studying the membrane skeleton and its components in vivo. Considered here in brief detail are a variety of genetic techniques that have already been used to study cytoskeletal proteins. These techniques should also prove useful for future study of the membrane skeleton.

Human lamin B contains a farnesylated cysteine residue.

We recently showed that HeLa cell lamin B is modified by a mevalonic acid derivative. Here we identified the modified amino acid, determined its mode of linkage to the mevalonic acid derivative, and established the derivative's structure. A cysteine residue is modified because experiments with lamin B that had been biosynthetically labeled with [3H]mevalonic acid or [35S]cysteine and then extensively digested with proteases yielded 3H- or 35S-labeled products that co-chromatographed in five successive systems. A thioether linkage rather than a thioester linkage is involved because the mevalonic acid derivative could be released from the 3H-labeled products in a pentane-extractable form by treatment with Raney nickel but not with methanolic KOH. The derivative is a farnesyl moiety because the Raney nickel-released material was identified as 2,6,10-trimethyl-2,6,10-dodecatriene by a combination of gas chromatography and mass spectrometry. The thioether-modified cysteine residue appears to be located near the carboxyl end of lamin B because treatment of 3H-labeled lamin B with cyanogen bromide yielded a single labeled polypeptide that mapped toward this end of the cDNA-inferred sequence of human lamin B.

Evidence for modification of lamin B by a product of mevalonic acid.

Previous work from this laboratory has shown that a derivative of mevalonic acid is post-translationally incorporated into a number of specific proteins in Swiss 3T3 cells. Neither the nature of the modification nor the identities of the modified proteins have been determined to date. Here we describe results concerning modified proteins of approximately 67 kDa from HeLa cells and Chinese hamster ovary cells. We show that these proteins are specific to the nucleus and remain associated with a Triton/salt-insoluble nuclear fraction. Furthermore, immunological studies demonstrate that one of the modified proteins comigrates on two-dimensional gels with lamin B, a structural protein associated with the nuclear envelope. Using antibodies directed against lamin B in an immunoprecipitation experiment, we further show that this mevalonic acid-modified protein specifically coprecipitates with lamin B. These results support the hypothesis that lamin B is modified by a derivative of mevalonic acid.