Gene and stem cell therapies.

Gene and stem cell therapies hold promise for the treatment of a wide variety of inherited and acquired human diseases. Identification of genes involved in human disease and development of novel vectors and devices for delivering therapeutic genes to different tissues in vivo have resulted in significant progress in the area of gene therapy. Isolation of stem cells from organs formerly thought to have no regenerative potential, the demonstration of stem cell plasticity, and the creation of human embryonic stem cells clearly demonstrate the feasibility of human stem cell therapy. Much additional work remains to be done in the areas of vector development and stem cell biology before the full therapeutic potential of these approaches can be realized. Of equal importance, the ethical issues surrounding gene- and cell-based therapies must be confronted.

Inhibition of the generation of thrombin and factor Xa by a fucoidan from the brown seaweed Ecklonia kurome.

The effects of a fucoidan (C-II), which was purified from the brown seaweed Ecklonia kurome, on the generation of thrombin and factor Xa have been investigated by measuring the amidolytic activities by using the respective specific chromogenic substrates in both plasma and purified systems. C-II inhibited significantly the generation of thrombin in both the intrinsic and the extrinsic pathways, although the intrinsic inhibitory effect by C-II was more remarkable than the extrinsic one. On the other hand, C-II was a good inhibitor of the factor Xa generation in the intrinsic pathway, while it was a poor one in the extrinsic pathway. In the purified systems C-II also inhibited the formation of prothrombin-activating complex (i.e., prothrombinase), but not its activity. The concentration of C-II required for 50% inhibition of thrombin generation was about one-tenth to one-seventh of that of the activity of the generated thrombin in plasma. These results indicate that C-II has an inhibitory effect on the generation of thrombin by blocking the formation of prothrombinase and by preventing the generation of intrinsic factor Xa in addition to its antithrombin activity, and also that the generation-inhibitory effect is more remarkable than C-II's enhancement effect on the antithrombin activity by heparin cofactor II in plasma.

Objective evaluation of regional left ventricular wall motion during dobutamine stress echocardiographic studies using segmental analysis of color kinesis images.

OBJECTIVES: To test the feasibility of objective and automated evaluation of echocardiographic stress tests, we studied the ability of segmental analysis of color kinesis (CK) images to detect dobutamine-induced wall motion abnormalities and compared this technique with inexperienced reviewers of conventional gray-scale images. BACKGROUND: Conventional interpretation of stress echocardiographic studies is subjective and experience dependent. METHODS: CK images were obtained in 89 of 104 consecutive patients undergoing clinical dobutamine stress studies and were analyzed using custom software to calculate regional fractional area change in 22 segments in four standard views. Each patient's data obtained at rest was used as a control for automated detection of dobutamine-induced wall motion abnormalities. Independently, studies were reviewed without CK overlays by two inexperienced readers who classified each segment's response to dobutamine. A consensus reading of two experienced reviewers was used as the gold standard for comparisons. In a subgroup of 16 patients, these consensus readings and CK detection of wall motion abnormalities were compared with coronary angiography. RESULTS: The consensus reading detected ischemic response to dobutamine in 43 of 1958 segments in 23 of 89 patients. Automated detection of stress-induced wall motion abnormalities correlated more closely with the standard technique than the inexperienced reviewers (sensitivity 0.76 vs. 0.55, specificity 0.98 vs. 0.94 and accuracy 0.97 vs. 0.92). When compared with coronary angiography in a subgroup of patients, analysis of CK images differentiated between normal and abnormal wall motion more accurately than expert readers of gray-scale images (accuracy of 0.93 vs. 0.82). CONCLUSIONS: Analysis of CK images allows fast, objective and automated evaluation of regional wall motion, sensitive enough for clinical dobutamine stress data and more accurate than inexperienced readers. This method may result in a valuable adjunct to conventional visual interpretation of dobutamine stress echocardiography.

Transesophageal echocardiographic diagnosis of a mycotic ascending aortic pseudoaneurysm as a source of embolism.

Pseudoaneurysm of the ascending aorta may develop after aortic injury from trauma or surgery. We report a case of dehiscence of an aortic graft leading to ascending aortic pseudoaneurysm that was diagnosed by transesophageal echocardiography. In addition, the transesophageal echocardiogram identified the pseudoaneurysm as the likely cause of a saddle embolus to the iliac bifurcation of the aorta. This case is also unique in that the patient's pseudoaneurysm was infected with fungus.

An alternative access to a trisaccharide repeating unit of the capsular polysaccharide of Streptococcus pneumoniae serotype 19A.

A chemical synthesis has been achieved for beta-D-ManNAc-(1-->4)-alpha-D-Glc-(1-->3)-L-Rha, a trisaccharide repeating unit of the capsular polysaccharide of Streptococcus pneumoniae serotype 19A, by stepwise link-up of the suitably functionalized, constituent sugar units. A beta-selective glycosylation of trimethylsilylethyl glucoside having free 4-OH with 2-(benzoyloxyimino)-2-deoxyglycosyl bromide, followed by manno-selective hydroboration, N-acetylation, and functionalization of the anomeric center (1-OSE-->1-OH-->1-F), gave a key disaccharide donor, beta-D-ManNAc-(1-->4)-alpha-D-Glc-(1-->F. Ensuing glycosylation of an L-rhamnosyl acceptor with the donor substrate afforded, after deblocking, the target trisaccharide in 6.5% yield over 13 steps from D-glucose.

Synthesis and utility of 2-(benzoyloxyimino)-2-deoxy-alpha-D-lyxo-hexopyranosyl bromide as a novel alpha-D-talosaminide building block.

A novel alpha-D-talosaminyl donor, 2-(benzoyloxyimino)-2-deoxy-alpha-D-lyxo-hexopyranosyl bromide has been synthesized in a total yield of 32% over 6 steps from D-galactose. The utility of the donor was evaluated for the elaboration of alpha-D-TalNAc-(1-->6)-alpha-D-Gal, alpha-D-TalNAc-(1-->6)-alpha-D-Glc, and alpha-D-TalNAc-(1-->3)-L-serine derivatives by a simple 3-step sequence, comprising alpha-selective glycosylation of appropriately protected acceptors of D-galactose, D-glucose, and L-serine, talo-selective hydroboration of the oxyimino function to an amino group, and N-acetylation.

Syntheses of three interglycosidic isomers of N-acetyl-beta-D-mannosaminyl-L-rhamnoses associated with O-antigens of several gram-negative opportunistic pathogens.

We achieved practical, highly stereoselective syntheses of three interglycosidic isomers of N-acetyl-beta-D-mannosaminyl-L-rhamnoses, among which a beta(1-->4)-isomer corresponds to the repeating unit of the O-antigen of lipopolysaccharide (LPS) from the opportunistic pathogens Pseudomonas cepacia O5 and Pseudomonas aeruginosa X (Meitert). The other isomers are a beta(1-->2)-disaccharide, a constituent of LPS from Escherichia coli O1A, and an artificial beta(1-->3)-isomer. The disaccharides were obtained by simple three-step reaction sequences from 2-(benzoyloxyimino)-2-deoxyglycosyl halides (mannosamine progenitor). beta-Selective glycosylations of appropriately protected L-rhamnosyl acceptors were performed. Subsequent reduction of the 2-acyloxyimino function to an amino group, N-acetylation, and removal of the protecting groups provided the target disaccharides. 13C-NMR and nuclear Overhauser effect spectra proved to be useful for structural determination of the positional isomers of the disaccharides.

Unfolding of newly made retinol-binding protein by dithiothreitol. Sensitivity to retinoids.

In cultured hepatoma HepG2 cells, serum retinol-binding protein (RBP) is secreted more rapidly in the presence of retinol than in its absence (Tosetti, F., Ferrari, N., Pfeffer, U., Brigati, C., and Vidali, G. (1992) Exp. Cell Res. 200, 467-472). In the presence of millimolar concentration of DTT, HepG2 cells synthesize fully reduced RBP within the endoplasmic reticulum (ER) which, upon removal of DTT, forms disulfide bonds post-translationally. Secretion of this post-translationally folded RBP is also dependent on the presence of retinol. Using nonreducing gel electrophoresis, we resolved disulfide-bonded RBP folding intermediates. In addition, two other intracellular folding intermediates, compact I and II, which co-migrate with mature RBP were resolved by their different sensitivity to DTT-induced unfolding. Retinol, as well as retinoic acid, stabilized both compact I and II RBP intermediates to DTT-induced unfolding, suggesting that RBP assumes different conformations in the ER in the presence and absence of a ligand. However, only RBP synthesized in the presence of retinol is rapidly secreted, indicating that the ER export quality control system recognizes RBP containing retinol, but not retinoic acid, as fully folded and competent for export. Folding of RBP so that it is stabilized to DTT reduction is not a sufficient condition for ER exit.

In vitro unfolding of retinol-binding protein by dithiothreitol. Endoplasmic reticulum-associated factors.

HepG2 cells in the presence of DTT synthesize fully reduced serum retinol-binding protein (RBP) within the endoplasmic reticulum (ER). Upon removal of DTT, RBP forms disulfide bonds and a folding intermediate, compact II, accumulates within the ER. Compact II RBP co-migrates on nonreducing gel electrophoresis with the secreted form of RBP but is differentiated from secreted RBP by its sensitivity to DTT-induced unfolding (see accompanying article; Kaji, E. H., and Lodish, H. F. (1993) J. Biol. Chem. 268, 22188-22194). Here, we have reconstituted DTT-induced unfolding of compact II RBP in a broken cell system and demonstrate that ER-associated factors enhance the unfolding of RBP by DTT. Protein disulfide isomerase is likely to be one such factor since it enhances the rate of RBP unfolding by DTT in vitro; protein disulfide isomerase-induced unfolding requires the absence of retinoids, similar to the DTT-induced unfolding in vivo. ATP enhances the unfolding of RBP in the absence but not in the presence of retinol, both in intact and broken cells. Thus, protein disulfide isomerase and other ATP-dependent factors can unfold partly folded (or misfolded) RBP in the ER, suggesting how improperly folded proteins might be correctly refolded in vivo.

Expression cloning of an adenylate cyclase-coupled calcitonin receptor.

A calcitonin receptor complementary DNA (cDNA) was cloned by expression of a cDNA library from a porcine kidney epithelial cell line in COS cells. The 482-amino acid receptor has high affinity for salmon calcitonin (dissociation constant Kd approximately 6 nM) and is functionally coupled to increases in intracellular cyclic adenosine monophosphate (cAMP). The receptor shows no sequence similarity to other reported G protein-coupled receptors but is homologous to the parathyroid hormone-parathyroid hormone-related peptide (PTH-PTHrP) receptor, indicating that the receptors for these hormones, which regulate calcium homeostasis, represent a new family of G protein-coupled receptors.

Cloning and functional expression of a vascular smooth muscle endothelin 1 receptor.

By screening a cDNA library derived from the A10 rat vascular smooth muscle cell line for functional expression in COS cells, we have isolated a high-affinity receptor for endothelin 1 (Kd = 476 pM) and endothelin 2. The affinity of the cloned endothelin receptor for endothelin 3 is greater than 100 times less in A10 cells and in a CHO cell line stably transformed by the endothelin receptor cDNA. The 426-amino acid receptor polypeptide has seven putative hydrophobic transmembrane domains and is presumed to be a member of the family of guanine nucleotide-binding regulatory (G) protein-coupled receptors. Microinjection of in vitro transcripts of the cloned cDNA into CHO cells confers a transient increase in intracellular calcium in response to endothelin 1, indicating that the receptor is functional and couples to the appropriate G protein(s). RNA analysis reveals high expression in rat lung and heart, tissues known to exhibit binding to iodinated endothelin 1.

Inhibition of mouse oocyte cyclic AMP phosphodiesterase by steroid hormones: a possible mechanism for steroid hormone inhibition of oocyte maturation.

Testosterone, progesterone, and estradiol-17 beta each inhibit cAMP phosphodiesterase activity of mouse oocyte extracts in a concentration-dependent manner. This finding provides an explanation for the inhibitory effect of steroid hormones on germinal vesicle breakdown (GVBD) of mouse oocytes in vitro. Furthermore, it raises the possibility that steroid hormones present in follicular fluid participate in maintaining meiotic arrest in vivo by acting in a nonclassical manner.

Development of activatable adenylate cyclase in the preimplantation mouse embryo and a role for cyclic AMP in blastocoel formation.

Forskolin- and cholera toxin-activated adenylate cyclase activity increases between the morula and blastocyst stages of mouse preimplantation development, as assessed by the ability of these agents to increase embryonic cAMP levels. Development of activatable adenylate cyclase requires transcription but is independent of the fifth nuclear replication, cell division, and compaction. Early cavitating embryos treated with cholera toxin and forskolin or with N6-monobutyryl-cAMP display an increase in the rate of fluid accumulation in comparison with untreated controls. The stimulatory effect is specific for cAMP, since neither the inactive cAMP analogue N6-monobutyryl-2'-deoxy-cAMP nor N2-monobutyryl-cGMP stimulates the rate of fluid accumulation. These results constitute the first report of a possible physiological function for cAMP in preimplantation development, namely, in blastocoel formation.

Influences of verapamil, X-537A, A-23187 and adenosine 3',5'-cyclic monophosphate on release of 5-hydroxytryptamine from rat brain slices.

Influences of verapamil, X-537A, A-23187 and cyclic-AMP on the release of [3H]-5-HT taken up into rat brain slices, were examined. Incubation with 40 mM KCl induced tritium release which was dependent on the presence of Ca2+. Verapamil, which blocks Ca2+ influx in excitable tissues, decreased potassium-induced release of 5-HT. Tritium release induced by ionophore X-537A was not dependent on extracellular Ca2+ while that induced by A-23187 required Ca2+. Cyclic-AMP, dibutyryl cyclic-AMP and theophylline did not significantly stimulate 5-HT release either in the presence or absence of Ca2+.