Erratum: Cloning and sequencing of the phycocyanin gene from Spirulina maxima and its evolutionary analysis

The genes were cloned for the two apoprotein subunits, and ,of phycocyanin from the cyanobacterium Spirulina maxima = Arthrospiramaxima) strain F3. The - and -subunit gene-coding regionscontain 489 bp and 519 bp, respectively. The -subunit gene is upstreamfrom the -subunit gene, with a 111-bp segment separating them.Similarities between the -subunits of S. maxima and nine othercyanobacteria were between 58% and 99%, as were those between the -subunits. The maximum similarity between the - and -subunits from S. maxima was 27%.     

Design and synthesis of factor Xa inhibitors and their prodrugs

In addition to our previously reported fluoro acrylamides Xa inhibitors 2 and 3, a series of potent and novel cyclic diimide amidine compounds has been identified. In efforts to improve their oral bioavailability, replacement of the amidine group with methyl amidrazone gives compounds of moderate potency (14, IC(50)=0.028 microM). In the amidoxime prodrug approach, the amidoxime compounds show good oral bioavailability in rats and dogs. High plasma level of prodrug 26 and significant concentration of active drug 26a were obtained upon oral administration of prodrug 26 in rats.     

Dysregulation of EGF family of growth factors and COX-2 in the uterus during the preattachment and attachment reactions of the blastocyst with the luminal epithelium correlates with implantation failure in LIF-deficient mice

Various mediators, including cytokines, growth factors, homeotic gene products, and prostaglandins (PGs), participate in the implantation process in an autocrine, paracrine, or juxtacrine manner. However, interactions among these factors that result in successful implantation are not clearly understood. Leukemia inhibitory factor (LIF), a pleiotropic cytokine, was shown to be expressed in uterine glands on day 4 morning before implantation and is critical to this process in mice. However, the mechanism by which LIF executes its effects in implantation remains unknown. Moreover, interactions of LIF with other implantation-specific molecules have not yet been defined. Using normal and delayed implantation models, we herein show that LIF is not only expressed in progesterone (P4)-primed uterine glands before implantation in response to nidatory estrogen, it is also induced in stromal cells surrounding the active blastocyst at the time of the attachment reaction. This suggests that LIF has biphasic effects: first in the preparation of the receptive uterus and subsequently in the attachment reaction. The mechanism by which LIF participates in these events was addressed using LIF-deficient mice. We observed that while uterine cell-specific proliferation, steroid hormone responsiveness, and expression patterns of several genes are normal, specific members of the EGF family of growth factors, such as amphiregulin (Ar), heparin-binding EGF-like growth factor (HB-EGF), and epiregulin, are not expressed in LIF(-/-) uteri before and during the anticipated time of implantation, although EGF receptor family members (erbBs) are expressed correctly. Furthermore, cyclooxygenase-2 (COX-2), an inducible rate-limiting enzyme for PG synthesis and essential for implantation, is aberrantly expressed in the uterus surrounding the blastocyst in LIF(-/-) mice. These results suggest that dysregulation of specific EGF-like growth factors and COX-2 in the uterus contributes, at least partially, to implantation failure in LIF(-/-) mice. Since estrogen is essential for uterine receptivity, LIF induction, and blastocyst activation, it is possible that the nidatory estrogen effects in the P4-primed uterus for implantation are mediated via LIF signaling. However, we observed that LIF can only partially resume implantation in P4-primed, delayed implanting mice in the absence of estrogen, suggesting LIF induction is one of many functions that are executed by estrogen for implantation.     

Dynamics of intracellular movement and nucleocytoplasmic recycling of the ligand-activated androgen receptor in living cells

An expression construct containing the cDNA encoding a modified aequorea green fluorescent protein (GFP) ligated to the 5'-end of the rat androgen receptor (AR) cDNA (GFP-AR) was used to study the intracellular dynamics of the receptor movement in living cells. In three different cell lines, ie. PC3, HeLa, and COS1, unliganded GFP-AR was seen mostly in the cytoplasm and rapidly (within 15-60 min) moved to the nuclear compartment after androgen treatment. Upon androgen withdrawal, the labeled AR migrated back to the cytoplasmic compartment and maintained its ability to reenter the nucleus on subsequent exposure to androgen. Under the condition of inhibited protein synthesis by cycloheximide (50 microg/ml), at least four rounds of receptor recycling after androgen treatment and withdrawal were recorded. Two nonandrogenic hormones, 17beta-estradiol and progesterone at higher concentrations (10(-7)/10(-6) M), were able to both transactivate the AR-responsive promoter and translocate the GFP-AR into the nucleus. Similarly, antiandrogenic ligands, cyproterone acetate and casodex, were also capable of translocating the cytoplasmic AR into the nucleus albeit at a slower rate than the androgen 5alpha-dihydrotestosterone (DHT). All AR ligands with transactivation potential, including the mixed agonist/antagonist cyproterone acetate, caused translocation of the GFP-AR into a subnuclear compartment indicated by its punctate intranuclear distribution. However, translocation caused by casodex, a pure antagonist, resulted in a homogeneous nuclear distribution. Subsequent exposure of the casodex-treated cell to DHT rapidly (15-30 min) altered the homogeneous to punctate distribution of the already translocated nuclear AR. When transported into the nucleus either by casodex or by DHT, GFP-AR was resistant to 2 M NaCl extraction, indicating that the homogeneously distributed AR is also associated with the nuclear matrix. Taken together, these results demonstrate that AR requires ligand activation for its nuclear translocation where occupancy by only agonists and partial agonists can direct it to a potentially functional subnuclear location and that one receptor molecule can undertake multiple rounds of hormonal signaling; this indicates that ligand dissociation/inactivation rather than receptor degradation may play a critical role in terminating hormone action.     

Role of aquaporin-4 in airspace-to-capillary water permeability in intact mouse lung measured by a novel gravimetric method

The mammalian peripheral lung contains at least three aquaporin (AQP) water channels: AQP1 in microvascular endothelia, AQP4 in airway epithelia, and AQP5 in alveolar epithelia. In this study, we determined the role of AQP4 in airspace-to-capillary water transport by comparing water permeability in wild-type mice and transgenic null mice lacking AQP1, AQP4, or AQP1/AQP4 together. An apparatus was constructed to measure lung weight continuously during pulmonary artery perfusion of isolated mouse lungs. Osmotically induced water flux (J(v)) between the airspace and capillary compartments was measured from the kinetics of lung weight change in saline-filled lungs in response to changes in perfusate osmolality. J(v) in wild-type mice varied linearly with osmotic gradient size (4.4 x 10(-5) cm(3) s(-1) mOsm(-1)) and was symmetric, independent of perfusate osmolyte size, weakly temperature dependent, and decreased 11-fold by AQP1 deletion. Transcapillary osmotic water permeability was greatly reduced by AQP1 deletion, as measured by the same method except that the airspace saline was replaced by an inert perfluorocarbon. Hydrostatically induced lung edema was characterized by lung weight changes in response to changes in pulmonary arterial inflow or pulmonary venous outflow pressure. At 5 cm H(2)O outflow pressure, the filtration coefficient was 4.7 cm(3) s(-1) mOsm(-1) and reduced 1.4-fold by AQP1 deletion. To study the role of AQP4 in lung water transport, AQP1/AQP4 double knockout mice were generated by crossbreeding of AQP1 and AQP4 null mice. J(v) were (cm(3) s(-1) mOsm(-1) x 10(-5), SEM, n = 7-12 mice): 3.8 +/- 0. 4 (wild type), 0.35 +/- 0.02 (AQP1 null), 3.7 +/- 0.4 (AQP4 null), and 0.25 +/- 0.01 (AQP1/AQP4 null). The significant reduction in P(f) in AQP1 vs. AQP1/AQP4 null mice was confirmed by an independent pleural surface fluorescence method showing a 1.6 +/- 0.2-fold (SEM, five mice) reduced P(f) in the AQP1/AQP4 double knockout mice vs. AQP1 null mice. These results establish a simple gravimetric method to quantify osmosis and filtration in intact mouse lung and provide direct evidence for a contribution of the distal airways to airspace-to-capillary water transport.     

Introducing foreign DNA into tiger shrimp (Penaeus monodon) by electroporation

Electroporation was used to introduce pFLAG-CMV-1-BAP, a DNA fragment that includes a bacterial alkaline phosphatase gene driven by a human cytomegalovirus (CMV) promoter, into Penaeus monodon zygotes. The transgenic tiger shrimp was achievedby using 10kV, 28 pulses, 120 g sec pulse time, 10 cycles, and a DNA concentration of 37.5 microg/mL. The hatching rate of electroporated zygotes (46%) was significantly lower than that of zygotes in the untreated group (89%). The survival rate of postlarvae in the electroporated group using a DNA concentration of 37.5 microg/mL decreased from 0.6% for postlarva 45 to 0.4% for postlarva 120. Based on dot blot analysis, the rate of gene transfer was 37% in mysis-stage, 23% postlarva 15(PL15), 19% postlarva 45(PL45), and 21% 4-month-old (about PL120). Genomic Southern blotting demonstrated that DNA from transgenic tiger shrimp contained fragments of exogenous DNA that were smaller, larger and of the same molecular size as pFLAG-CMV-1-BAP. Transferred DNA fragments were integrated into the genomes of 31% of the transgenic tiger shrimp. The exogenous DNA was mosaically distributed in a wide variety of tissues. Immunohistochemical staining revealed that the FLAG-BAP fused-protein encoded by pFLAG-CMV-1-BAP was present in the ovaries of some transgenic tiger shrimp.     

Aminopeptidase-A. I. CDNA cloning and expression and localization in rat tissues

Aminopeptidase-A (APA) is an ectoenzyme that selectively hydrolyzes acidic residues from the amino terminus of oligopeptides, including biologically active [Asp(1)]ANG II and [Asp(1)]CCK-8. We sought to characterize rat APA by cDNA cloning and expression and to determine its tissue distribution by in situ hybridization and immunohistochemistry. Sequence analysis of overlapping cDNA clones isolated from rat kidney cDNA libraries indicated that the full-length cDNA encoded a 945-amino acid protein with a predicted molecular mass of 108 kDa; the size was confirmed by in vitro translation of a full-length cDNA construct. Transient transfection of the full-length cDNA construct in mammalian cells yielded a protein approximately 140 kDa in size, a size that agrees with the immunoblots of APA from rat tissue and is consistent with APA being known as a glycosylated protein. Tissue APA activity and mRNA expression were highest in the kidney and ileum. Localization of APA by in situ hybridization and immunohistochemistry indicated that, with the exception of the kidney and ileum, where APA was localized to the luminal brush border of proximal tubules and enterocytes, respectively, APA was associated with either capillaries or the lining of sinusoids. Areas known to be physiological targets for ANG II, including glomeruli, the zona glomerulosa, and anterior pituitary, had high levels of APA. The localization pattern suggests that APA may subserve multiple functions, i.e., a generalized role in peptide scavenging and perhaps a more specific role in metabolism of circulating or locally produced ANG II or CCK-8.