Isolation and characterization of the cDNA for pulmonary surfactant-associated protein-B (SP-B) in the rabbit

Pulmonary surfactant contains phospholipids including dipalmitoyl-phosphatidylcholine and three surfactant-associated proteins designated SP-A, SP-B and SP-C. A cDNA for rabbit SP-B has been isolated from a fetal (30 days gestation) rabbit lung cDNA library constructed in lambda gt11. The cDNA and deduced amino acid sequences show strong homology with the cDNAs and predicted 40 kDa proproteins for human and canine SP-B. Strong homology is also observed with the amino acid sequences directly determined for the mature 8 kDa bovine and porcine SP-B isolated from lung lavage. SP-B is remarkable for its high cysteine and proline content and for the hydrophobic nature of the organic solvent-soluble, mature protein. In vitro translation of sense but not antisense RNA transcribed from the cDNA led to the production of 40 kDa and 32 kDa proteins. These proteins were immunoprecipitated by an antibody raised against bovine SP-B. Northern blot analysis revealed the mRNA for rabbit SP-B appears in fetal rabbit lung late in gestation and falls slightly in the neonate.     

Design and synthesis of orally bioavailable serum and glucocorticoid-regulated kinase 1 (SGK1) inhibitors

The lead serum and glucocorticoid-related kinase 1 (SGK1) inhibitors 4-(5-phenyl-1H-pyrrolo[2,3-b]pyridin-3-yl)benzoic acid (1) and {4-[5-(2-naphthalenyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]phenyl}acetic acid (2) suffer from low DNAUC values in rat, due in part to formation and excretion of glucuronic acid conjugates. These PK/glucuronidation issues were addressed either by incorporating a substituent on the 3-phenyl ring ortho to the key carboxylate functionality of 1 or by substituting on the group in between the carboxylate and phenyl ring of 2. Three of these analogs have been identified as having good SGK1 inhibition potency and have DNAUC values suitable for in vivo testing.     

Dynein light chain 1 and a spindle-associated adaptor promote dynein asymmetry and spindle orientation

The cytoplasmic dynein motor generates pulling forces to center and orient the mitotic spindle within the cell. During this positioning process, dynein oscillates from one pole of the cell cortex to the other but only accumulates at the pole farthest from the spindle. Here, we show that dynein light chain 1 (DYNLL1) is required for this asymmetric cortical localization of dynein and has a specific function defining spindle orientation. DYNLL1 interacted with a spindle-microtubule–associated adaptor formed by CHICA and HMMR via TQT motifs in CHICA. In cells depleted of CHICA or HMMR, the mitotic spindle failed to orient correctly in relation to the growth surface. Furthermore, CHICA TQT motif mutants localized to the mitotic spindle but failed to recruit DYNLL1 to spindle microtubules and did not correct the spindle orientation or dynein localization defects. These findings support a model where DYNLL1 and CHICA-HMMR form part of the regulatory system feeding back spindle position to dynein at the cell cortex.     

Neighbourhood Factors and Depression among Adolescents in Four Caribbean Countries

Background

Past research suggests that perceived neighbourhood conditions may influence adolescents'' emotional health. Relatively little research has been conducted examining the association of perceived neighbourhood conditions with depressive symptoms among Caribbean adolescents. This project examines the association of perceived neighbourhood conditions with levels of depressive symptoms among adolescents in Jamaica, the Bahamas, St. Kitts and Nevis, and St. Vincent.

Methods

Adolescents attending grade ten of the academic year 2006/2007 in Jamaica, the Bahamas, St. Vincent, and St. Kitts and Nevis were administered the Neighbourhood Characteristics Questionnaire along with the BDI-II. Social cohesion, attachment to the neighbourhood, neighbourhood quality, neighbourhood crime, and neighbourhood disorder scales were created by summing the relevant subscales of the Neighbourhood Characteristics Questionnaire. Multiple regression analyses were used to examine the relationships of perceived neighbourhood conditions to depressive symptoms.

Results

A wide cross-section of tenth grade students in each nation was sampled (n = 1955; 278 from Jamaica, 217 from the Bahamas, 737 St. Kitts and Nevis, 716 from St. Vincent; 52.1% females, 45.6% males and 2.3% no gender reported; 12 to 19 years, mean = 15.3 yrs, sd = .95 yr). Nearly half (52.1%) of all adolescents reported mild to severe symptoms of depression with 29.1% reporting moderate to severe symptoms of depression. Overall, Jamaican adolescents perceived their neighbourhoods in a more positive manner than those in the Bahamas, St. Vincent and St. Kitts and Nevis. Results of a series of hierarchical multiple regression analyses suggested that a different pattern of neighbourhood factors for each island were associated with depressive symptoms. However, neighbourhood factors were more highly associated with depressive symptoms for Jamaican students than for students in the other three islands.

Conclusions

Neighbourhood factors appear to be partially associated with adolescents'' self-reports of depressive symptoms. However, other factors may mitigate this relationship.     

Regulation of voltage-gated potassium channels by PI(4,5)P2

Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P₂) regulates activities of numerous ion channels including inwardly rectifying potassium (K_ir) channels, KCNQ, TRP, and voltage-gated calcium channels. Several studies suggest that voltage-gated potassium (K_V) channels might be regulated by PI(4,5)P₂. Wide expression of K_V channels in different cells suggests that such regulation could have broad physiological consequences. To study regulation of K_V channels by PI(4,5)P₂, we have coexpressed several of them in tsA-201 cells with a G protein–coupled receptor (M₁R), a voltage-sensitive lipid 5-phosphatase (Dr-VSP), or an engineered fusion protein carrying both lipid 4-phosphatase and 5-phosphatase activity (pseudojanin). These tools deplete PI(4,5)P₂ with application of muscarinic agonists, depolarization, or rapamycin, respectively. PI(4,5)P₂ at the plasma membrane was monitored by Förster resonance energy transfer (FRET) from PH probes of PLCδ1 simultaneously with whole-cell recordings. Activation of Dr-VSP or recruitment of pseudojanin inhibited K_V7.1, K_V7.2/7.3, and K_ir2.1 channel current by 90–95%. Activation of M₁R inhibited K_V7.2/7.3 current similarly. With these tools, we tested for potential PI(4,5)P₂ regulation of activity of K_V1.1/K_Vβ1.1, K_V1.3, K_V1.4, and K_V1.5/K_Vβ1.3, K_V2.1, K_V3.4, K_V4.2, K_V4.3 (with different KChIPs and DPP6-s), and hERG/KCNE2. Interestingly, we found a substantial removal of inactivation for K_V1.1/K_Vβ1.1 and K_V3.4, resulting in up-regulation of current density upon activation of M₁R but no changes in activity upon activating only VSP or pseudojanin. The other channels tested except possibly hERG showed no alteration in activity in any of the assays we used. In conclusion, a depletion of PI(4,5)P₂ at the plasma membrane by enzymes does not seem to influence activity of most tested K_V channels, whereas it does strongly inhibit members of the K_V7 and K_ir families.     

2-Aminoethylphosphonate utilization in Pseudomonas putida BIRD-1 is controlled by multiple master regulators

Bacteria possess various regulatory mechanisms to detect and coordinate a response to elemental nutrient limitation. In pseudomonads, the two-component system regulators CbrAB, NtrBC and PhoBR, are responsible for regulating cellular response to carbon (C), nitrogen (N) and phosphorus (P) respectively. Phosphonates are reduced organophosphorus compounds produced by a broad range of biota and typified by a direct C-P bond. Numerous pseudomonads can use the environmentally abundant phosphonate species 2-aminoethylphosphonate (2AEP) as a source of C, N, or P, but only PhoBR has been shown to play a role in 2AEP utilization. On the other hand, utilization of 2AEP as a C and N source is considered substrate inducible. Here, using the plant-growth-promoting rhizobacterium Pseudomonas putida BIRD-1 we present evidence that 2AEP utilization is under dual regulation and only occurs upon depletion of C, N, or P, controlled by CbrAB, NtrBC, or PhoBR respectively. However, the presence of 2AEP was necessary for full gene expression, i.e. expression was substrate inducible. Mutation of a LysR-type regulator, termed AepR, upstream of the 2AEP transaminase-phosphonatase system (PhnWX), confirmed this dual regulatory mechanism. To our knowledge, this is the first study identifying coordination between global stress response and substrate-specific regulators in phosphonate metabolism.     

Leaf Starch Metabolism during the Growth of Pepper (Capsicum annuum) Plants

Levels of starch and sugars, and the activities of amylase and starch phosphorylase were measured in expanding leaves harvested in early morning and early evening during the growth of pepper (Capsicum annuum L.; cv `Bellboy') plants. The differences between starch levels 1 hour after dawn and 1 hour after dusk increased during the period of initial fruit expansion. This diurnal starch difference was strongly correlated with post-dusk amylase activities in leaves at both stages of expansion. There was also a strong correlation between levels of amylase in immature and those in mature leaves throughout the experiment. Phosphorylase activity showed no direct relationship to leaf starch levels, and there was no similarity between activities in immature and mature leaves. An increase in photosynthesis during plant development was observed which could account for the increased starch synthesis at initial fruit expansion.     

Tandem quadruplication of HMA4 in the zinc (Zn) and cadmium (Cd) hyperaccumulator Noccaea caerulescens

Zinc (Zn) and cadmium (Cd) hyperaccumulation may have evolved twice in the Brassicaceae, in Arabidopsis halleri and in the Noccaea genus. Tandem gene duplication and deregulated expression of the Zn transporter, HMA4, has previously been linked to Zn/Cd hyperaccumulation in A. halleri. Here, we tested the hypothesis that tandem duplication and deregulation of HMA4 expression also occurs in Noccaea.A Noccaea caerulescens genomic library was generated, containing 36,864 fosmid pCC1FOS™ clones with insert sizes ∼20–40 kbp, and screened with a PCR-generated HMA4 genomic probe. Gene copy number within the genome was estimated through DNA fingerprinting and pooled fosmid pyrosequencing. Gene copy numbers within individual clones was determined by PCR analyses with novel locus specific primers. Entire fosmids were then sequenced individually and reads equivalent to 20-fold coverage were assembled to generate complete whole contigs.Four tandem HMA4 repeats were identified in a contiguous sequence of 101,480 bp based on sequence overlap identities. These were flanked by regions syntenous with up and downstream regions of AtHMA4 in Arabidopsis thaliana. Promoter-reporter β-glucuronidase (GUS) fusion analysis of a NcHMA4 in A. thaliana revealed deregulated expression in roots and shoots, analogous to AhHMA4 promoters, but distinct from AtHMA4 expression which localised to the root vascular tissue.This remarkable consistency in tandem duplication and deregulated expression of metal transport genes between N. caerulescens and A. halleri, which last shared a common ancestor >40 mya, provides intriguing evidence that parallel evolutionary pathways may underlie Zn/Cd hyperaccumulation in Brassicaceae.     

37-kDa laminin receptor precursor mediates GnRH-II-induced MMP-2 expression and invasiveness in ovarian cancer cells

GnRH-II enhances ovarian cancer cell invasion in an autocrine manner. We have now found that GnRH-II increases 37-kDa laminin receptor precursor (LRP) production in GnRH receptor (GnRHR)-positive OVCAR-3 and CaOV-3 ovarian cancer cells, while small interfering RNA (siRNA)-mediated depletion of GnRH-II or GnRHR mRNA abrogates this. The invasiveness of ovarian cancer cells is also reduced >85% by siRNA-mediated knockdown of LRP levels and >50% by pretreatment of Matrigel with a synthetic peptide that blocks interactions between laminin and the 67-kDa nonintegrin laminin receptor which comprises two LRP subunits. Conversely, overexpressing LRP in CaOV-3 cells increases their invasiveness 5-fold, while overexpressing LRP with a nonfunctional laminin-binding site does not. Depletion of LRP by siRNA treatment reduces CaOV-3 cell attachment to laminin-coated plates by ～80% but only reduces their binding to Matrigel by ～20%. Thus, while LRP influences CaOV-3 cell adhesion to laminin, LRP must act in other ways to enhance invasion. Matrix metalloproteinases (MMPs) are key mediators of invasion, and LRP siRNA treatment of OVCAR-3 and CaOV-3 cells inhibits MMP-2 but not MMP-9 mRNA levels. Overexpressing LRP in these cells increases MMP-2 production specifically, while a laminin-binding deficient LRP does not. Importantly, LRP siRNA treatment abolishes GnRH-II-induced MMP-2 production, and invasion in OVCAR-3 and CaOV-3 cells, which was also seen after MMP-2 siRNA treatment. These results suggest that GnRH-II-induced LRP expression increases the amount of the 67-kDa nonintegrin laminin receptor, which appears to interact with laminin in the extracellular matrix to promote MMP-2 expression and enhance ovarian cancer cell invasion.     

Muscle metaboreflex attenuates spontaneous heart rate baroreflex sensitivity during dynamic exercise

Hypoperfusion of active skeletal muscle elicits a reflex pressor response termed the muscle metaboreflex. Dynamic exercise attenuates spontaneous baroreflex sensitivity (SBRS) in the control of heart rate (HR) during rapid, spontaneous changes in blood pressure (BP). Our objective was to determine whether muscle metaboreflex activation (MRA) further diminishes SBRS. Conscious dogs were chronically instrumented for measurement of HR, cardiac output, mean arterial pressure, and left ventricular systolic pressure (LVSP) at rest and during mild (3.2 km/h) or moderate (6.4 km/h at 10% grade) dynamic exercise before and after MRA (via partial reduction of hindlimb blood flow). SBRS was evaluated as the slopes of the linear relations (LRs) between HR and LVSP during spontaneous sequences of at least three consecutive beats when HR changed inversely vs. pressure (expressed as beats x min(-1) x mmHg(-1)). During mild exercise, these LRs shifted upward, with a significant decrease in SBRS (-3.0 +/- 0.4 vs. -5.2 +/- 0.4, P<0.05 vs. rest). MRA shifted LRs upward and rightward and decreased SBRS (-2.1 +/- 0.1, P<0.05 vs. mild exercise). Moderate exercise shifted LRs upward and rightward and significantly decreased SBRS (-1.2 +/- 0.1, P<0.05 vs. rest). MRA elicited further upward and rightward shifts of the LRs and reductions in SBRS (-0.9 +/- 0.1, P<0.05 vs. moderate exercise). We conclude that dynamic exercise resets the arterial baroreflex to higher BP and HR as exercise intensity increases. In addition, increases in exercise intensity, as well as MRA, attenuate SBRS.