Ca signaling and fluid secretion by secretory cells of the airway epithelium

Cytoplasmic Ca²⁺ is a master regulator of airway physiology; it controls fluid, mucus, and antimicrobial peptide secretion, ciliary beating, and smooth muscle contraction. The focus of this review is on the role of cytoplasmic Ca²⁺ in fluid secretion by airway exocrine secretory cells. Airway submucosal gland serous acinar cells are the primary fluid secreting cell type of the cartilaginous conducting airways, and this review summarizes the current state of knowledge of the molecular mechanisms of serous cell ion transport, with an emphasis on their regulation by intracellular Ca²⁺. Many neurotransmitters that regulate secretion from serous acinar cells utilize Ca²⁺ as a second messenger. Changes in intracellular Ca²⁺ concentration regulate the activities of ion transporters and channels involved in transepithelial ion transport and fluid secretion, including Ca²⁺-activated K⁺ channels and Cl⁻ channels. We also review evidence of interactions of Ca²⁺ signaling with other signaling pathways (cAMP, NO) that impinge upon different ion transport pathways, including the cAMP/PKA-activated cystic fibrosis (CF) transmembrane conductance regulator (CFTR) anion channel. A better understanding of Ca²⁺ signaling and its targets in airway fluid secretion may identify novel strategies to intervene in airway diseases, for example to enhance fluid secretion in CF airways.     

Distinct determinants of tRNA recognition by the TrmD and Trm5 methyl transferases

TrmD and Trm5 are, respectively, the bacterial and eukarya/archaea methyl transferases that catalyze transfer of the methyl group from S-adenosyl methionine (AdoMet) to the N1 position of G37 in tRNA to synthesize m1G37-tRNA. The m1G37 modification prevents tRNA frameshifts on the ribosome by assuring correct codon-anticodon pairings, and thus is essential for the fidelity of protein synthesis. Although TrmD and Trm5 are derived from unrelated AdoMet families and recognize the cofactor using distinct motifs, the question of whether they select G37 on tRNA by the same, or different, mechanism has not been answered. Here we address this question by kinetic analysis of tRNA truncation mutants that lack domains typically present in the canonical L shaped structure, and by evaluation of the site of modification on tRNA variants with an expanded or contracted anticodon loop. With both experimental approaches, we show that TrmD and Trm5 exhibit separate and distinct mode of tRNA recognition, suggesting that they evolved by independent and non-overlapping pathways from their unrelated AdoMet families. Our results also shed new light onto the significance of the m1G37 modification in the controversial quadruplet-pairing model of tRNA frameshift suppressors.     

Mutational analysis of S12 protein and implications for the accuracy of decoding by the ribosome

The fidelity of aminoacyl-tRNA selection by the ribosome depends on a conformational switch in the decoding center of the small ribosomal subunit induced by cognate but not by near-cognate aminoacyl-tRNA. The aminoglycosides paromomycin and streptomycin bind to the decoding center and induce related structural rearrangements that explain their observed effects on miscoding. Structural and biochemical studies have identified ribosomal protein S12 (as well as specific nucleotides in 16S ribosomal RNA) as a critical molecular contributor in distinguishing between cognate and near-cognate tRNA species as well as in promoting more global rearrangements in the small subunit, referred to as “closure.” Here we use a mutational approach to define contributions made by two highly conserved loops in S12 to the process of tRNA selection. Most S12 variant ribosomes tested display increased levels of fidelity (a “restrictive” phenotype). Interestingly, several variants, K42A and R53A, were substantially resistant to the miscoding effects of paromomycin. Further characterization of the compromised paromomycin response identified a probable second, fidelity-modulating binding site for paromomycin in the 16S ribosomal RNA that facilitates closure of the small subunit and compensates for defects associated with the S12 mutations.     

<Emphasis Type="Italic">ASYMMETRIC LEAVES2-LIKE1</Emphasis>gene,a member of the AS2/LOB family,controls proximal–distal patterning in <Emphasis Type="Italic">Arabidopsis</Emphasis> petals

The formation and the development of the floral organs require an intercalate expression of organ-specific genes. At the same time, meristem-specific genes are repressed to complete the differentiation of the organs in the floral whorls. In an Arabidopsis activation tagging population, a mutant affected in inflorescence architecture was identified. This gain-of-function mutant, designateddownwards siliques1 (dsl1-D), has shorter internodes and the lateral organs such as flowers are bending downwards, similar to the loss-of-function brevipedicellus (bp) mutant. The affected gene in dsl1-D appeared to be ASYMMETRIC LEAVES2-LIKE1 (ASL1)/LATERAL ORGAN BOUNDARIESdomain gene 36 (LBD36), which is a member of the ASYMMETRIC LEAVES2 (AS2)/LATERAL ORGAN BOUNDARIES (LOB) domain gene family. Analysis of the loss-of-function mutant asl1/lbd36 did not show morphological aberration. Double mutant analysis of asl1/lbd36 together with as2, the ASL1/LBD36 closest homologue, demonstrates that these two members of the AS2/LOB family act partially redundant to control cell fate determination in Arabidopsis petals. Moreover, molecular analysis revealed that overexpression of ASL1/LBD36 leads to repression of the homeobox gene BP, which supports the model that an antagonistic relationship between ASL/LBD and homeobox members is required for the differentiation of lateral organs.     

Sodium and chloride concentrations, pH, and depth of airway surface liquid in distal airways

The composition and depth of the airway surface liquid (ASL) are key parameters in airway physiology that are thought to be important in the pathophysiology of cystic fibrosis and other diseases of the airways. We reported novel fluorescent indicator and microscopy methods to measure [Na+], [Cl-], pH, and depth of the ASL in large airways (Jayaraman, S., Y. Song, L. Vetrivel, L. Shankar, and A.S. Verkman. 2001. J. Clin. Invest. 107:317-324.). Here we report a stripped-lung preparation to measure ASL composition and depth in small distal airways. Distal ASL was stained with ion- or pH-sensitive fluorescent indicators by infusion into mouse trachea of a perfluorocarbon suspension of the indicator. After stripping the pleura and limited microdissection of the lung parenchyma, airways were exposed for measurement of ASL [Na+], [Cl-], and pH by ratio imaging microscopy, and depth by confocal microscopy. The stripped-lung preparation was validated in stability and tissue viability studies. ASL [Na+] was 122 +/- 2 mM, [Cl-] was 123 +/- 4 mM and pH was 7.28 +/- 0.07, and not dependent on airway size (<100- to >250-mum diameter), ENaC inhibition by amiloride, or CFTR inhibition by the thiazolidinone CFTRinh-172. ASL depth was 8-35 mum depending on airway size, substantially less than that in mouse trachea of approximately 55 mum, and not altered significantly by amiloride. These results establish a novel lung preparation and fluorescence approach to study distal airway physiology and provide the first data on the composition and depth of distal ASL.     

Prelinguistic development of children and chimpanzees

What was unique about our laboratory was the detailed simulation of a human rearing environment for infant chimpanzees, together with continuous, intensive observation and recording of behavioral development. Communication in American Sign Language (ASL) was integrated into this procedure in a way that simulates the teaching of vocal and gestural languages to human children. The result is a comprehensive longitudinal record of the stage by stage development of two-way communication in sign language and its relation to basic aspects of behavioral development such as: locomotion, manipulative skills, observational learning, and social play. In the longitudinal records of five infant chimpanzees, we can trace the patterns of development and also examine the consistency of individual differences through the first five years. The central question is the relation between developing skills in the use of American Sign Language and the rest of behavioral development. To analyze factors that influence development, we have compared records for infant chimpanzees reared in the crossfostering laboratory with records for chimpanzees reared in the wild by their own mothers and chimpanzees reared in cages under conventional laboratory conditions, as well as with records of development for human children, reared in homes and reared in institutions.     

Argininosuccinate synthetase and argininosuccinate lyase: two ornithine cycle enzymes from Agaricus bisporus

Accumulation of high quantities of urea in fruiting bodies is a known feature of larger basidiomycetes. Argininosuccinate synthetase (ASS) and argininosuccinate lyase (ASL) are two ornithine cycle enzymes catalysing the last two steps in the arginine biosynthetic pathway. Arginine is the main precursor for urea formation. In this work the nucleotide sequences of the genes and corresponding cDNAs encoding argininosuccinate synthetase (ass) and argininosuccinate lyase (asl) from Agaricus bisporus were determined. Eight and six introns were present in the ass and asl gene, respectively. The location of four introns in the asl gene were conserved among vertebrate asl genes. Deduced amino acid sequences, representing the first homobasidiomycete ASS and ASL protein sequences, were analysed and compared with their counterparts in other organisms. The ass ORF encoded for a protein of 425 amino acids with a calculated molecular mass of 47 266 Da. An alignment with ASS proteins from other organisms revealed high similarity with fungal and mammalian ASS proteins, 61–63 % and 51–55 % identity, respectively. The asl open reading frame (ORF) encoded a protein of 464 amino acids with an calculated mass of 52 337 Da and similar to ASS shared the highest similarity with fungal ASL proteins, 59–60 % identity.Northern analyses of ass and asl during fruiting body formation and post-harvest development revealed that expression was significantly up-regulated from developmental stage 3 on for all the tissues studied. The expression reached a maximum at the later stages of fruiting body growth, stages 6 and 7. Both ass and asl genes were up-regulated within 3 h after harvest showing that the induction mechanism is very sensitive to the harvest event and emphasizes the importance of the arginine biosynthetic pathway/ornithine cycle in post-harvest physiology.     

Functional interactions by transfer RNAs in the ribosome

Recent X-ray crystal structures of the ribosome have revolutionized the field by providing a much-needed structural framework to understand ribosome function. Indeed, the crystal structures rationalize much of the genetic and biochemical data that have been meticulously gathered over 50 years. Here, we focus on the interactions between tRNAs and the ribosome and describe some of the insights that the structures provide about the mechanism of translation. Both high-resolution structures and functional studies are essential for fully appreciating the complex process of protein synthesis.