Supramolecular structures of peptide assemblies in membranes by neutron off-plane scattering: method of analysis

In a previous paper (Yang et al., Biophys. J. 75:641-645, 1998), we showed a simple, efficient method of recording the diffraction patterns of supramolecular peptide assemblies in membranes where the samples were prepared in the form of oriented multilayers. Here we develop a method of analysis based on the diffraction theory of two-dimensional liquids. Gramicidin was used as a prototype model because its pore structure in membrane in known. At full hydration, the diffraction patterns of alamethicin and magainin are similar to gramicidin except in the scale of q (the momentum transfer of scattering), clearly indicating that both alamethicin and magainin form pores in membranes but of different sizes. When the hydration of the multilayer samples was decreased while the bilayers were still fluid, the in-plane positions of the membrane pores became correlated from one bilayer to the next. We believe that this is a new manifestation of the hydration force. The effect is most prominent in magainin patterns, which are used to demonstrate the method of analysis. When magainin samples were further dehydrated or cooled, the liquid-like diffraction turned into crystal-like patterns. This discovery points to the possibility of investigating the supramolecular structures with high-order diffraction.     

daf-1, a C. elegans gene controlling dauer larva development, encodes a novel receptor protein kinase

The dauer larva is a developmentally arrested, non-feeding dispersal stage normally formed in response to overcrowding and limited food. The daf-1 gene specifies an intermediate step in a hierarchy of genes thought to specify a pathway for neural transduction of environmental cues. Mutations in daf-1 result in constitutive formation of dauer larvae even in abundant food. This gene has been cloned by Tc1-transposon tagging, and it appears to encode a new class of serine/threonine kinase. A daf-1 probe detects a 2.5 kb mRNA of low abundance, and the DNA sequence indicates that the gene encodes a 669 amino acid protein, with a putative transmembrane domain and a C-terminal protein kinase domain most closely related to the cytosolic, raf proto-oncogene family. Hence, the daf-1 product appears to be a cell-surface receptor required for transduction of environmental signals into an appropriate developmental response.     

A Ca2+-activated ATPase specifically released by Ca2+ shock from Paramecium tetraurelia

Deciliation of Paramecium tetraurelia by a Ca²⁺ shock procedure releases a discrete set of proteins which represent about 1% of the total cell protein. Marker enzymes for cytoplasm (hexokinase), endoplasmic reticulum (glucose-6-phosphatase), peroxisomes (catalase), and lysosomes (acid phosphatase) were not released by this treatment. Among the proteins selectively released is a Ca²⁺-dependent ATPase. This enzyme has a broad substrate specificity which includes GTP, ATP, and UTP, and it can be activated by Ca²⁺, Sr²⁺, or Ba²⁺, but not by Mg²⁺ or by monovalent cations. The crude enzyme has a specific activity of 2–3 μmol/min per mg; the optimal pH for activity is 7.5. ATPase, GTPase, and UTPase all reside in the same protein, which is inhibited by ruthenium red, is irreversibly denatured at 50°C, and which has a sedimentation coefficient of 8–10 S. This enzyme is compared with other surface-derived ATPases of ciliated protozoans, and its possible roles are discussed.     

LG II balancer chromosomes in Caenorhabditis elegans: mT1(II;III) and the mIn1 set of dominantly and recessively marked inversions

Two new genetic balancers for chromosome II of Caenorhabditis elegans were isolated and characterized. mIn1 was shown to be an inversion of a large central portion of the chromosome, extending from lin-31 to rol-1, that includes most of the genes on the chromosome. It balances a region to the left of the gene cluster that was previously not covered by any of the available balancers. mIn1 recombines efficiently with the normal chromosome II in regions outside the rearrangement at both ends, and appears to enhance recombination frequency adjacent to the inversion breakpoints. Eight variant strains of mIn1 were isolated, including forms that carry recessive morphological or lethal markers, an unmarked form, and one that carries an integrated transgene that confers a semi-dominant green fluorescent protein (GFP) phenotype. This set of variants makes mIn1 useful for a wide variety of applications. The second balancer, mT1, was shown to be a II;III translocation that suppresses recombination on the right arms of chromosomes II and III. It balances chromosome II from the region between bli-2 and dpy-10 to the right end of the chromosome, and chromosome III from the region between daf-2 and unc-93 to the right end. These rearrangements provide the means to stabilize efficiently most of the genes on chromosome II, and may be useful for studies of chromosome pairing and recombination.     

The relationship between narrow calcium-phosphorus ratio and reproductive problems in a daitry herd: A case report

A dairy herd was examined because of an increased incidence of dystocia, retained placenta, and post-partum metritis. The common infectious diseases were eliminated as potential causes of the reproductive problems. The cattle were found to have a mean serum calcium (Ca) of 8.98 mg% and a mean serum phosphorus (P) of 8.25 mg% for a ratio of 1.1:1. Feed analysis revealed low Ca and P in the ration. The cattle were supplemented with steamed bone meal for 3 months. At the end of this time incidence of dystocia had been reduced from 75% to 10%; the incidence of retained placenta from 35% to 8%; and the incidence of postpartum metritis from 70% to 10%. The mean serum Ca had increased to 10.26 mg% and the mean serum P was 6.72 mg% for a mean ratio of 1.53:1.     

Comparative physiological disposition of melatonin and its benzo(b)thiophene analog in the rat

Melatonin and its benzo[b]thiophene analog were labeled by acetylation of the corresponding 5-methoxyarylethylamines with ³H-acetic anhydride. The benzo[b]thiophene analog had a much higher lipid solubility. When administered to rats, both compounds disappeared from plasma and tissues by first-order decay. The dispositions were similar, with the higher lipid solubility of the benzo[b]thiophene analog resulting in higher tissue: plasma ratios, especially in adipose tissue, kidney and liver, and longer half-lives in plasma and tissues.     

Localization of a guanylyl cyclase to chemosensory cilia requires the novel ciliary MYND domain protein DAF-25

In harsh conditions, Caenorhabditis elegans arrests development to enter a non-aging, resistant diapause state called the dauer larva. Olfactory sensation modulates the TGF-β and insulin signaling pathways to control this developmental decision. Four mutant alleles of daf-25 (abnormal DAuer Formation) were isolated from screens for mutants exhibiting constitutive dauer formation and found to be defective in olfaction. The daf-25 dauer phenotype is suppressed by daf-10/IFT122 mutations (which disrupt ciliogenesis), but not by daf-6/PTCHD3 mutations (which prevent environmental exposure of sensory cilia), implying that DAF-25 functions in the cilia themselves. daf-25 encodes the C. elegans ortholog of mammalian Ankmy2, a MYND domain protein of unknown function. Disruption of DAF-25, which localizes to sensory cilia, produces no apparent cilia structure anomalies, as determined by light and electron microscopy. Hinting at its potential function, the dauer phenotype, epistatic order, and expression profile of daf-25 are similar to daf-11, which encodes a cilium-localized guanylyl cyclase. Indeed, we demonstrate that DAF-25 is required for proper DAF-11 ciliary localization. Furthermore, the functional interaction is evolutionarily conserved, as mouse Ankmy2 interacts with guanylyl cyclase GC1 from ciliary photoreceptors. The interaction may be specific because daf-25 mutants have normally-localized OSM-9/TRPV4, TAX-4/CNGA1, CHE-2/IFT80, CHE-11/IFT140, CHE-13/IFT57, BBS-8, OSM-5/IFT88, and XBX-1/D2LIC in the cilia. Intraflagellar transport (IFT) (required to build cilia) is not defective in daf-25 mutants, although the ciliary localization of DAF-25 itself is influenced in che-11 mutants, which are defective in retrograde IFT. In summary, we have discovered a novel ciliary protein that plays an important role in cGMP signaling by localizing a guanylyl cyclase to the sensory organelle.     

Osteopontin is an endogenous modulator of the constitutively activated phenotype of pulmonary adventitial fibroblasts in hypoxic pulmonary hypertension

Increased cell proliferation and migration, of several cell types are key components of vascular remodeling observed in pulmonary hypertension (PH). Our previous data demonstrate that adventitial fibroblasts isolated from pulmonary arteries of chronically hypoxic hypertensive calves (termed PH-Fibs) exhibit a "constitutively activated" phenotype characterized by high proliferative and migratory potential. Osteopontin (OPN) has been shown to promote several cellular activities including growth and migration in cancer cells. We thus tested the hypothesis that elevated OPN expression confers the "activated" highly proproliferative and promigratory/invasive phenotype of PH-Fibs. Our results demonstrate that, both in vivo and ex vivo, PH-Fibs exhibited increased expression of OPN, as well as its cognate receptors, α(V)β(3) and CD44, compared with control fibroblasts (CO-Fibs). Augmented OPN expression in PH-Fibs corresponded to their high proliferative, migratory, and invasive properties and constitutive activation of ERK1/2 and AKT signaling. OPN silencing via small interfering RNA or sequestering OPN production by specific antibodies led to decreased proliferation, migration, invasion, and attenuated ERK1/2, AKT phosphorylation in PH-Fibs. Furthermore, increasing OPN levels in CO-Fibs via recombinant OPN resulted in significant increases in their proliferative, migratory, and invasive capabilities to the levels resembling those of PH-Fibs. Thus our data suggest OPN as an essential contributor to the activated (highly proliferative, migratory, and proinvasive) phenotype of pulmonary adventitial fibroblasts in hypoxic PH.