Immunofluorescence microscopic demonstration of vimentin filaments in asteroid bodies of sarcoidosis. A comparison with electron microscopic findings

Asteroid bodies in multinucleate giant cells from sarcoid granulomas were investigated by immunofluorescence and electron microscopy. The following points have been established: 1. Asteroid bodies are made up of individual components of the so-called cytoskeleton, predominantly vimentin filaments. Microtubules are involved in smaller amounts in the formation of the asteroid bodies. 2. They arise within the area of the cytosphere. The body of the asteroid includes the centrioles while the arms of the asteroid usually extend into the Golgi area and occasionally up to the cell nuclei. 3. Asteroid bodies result from aggregation of the flexible filamentous and microtubular systems of the centrosphere. The processes of aggregation probably result from local fluid shifts and sol-gel transformations. 4. The stellate form of the aggregations is determined by the preexistent radial arrangement of the elements of the cytosphere. 5. The prevailing specific environment of the underlying granulomatous disease, together with the internal characteristics of the structure and function of the giant cells, in particular in states of exhaustion may play a part in their development.     

Role of the fusogenic peptide sequence in syncytium induction and infectivity of human immunodeficiency virus type 2.

Syncytium induction is a characteristic feature of infection by human immunodeficiency virus (HIV) in vitro. The hydrophobic amino terminus of the transmembrane glycoprotein of HIV type 1 is an essential determinant of virus entry into the target cell population and the formation of syncytia in cell culture. To define the role of the HIV type 2 fusion peptide during infection and syncytium formation, we introduced 8 amino acid substitutions into the hydrophobic amino terminus of gp41, changing either the hydrophobicity, the charge, or the polarity of the amino acid. Viruses containing the envelope mutations were analyzed for their syncytium-inducing capacities, levels of infectivity, and envelope processing and expression. Mutations that increased the hydrophobic nature of the fusion peptide increased syncytium formation, whereas mutations which increased the charge and the polarity and/or decreased the hydrophobicity of the fusion domain severely reduced the capacity of the virus to induce syncytia. However, viruses severely compromised for syncytium formation exhibit only slightly lower levels of infectivity.     

Rat cystathionine beta-synthase. Gene organization and alternative splicing.

We elucidated the structure and alternative splicing patterns of the rat cystathionine beta-synthase gene. The gene is 20-25 kilobase pairs long, and its coding region is divided into 17 exons. These are alternatively spliced, forming four distinct mRNAs (types I through IV). The predicted open reading frames encode proteins of 61.5, 39, 60, and 52.5 kDa, respectively. Exons 13 and 16 are used alternatively and mutually exclusively. Exon 13 includes a stop codon and encodes the unique carboxyl-terminal sequence found in types II and IV. Exon 16 is present only in type I. Types I and III, which differ by 42 nucleotides (exon 16), are the predominant synthase mRNA forms in rat liver. Seventeen arginine peptides from pure liver synthase matched the deduced amino acid sequences of types I and III. These two polypeptides are detectable in liver extracts; each exhibits enzymatic activity when expressed in transfected Chinese hamster cells. Synthase shows substantial sequence similarity with pyridoxal 5'-phosphate dependent enzymes from lower organisms. Similarity of synthase to Escherichia coli O-acetylserine (thiol)-lyase (cysK) is 52%; E. coli tryptophan synthase beta chain (trpB), 36%; yeast serine deaminase, 33%. Lysine 116 in synthase aligns with the established pyridoxyllysine residue of these enzymes suggesting that it is the pyridoxal 5'-phosphate binding residue.     

Two approaches to obtaining low,extracellular deoxyribonuclease activity in cultures of heterocyst-forming cyanobacteria

Six out of 158 axenic strains of heterocyst-forming cyanobacteria consistently failed to produce circles of clearing in agar medium containing DNA-methyl green. When tested with [³H]DNA and coliphage DNA, supernatant fluids from cultures of two of these strains [University of Texas Culture Collection (UTEX) strain 2014 and 19-6C-C] showed no detectable deoxyribonuclease activity, and such fluids from another two of the six, and four others, showed low but detectable deoxyribonuclease activity. Covalently closed circular (plasmid) DNA was not detectably degraded by supernatant fluids from UTEX 2014 and 19-6C-C and from four of the other strains. When DNA was incubated with whole cells of certain strains, a sereis of fragments of discrete size was produced, perhaps by cell-bound, periplasmic, restriction endonucleases. Inclusion of one-tenth strength saline sodium citrate (SSC) in an eight-fold dilution of the medium of Allen and Arnon had little effect on growth of Anabaena variabilis American Type Culture Collection (ATCC) strain 29413 yet prevented all but slight degradation of plasmid pBR322 or of DNA.     

Inhibition of cellular protein synthesis by simultaneous pretreatment of host cells with fowl plague virus and actinomycin D: a method for studying early protein synthesis of several RNA viruses.

A method is described for analysis of viral protein synthesis early after infection when minute amounts of viral proteins are effectively concealed by large amounts of produced host-specific proteins. The method is superior to a radioimmune assay, since all virus-induced proteins can be measured independent of their immunological reactivity. Host-specific protein synthesis can be suppressed by infection with fowl plague virus. Addition of actinomycin C 1.25 h postinfection does not prevent this suppression, but it does block effectively the formation of fowl plague virus-specific proteins. Such cells synthesize only small amounts of cellular proteins, as revealed by polyacrylamide electrophoresis. They can be superinfected with several different enveloped viruses, however, without significant diminution of virus yeilds. In pretreated cells the eclipse is shortened for Semliki Forest virus, Sindbis virus, and vesicular stomatitis virus, but prolonged for Newcastle disease virus. The onset of protein synthesis, specific for the superinfecting virus, could be clearly demonstrated within 1 h after superinfection. At this time, in cells superinfected with Semliki Forest virus, great amounts of NSP 75 (nonstructural protein; molecular weight, 75 X 10(3)) and reduced amounts of the core protein C could be deomonstrated. The precursor glycoprotein NSP 68 is followed by a new polypeptide, NSP 65: three proteins with molecular weights exceeding 100 X 10(3) were observed which are missing later in the infectious cycle. Similar results were obtained after superinfection with Sindbis virus. The formation of a new polypeptide with a molecular weight of about 80 X 10(3) was detected. After superinfection with vesicular stomatis virus or Newcastle disease virus the formation of new proteins, characteristic for the early stage of infeciton, was not observed.     

Demography of an alpine population of Savannah Sparrows

ABSTRACT.   Although Savannah Sparrows ( Passerculus sandwichensis ) have been well studied across their North American range, few data are available for populations that breed in high-elevation habitats. We collected data over six years on the demography of a population of Savannah Sparrows ( P. s. anthinus ) breeding in alpine tundra and sub-alpine meadows in northern British Columbia, Canada. The mean duration of the breeding season at our site was 45.5 d, and pairs produced a maximum of one brood per season. Clutch sizes varied annually (mean = 4.37, range = 3.90 – 4.71 eggs). Nest fate also varied among years (range = 33 – 92%) due to variation in abiotic (weather) and biotic (predators) conditions. Uncorrected return rates of banded birds were 68% for adults and 17% for juveniles ( N = 22 and 102, respectively). However, when resighting probability was taken into account, apparent annual survival was 75% for adults and 34% for juveniles. Compared to populations at lower elevations, Savannah Sparrows in our study had shorter breeding seasons, fewer broods per season, larger clutches, and higher adult and juvenile return rates. Our results suggest that Savannah Sparrows that breed in high-elevation habitats have adopted a low fecundity, high survival life history strategy that enables their persistence in these challenging environments.     

Domain Organization,Catalysis and Regulation of Eukaryotic Cystathionine Beta-Synthases

Cystathionine beta-synthase (CBS) is a key regulator of sulfur amino acid metabolism diverting homocysteine, a toxic intermediate of the methionine cycle, via the transsulfuration pathway to the biosynthesis of cysteine. Although the pathway itself is well conserved among eukaryotes, properties of eukaryotic CBS enzymes vary greatly. Here we present a side-by-side biochemical and biophysical comparison of human (hCBS), fruit fly (dCBS) and yeast (yCBS) enzymes. Preparation and characterization of the full-length and truncated enzymes, lacking the regulatory domains, suggested that eukaryotic CBS exists in one of at least two significantly different conformations impacting the enzyme’s catalytic activity, oligomeric status and regulation. Truncation of hCBS and yCBS, but not dCBS, resulted in enzyme activation and formation of dimers compared to native tetramers. The dCBS and yCBS are not regulated by the allosteric activator of hCBS, S-adenosylmethionine (AdoMet); however, they have significantly higher specific activities in the canonical as well as alternative reactions compared to hCBS. Unlike yCBS, the heme-containing dCBS and hCBS showed increased thermal stability and retention of the enzyme’s catalytic activity. The mass-spectrometry analysis and isothermal titration calorimetry showed clear presence and binding of AdoMet to yCBS and hCBS, but not dCBS. However, the role of AdoMet binding to yCBS remains unclear, unlike its role in hCBS. This study provides valuable information for understanding the complexity of the domain organization, catalytic specificity and regulation among eukaryotic CBS enzymes.     

Molecular phylogeny of three ambiguous ciliate genera: Kentrophoros,Trachelolophos and Trachelotractus (Alveolata,Ciliophora)

Gao, S., Strüder‐Kypke, M.C., Al‐Rasheid, K.A.S., Lin, X. & Song, W. (2010). Molecular phylogeny of three ambiguous ciliate genera: Kentrophoros, Trachelolophos and Trachelotractus (Alveolata, Ciliophora).—Zoologica Scripta, 39, 305–313. Very few molecular studies on the phylogeny of the karyorelictean ciliates have been carried out because data of this highly ambiguous group are extremely scarce. In the present study, we sequenced the small subunit ribosomal RNA genes of three morphospecies representing two karyorelictean genera, Kentrophoros, Trachelolophos, and one haptorid, Trachelotractus, isolated from the South and East China Seas. The phylogenetic trees constructed using Bayesian inference, maximum likelihood, maximum parsimony and neighbor‐joining methods yielded essentially similar topologies. The class Karyorelictea is depicted as a monophyletic clade, closely related to the class Heterotrichea. The generic concept of the family Trachelocercidae is confirmed by the clustering of Trachelolophos and Tracheloraphis with high bootstrap support; nevertheless, the order Loxodida is paraphyletic. The transfer of the morphotype Trachelocerca entzi Kahl, 1927 to the class Litostomatea and into the new haptorid genus Trachelotractus, as suggested by previous researchers based on morphological studies, is consistently supported by our molecular analyses. In addition, the poorly known species Parduczia orbis occupies a well‐supported position basal to the Geleia clade, justifying the separation of these genera from one another.