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.     

Short-term progressive early diagenesis in density bands of recent corals:Porites Colonies,Mauritius Island,Indian Ocean

Summary The growth history of some recentPorites colonies of Mauritius Island (Indian Ocean) was dated by sclerochronological methods. Couples of high-density and low-density bands represent the annual growth rate of the corals and allow the growth pattern of every year in the corallum to be counted. The growth and structure of the skeletons ofPorites solida andPorites lutea were investigated. Older parts of the aragonitic skeleton in these 10 to 20 year old corals show various secondary microstructures resulting from alterations and thickenings of the elements of the skeleton. The primary needle-like aragonite crystals are absent in older parts of the corallum and blocky aragonitic cements can occur. Pores and primary skeletal elements are overgrown by new microstructures. These microstructures are caused by secondary cementation and exhibit frontal zones (Stirnzonen), zigzag-like and pseudolamellar-structures. The lamellar structures can be compared with similar structures in the exoskeleton of some Rugosa. A very short early diagenesis within the recent corals is responsible for the thickening and alteration of skeletal elements. It occurs only 4 to 5 years after the formation of the skeleton and tends to increase in importance in older parts of the corallum. Nevertheless, there is no proof for any alteration of aragonite to calcite.     

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.     

FURTHER STUDIES ON MOBILIZATION OF CFUs

Mobilization of CFUs from haemopoietic tissues into circulation was studied after injection of different bacterial lipopolysaccharides (LPS), zymosan, phytohaemagglutinin (PHA), concanavalin A (Con A), trypsin and di-isopropyl-fluorophosphate-inhibited trypsin. All bacterial LPS used gave an increase of CFUs in the peripheral blood at 1 h after i.v. injection. Some variation in activity could not be excluded. As with Salmonella typhosa LPS, zymosan gave an increase in circulating CFUs during the first few hr and a second peak a few days later. After injection of zymosan as well as S. typhosa LPS the second peak in the blood was accompanied by a large increase in CFUs numbers in the spleen. PHA gave an immediate mobilization of CFUs, but the mobilization after injection of Con A during the first few hr occurred more slowly. After injection of S. typhosa LPS, zymosan and PHA the blood C3 level was found to be depressed considerably. This might indicate that the complement system is involved in the early mobilization of CFUs. Dexamethasone, a synthetic hormone which has been reported to give sequestration of several cell types in the bone marrow, did not inhibit the early and late mobilization of CFUs which normally occurs after injection of S. typhosa LPS.     

The Histochemical Localization of Cholesterol in Formalin-Fixed and Fresh Frozen Sections

The premixed cholesterol reagent (acetic acid, sulphuric acid, ferric chloride and phosphoric acid) employed in a spectrophotometric method for serum cholesterol determinations was applied to frozen sections of various rat and rabbit tissues. The histochemical reaction was compared with the results obtained by the classical Schultz reaction. The same bluish-green color was observed after treatment of formalin-fixed and fresh cryostat sections incubated in 2.5% iron alum at 37 C for 3 days. The premixed cholesterol reagent is stable for years at room temperature if stored in a brown bottle and is thus readily available for routine use.     

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.     

Influence of group size on water imbibition by Hodotermes mossambicus alate termites

When newly emerged Hodotermes mossambicus alates are confined in pairs they drink almost half of their weight in water. Groups on the other hand do not exhibit this behaviour. The results obtained with individuals held in solitary confinement were intermediate between those obtained with pairs and groups. The imbibed water is stored in two large ‘water-sacs’ which extend well into the abdomen. Samples of the fluid from the ‘water-sacs’ had a relatively high freezing point and also contained far less Folin phenol positive material and Na⁺ and K⁺ than haemolymph.     

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.