Dimorphism in methane seep-dwelling ecotypes of the largest known bacteria

We present evidence for a dimorphic life cycle in the vacuolate sulfide-oxidizing bacteria that appears to involve the attachment of a spherical Thiomargarita-like cell to the exteriors of invertebrate integuments and other benthic substrates at methane seeps. The attached cell elongates to produce a stalk-like form before budding off spherical daughter cells resembling free-living Thiomargarita that are abundant in surrounding sulfidic seep sediments. The relationship between the attached parent cell and free-living daughter cell is reminiscent of the dimorphic life modes of the prosthecate Alphaproteobacteria, but on a grand scale, with individual elongate cells reaching nearly a millimeter in length. Abundant growth of attached Thiomargarita-like bacteria on the integuments of gastropods and other seep fauna provides not only a novel ecological niche for these giant bacteria, but also for animals that may benefit from epibiont colonization.     

An ultrastructural study of the spermatozoa ofEulalia sp. (Phyllodocidae),Lepidonotus sp. (Polynoidae),Lumbrineris sp. (Lumbrineridae) andOwenia fusiformis (Oweniidae)

The ultrastructure of the mature spermatozoa of four polychaetes is described:Eulalia sp. (Phyllodocidae),Lepidonotus sp. (Polynoidae),Lumbrineris sp. (Lumbrineridae) andOwenia fusiformis (Oweniidae). All the sperm show features typical of externally fertilizing sperm in having a rounded nucleus, a short unmodified midpiece, and a simple flagellum with a 9+2 axoneme.Owenia fusiformis andLepidonotus sp. have a nuclear cone extending into the subacrosomal space that may act to present the inner acrosomal membrane to the egg during fertilization. The acrosome ofLumbrineris sp. is flattened and crenulated. The sperm ofEulalia sp. is unusual in having the four mitochondria of the midpiece ensheathed by a membrane. Comparisons are made with other polychaete sperm, and the use of sperm ultrastructure as a taxonomic tool within the Polychaeta is discussed.     

Target-sensitive immunoliposomes: preparation and characterization

A novel target-sensitive immunoliposome was prepared and characterized. In this design, target-specific binding of antibody-coated liposomes was sufficient to induce bilayer destabilization, resulting in a site-specific release of liposome contents. Unilamellar liposomes were prepared by using a small quantity of palmitoyl-immunoglobulin G (pIgG) to stabilize the bilayer phase of the unsaturated dioleoylphosphatidylethanolamine (PE) which by itself does not form stable liposomes. A mouse monoclonal IgG antibody to the glycoprotein D of Herpes simplex virus (HSV) and PE were used in this study. A minimal coupling stoichiometry of 2.2 palmitic acids per IgG was essential for the stabilization activity of pIgG. In addition, the minimal pIgG to PE molar ratio for stable liposomes was 2.5 X 10(-4). PE immunoliposomes bound with HSV-infected mouse L929 cells with an apparent Kd of 1.00 X 10(-8) M which was approximately the same as that of the native antibody. When 50 mM calcein was encapsulated in the PE immunoliposomes as an aqueous marker, binding of the liposomes to HSV-infected cells resulted in a cell concentration dependent lysis of the liposomes as detected by the release of the encapsulated calcein. Neither uninfected nor Sendai virus infected cells caused a significant amount of calcein release. Therefore, the release of calcein from PE immunoliposomes was target specific. Dioleoylphosphatidylcholine immunoliposomes were not lysed upon contact with infected cells under the same conditions, indicating that PE was essential for the target-specific liposome destabilization.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of a two-signal-dependent, Ia+ mononuclear phagocyte progenitor subpopulation that is sensitive to inhibition by ferritin

Evidence is presented that the ferritin-inhibitable, Ia+ monocyte progenitor in murine marrow requires two signals for stimulation of clonal proliferation. Escherichia coli K235 lipopolysaccharide (LPS) at 0.1 ng/ml enhanced macrophage colony formation by 25 to 70% in murine marrow cultures stimulated with colony-stimulating factor (CSF-1). The progenitors which responded to LPS and CSF-1 represented a distinct subpopulation. Pretreatment of marrow cells with complement plus anti-Ia, anti-H2, anti-asialo GM1, and anti-Mac-1 antibodies specifically depleted the two-signal-requiring progenitors. In addition, the same progenitors were depleted by preincubation with hydroxyurea, indicating that these cells were in cell cycle when removed from the marrow. When compared with the quiescent progenitors, the Ia+, cycling cells were more sensitive to the antiproliferative effects of interferon alpha/beta but were more resistant to inhibition by E prostaglandins. Pretreatment with T cell-specific antibodies and complement specifically enhanced cloning of quiescent progenitors without affecting cloning of the Ia+, cycling subpopulation. Moreover, rat liver ferritin at 10(-8) to 10(-10) M specifically inhibited clonal proliferation of the Ia+ progenitors. Finally, the requirement for LPS as the additional stimulant could be replaced by the addition of haplotype-specific anti-Ia antibody to CSF-stimulated cultures. In contrast to LPS, anti-IA was competitive with inhibitory ferritin in clonal proliferation of the Ia+ progenitors. The significance of these observations in regulation of monocytopoiesis is discussed.     

Viral replication is required for induction of ocular immunopathology by herpes simplex virus.

Corneal infection of BALB/c mice with herpes simplex virus type 1 results in a chronic inflammatory response in the stroma termed herpetic stromal keratitis (HSK). This disease is considered to be immunopathological and mediated primarily by CD4+ T cells of the type 1 cytokine profile. However, the nature of the antigens, virus or host derived, which drive the inflammatory response remains in doubt. In this study, the relevance of infection with replicating virus for the subsequent development of HSK was evaluated with immunocompetent mice as well as with SCID mice reconstituted with herpes simplex virus-immune CD4+ T cells. In the corneas of immunocompetent mice, infectious virus, viral antigen, and mRNA expression were detectable for only a brief period of time (< or = 7 days postinfection), and all were undetectable by the time clinical lesions were evident (10 to 15 days). Viral replication, however, was necessary for the development of HSK in both models, since infection with UV-inactivated virus or with mutant viruses which were incapable of multiple rounds of replication in vivo failed to induce HSK. The inactivated and mutant viral preparations did, however, stimulate T-cell immune responses in immunocompetent mice. The results are discussed in terms of possible involvement of host antigens exposed in response to transient progeny virion replication in the immune-privileged cornea.     

Herpetic stromal keratitis in the reconstituted scid mouse model.

Infections of the cornea with herpes simplex virus type 1 cause inflammatory lesions which frequently lead to blindness. The disease is suspected to be immunopathological in nature. To establish this point and to study possible mechanisms involved, corneal infections in C.B-17 scid/scid and cell-reconstituted scid mice were investigated. Whereas unreconstituted scid mice failed to develop herpetic stromal keratitis (HSK) and died of encephalitis, mice reconstituted with T lymphocytes generated severe lesions. T cells of the CD4+ subset were found to be essential mediators of the HSK lesion, while T cells of the CD8+ subset protected mice from lethality. The results confirm that HSK is an immunopathological disease and that scid mice provide a convenient model that should prove valuable in establishing the biochemical mechanisms by which HSK is mediated.     

Site-directed mutagenesis of the katG gene of Mycobacterium tuberculosis: effects on catalase–peroxidase activities and isoniazid resistance

Recent studies examining the molecular mechanisms of isoniazid (INH) resistance in Mycobacterium tuberculosis have demonstrated that a significant percentage of drug-resistant strains are mutated in the katG gene which encodes a catalase–peroxidase, and the majority of these alterations are missense mutations which result in the substitution of a single amino acid. In previous reports, residues which may be critical for enzymatic activity and the drug-resistant phenotype have been identified by evaluating INH-resistant clinical isolates and in vitro mutants. In this study, site-directed mutagenesis techniques were utilized to alter the wild-type katG gene from M. tuberculosis at 13 of these codons. The effects of these mutations were determined using complementation assays in katG -defective, INH-resistant strains of Mycobacterium smegmatis and Mycobacterium bovis BCG. This mutational analysis revealed that point mutations in the katG gene at nine of the 13 codons can cause drug resistance, and that enzymatic activity and resistance to INH are inversely related. In addition, mutations in the mycobacterial catalase–peroxidase which reduce catalase activity also decrease peroxidase activity.     

Mouse lymph node germinal centers contain a selected subset of T cells--the helper phenotype

Cells staining for Lyt-1 are more frequent than cells staining for Lyt-2 in both primary follicles and the cuffs of secondary follicles; there is an even more striking predominance of cells bearing only Lyt-1 in germinal centers. In addition, there is an increase in the total percentage of cells bearing T cell antigens in germinal centers compared to primary follicles. These differences in phenotype and distribution of T cell populations indicate the T cells in B cell areas, and especially in germinal centers, are not randomly selected, but rather represent a specific subpopulation of T cells enriched for the helper phenotype (Lyt-1+2-), perhaps involved in the development and/or function of germinal centers.