Distribution of actin and myosin in a rat neuronal cell line infected with herpes simplex virus

Summary The indirect immunofluorescence technique was used to study alterations in the distribution of actin and myosin filaments in a rat B 103 neuronal cell line infected with herpes simplex virus type 1 (HSV-1). In uninfected cells, actin filaments were arranged in parallel and ran lengthwise from one end of the cell to the other; although myosin filaments were closely associated with actin filaments, additional myosin formed a netlike distribution which did not stain for actin. In infected cells, actin filaments became more randomly aligned and were concentrated along with myosin in close association with rosette-like formations of nuclei in syncytial cells; structural organization of actin and myosin within these intensely staining areas was no longer evident. The possible role of contractile proteins (actin and myosin) in viral infections of neural tissue is raised.With 3 Figures     

Sterilitätsbehandlung mit donogener Insemination

Donor inseminations (DI) have been performed for decades. Most of the publications on this topic deal only with problems of tolerance and acceptance of this treatment for sterility. We already reported on them in parts I and II. In the present third and last part, we discuss the indications for DI: male infertility, genetic disorders, and unsuccessful assisted reproduction therapy. Which conditions do affect the success of therapy? Which methods are recommended? Our treatment results verify realistically that in effect DI only produces the desired child in about 50 % of the couples. As a complementary therapy, in vitro fertilization (IVF) with donor spermatozoa offers a real chance for pregnancy even for women whose husbands are infertile and who themselves suffer from impaired fertility such as pathological conditions of the fallopian tube or when simple inseminations have not resulted in pregnancy. After receiving consent from the State Physicians’ Chamber, we treated 19 women by donor IVF in our group practice and fulfilled their desire to bear their own child.     

Ultrastructural morphometric investigation of early lesions in the pulmonary alveolar region of pigs during experimental swine influenza infection.

Experimental infection of specific-pathogen-free pigs with swine influenza virus by the intratracheal route resulted in a severe respiratory disease that closely resembled natural swine influenza in clinical course and pathologic lesions. Alveolar epithelial necrosis with sloughing of necrotic cells occurred from 24 to 96 hours after inoculation (p.i.) and was associated with alveolar edema and diffuse interstitial pneumonitis. The latter, initially of neutrophilic character, became histiocytic 48 hours p.i. Ultrastructural analysis of alveolar parenchyma disclosed viral replication in epithelial cells beginning at 5 hours p.i. and lasting to 96 hours. Budding of pleomorphic virus particles from the surface of alveolar epithelial cells and accumulation of viral proteins within the nucleus and cytoplasm of epithelial cells were seen. The extent of parenchymal lesions as quantified by stereologic morphometry within the whole lung was characterized by a marked relative and absolute volume increase of interalveolar septa and increased air-blood tissue barrier thickness. The volume increase of interalveolar septa was due to an increase of interstitial tissue volume by 85% in pigs at 96 hours p.i., compared with control pigs with similar lung volumes.     

Identification of a galactose-binding lectin on Fusobacterium nucleatum FN-2.

A previous study has suggested that Fusobacterium nucleatum FN-2 contains a galactose-binding protein (lectin) on the cell surface (P. A. Murray, V. Matarese, C. I. Hoover, and J. R. Winkler, FEMS Microbiol. Lett. 40:123-127, 1987). In the present study, the molecular specificity and size of this lectin were investigated by several techniques. Whole-cell affinity chromatography with asialofetuin covalently coupled to Sepharose 6MB demonstrated that 81% of 3H-labeled F. nucleatum were specifically eluted by 0.5 M galactose. Specific binding was calcium dependent and did not occur in the presence of calcium chelators. Binding was inhibited by preincubation with galactose. Agglutination of human parotid saliva by F. nucleatum was also inhibited by galactose and its structural analogs. Inhibition by lactose was 2 times that of galactose, inhibition by p-aminophenyl galactosides was 4 times that of galactose, and inhibition by asialoglycopeptides was 100 times that of galactose. Similar inhibition results were obtained for hemagglutination of neuraminidase-treated erythrocytes. These findings suggest that the binding specificity of F. nucleatum FN-2 is more complex than simply the recognition of the monosaccharide galactose. This is consistent with the concept that lectins considered identical in terms of monosaccharide specificity can recognize fine differences in more complex structures. To identify the specific bacterial component(s) involved in galactose recognition, proteins of F. nucleatum FN-2 were separated on a 4 to 11% gradient sodium dodecyl sulfate slab gel, transferred to nitrocellulose paper to renature bacterial binding sites, and then incubated with 125I-labeled asialofetuin. Autoradiographs of the nitrocellulose revealed a band at a range of Mr 300,000 to 330,000 which was not present when the blots were preincubated with galactose. These data support the concept that F. nucleatum FN-2 possesses a lectin that recognizes galactose and galactose-containing substrates.     

Reciprocal modification of Fas activation and stress protein response decides apoptosis or resistance development of cells

Activation of heat shock factor (HSF)-1 DNA binding and heat shock protein (hsp)-70 expression enable resistance of cells to various forms of stress and maintain cell survival. Fas, a membrane-bound protein, is a central pro-apoptotic factor. Its activation leads to a cascade of events resulting in programmed cell death. Herein, these two mechanisms with contrary functions, promoting either cell survival or death, were addressed for their potential to inhibit each other's activation. Induction of Fas-mediated signalling was followed by a rapid decrease of HSF1 DNA binding and inducible hsp70 expression. Inhibition of HSF1 DNA binding was demonstrated to be based on absent hyperphosphorylation of HSF1 during FAS-signalling. These effects of Fas-activation on the HSF1/hsp70 stress response were blocked by ICE (caspase 1)-inhibitors, suggesting an ICE-mediated process. Furthermore, inhibition of HSF1/hsp70 was accompanied by an increase of apoptosis rates from 20% to 50% in response to heat stress. When analyzing Fas-mediated apoptosis in the presence of HSF1/hsp70 activation, decreased apoptosis rates were detected with induced expression of hsp70 but not with activation of HSF1-DNA binding alone. Thus, we conclude that inhibition of the HSF1/hsp70 stress response during Fas-mediated apoptosis and vice versa may facilitate a cell to pass a previously chosen pathway, stress resistance or apoptosis.     

Effect of mouse lymphokines and cloned mouse interferon-gamma on the interaction of Rickettsia prowazekii with mouse macrophage-like RAW264.7 cells.

We studied the effects of crude mouse lymphokines and cloned mouse interferon-gamma on the interaction of Rickettsia prowazekii with mouse macrophage-like RAW264.7 cells. Treatment of RAW264.7 cells with lymphokines before infection, after infection, or both before and after infection with R. prowazekii led to killing of a substantial proportion of the RAW264.7 cells. Such cytotoxicity required both lymphokines and viable R. prowazekii and did not occur in mouse fibroblastic L929 cells. Untreated cultures of RAW264.7 cells supported good growth of the Breinl strain of R. prowazekii, but in lymphokine-treated cultures, little or no rickettsial growth occurred in the cells that survived the cytotoxic reaction. In addition, treatment of RAW264.7 cells with lymphokines before rickettsial infection was associated with suppression of the initial infection. The effects of cloned mouse interferon-gamma were similar to the effects of crude mouse lymphokines. Assessment of cytotoxicity, inhibition of the initial infection, and inhibition of rickettsial growth in RAW264.7 cells pretreated with various concentrations of interferon-gamma indicated that the effects of the lymphokines could be explained by the interferon-gamma that was present in these preparations. Treatment of RAW264.7 cells with interferon-gamma makes them unsuitable host cells for R. prowazekii.     

Aspirin downregulates homocysteine formation in stimulated human peripheral blood mononuclear cells

Moderate hyperhomocysteinaemia is established as an independent risk factor for atherosclerosis, thrombosis, stroke and dementia. Hyperhomocysteinaemia is mostly caused by the deficiency of B-vitamins folate and vitamin B12, which are essential cofactors in the remethylation of homocysteine to methionine. Interestingly, moderate hyperhomocysteinaemia is also often observed in chronic diseases, in which also elevated immune activation markers such as neopterin or sTNFR-II are found. In order to simulate immune activation in vitro, human peripheral blood mononuclear cells (PBMC) were stimulated with mitogens. Stimulation significantly increased homocysteine production in comparison with unstimulated PBMC; in parallel also neopterin formation was induced. Homocysteine formation was due to cell proliferation, proliferating T lymphocytes, and also the myelomonocytic cell line U-937 produced homocysteine. Treatment with the anti-inflammatory drug aspirin dose-dependently inhibited homocysteine production and also neopterin formation in human PBMC. Treatment with salicylic acid showed similar effects as aspirin; FACS analysis showed that both compounds inhibited cell proliferation by arresting cells in the G0/G1-phase. In U-937, both compounds also slightly induced apoptosis at 5 mm. Proliferation-induced homocysteine formation and in parallel also monocyte activation can be suppressed effectively by aspirin and salicylic acid in vitro, suggesting that also in vivo aspirin may downregulate not only inflammation but also formation of homocysteine.     

NPHS2 gene, nephrotic syndrome and focal segmental glomerulosclerosis: a HuGE review.

Nephrotic syndrome, characterized by edema, proteinuria, hyperlipidemia and low serum albumin, is a manifestation of kidney disease involving the glomeruli. Nephrotic syndrome may be caused by primary kidney disease such as focal segmental glomerulosclerosis. Mutations in the podocin gene, NPHS2, have been shown in familial and sporadic forms of steroid-resistant nephrotic syndrome, including focal segmental glomerulosclerosis. Podocin is an integral membrane protein located at the slit diaphragm of the glomerular permeability barrier. Complete information is lacking for the population frequency of some NPHS2 variants for all racial and ethnic groups. The most frequently reported variant, R229Q, is more common among European-derived populations than African-derived populations. We calculated crude odds ratios and 95% confidence intervals of childhood nephrotic syndrome and focal segmental glomerulosclerosis associated with R229Q heterozygosity using data from five studies. The R229Q variant is not associated with focal segmental glomerulosclerosis in the US population of African descent. In contrast, the R229Q variant is associated with a trend toward increased focal segmental glomerulosclerosis risk in European-derived populations, with an estimated increased risk of 20-40%. Our insight into the association between NPHS2 variants and nephrotic disease is hampered by the limitations of the existing studies, including small numbers of affected individuals and suboptimal control groups. Nevertheless, the available data suggest that large epidemiological case-control studies to examine the association between NPHS2 variants and nephrotic syndrome are warranted.     

Neutralization of lymphokine-mediated antirickettsial activity of fibroblasts and macrophages with monoclonal antibody specific for murine interferon gamma.

Lymphokine-mediated inhibition of Rickettsia prowazekii multiplication in L929 fibroblasts was eliminated by treatment of the lymphokine with a monoclonal antibody specific for interferon-gamma. Soluble monoclonal antibody and antibody conjugated to Sepharose beads were equally effective. Macrophage activation to limit the multiplication of Rickettsia conorii was eliminated with antibody-conjugated beads; however, neutralization of the ability to activate macrophages with soluble antibody was not complete and required more antibody than did neutralization of antiviral activity.