An RT-PCR analysis of mRNA for growth factor receptors in damaged and control sensory epithelia of rat utricles.

Sensory epithelia from normal rat utricles and those cultured with and without neomycin treatment were assayed for the presence of growth factor receptor mRNAs by RT-PCR (reverse transcriptase-polymerase chain reaction). Both undamaged and damaged utricles showed mRNA for Insulin receptor, IGF-I receptor, FGF receptor 1, EGF receptor, and PDGF alpha receptor. Neomycin-damaged sensory epithelia showed less PDGF alpha receptor mRNA than undamaged epithelia, suggesting that this message by expressed at higher copy levels in hair cells than in supporting cells. Consistent with that hypothesis, immunohistochemistry revealed much stronger PDGF alpha receptor staining in the hair cells than in the supporting cells. Preliminary evidence suggests that IGF-I receptor message also may be lowered in neomycin-damaged epithelia.

Growth factors as potential drugs for the sensory epithelia of the ear.

The highly ordered structures of the hearing and balance organs of vertebrate ears go through a coordinated sequence of cellular and morphogenetic events. It is to be expected that protein growth factors and other extracellular signals will regulate many events during embryonic development of the ear, including the induction of the ear, the specific induction of sensory epithelia, the proliferation of the cells that form the sensory epithelia, the differentiation of the sensory and supporting cells, and the attraction and maintenance of innervation. After embryonic development, growth factors will support cell survival and innervation of new sensory cells. In damaged sensory epithelia, supplementation of the normal growth factors in these tissues has the potential to influence cellular responses to trauma, to reduce cell death and to promote the replacement of dead cells through renewed proliferation and differentiation, so as to improve hearing and balance health via preventive and restorative treatments. Assessment of the influences of specific growth factors on the sensory epithelia of vertebrate ears is at an early stage: this paper provides a brief account of what we know from studies of normal and experimentally manipulated epithelia, discusses the current questions and suggests directions for future studies.

Localization of a Toxoplasma gondii rhoptry protein by immunoelectron microscopy during and after host cell penetration.

We immunolocalized a Toxoplasma gondii rhoptry protein (ROP1) before and after parasite host cell invasion of human fibroblasts and TG180 murine sarcoma cells by electron microscopy and immunogold labeling using either a monoclonal antibody (Tg49) or a monospecific rabbit antiserum (alpha 249). At all stages of parasite growth ROP1 was found within the body but rarely within the peduncle of rhoptries, even in those that appeared empty. Immediately after host cell invasion ROP1 was associated with the parasitophorous vacuole membrane. Within hours after invasion the amount of ROP1 immunodetectable on the parasitophorous vacuole membrane was markedly decreased. The localization of ROP1 suggests a role in the early establishment of infection in host cells, consistent with previous work that has indicated that monoclonal antibodies to ROP1 (including the one used in these studies) interfere with the phenomenon of penetration enhancement.

Role of the galactose lectin of Entamoeba histolytica in adherence-dependent killing of mammalian cells.

Entamoeba histolytica extracellular killing of host cells is contact dependent. Adherence to human colonic epithelial cells and mucins is mediated by a galactose-specific lectin. The effect on cytotoxicity of a panel of monoclonal antibodies (MAb) directed against the galactose lectin was tested. As expected, those MAb which inhibited adherence also decreased cytotoxicity. However, one antilectin MAb blocked cytotoxicity after adherence had occurred, indicating that the lectin has a role in cell killing that is distinct from its adherence function.

A soluble phospholipase of Toxoplasma gondii associated with host cell penetration.

We previously reported that phospholipase increases host cell penetration by Toxoplasma gondii. Here we show that calcium-dependent phospholipase A (PLA) activity is found in the supernatant of sonically disrupted T. gondii. When fractions of disrupted T. gondii were incubated with host cells, the release of fatty acids and lysolipids was detected. Fractions of sonically disrupted T. gondii with PLA activity increased T. gondii host cell penetration in a bioassay. In addition, a protein of approximately 20 kDa was detected by immunoblot of T. gondii antigens with horse antiserum to snake venom, the major antibody of which recognizes PLA2. Incubation of T. gondii with exogenous PLA2 resulted in increased solubility of a rhoptry protein. This protein, which we previously characterized as involved with enhanced parasite invasion of host cells and which is recognized by monoclonal antibody Tg49, was detected in increased amounts in supernatant fractions of extracellular parasites treated with PLA2. Whereas without PLA2 treatment, it is only slightly soluble under physiological conditions. This raises the possibility that PLA may be implicated in the release of rhoptry proteins.

Pathogenic and nonpathogenic strains of Entamoeba histolytica can be differentiated by monoclonal antibodies to the galactose-specific adherence lectin.

Entamoeba histolytica infection results in either asymptomatic colonization or invasive colitis and liver abscess. E. histolytica isolates from patients with invasive disease have characteristic isoenzyme profiles (pathogenic zymodemes), suggesting a role for parasite factors in determining the severity of infection. A galactose-specific cell surface lectin from a pathogenic zymodeme was shown to mediate in vitro adherence to human colonic mucins and contact-dependent killing of target cells. Six nonoverlapping antigenic determinants were identified on the 170-kilodalton heavy subunit of the pathogenic lectin. Anti-lectin monoclonal antibodies (MAb) directed against epitopes 1 and 2 enhanced adherence whereas MAb to epitopes 3 through 6 either inhibited or had no effect on adherence. We tested 50 pathogenic and nonpathogenic strains for reactivity to these anti-lectin MAb by radioimmunoassay. MAb to epitopes 1 through 6 reacted in the radioimmunoassay with all 16 pathogenic zymodeme strains tested. In contrast, only MAb to epitopes 1 and 2 bound to the lectin from nonpathogenic strains. Western immunoblots with anti-lectin antibodies showed that the 170-kilodalton heavy subunit was present in the nonpathogenic amebae. Adherence of the nonpathogenic SAW 760 strain to human erythrocytes was enhanced by MAb to epitope 1 and blocked by galactose, confirming the presence of a functionally active lectin. A lectin radioimmunoassay based on MAb to epitopes 1 and 3 proved to be a simple and rapid method to distinguish pathogenic from nonpathogenic amebae in culture. Further exploration of the functional consequences of the antigenic differences demonstrated for the lectin may lead to a better understanding of its role in pathogenesis.

The role of phospholipase in host cell penetration by Toxoplasma gondii.

Phospholipase A2 (PLA2) plays an important pathogenic role in infections caused by several microorganisms and has been implicated in host cell invasion. The mechanism of host cell penetration by the intracellular protozoan Toxoplasma gondii involves several steps; we have investigated the role of PLA2 in cellular invasion by the tachyzoite stage of this parasite. We assayed T. gondii invasion of human fibroblast monolayers by measurement of the selective incorporation of 3H-uracil into growing intracellular parasites. Exogenous PLA2 from snake venom (Naja naja) increased the penetration of fibroblasts by T. gondii, while horse antiserum to Naja hannah venom inhibited penetration. An irreversible PLA2 inhibitor, p-bromophenacyl bromide, blocked penetration without metabolically disabling the parasite. When host fibroblasts were preincubated with this drug, penetration was not affected, supporting a role for parasite rather than host cell PLA2 in the penetration process. Another PLA2 inhibitor, nordihydroguaiaretic acid, also inhibited penetration. We assayed extracellular T. gondii tachyzoites, purified from host cell debris, for PLA2 activity by radiometric detection of fatty acid release from labeled Escherichia coli membranes. Sonically disrupted parasites contained a low level of calcium-dependent PLA2 with maximum activity at pH 8.5-9.0. These experiments suggest that a phospholipase is implicated in T. gondii host cell invasion.

Electron microscopic study of Saccharomyces cerevisiae rDNA chromatin replication.

An electron microscopic study was made of the replication of rDNA chromatin of Saccharomyces cerevisiae. Two different methods were used to synchronize cells. cdc7-1 cells were raised to a restrictive temperature, whereas A364a cells were blocked with mating factor. Replication bubbles typically opened in the nontranscribed spacers of rDNA repeats in both cell types. The mean position of the center of these bubbles corresponds closely to a position where an autonomously replicating sequence previously has been mapped in an rDNA repeat. Clusters of replication bubbles containing up to four bubbles spaced one to three genes apart were seen opening in early S phase.

In vivo and in vitro antimicrobial activity of silver sulfadiazine and cerium nitrate.

The antimicrobial activity of cerium nitrate and silver sulfadiazine was assessed in vitro and in vivo using Pseudomonas aeruginosa. In vitro, the activity of silver sulfadiazine was significantly greater than that of cerium nitrate. Synergism between silver sulfadiazine and cerium nitrate was observed in water or saline solution suspensions, but not in broth. In vivo, cerium nitrate offered no therapeutic benefit in reducing wound infection in contaminated wounds. Treatment of similar wounds with silver sulfadiazine resulted in a significant decrease in wound infection and in the level of viable bacteria when compared with that for untreated controls. The addition of cerium nitrate to silver sulfadiazine in aqueous soultion reduced the therapeutic benefit of silver sulfadiazine.