Stress Response and Virulence Potential Modulating Effect of Peppermint Essential Oil in Campylobacter jejuni.

Campylobacter jejuni is one of the most common food-borne bacteria that causes gastrointestinal symptoms. In the present study we have investigated the molecular basis of the anti-Campylobacter effect of peppermint essential oil (PEO), one of the oldest EO used to treat gastrointestinal diseases. Transcriptomic, quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and proteomic, two-dimensional polyacryl amid gel electrophoresis (2D-PAGE) methods have revealed that, in the presence of a sublethal concentration of PEO, the expression of several virulence-associated genes was decreased (cheY 0.84x; flhB 0.79x; flgE 0.205x; cadF 0.08x; wlaB 0.89x; porA 0.25x; cbf2 4.3x) while impaired motility was revealed with a functional analysis. Scanning electron micrographs of the exposed cells showed that, unlike in the presence of other stresses, the originally curved C. jejuni cells straightened upon PEO exposure. Gaining insight into the molecular background of this stress response, we have revealed that in the presence of PEO C. jejuni dominantly exerts a general stress response that elevates the expression of general stress genes like dnaK, groEL, groES (10.41x, 3.63x, and 4.77x). The most important genes dps, sodB, and katA involved in oxidative stress responses showed however moderate transcriptional elevations (1,58x, 1,55x, and 1,85x).

Hematin polymerization assay as a high-throughput screen for identification of new antimalarial pharmacophores.

Hematin polymerization is a parasite-specific process that enables the detoxification of heme following its release in the lysosomal digestive vacuole during hemoglobin degradation, and represents both an essential and a unique pharmacological drug target. We have developed a high-throughput in vitro microassay of hematin polymerization based on the detection of (14)C-labeled hematin incorporated into polymeric hemozoin (malaria pigment). The assay uses 96-well filtration microplates and requires 12 h and a Wallac 1450 MicroBeta liquid scintillation counter. The robustness of the assay allowed the rapid screening and evaluation of more than 100, 000 compounds. Random screening was complemented by the development of a pharmacophore hypothesis using the "Catalyst" program and a large amount of data available on the inhibitory activity of a large library of 4-aminoquinolines. Using these methods, we identified "hit" compounds belonging to several chemical structural classes that had potential antimalarial activity. Follow-up evaluation of the antimalarial activity of these compounds in culture and in the Plasmodium berghei murine model further identified compounds with actual antimalarial activity. Of particular interest was a triarylcarbinol (Ro 06-9075) and a related benzophenone (Ro 22-8014) that showed oral activity in the murine model. These compounds are chemically accessible and could form the basis of a new antimalarial medicinal chemistry program.

4-aminoquinoline analogs of chloroquine with shortened side chains retain activity against chloroquine-resistant Plasmodium falciparum.

We have synthesized several 4-aminoquinolines with shortened side chains that retain activity against chloroquine-resistant isolates of Plasmodium falciparum malaria (W. Hofheinz, C. Jaquet, and S. Jolidon, European patent 94116281.0, June 1995). We report here an assessment of the activities of four selected compounds containing ethyl, propyl, and isopropyl side chains. Reasonable in vitro activity (50% inhibitory concentration, < 100 nM) against chloroquine-resistant P. falciparum strains was consistently observed, and the compounds performed well in a variety of plasmodium berghei animal models. However, some potential drawbacks of these compounds became evident upon in-depth testing. In vitro analysis of more than 70 isolates of P. falciparum and studies with a mouse in vivo model suggested a degree of cross-resistance with chloroquine. In addition, pharmacokinetic analysis demonstrated the formation of N-dealkylated metabolites of these compounds. These metabolites are similarly active against chloroquine-susceptible strains but are much less active against chloroquine-resistant strains. Thus, the clinical dosing required for these compounds would probably be greater for chloroquine-resistant strains than for chloroquine-susceptible strains. The clinical potential of these compounds is discussed within the context of chloroquine's low therapeutic ratio and toxicity.

Immunohistochemical analysis of thiol: protein disulfide oxidoreductase in hypothalamic neurons of Brattleboro rat.

The immunolocalization of thiol: protein disulfide oxidoreductase (TPO) in CNS of Wistar rats and homozygous Brattleboro rats was investigated by use of monospecific antiserum and Sternberger's unlabelled immunoenzyme technique. It was revealed that TPO is present in hypothalamic neurons belonging to nucleus supraopticus and paraventricularis. The number of immunopositive nerve cells was reduced in Brattleboro rats as compared to Wistar rats. It was concluded that TPO must have tasks in CNS unrelated to the management of vasopressin production and/or processing.

Distribution of cathepsin D immunoreactivity in the central nervous system of rat and selected brain regions of man.

The regional distribution and cellular localization of cathepsin D immunoreactivity was demonstrated at the light microscopic level in the CNS of rat and man by use of unlabelled immunoenzyme technique. A wide but uneven distribution was substantiated for the rat brain. Furthermore, we present evidence that antiserum produced against rat liver enzyme is capable of recognizing cathepsin D in human brain.

Proctolin-related material in the mouse brain as revealed by immunohistochemistry.

By use of a specific antiserum against the insect peptide proctolin we were able to identify proctolin-immunoreactive neurons in the mouse brain. These nerve cells belong to the nuc. mesencephalicus n. trigemini. Furthermore, the antiserum stained very few nerve fibers with varicosities in the immediate neighborhood of the roof of the third ventricle. The chemical identity of the immunoreactive material with genuine proctolin remains to be elucidated.

Cerebral insulin-like immunoreactivity in rats and mice. Drastic decline during postnatal ontogenesis.

We studied the behavior of the insulin-like immunoreactivity in brains of rats and mice during the first 20 d post natum by immunohistochemistry and radioimmunoassay. It was found that a dramatic decline in the concentration of the peptide, accompanied by a strong reduction of immunoreactive cells, takes place during this period. A possible role of cerebral insulin as a promoter of nerve cell growth and development is briefly discussed.

Insulin-like immunoreactivity in the human brain- A preliminary report.

The localization and regional distribution of insulin-like immunoreactivity (IRI) was studied in human brain autopsy material using the indirect immunofluorescence technique. A positive reaction for IRI could be observed in many neurons of the hypothalamus, the hippocampus, corpus amygdaloideum, medulla oblongata (especially within the nuclei of cranial nerves IX, X and XII), and the cerebral cortex, whereas the cerebellar cortex was lacking in immunohistochemically detectable insulin-like material. No nerve fibres containing polypeptides could be revealed. Additionally, the insulin content of various brain regions was estimated by radioimmunoassay. Insulin concentrations in human nervous tissue were found to be elevated in comparison to blood plasma levels.

Immunoreactive glucagon in neurons of various parts of the human brain. Demonstration by immunofluorescence technique.

The presence of glucagon-like immunoreactivity in nerve cells of different parts of the human brain was demonstrated by the indirect immunofluorescence technique. A bright fluorescent reaction was observed in the pyramidal cells of lamina V of the Neocortex. Less prominent concentrations of the glucagon-like material were detected in a few pyramidal cells of the Hippo-campus and in some neurons of the Presubiculum and Subiculum. Within the Corpus amygdaloideum, only a few magnocellular neurons showed a positive reaction. The Hypothalamus was evidenced by a moderate, but widely distributed, reaction in magnocellular and medium-sized nerve cells in different nuclei (especially Nuc. ventromedialis and Nuc. arcuatus). A strong immunofluorescence was localized to some neurocytes in the Nuc. amibigus, and Nuc. n. hypoglassi. The Purkinje cells of the cerebellar cortex were free from immunoreactive material, but fluorescence occurred in some very small nerve cells of the Cerebellum (probably granular cells). A dependence of the strength of immunofluorescence of the time delay between autopsy and death is shown.

Thin sectioning and freeze-etching studies of isolated islets of Langerhans of rats in gestation and after hypothalamic lesion.

Morphometrical studies of B-cell granules have not demonstrated marked morphological changes in various states of hyperinsulinemia.

Are certain peculiarities in the distribution of somatostatin-like immunoreactivity (SLI) in the brain of sand rats (Psammomys obesus) possibly connected with an altered carbohydrate metabolism?

The hypothalamic and extrahypothalamic distribution of somatostatin-like immunoreactivity (SLI) was investigated in fed adult sand rats (Psammomys obesus) of both sexes using the immunofluorescence technique. Blood glucose and insulin concentrations were determined in these animals prior to decapitation. In a blind study, the amount of immunofluorescence of certain CNS areas and the glucose: Insulin ratio were compared and found to be connected: High amounts of SLI were detected in the dorsal hippocampus, in some hypothalamic nuclei and the median eminence of rats displaying low glucose levels. Sand rats with high blood glucose values did not show SLI in the hippocampal formation and the immunofluorescence of the circumventricular (hypothalamic) regions and the Eminentia mediana was drastically reduced. A possible correlation of SLI with insulin concentrations in the blood could not be revealed. The data obtained are discussed as a possible expression of a CNS influence on glucoregulation in this species.

Regional distribution of glucagon-like immunoreactive material in the brain of rats and sand rats. An immunohistochemical investigation.

Glucagon-like immunoreactivity was demonstrated in different parts of rat and sand rat central nervous system by the indirect immunofluorescence method. In hyperglycemic animals the level of immunoreactive material was reduced to a great extent.

An immunofluorescent reaction appears to insulin-antiserum in different CNS regions of two rat species.

Using immunofluorescence technique a positive reaction to insulin antiserum could be revealed in different parts of the CNS of Wistar rats and sand rats.