Binding of an antimicrobial peptide to bacterial cells: Interaction with different species, strains and cellular components.

Antimicrobial peptides (AMPs) selectively kill bacteria by disrupting their cell membranes, and are promising compounds to fight drug-resistant microbes. Biophysical studies on model membranes have characterized AMP/membrane interactions and the mechanism of bilayer perturbation, showing that accumulation of cationic peptide molecules in the external leaflet leads to the formation of pores ("carpet" mechanism). However, similar quantitative studies on real cells are extremely limited. Here, we investigated the interaction of the dansylated PMAP23 peptide (DNS-PMAP23) with a Gram-positive bacterium, showing that 107 bound peptide molecules per cell are needed to kill it. This result is consistent with our previous finding for Gram-negative strains, where a similar high threshold for killing was determined, demonstrating the general relevance of the carpet model for real bacteria. However, in the case of the Gram-positive strain, this number of molecules even exceeds the total surface available on the bacterial membrane. The high affinity of DNS-PMAP23 for the anionic teichoic acids of the Gram-positive cell wall, but not for the lipopolysaccharides of Gram-negative bacteria, provides a rationale for this finding. To better define the role of anionic lipids in peptide/cell association, we studied DNS-PMAP23 interaction with E. coli mutant strains lacking phosphatidylglycerol and/or cardiolipin. Surprisingly, these strains showed a peptide affinity similar to that of the wild type. This finding was rationalized by observing that these bacteria have an increased content of other anionic lipids, thus maintaining the total membrane charge essentially constant. Finally, studies of DNS-PMAP23 association to dead bacteria showed an affinity an order of magnitude higher compared to that of live cells, suggesting strong peptide binding to intracellular components that become accessible after membrane perturbation. This effect could play a role in population resistance to AMP action, since dead bacteria could protect the surviving cells by sequestering significant amounts of peptide molecules. Overall, our data indicate that quantitative studies of peptide association to bacteria can lead to a better understanding of the mechanism of action of AMPs.

Monoglyceride lipase deficiency causes desensitization of intestinal cannabinoid receptor type 1 and increased colonic µ-opioid receptor sensitivity.

BACKGROUND AND PURPOSE: Monoglyceride lipase (MGL) degrades 2-arachidonoyl glycerol (2-AG), an endogenous agonist of cannabinoid receptors (CB1/2 ). Because the CB1 receptor is involved in the control of gut function, we investigated the effects of pharmacological inhibition and genetic deletion of MGL on intestinal motility. Furthermore, we determined whether defective 2-AG degradation affects µ-opioid receptor (µ receptor) signalling, a parallel pathway regulating gut motility. EXPERIMENTAL APPROACH: Gut motility was investigated by monitoring Evans Blue transit and colonic bead propulsion in response to MGL inhibition and CB1 receptor or µ receptor stimulation. Ileal contractility was investigated by electrical field stimulation. CB1 receptor expression in ileum and colon was assessed by immunohistochemical analyses. KEY RESULTS: Pharmacological inhibition of MGL slowed down whole gut transit in a CB1 receptor-dependent manner. Conversely, genetic deletion of MGL did not affect gut transit despite increased 2-AG levels. Notably, MGL deficiency caused complete insensitivity to CB1 receptor agonist-mediated inhibition of whole gut transit and ileal contractility suggesting local desensitization of CB1 receptors. Accordingly, immunohistochemical analyses of myenteric ganglia of MGL-deficient mice revealed that CB1 receptors were trapped in endocytic vesicles. Finally, MGL-deficient mice displayed accelerated colonic propulsion and were hypersensitive to µ receptor agonist-mediated inhibition of colonic motility. This phenotype was reproduced by chronic pharmacological inhibition of MGL. CONCLUSION AND IMPLICATIONS: Constantly elevated 2-AG levels induce severe desensitization of intestinal CB1 receptors and increased sensitivity to µ receptor-mediated inhibition of colonic motility. These changes should be considered when cannabinoid-based drugs are used in the therapy of gastrointestinal diseases.

Effects of new bisphosphonic acids on tumor-induced bone destruction in the rat.

This report is concerned with therapeutic studies utilizing new bisphosphonic acids on tumor-induced osteolytic metastases. The bone metastases on SD rats were induced by intraarterial and intraosseous transplantation of Walker carcinosarcoma 256 B ascites cells. The treatment was carried out using disodium-3-amino-1-hydroxypropylidene-1,1-bisphosphonate (ADP), diglycidyl-[3-(3, 3-bisphosphono-3-hydroxy-propylamino)-2-hydroxypropyl-]urazol++ +-Na2 (DDU) and 1,2,4-triglycidylurazol (TGU). The extent of bone metastases was determined by X-ray on the 5th and 10th days following tumor inoculation, as well as both microradiographically and histologically upon termination of the experiment. High dose DDU produced a clear reduction of the tumor osteolysis, but these positive results were surpassed using APD. The best results were achieved by pretreatment with APD 24 h prior to tumor inoculation.

Differentiation of the mammalian retinal pigment epithelium in vitro: influence of presumptive retinal neuroepithelium and head mesenchyme.

The ancestor cells of the pigment epithelium of the mammalian eye are derived from the neuroepithelial cells of the neural plate. They are neurally determined in the process of neurulation but finally decide to follow the pigment cell lineage, whereas the adjacent tissue develops into the neuroretina and the optic stalk. This decision is most probably made in the developmental stage of eye cup formation. The pigment epithelium becomes restricted to the outer leaf of the eye cup and does not encroach on the adjacent neuroepithelial tissues of the internal leaf and the eye stalk. It is therefore supposed to be channelled by a locally confined determinant factor that has not yet been identified. In the present study, development of the mammalian eye and the neural versus pigment cell decision were investigated in mouse embryos. Three approaches were used to discover the source of the putative determinant involved in the process of neuroepithelial decision. First, eye primordia were cultured from stage 11 embryos (0 somites, early neural plate stage, embryonic day 7 1/2-8) to stage 16 embryos (34 somites, neural tube stage, ed 10); this is prior to pigment cell induction. The eye primordia were first cultured in head segments and their natural position. In these experiments, 50% of the ocular neuroepithelia developed along the nerve cell and glial cell lineage. However, the other 50% of the cultured specimens partly developed into pigment epithelia. In these specimens the determinant factors had obviously remained functionally intact in vitro. In the second type of experiment, the eye primordia were also cultured within the head segments, but with the prospective neuroretina selectively removed. This experiment should show whether the inner layer of the eye cup (the prospective neuroretina) is involved in the neuroepithelial lineage decision. In these experiments 90% of the cultured eye primordia failed to develop pigmented cells. The prospective neuroretina was therefore considered as a candidate for the production of an inductive factor. Finally, eye primordia from stage 14-15 embryos (13-29 somites, ed 9-9 1/2) were either transplanted into heterotopic tissues, such as mesenchymal organs, neuroepithelium or heterochronic muscle, or grown as controls in their natural position and tissue environment. In these conditions both transplanted eye primordia and controls bore pigmented epithelium. Hence, the lineage decision, whether to form neural or pigment cell, remained undisturbed in all epitopes tested. On the basis of these experiments, it seemed unlikely that the development of pigment cells was initiated by a mesenchyme-derived factor exclusively produced near the eye vesicle.

Metastatic low-grade inflammatory myofibroblastic tumor (IMT) in the central nervous system of a 29-year-old male patient.

A case of myofibrosarcoma (IMT) of the brain and lung as well as the spinal cord is described. A 29-year-old male patient presented with fever (40 degrees C), malaise, vomitus, meningism and leukocytosis. Computer tomography identified a bleeding in the left frontal lobe. A bleeding angioma was suspected and an operation was performed. The histological examination could not reveal an exact diagnosis. Eight months after complete recovery from the first bleeding, the patient had a second intracranial temporo-occipital bleeding on the right side which has been removed operatively. A new lesion was seen in the left parietal white matter of the brain. A growing cavernoma was suspected and resection of the lesion was planned. Pre-operatively the patient suffered from hemoptysis and fever. The X-ray of the chest showed a pulmonary lesion in the left lower lobe. In the CT of the chest a large tumor in the left lower lobe of the lung and additionally a cystic structure in the mediastinum was seen. The histological examination of this tumor identified an inflammatory myofibroblastic tumor (IMT). The left parietal lesion has been resected after the thoracic operation. The brain lesions were estimated to be metastases of the IMT of the lung. In the further clinical history the patient developed a large spinal cord metastasis of the thoracic spine. The metastatic development of the tumor reported in this case is unusual. The current therapy of these tumors consists of complete tumor resection and further clinical controls. However, due to the localization and the extension of some lesions in the present case, the complete resection has not been possible. There is no proven role of chemotherapy and radiation therapy. The patient died due to the pulmonary deterioration.

The preparation of native livers for morphological studies.

We describe experiences with a perfusion fixation apparatus that was used for studies on approximately 500 native livers. Immediately after excision of the specimen, small samples from needle biopsy specimens are obtained for snap-freezing and the remaining portions of the specimens are fixed and embedded in paraffin. In the laboratory, the portal vein and, if possible, the hepatic artery or common hepatic duct are then cannulated and the livers are perfused for 3 days with Kaiserling's solution. An electric pump drives the perfusion apparatus and allows the formalin to cascade through stacked plastic containers, with the specimens attached to the inflow nozzles for the fixative. Eight or more livers (or other organs and specimens) can be accommodated simultaneously. Angiograms or cholangiograms can be prepared before or after fixation; we prefer the latter. The livers are then sliced with an extra-long knife, which minimizes cutting marks. Most preparations are thoroughly fixed and yield excellent specimens, not only for routine microscopic study but also for special methods such as scanning electron microscopy and trace metal analysis. The liver slices can be stored indefinitely, which allows long-range collection for routine review or research purposes. In approximately 5% of the cases, specimens cannot be perfused properly and thus are unsuitable for this type of preparation. With autopsy specimens this percentage is higher, probably because of postmortem clotting. Gravity perfusion of the livers before placement into the apparatus generally enables identification of specimens with incomplete filling of the vasculature.

[Primary traumatic midbrain syndrome--follow-up and prognosis of acute primary brain stem damage]. / Das primär traumatische Mittelhirnsyndrom--Verlaufsformen und Prognose einer akuten primären Hirnstammschädigung.

Within 27 months 122 patients with severe head injury were treated at our clinic. Of these patients twelve (9.8%) were categorized as having a primary brain stem lesion (9 male and 3 female, mean age 28.3 years (17 to 73 years). Their injuries were caused primarily by traffic accidents. Initial and follow-up CT ruled out mass lesions or other causes for transtentorial herniation, supporting the diagnosis of primary brain stem lesion. Respiratory insufficiency and control of vegetative function demanded artificial ventilation and analog-sedation for up to 32 days (mean 18 days) on our Intensive Care Unit. In all patients we performed initial and follow-up CT scans, ICP monitoring, evoked potentials (AEP, SSEP) and TCD. MRI was carried out in four patients. One patient died during the acute hospital phase, 7 were transferred in poor and four in good condition. During rehabilitation one patient died, two, one in a vegetative state and one in poor condition were transferred to a caring facility. Eight patients with a good or moderate recovery were dismissed home, subsequently regaining their prior social function. The primary traumatic brain stem lesion presents as a dramatic clinical picture. As shown in our series the prognosis is good independent of the duration of coma. The important prognostic factors were the primary neurological state according to the Gerstenbrand and Luecking classification, the degree of the brain stem lesion in CT scan and MRI, and normal evoked potentials, indicating a favourable outcome.

Protective effects of a prophylactic treatment with the bisphosphonate 3-amino-1-hydroxypropane-1,1-bisphosphonic acid on the development of tumor osteopathies in the rat: experimental studies with the Walker carcinosarcoma 256.

The present report primarily describes protective effects of a long-term prophylactic treatment with 3-amino-1-hydroxypropane-1,1-bisphosphonic acid (APD) on the development of tumor-induced osteolytic bone destructions. Pretreatment with daily intravenous doses of APD 9.5 mg/kg for 1 week resulted in a significant reduction of Walker carcinosarcoma 256-induced bone destruction, when Walker cells were transplanted intraosseously (2 x 10(6) tumor cells/rat) 7 weeks later. Shorter pretreatment periods (4, 2 or 1 week prior to tumor inocculation) resulted in a nearly total inhibition of bone destruction as well as tumor-induced hypercalcemia. Tumor growth itself was not inhibited by APD pretreatment. Histological and microradiographical findings are reported. Consistent to our experiments and with regard to new immunocytological methods to detect single metastatic tumor cells in the bone marrow, potential risk groups may be defined which may profit from the prophylactic APD treatment to inhibit tumor-induced bone destructions.