Analgesic properties of N-terminal substituted phenylalanyl-alanine

Phenylalanylalanine, an in vitro inhibitor of enkephalinase, and some of its N alpha-derivatives are shown to possess an analgesic action when injected i.p. and i.c.v. into mice in the presence or absence of Leu5-enkephalin. In the second case a synergistic response is observed. The intensity of the analgesic response depends markedly on the nature of the N-terminal substituent which affects the hydrophobic character of the resulting dipeptide, its subsequent transport and probably its rate of biotransformation by cleaving enzymes.     

Mesencephalic dopaminergic neurons express a repertoire of olfactory receptors and respond to odorant-like molecules

Background

The mesencephalic dopaminergic (mDA) cell system is composed of two major groups of projecting cells in the Substantia Nigra (SN) (A9 neurons) and the Ventral Tegmental Area (VTA) (A10 cells). Selective degeneration of A9 neurons occurs in Parkinson’s disease (PD) while abnormal function of A10 cells has been linked to schizophrenia, attention deficit and addiction. The molecular basis that underlies selective vulnerability of A9 and A10 neurons is presently unknown.

Results

By taking advantage of transgenic labeling, laser capture microdissection coupled to nano Cap-Analysis of Gene Expression (nanoCAGE) technology on isolated A9 and A10 cells, we found that a subset of Olfactory Receptors (OR)s is expressed in mDA neurons. Gene expression analysis was integrated with the FANTOM5 Helicos CAGE sequencing datasets, showing the presence of these ORs in selected tissues and brain areas outside of the olfactory epithelium. OR expression in the mesencephalon was validated by RT-PCR and in situ hybridization. By screening 16 potential ligands on 5 mDA ORs recombinantly expressed in an heterologous in vitro system, we identified carvone enantiomers as agonists at Olfr287 and able to evoke an intracellular Ca²⁺ increase in solitary mDA neurons. ORs were found expressed in human SN and down-regulated in PD post mortem brains.

Conclusions

Our study indicates that mDA neurons express ORs and respond to odor-like molecules providing new opportunities for pharmacological intervention in disease.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-729) contains supplementary material, which is available to authorized users.     

In vitro assessment of some sperm function following exposure to levonorgestrel in human fallopian tubes

Background  

The mechanism of action of levonorgestrel (LNG) as emergency contraception (EC) remains a subject of debate and its effect on sperm function has been only partially explained. The aim of this study was to assess whether LNG at a similar dose to those found in serum following oral intake for EC could affect spermatozoa when exposed to human fallopian tubes in vitro.     

The miR-17-5p microRNA is a key regulator of the G1/S phase cell cycle transition

Background

MicroRNAs are modifiers of gene expression, acting to reduce translation through either translational repression or mRNA cleavage. Recently, it has been shown that some microRNAs can act to promote or suppress cell transformation, with miR-17-92 described as the first oncogenic microRNA. The association of miR-17-92 encoded microRNAs with a surprisingly broad range of cancers not only underlines the clinical significance of this locus, but also suggests that miR-17-92 may regulate fundamental biological processes, and for these reasons miR-17-92 has been considered as a therapeutic target.

Results

In this study, we show that miR-17-92 is a cell cycle regulated locus, and ectopic expression of a single microRNA (miR-17-5p) is sufficient to drive a proliferative signal in HEK293T cells. For the first time, we reveal the mechanism behind this response - miR-17-5p acts specifically at the G1/S-phase cell cycle boundary, by targeting more than 20 genes involved in the transition between these phases. While both pro- and anti-proliferative genes are targeted by miR-17-5p, pro-proliferative mRNAs are specifically up-regulated by secondary and/or tertiary effects in HEK293T cells.

Conclusion

The miR-17-5p microRNA is able to act as both an oncogene and a tumor suppressor in different cellular contexts; our model of competing positive and negative signals can explain both of these activities. The coordinated suppression of proliferation-inhibitors allows miR-17-5p to efficiently de-couple negative regulators of the MAPK (mitogen activated protein kinase) signaling cascade, promoting growth in HEK293T cells. Additionally, we have demonstrated the utility of a systems biology approach as a unique and rapid approach to uncover microRNA function.     

Haemocytes of the clam Tapes philippinarum (Adams & Reeve, 1850): morphofunctional characterisation

Tapes philippinarum is a bivalve mollusc of the Pacific Ocean, successfully imported for human consumption into the northern Adriatic Sea (Europe). For better knowledge of its considerable adaptive ability in comparison with similar autochthonous species, a morpho-functional characterisation of its haemocytes was carried out with the establishment of short-term cell cultures (60 min at 25 degrees C). Various methods of cytochemical staining identified four cell types in the haemolymph: granulocytes (48.05% +/- 1.43), hyalinocytes (32.18% +/- 0.99), haemoblasts (18.97% +/- 0.63) and serous cells (0.8% +/- 0.19). The granulocytes, possessing cytoplasmic granules with differing dye affinity, included basophils, neutrophils and acidophils. Such granules stained vitally with Neutral Red, and correspond to lysosomes. Hydrolytic and oxidative enzymes were mainly detectable after stimulation in the presence of yeast cells. Both granulocytes and hyalinocytes were positive for alkaline phosphatase, non-specific esterase, peroxidase, and cytochrome C oxidase, whereas only granulocytes were positive for beta-glucuronidase, acid esterase, and arylsulphatase. Both cell types were competent phagocytes towards yeast and plasma had an opsonising effect. Moreover, the respiratory burst accompanied phagocytosis with superoxide anion production, recognisable through cytoplasmic deposits of formazan after treatment with nitro blue tetrazolium. Haemoblasts were small undifferentiated cells which, due to their morphology and positivity to the anti-CD34 antibody, show the typical features of stem cells. Serous cells, probably arising from Keber's gland and belonging to another differentiation pathway, contained non-sulphate acid mucopolysaccharides and play an important role in early defence mechanisms, taking part in the formation of clots.     

Nondestructive and on-line monitoring of tablets using light-induced fluorescence technology

A system using light-induced fluorescence (LIF) technology was developed for rapid and nondestructive analysis of active pharmaceutical ingredients on tablet surfaces. Nonhomogenous tablets with defined layer of active ingredients were made by 3-Dimensional Printing technology to determine penetration depths of the light source and the resultant fluorescence responses. The LIF method of analysis showed penetration to depths of up to 3 mm into tablets. A correlation between LIF signals from analysis of tablet surfaces and the total drug content of the respective tablets was established. This method of surface analysis was verified with UV spectrometric methods for the total drug content of each respective tablet. The results from a small sample population of tablets made from both homogeneous and nonhomogeneous powder mixtures established good correlation between LIF surface monitoring and total tablet content. The use of on-line monitoring of the individual tablet for surface content demonstrated consistent LIF profiles from simulated production rates up to 3000 tablets a minute. The instrument was also field tested successfully on a tablet analyzer.     

Alimentary tract of Kowalevskiidae (Appendicularia, Tunicata) and evolutionary implications

The alimentary tract of Kowalevskia tenuis and K. oceanica, the only species of the appendicularian family Kowalevskiidae, was studied both at the light and electron microscope levels and compared with species belonging to the other two families of the class. Kowalevskids show interesting specializations: 1) the pharynx opens on both sides through two opposing spiracles, modified into long ciliated fissures, and possesses an original filtering system of ciliated combs arranged in two pairs of opposing longitudinal rows; 2) the endostyle is absent, its place being taken by a ciliated groove without any glandular cell; 3) posterior to the esophagus, the globular stomach and rectum form a digestive nucleus comprising a few, large cells including two well-developed, specialized valves, cardiac and pyloric; 4) special apical junctions bearing characteristics of both gap and adherens junctions are diffuse along the gut epithelium; 5) the heart is absent. Our data suggest that Kowalevskiidae underwent a high degree of specialization for food filtering and are more closely related to Fritillariidae, with which they share several characters, rather than Oikopleuridae, the latter probably representing the most primitive family of appendicularians.     

Synthesis, conformational and pharmacological studies on dermorphin N-terminal tetrapeptide analogues

Dermorphin structure–activity relationships toward and opioid receptors were investigated using a series of synthetic peptides, in which the aromatic residues at positions 1 or/and 3 of the N-terminal tetrapeptide analogue H-Tyr-d-Arg-Phe--Ala-NH₂ were replaced by unnatural or constrained amino acids.     

Ontogeny of the retina and optic nerve of Xenopus laevis: IV. Ultrastructural evidence of early ganglion cell differentiation

Ultrastructural evidence indicates that Xenopus retinal ganglion cell axons differentiate early, between stages 28 and 32. Light microscope studies indicated the presence of argryophilic material in the ventral retina and optic stalk of early embryos. Ultrastructural analysis of this region confirmed the presence of axons in the stalk and interstices of ventral retinal cells. Axons containing aligned microtubules and neurofilaments and elongated mitochondria with a paucity of other cell inclusions are found with increasing frequency in the ventral retina from stages 28 through $\text{33}\text{34}$. Central and dorsal regions of the retinas examined show little or no evidence of axons. A discrete, small bundle of axons is found in the optic stalk of stage 28 embryos and by stage $\text{30}\text{31}$ the number of axons in bundles has increased, suggesting early fasciculation. Between stages 28 and $\text{33}\text{34}$ (± 12 hr) extracellular space surrounding early axons diminishes and processes from neuroretinal cells in contact with axons surround developing axon bundles. The evidence presented suggests that axon initiation occurs in stages much earlier than previously reported. Other investigators have failed to detect ganglion cell differentiation prior to stage 32 possibly because they examined regions of the retina with few axons. Thus, experiments which rotate the retina in the orbit may have to be reevaluated since regenerating axons may use previously established pathways to organize and “home in” on tectal target cells.     

Rapid, sequential changes in surface morphology of PC12 pheochromocytoma cells in response to nerve growth factor

The effect of nerve growth factor (NGF), a substance that promotes the differentiation and maintenance of certain neurons, was studied via scanning electron microscopy utilizing the PC12 clonal NGF-responsive pheochromocytoma cell line. After 2-4 d of exposure to NGF, these cells acquire many of the properties of normal sympathic neurons. However, by phase microscopy, no changes are discernible within the first 12-18 h. Since the primary NGF receptor appears to be a membrane receptor, it seemed likely that some of the initial responses to the factor may be surface related. PC12 cells maintained without NGF are round to ovoid and have numerous microvilli and small blebs. After the addition of NGF, there is a rapidly initiated sequential change in the cell surface. Ruffles appear over the dorsal surface of the cells with 1 min, become prominent by 3 min, and almost disappear by 7 min. Microvilli, conversely, disappear as the dorsal ruffles become prominent. Ruffles are seen at the the periphery of cell at 3 min, are prominent on most of the cells by 7 min and are gone by 15 min. The surface remains smooth from 15 min until 45 min when large blebs appear. The large blebs are present on most cells at 2 h and are gone by 4 h. The surface remains relatively smooth until 6-7 h of NGF treatment, when microvilli reappear as small knobs. These microvilli increase in both number and length to cover the cell surface by 10 h. These changes were not observed with other basic proteins, with α-bungarotoxin (which binds specifically to PC12 membranes), and were not affected by an RNA synthesis inhibitor that blocks initiation of neurite outgrowth. Changes in the cell surface architecture appear to be among the earlist NGF responses yet detected and may represent or reflect primary events in the mechanism of the factor’s action.