Increased expression of rat synuclein in the substantia nigra pars compacta identified by mRNA differential display in a model of developmental target injury

Human alpha-synuclein was identified on the basis of proteolytic fragments derived from senile plaques of Alzheimer's disease, and it is the locus of mutations in some familial forms of Parkinson's disease. Its normal function and whether it may play a direct role in neural degeneration remain unknown. To explore cellular responses to neural degeneration in the dopamine neurons of the substantia nigra, we have developed a rodent model of apoptotic death induced by developmental injury to their target, the striatum. We find by mRNA differential display that synuclein is up-regulated in this model, and thus it provides an opportunity to examine directly whether synuclein plays a role in the death of these neurons or, alternatively, in compensatory responses. Up-regulation of mRNA is associated with an increase in the number of neuronal profiles immunostained for synuclein protein. At a cellular level, synuclein is almost exclusively expressed in normal neurons, rather than apoptotic profiles. Synuclein is up-regulated throughout normal postnatal development of substantia nigra neurons, but it is not further up-regulated during periods of natural cell death. We conclude that up-regulation of synuclein in the target injury model is unlikely to mediate apoptotic death and propose that it may be due to a compensatory response in neurons destined to survive.     

Calreticulin affects focal contact-dependent but not close contact-dependent cell-substratum adhesion.

We used two cell lines expressing fast (RPEfast) and slow (RPEslow) attachment kinetics to investigate mechanisms of cell-substratum adhesion. We show that the abundance of a cytoskeletal protein, vinculin, is dramatically decreased in RPEfast cells. This coincides with the diminished expression level of an endoplasmic reticulum chaperone, calreticulin. Both protein and mRNA levels for calreticulin and vinculin were decreased in RPEfast cells. After RPEfast cells were transfected with cDNA encoding calreticulin, both the expression of endoplasmic reticulum-resident calreticulin and cytoplasmic vinculin increased. The abundance of other adhesion-related proteins was not affected. RPEfast cells underexpressing calreticulin displayed a dramatic increase in the abundance of total cellular phosphotyrosine suggesting that the effects of calreticulin on cell adhesiveness may involve modulation of the activities of protein tyrosine kinases or phosphatases which may affect the stability of focal contacts. The calreticulin and vinculin underexpressing RPEfast cells lacked extensive focal contacts and adhered weakly but attached fast to the substratum. In contrast, the RPEslow cells that expressed calreticulin and vinculin abundantly developed numerous and prominent focal contacts slowly, but adhered strongly. Thus, while the calreticulin overexpressing RPEslow cells "grip" the substratum with focal contacts, calreticulin underexpressing RPEfast cells use close contacts to "stick" to it.     

Effects of chronic hypogonadism on corticosterone secretion and cyclic AMP production in male rat adrenocortical cells

AIM: To determine the secretion of corticosterone (CCS) both in vivo and in vitro during different intervals after orchidectomy in male rats. METHODS: Three- and 12-month-old rats had been orchidectomized 0, 3, 6, or 9 months before decapitation. RESULTS: Orchidectomy increased the concentrations of plasma CCS, the basal release of CCS, and the adenosine 3', 5'-cyclic monophosphate (cAMP) production in rat zona fasciculata reticularis (ZFR) cells. The forskolin/3-isobutyl-l-methylxanthine-stimulated releases of CCS and cAMP production by ZFR cells were higher in rats with chronic hypogonadism. The CCS release from ZFR cells of orchidectomized rat was not altered by 8-bromo-cAMP treatment. Orchidectomy enhanced the stimulatory effect of deoxycorticosterone on CCS release in ZFR cells. CONCLUSION: These results suggest that orchidectomy-related increases of CCS secretion in rats are associated with an increase of adenylate cyclase activity, cAMP generation, and 11-beta-hydroxylase activity in ZFR cells.     

ERM proteins of the lens

Ezrin and radixin and protein 4.1 were detected in the lens of the eye. These proteins were mainly present in the young elongating cortical fiber cells and localized to the plasma membranes. Moesin was not detected. Ezrin, radixin, and protein 4.1 provide another means whereby actin is linked to the plasma membrane in addition to the known adherens junctions in the lens.     

Direct heterogeneous electron transfer of theophylline oxidase

Direct electron transfer (DET) was shown between the heme containing enzyme theophylline oxidase (ThO) and the surface of both graphite and gold electrodes. As proof on graphite a steady state current for theophylline was recorded using the electrode modified with adsorbed ThO. The electrode showed a Michaelis-Menten-like response to theophylline with a detection limit of 0.2 mM and a Michaelis-Menten constant equal to 3.2 mM. These initial results open up a possibility for the development of reagentless third generation biosensor based on heterogeneous DET between ThO and an electrode. On gold DET between ThO and the surface of aldrithiol modified gold was studied with spectroelectrochemical measurements. DET was observed for soluble ThO as a change of its spectrum in a gold capillary responding to a change in the applied potential. It was shown that the redox conversion of the heme domain of the enzyme is directly (mediatorlessly) driven by the potential applied at the gold electrode. The measurements enabled an estimation of the formal potential (E degrees ') of the redox process equal to -275 +/- 50 mV versus Ag|AgClsat at pH 7.0. The experimentally determined number of the electrons involved in this heterogeneous electron transfer process was estimated to be equal to 0.53. The low precision in determination of the E degrees ' and the value of the number of electrons lower than one indicate that kinetic restrictions disturbed the evaluation of the true thermodynamic values from relatively fast spectroelectrochemical measurements.     

Natural ligands of hamster aphrodisin

The chemical nature of vertebrate pheromones remains largely to be deciphered. Hamster aphrodisin is a rare instance of mammal proteinaceous sexual pheromone. This protein, found in vaginal secretions, facilitates the mounting behaviour of males via activation of a specialized sensory structure named the vomeronasal organ, which activates the accessory olfactory bulb. Since it might carry small pheromonal ligands due to its lipocalin structure, we analysed organic extracts from natural aphrodisin. We identified five predominant compounds specifically bound onto natural aphrodisin as 1-hexadecanol (44.7%), 1-octadecanol (19.5%), Z-9-octadecen-1-ol (18.2%), E-9-octadecen-1-ol (15.4%) and hexadecanoic acid (2.2%). Interestingly, these compounds are also described as part of insect pheromone blends, disclosing the continuing story of amazing coincidences of chemical communication shared by mammals and insects.     

Characterization of an ultraviolet B-induced lipase in Arabidopsis

An Arabidopsis expressed sequence tag clone, 221D24, encoding a lipase has been characterized using an antisense approach. The lipase gene is expressed during normal growth and development of Arabidopsis rosette leaves but is down-regulated as the leaves senesce. When plants are exposed to sublethal levels of UV-B radiation, expression of the lipase is strongly up-regulated. The lipase protein is localized in the cell cytosol and is present in all organs of Arabidopsis plants. Recombinant lipase protein produced in Escherichia coli preferentially hydrolyzed phospholipids, indicating that the gene encodes a phospholipase. Transgenic plants in which lipase expression is suppressed showed enhanced tolerance to UV-B stress but not osmotic stress and were unable to up-regulate PR-1 expression when irradiated with UV-B. The observations collectively indicate that the lipase is capable of deesterifying membrane phospholipids and is up-regulated in response to UV-B irradiation.