The effect of protein conformational flexibility on the electronic properties of a chromophore

In this paper we address the question of how a protein environment can modulate the absorption spectrum of a chromophore during a molecular dynamics simulation. The effect of the protein is modeled as an external field acting on the unperturbed eigenstates of the chromophore. Using a first-principles method recently developed in our group, we calculated the perturbed electronic energies for each frame and the corresponding wavelength absorption during the simulation. We apply this method to a nanosencond timescale molecular dynamics simulation of the light-harvesting peridinin-chlorophyll-protein complex from Amphidinium carterae, where chlorophyll was selected among the chromophores of the complex for the calculation. The combination of this quantum-classical calculation with the analysis of the large amplitude motions of the protein makes it possible to point out the relationship between the conformational flexibility of the environment and the excitation wavelength of the chromophore. Results support the idea of the existence of a correlation between protein conformational flexibility and chlorophyll electronic transitions induced by light.     

Chromosomal and molecular characterization of Aethomys kaiseri from Zambia and Aethomys chrysophilus from Tanzania (Rodentia, Muridae)

Aethomys is a common and widespread rodent genus in the African savannas and grasslands. However, its systematics and taxonomy are still unclear as no study has covered the entire range. In fact it might not be a monophyletic genus and perhaps should be split into two subgenera, Micaelamys and Aethomys. In this paper, we present findings based on the cytogenetics and the entire cytochrome b sequence of two species from Zambia (A. kaiseri) and Tanzania (A. chrysophilus), and we compare them with the sequences of a South African species (A. namaquensis) and other allied muroid genera. Comparison of the banded chromosomes revealed complete G-band homology between the autosomes of the two species. However, the X and Y chromosomes clearly differ in size and in C- and G-banding, being much larger in A. kaiseri. Comparison of the cytochrome b sequences places the separation between A. kaiseri and A. chrysophilus at 4.49 Mya, a period of intense speciation in other African muroids. The resulting phylogeny strongly supports the idea of a paraphyletic group, suggesting the need to elevate the previously described subgenera to the genus rank.     

Selective modifications in the nucleus accumbens of dopamine synaptic transmission in rats exposed to chronic stress

Stressful events are accompanied by modifications in dopaminergic transmission in distinct brain regions. As the activity of the neuronal dopamine (DA) transporter (DAT) is considered to be a critical mechanism for determining the extent of DA receptor activation, we investigated whether a 3-week exposure to unavoidable stress, which produces a reduction in DA output in the nucleus accumbens shell (NAcS) and medial prefrontal cortex (mPFC), would affect DAT density and DA D1 receptor complex activity in the NAcS, mPFC and caudate-putamen (CPu). Rats exposed to unavoidable stress showed a decreased DA output in the NAcS accompanied by a decrease in the number of DAT binding sites, and an increase in the number of DA D1 binding sites and Vmax of SKF 38393-stimulated adenylyl cyclase. In the mPFC, stress exposure produced a decrease in DA output with no modification in DAT binding or in DA D1 receptor complex activity. Moreover, in the CPu stress exposure induced no changes in DA output or in the other neurochemical variables examined. This study shows that exposure to a chronic unavoidable stress that produces a decrease in DA output in frontomesolimbic areas induced several adaptive neurochemical modifications selectively in the nucleus accumbens.     

Chiasma Repatterning Across a Chromosomal Hybrid Zone Between Chromosomal Races of Mus Musculus Domesticus

Chiasma number and distribution were analysed in male house mice from a karyotypic hybrid zone between the CD race (2n=22) and the standard race (2n=40) located in central Italy. Chiasma repatterning occurs across the transect. The overall trend produces a diminution of chiasmata in the mice with CD chromosomal background. The progressive reduction of chiasmata indicates that genes could pass from one race to another in an asymmetrical way: from metacentric races to the standard population.     

Large and dissimilar repertoire of Melan-A/MART-1-specific CTL in metastatic lesions and blood of a melanoma patient

It is widely accepted that the repertoire of Melan-A-specific T cells naturally selected in melanoma patients is diverse and mostly nonoverlapping among different individuals. To date, however, no studies have addressed the TCR profile in different tumor sites and the peripheral blood from the same patient. We compared the TCR usage of Melan-A-specific T cells from different compartments of a single melanoma patient to evaluate possible clonotype expansion or preferential homing over a 4-mo follow-up period. Using HLA-A2 peptide tetramers, CD8(+) T cells recognizing the modified Melan-A immunodominant ELAGIGILTV peptide were isolated from four metastatic lesions resected from a single melanoma patient, and their TCR repertoire was studied. A panel of T cell clones was generated by cell cloning of tetramer-positive cells. Analysis of the TCR beta-chain V segment and the complementarity-determining region 3 (CDR3) length and sequence revealed a large diversity in the TCR repertoire, with only some of the clones showing a partial conservation in the CDR3. A similar degree of diversity was found by analyzing a number of T cell clones obtained after sorting a Melan-A-specific population derived from PBLs of the same patient after in vitro culture with the immunodominant epitope. Moreover, clonotypes found at one site were not present in another, suggesting the lack of expansion and circulation of one or more clonotypes. Taken together, these results buttress the notion that the CTLs recognizing the immunodominant Ag of Melan-A comprise a high number of different clonotypic TCR, of which only some exhibit common features in the CDR3.     

Copper amine oxidase expression in defense responses to wounding and Ascochyta rabiei invasion

Wounding chickpea (Cicer arietinum) internodes or cotyledons resulted in an increase in the steady-state level of copper amine oxidase (CuAO) expression both locally and systemically. Dissection of the molecular mechanisms controlling CuAO expression indicated that jasmonic acid worked as a potent inducer of the basal and wound-inducible CuAO expression, whereas salicylic acid and abscisic acid caused a strong reduction of the wound-induced CuAO expression, without having any effect on the basal levels. Epicotyl treatment with the CuAO mechanism-based inhibitor 2-bromoethylamine decreased hydrogen peroxide (H(2)O(2)) levels in all the internodes, as evidenced in vivo by 3,3'-diaminobenzidine oxidation. Moreover, inhibitor pretreatment of wounded epicotyls resulted in a lower accumulation of H(2)O(2) both at the wound site and in distal organs. In vivo CuAO inhibition by 2-bromoethylamine after inoculation of resistant chickpea cv Sultano with Ascochyta rabiei resulted in the development of extended necrotic lesions, with extensive cell damage occurring in sclerenchyma and cortical parenchyma tissues. These results, besides stressing the fine-tuning by key signaling molecules in wound-induced CuAO regulation, demonstrate that local and systemic CuAO induction is essential for H(2)O(2) production in response to wounding and indicate the relevance of these enzymes in protection against pathogens.     

Increase in ceramide level alters the lysosomal targeting of cathepsin D prior to onset of apoptosis in HT-29 colon cancer cells

Ceramide has been suggested as an important mediator of apoptosis. In HT-29 colorectal cancer cells increased ceramide levels, induced by exogenous N-acetylsphingosine (NAS, also known as C2-ceramide) or by 1-phenyl-2-(decanoylamino)-3-morpholino-1-propanol (PDMP), inhibited the transport and processing of cathepsin D (CD), a lysosomal protease implicated in apoptosis of tumour cells. C2-dihydroceramide (DH-C2), an inactive analogue of NAS, had no effect on CD transport and maturation. The treatment with either NAS or PDMP was revealed to be cytotoxic for HT-29 cells and led to cell death with classical features of apoptosis. Morphological signs of apoptosis and DNA fragmentation became apparent only between 24 and 48 h of incubation and poly(ADP ribose)-polymerase cleavage, a hallmark of caspase 3 activity, occurred no earlier than 8 h from incubation. Secretion of proCD was almost abolished and the formation of double-chain mature CD was reduced and delayed by NAS, whereas PDMP largely inhibited the lysosomal targeting and maturation of proCD. NAS- and PDMP-induced alteration of proCD transport and maturation were apparent already 2 h after incubation with the drugs, which is much earlier than when classical biochemical and morphological evidence of apoptosis could be detected. These data indicate that alteration of CD (and possibly of other glycoproteins) transport along the secretory pathway due to increased levels of cell-associated ceramide is an early event in cells undergoing apoptosis.     

Pressure drops in a distensible model of end-to-side anastomosis in systemic-to-pulmonary shunts

The modified Blalock-Taussig shunt is a surgical procedure used as a palliation to treat complex congenital heart defects. It consists of an interposing prosthetic tube between the innominate/subclavian artery and the right pulmonary artery. Previous experience indicates that the pressure drop across the shunt is affected by the pulmonary pressure at the distal anastomosis combined with the distensibility of the anastomosis. In this study, a computational fluid-structure interaction approach is presented to investigate the haemodynamic behaviour. Steady-state fluid dynamics and structural analyses were carried out using commercial codes based on the finite element method (FIDAP and ABAQUS) coupled by means of a purposely-developed procedure to transfer boundary conditions. Both prosthetic tube and artery walls were characterised by non-linear material properties. Three different pulmonary pressures (2, 5 and 15 mmHg) and two volume flow rates (0.4 and 0.8 l/min) were investigated. Results indicate that the effects of distensibility at the distal anastomosis on the shunt pressure drop are relevant only when the distal anastomosis on the shunt pressure drop are relevant only when the distal anastomosis is not fully distended, which occurs when the pulmonary pressure is lower than 5 mmHg.