Bulla gouldiana period exhibits unique regulation at the mRNA and protein levels

The authors cloned the period (per) gene from the marine mollusk Bulla gouldiana, a well-characterized circadian model system. This allowed them to examine the characteristics of the per gene in a new phylum, and to make comparisons with the conserved PER domains previously characterized in insects and vertebrates. Only one copy of the per gene is present in the Bulla genome, and it is most similar to PER in two insects: the cockroach, Periplaneta americana, and silkmoth, Antheraea pernyi. Comparison with Drosophila PER (dPER) and murine PER 1 (mPER1) sequence reveals that there is greater sequence homology between Bulla PER (bPER) and dPER in the regions of dPER shown to be important to heterodimerization between dPER and Drosophila timeless. Although the structure suggests conservation between dPER and bPER, expression patterns differ. In all cells and tissues examined that are peripheral to the clock neurons in Bulla, bPer mRNA and protein are expressed constitutively in light:dark (LD) cycles. In the identified clock neurons, the basal retinal neurons (BRNs), a rhythm in bPer expression could be detected in LD cycles with a peak at zeitgeber time (ZT) 5 and trough expression at ZT 13. This temporal profile of expression more closely resembles that of mPER1 than that of dPER. bPer rhythms in the BRNs were not detected in continuous darkness. These analyses suggest that clock genes may be uniquely regulated in different circadian systems, but lead to similar control of rhythms at the cellular, tissue, and organismal levels.     

Photoinduced electron transfer and chemical alpha-deoxygenation of D-galactono-1,4-lactone. Synthesis of 2-deoxy-D-lyxo-hexofuranosides

Two simple procedures for the synthesis of 2-deoxy-D-lyxo-hexono-1,4-lactone are described. Reductive cleavage of a 2-O-tosyl derivative of D-galactono-1,4-lactone in the presence of sodium iodide afforded the 2-deoxy derivative. On the other hand, alpha-deoxygenation of D-galactono-1,4-lactone was easily achieved by photochemical electron transfer deoxygenation of HO-2 as the 3-(trifluoromethyl)benzoate. Methyl 2-deoxy-beta-D-lyxo-hexafuranoside ('methyl 2-deoxy-beta-D-galactofuranoside') was synthesized and tested as substrate for exo beta-D-galactofuranosidase from Penicillium fellutanum. The reaction was followed by HPAEC, showing that methyl 2-deoxy-beta-D-galactofuranoside was not hydrolyzed by incubation with the enzyme. Neither the 2-deoxy lactone, nor the 2-deoxy-beta-D-galactofuranoside acted as inhibitors of the reaction with the 4-nitrophenyl beta-D-galactofuranoside. The present and our previous results show that the hydroxyl groups at C-2, C-3 and C-6 of the galactofuranoside are essential for interaction with the exo beta-D-galactofuranosidase.     

Thiol protecting agents and antioxidants inhibit the mitochondrial permeability transition promoted by etoposide: implications in the prevention of etoposide-induced apoptosis

Etoposide (VP-16) is known to promote cell apoptosis either in cancer or in normal cells as a side effect. This fact is preceded by the induction of several mitochondrial events, including increase in Bax/Bcl-2 ratio followed by cytochrome c release and consequent activation of caspase-9 and -3, reduction of ATP levels, depolarization of membrane potential (DeltaPsi) and rupture of the outer membrane. These events are apoptotic factors essentially associated with the induction of the mitochondrial permeability transition (MPT). VP-16 has been shown to stimulate the Ca2+-dependent MPT induction similarly to prooxidants and to promote apoptosis by oxidative stress mechanisms, which is prevented by glutathione (GSH) and N-acetylcysteine (NAC). Therefore, the aim of this work was to study the effects of antioxidants and thiol protecting agents on MPT promoted by VP-16, attempting to identify the underlying mechanisms on VP-16-induced apoptosis. The increased sensitivity of isolated mitochondria to Ca2+-induced swelling, Ca2+ release, depolarization of DeltaPsi and uncoupling of respiration promoted by VP-16, which are prevented by cyclosporine A proving that VP-16 induces the MPT, are also efficiently prevented by ascorbate, the primary reductant of the phenoxyl radicals produced by VP-16. The thiol reagents GSH, dithiothreitol and N-ethylmaleimide, which have been reported to prevent the MPT induction, also protect this event promoted by VP-16. The inhibition of the VP-16-induced MPT by antioxidants agrees with the prevention of etoposide-induced apoptosis by GSH and NAC and suggests the generation of oxidant species as a potential mechanism underlying the MPT that may trigger the release of mitochondrial apoptogenic factors responsible for apoptotic cascade activation.     

In vitro chloroquine resistance modulation study on fresh isolates of Brazilian Plasmodium falciparum: intrinsic antimalarial activity of phenothiazine drugs

Phenothiazine drugs - fluphenazine, chlorpromazine, methotrimeprazine and trifluoperazine - were evaluated as modulating agents against Brazilian chloroquine-resistant fresh isolates of Plasmodium falciparum. Aiming to simulate therapeutic schedules, chloroquine was employed at the concentration used for sensitive falciparum malaria treatment and anti-psychotic therapeutic concentrations of the phenothiazine drugs were adopted in two-fold serial dilutions. The in vitro microtechnique for drug susceptibility was employed. Unlike earlier reported data, the phenothiazine modulating effect was not observed. However, all the drugs demonstrated intrinsic antiplasmodial activity in concentrations lower than those described in the literature. In addition, IC50 estimates have been shown to be inferior to the usual anti-psychotic therapeutic concentrations. Statistical analysis also suggested an increase in the parasitaemia rate or, even, a predominant antiparasitic effect of phenothiazine over chloroquine when used in combination.     

Niemann-Pick type C disease: mutations of NPC1 gene and evidence of abnormal expression of some mutant alleles in fibroblasts

We analyzed Niemann-Pick type C disease 1 (NPC1) gene in 12 patients with Niemann-Pick type C disease by sequencing both cDNA obtained from fibroblasts and genomic DNA. All the patients were compound heterozygotes. We found 15 mutations, eight of which previously unreported. The comparison of cDNA and genomic DNA revealed discrepancies in some subjects. In two unrelated patients carrying the same mutations (P474L and nt 2972del2) only one mutant allele (P474L), was expressed in fibroblasts. The mRNA corresponding to the other allele was not detected even in cells incubated with cycloheximide. The promoter variants (-1026T/G and -1186T/C or -238 C/G), found to be in linkage with 2972del2 allele do not explain the lack of expression of this allele, as they were also found in control subjects. In another patient, (N1156S/Q922X) the N1156S allele was expressed in fibroblasts while the expression of the other allele was hardly detectable. In a fourth patient cDNA analysis revealed a point mutation in exon 20 (P1007A) and a 56 nt deletion in exon 22 leading to a frameshift and a premature stop codon. The first mutation was confirmed in genomic DNA; the second turned out to be a T-->G transversion in exon 22, predicted to cause a missense mutation (V1141G). In fact, this transversion generates a donor splice site in exon 22, which causes an abnormal pre-mRNA splicing leading to a partial deletion of this exon. In some NPC patients, therefore, the comparison between cDNA and genomic DNA may reveal an unexpected expression of some mutant alleles of NPC1 gene.     

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.     

Retention of antigen on follicular dendritic cells and B lymphocytes through complement-mediated multivalent ligand-receptor interactions: theory and application to HIV treatment

In HIV-infected patients, large quantities of HIV are associated with follicular dendritic cells (FDCs) in lymphoid tissue. During antiretroviral therapy, most of this virus disappears after six months of treatment, suggesting that FDC-associated virus has little influence on the eventual outcome of long-term therapy. However, a recent theoretical study using a stochastic model for the interaction of HIV with FDCs indicated that some virus may be retained on FDCs for years, where it can potentially reignite infection if treatment is interrupted. In that study, an approximate expression was used to estimate the time an individual virion remains on FDCs during therapy. Here, we determine the conditions under which this approximation is valid, and we develop expressions for the time a virion spends in any bound state and for the effect of rebinding on retention. We find that rebinding, which is influenced by diffusion, may play a major role in retention of HIV on FDCs. We also consider the possibility that HIV is retained on B cells during therapy, which like FDCs also interact with HIV. We find that virus associated with B cells is unlikely to persist during therapy.     

Glutamate-induced calcium increase in myotubes depends on up-regulation of a sodium-dependent transporter

We report a study on the regulation by 2-chloro adenosine (2CA) of a glutamate (Glu) transporter in myogenic C2C12 cells. Long-term 2CA exposition significantly increased the V(max) of the Glu transporter. Moreover, 2CA-treated cells responded to Glu challenge by a rapid and transient increase in their intracellular calcium level. The above reported effects were totally abolished by treating C2C12 cells with the Na(+)-dependent Glu transporter inhibitors DL-threo-b-hydroxyaspartic acid and L-trans-pyrrolidine-2,4-dicarboxylic acid. We propose that the possible link between the Glu uptake increase and the Glu induction of calcium rise could be the depolarizing currents carried by Na(+) coupled with transporter activity.