Separation of flavonoids from Aleurites moluccana leaves using chitosan modified with heptaldehyde

Heptaldehyde-modified chitosan (heptyl-chitosan, CH-Hp) was investigated as adsorbent for chromatograhic separation of the flavonoids from A. moluccana. The amount of 2"-O-rhamnosylswertisin isolated (30.0 mg) was approx. twice as high as swertisin (17.5 mg). The improved surface hydrophobicity effected by the heptyl groups promoted the separation of flavonoids. From the results obtained, CH-Hp seems to be more suitable for separation of glycosylated flavonoids than other flavonoids. Thus, modified chitosan described here can be used for hydrophobic interaction chromatography as sucessfully illustrated with flavonoids.     

Modifications of serotonergic and adrenergic receptor concentrations in the brain of aggressive Canis familiaris

The aim of the study was to measure beta-adrenergic (beta-AR) and serotonergic (5-HTR) receptor concentrations in different brain areas (frontal cortex, hippocampus, hypothalamus and thalamus) of normal and aggressive dogs. Eight adult male dogs, 4.2+/-0.6 years old, showing no clinical signs but aggression, were used for the study. Eight healthy male dogs, 4.4+/-0.8 years old, with no history of neurological and/or behavioural disorders and accidental death, were used as controls. The whole frontal cortex, hippocampus, thalamus and hypothalamus were collected after euthanasia and plasma membrane fractions obtained by ultracentrifugation. beta-AR and 5-HTR were measured by binding assays using specific radioligand [(-)[3H]CGP 12177 and 5-hydroxy[3H]-tryptamine trifluoroacetate, respectively]. A significant decrease in beta-AR levels was observed in the frontal cortex (P=0.001), hippocampus (P<0.0001), and thalamus (P<0.0001) of aggressive dogs compared to controls. As far as 5-HTR are concerned, two receptor subtypes were detected. The two subtypes were classified as low-affinity (5-HTR LA) and high-affinity (5-HTR HA) serotonergic receptors for [3H]-hydroxytryptamine, on the basis of their affinity for [3H]-hydroxytryptamine. 5-HTR LA significantly increased in the whole central nervous system (CNS) area of aggressive dogs (frontal cortex P=0.071; hippocampus P=0.0013; thalamus P<0.0001; hypothalamus P=0.0004); 5-HTR HA significantly increased only in the thalamus (P=0.0005) and hypothalamus (P=0.0002). Results suggest the possible role played by the catecholaminergic and serotonergic systems in canine aggressive behaviour. The understanding of the biological basis of canine aggression may enable the development of pharmacological treatments that would target specific neurotransmitter systems.     

Seizure Activity Produces Differential Changes in Adenosine A1 Receptors within Rat Hippocampus

Specific ligand binding to rat hippocampal adenosine A₁ receptor after administration of the convulsant drug 3-mercaptopropionic acid (MP) was studied by means of a quantitative autoradiographic method. 2-Chloro-N6-[cyclopentyl-2,3,4,5-³H adenosine] ([³H]CCPA), a potent and selective A₁ receptor ligand, was selected for binding studies. MP administration (150 mg/kg, i.p.), at seizure, caused significant increases in the following CA1 layers: pyramidal (45%), radiatum (18%) and lacunosum molecular (35%); in CA2 area, a significant decrease in stratum oriens (36%) and an increase in stratum radiatum (14%) and lacunosum molecular (33%) layers was observed. In CA3 area a rise in pyramidal (40%) and radiatum layers (26%), as well as in hillus (97%) was found. At postseizure, changes were restricted to CA1, CA2 and CA3 pyramidal layers and to CA1 lacunosum molecular layer, with increases ranging from 22 to 50%. These results show that [³H]CCPA binding is modified diversely in intrahippocampal layers and areas, thus indicating their dissimilar role in seizure activity.     

Differential Expression of Cerebellar Metabotropic Glutamate Receptors mGLUR2/3 and mGLUR4a after the Administration of a Convulsant Drug and the Adenosine Analogue Cyclopentyladenosine

Metabotropic glutamate receptors (mGluR) play a role in synaptic transmission, neuronal modulation and plasticity but their action in epileptic activity is still controversial. On the other hand adenosine acts as a neuromodulator with endogenous anticonvulsive properties. Since cerebellum from epileptic patients has shown neuronal damage, sometimes associated with Purkinje cells loss, we have explored the effect of repetitive seizures on two types of mGluR in the cerebellum. Seizures were induced by the convulsant drug 3-mercaptopropionic acid (MP) and the effect of the adenosine analogue cyclopentyladenosine (CPA) alone or before MP administration (CPA+MP) were also evaluated. The expression of the receptors subtypes 2/3 (mGluR2/3) and 4a (mGluR4a) was assessed by immunocitochemistry. Granular cell layer was labeled with mGluR2/3 antibody and increased immunoreactivity was observed after MP (60%), CPA (53%) and CPA + MP (85%) treatments. Control cerebellum slices showed mGluR4a reactivity around Purkinje cells, while MP, CPA and CPA+MP treatment decreased this immunostaining. Repetitive administration of MP and CPA induces an increased cerebellar mGluR2/3 and a decreased mGluR4a immunostaining, suggesting a distinct participation of both receptors that may be related to the type of cell involved. A protective action and /or an apoptotic effect may not be discarded. CPA repetitive administration although increase seizure latency, cannot prevent seizure activity.     

Retrospective investigation of an influenza A/H1N1pdm outbreak in an Italian military ship cruising in the Mediterranean Sea, May-September 2009

Background

Clinical surveillance may have underestimated the real extent of the spread of the new strain of influenza A/H1N1, which surfaced in April 2009 originating the first influenza pandemic of the 21^st century. Here we report a serological investigation on an influenza A/H1N1pdm outbreak in an Italian military ship while cruising in the Mediterranean Sea (May 24-September 6, 2009).

Methods

The contemporary presence of HAI and CF antibodies was used to retrospectively estimate the extent of influenza A/H1N1pdm spread across the crew members (median age: 29 years).

Findings

During the cruise, 2 crew members fulfilled the surveillance case definition for influenza, but only one was laboratory confirmed by influenza A/H1N1pdm-specific RT-PCR; 52 reported acute respiratory illness (ARI) episodes, and 183 reported no ARI episodes. Overall, among the 211 crew member for whom a valid serological result was available, 39.3% tested seropositive for influenza A/H1N1pdm. The proportion of seropositives was significantly associated with more crowded living quarters and tended to be higher in those aged <40 and in those reporting ARI or suspected/confirmed influenza A/H1N1pdm compared to the asymptomatic individuals. No association was found with previous seasonal influenza vaccination.

Conclusions

These findings underline the risk for rapid spread of novel strains of influenza A in confined environment, such as military ships, where crowding, rigorous working environment, physiologic stress occur. The high proportion of asymptomatic infections in this ship-borne outbreak supports the concept that serological surveillance in such semi-closed communities is essential to appreciate the real extent of influenza A/H1N1pdm spread and can constitute, since the early stage of a pandemic, an useful model to predict the public health impact of pandemic influenza and to establish proportionate and effective countermeasures.     

Genetic characterization of European-Zebu composite bovine using RFLP markers

A population of 370 European-Zebu composite beef heifers, consisting of six different breed compositions (A-F), were characterized genetically, using RFLP markers of luteinizing hormone receptor (LHR) and follicle-stimulating hormone receptor (FSHR) genes. Our objectives were to genetically characterize this population and to determine the structure and the genetic variability of this hybrid herd. The genotypes were determined through PCR, followed by digestion with restriction endonucleases. The PCR-RFLP analysis made it possible to identify the LHR and FSHR genotypes, as well as to characterize the degree of heterozygosis, which was high for all of the breed compositions, for both loci, except for two combinations for LHR (B and C). The observed heterozygosity (Ho) was lower than the expected heterozygosity (He) for compositions C (for LHR) and A and D (for FSHR); however, for the population as a whole, Ho was above He (with a mean of 57 versus 46%, respectively), reflecting the elevated genetic variability in this population and also the informative value of the RFLP markers, which could be useful for population genetic characterization studies. The analysis of the degree of genetic structure of this population, estimated by the Nei's statistic, for both loci, indicated an elevated total genetic diversity (HT = 47%), with most of this variability being due to intrapopulational diversity (HS = 46%), with a low degree of genetic differentiation among the six breed compositions (GST = 1.2%). The estimates generated by the Wright's F statistic indicated a non-endogamic population, with excess heterozygotes (FIT = -0.22), which was also observed at the intrapopulational level (FIS = -0.23). The results gave evidence that the genetic selection applied to this European-Zebu composite population did not affect the expected high genetic variability for this type of crossbreeding, which makes it possible to use these animals to obtain economically valuable productive and reproductive traits.     

Space and time-resolved gene expression experiments on cultured mammalian cells by a single-cell electroporation microarray

Single-cell experiments represent the next frontier for biochemical and gene expression research. Although bulk-scale methods averaging populations of cells have been traditionally used to investigate cellular behavior, they mask individual cell features and can lead to misleading or insufficient biological results. We report on a single-cell electroporation microarray enabling the transfection of pre-selected individual cells at different sites within the same culture (space-resolved), at arbitrarily chosen time points and even sequentially to the same cells (time-resolved). Delivery of impermeant molecules by single-cell electroporation was first proven to be finely tunable by acting on the electroporation protocol and then optimized for transfection of nucleic acids into Chinese Hamster Ovary (CHO-K1) cells. We focused on DNA oligonucleotides (ODNs), short interfering RNAs (siRNAs), and DNA plasmid vectors, thus providing a versatile and easy-to-use platform for time-resolved gene expression experiments in single mammalian cells.     

A 28,000 years old Cro-Magnon mtDNA sequence differs from all potentially contaminating modern sequences

Background

DNA sequences from ancient speciments may in fact result from undetected contamination of the ancient specimens by modern DNA, and the problem is particularly challenging in studies of human fossils. Doubts on the authenticity of the available sequences have so far hampered genetic comparisons between anatomically archaic (Neandertal) and early modern (Cro-Magnoid) Europeans.

Methodology/Principal Findings

We typed the mitochondrial DNA (mtDNA) hypervariable region I in a 28,000 years old Cro-Magnoid individual from the Paglicci cave, in Italy (Paglicci 23) and in all the people who had contact with the sample since its discovery in 2003. The Paglicci 23 sequence, determined through the analysis of 152 clones, is the Cambridge reference sequence, and cannot possibly reflect contamination because it differs from all potentially contaminating modern sequences.

Conclusions/Significance:

The Paglicci 23 individual carried a mtDNA sequence that is still common in Europe, and which radically differs from those of the almost contemporary Neandertals, demonstrating a genealogical continuity across 28,000 years, from Cro-Magnoid to modern Europeans. Because all potential sources of modern DNA contamination are known, the Paglicci 23 sample will offer a unique opportunity to get insight for the first time into the nuclear genes of early modern Europeans.     

Modification of [3H]MK801 Binding to Rat Brain NMDA Receptors after the Administration of a Convulsant Drug and an Adenosine Analogue: A Quantitative Autoradiographic Study

Specific [³H]MK801 binding to rat brain NMDA receptors after the administration of the convulsant drug 3-mercaptopropionic acid (MP) and the adenosine analogue cyclopentyladenosine (CPA) was studied by means of a quantitative autoradiographic method. MP administration (150 mg/kg, i.p.) caused significant decreases in [³H]MK801 binding in several hippocampus subareas and layers, mainly in CA1 and CA3 at seizure (11–27%) and postseizure (8–16%) and in cerebral occipital cortex at seizure (18–22%). In nucleus accumbens, a rise was observed at postseizure (44%) and a tendency to increase at seizure (24%). CPA (2mg/kg, i.p.) decreased ligand binding in hippocampus (CA1, CA2, CA3) (17–22%) and in occipital cerebral cortex (18–24%). When CPA was administered 30 minutes before MP (which delayed seizure onset) and rats were sacrified at seizure, decreases in [³H]MK801 binding in several layers of CA1 and CA3 of hippocampus (11–27%) and in CA1, CA2, CA3 (24–35%) after CPA+MP postseizure, and an increase in CA2 after CPA and CPA+MP postseizure (20–34%), were observed. A drop was found in the occipital subarea (18–24%) after CPA and in the frontal and occipital subarea after CPA+MP postseizure (24–34%) while no changes were observed in any treatment involving the other cerebral cortex regions, thalamic nuclei, caudate putamen and olfactory tubercle. These results show that [³H]MK801 binding changes according to drug treatment and the area being studied, thus indicating a different role in seizure activity.