Evaluation of the mutagenic activity of some aza-aromatic hydrocarbons

The mutagenic activity of 7 aza-aromatic hydrocarbons, which are suspected of being environmental pollutants, was assessed using the Salmonella assay. The compounds tested were: 1-azachrysene, 2-azachrysene, 4-azachrysene, 1-azabenz[a]anthracene, 2-azabenz[a]anthracene, 9-azabenz[a]anthracene, and 12-benzo[a]pyrene. None of the compounds was mutagenic in the absence of S9, but all were mutagenic in the presence of S9.     

AP180 Couples Protein Retrieval to Clathrin‐Mediated Endocytosis of Synaptic Vesicles

How clathrin‐mediated endocytosis (CME) retrieves vesicle proteins into newly formed synaptic vesicles (SVs) remains a major puzzle. Besides its roles in stimulating clathrin‐coated vesicle formation and regulating SV size, the clathrin assembly protein AP180 has been identified as a key player in retrieving SV proteins. The mechanisms by which AP180 recruits SV proteins are not fully understood. Here, we show that following acute inactivation of AP180 in Drosophila, SV recycling is severely impaired at the larval neuromuscular synapse based on analyses of FM 1‐43 uptake and synaptic ultrastructure. More dramatically, AP180 activity is important to maintain the integrity of SV protein complexes at the plasma membrane during endocytosis. These observations suggest that AP180 normally clusters SV proteins together during recycling. Consistent with this notion, SV protein composition and distribution are altered in AP180 mutant flies. Finally, AP180 co‐immunoprecipitates with SV proteins, including the vesicular glutamate transporter and neuronal synaptobrevin. These results reveal a new mode by which AP180 couples protein retrieval to CME of SVs. AP180 is also genetically linked to Alzheimer's disease. Hence, the findings of this study may provide new mechanistic insight into the role of AP180 dysfunction in Alzheimer's disease.      

Olive phenols efficiently inhibit the oxidation of serum albumin-bound linoleic acid and butyrylcholine esterase

Background

Olive phenols are widely consumed in the Mediterranean diet and can be detected in human plasma. Here, the capacity of olive phenols and plasma metabolites to inhibit lipid and protein oxidations is investigated in two plasma models.

Methods

The accumulation of lipid oxidation products issued from the oxidation of linoleic acid bound to human serum albumin (HSA) by AAPH-derived peroxyl radicals is evaluated in the presence and absence of phenolic antioxidants. Phenol binding to HSA is addressed by quenching of the Trp214 fluorescence and displacement of probes (quercetin, dansylsarcosine and dansylamide). Next, the esterase activity of HSA-bound butyrylcholine esterase (BChE) is used as a marker of protein oxidative degradation.

Results

Hydroxytyrosol, oleuropein, caffeic and chlorogenic acids inhibit lipid peroxidation as well as HSA-bound BChE as efficiently as the potent flavonol quercetin. Hydroxycinnamic derivatives bind noncompetitively HSA subdomain IIA whereas no clear site could be identified for hydroxytyrosol derivatives.

General significance

In both models, olive phenols and their metabolites are much more efficient inhibitors of lipid and protein oxidations compared to vitamins C and E. Low postprandial concentrations of olive phenols may help to preserve the integrity of functional proteins and delay the appearance of toxic lipid oxidation products.     

Synaptotagmin I stabilizes synaptic vesicles via its C2A polylysine motif

The synaptic vesicle protein, synaptotagmin I, is a multifunctional protein required for several steps in the synaptic vesicle cycle. It is primarily composed of two calcium‐binding domains, C₂A and C₂B. Within each of these domains, a polylysine motif has been identified that is proposed to mediate specific functions within the synaptic vesicle cycle. While the C₂B polylysine motif plays an important role in synaptic transmission in vivo, the C₂A polylysine motif has not previously been analyzed at an intact synapse. Here, we show that mutation of the C₂A polylysine motif increases the frequency of spontaneous transmitter release in vivo. The increased frequency is not a developmental consequence of disrupted synaptic transmission, as evoked transmitter release is unimpaired in the mutants. Our results demonstrate that synaptotagmin I plays a direct role in regulating spontaneous transmitter release, indicative of an active role in synaptic vesicle stabilization mediated by the C₂A polylysine motif. genesis 47:337–345, 2009. © 2009 Wiley‐Liss, Inc.     

C2B polylysine motif of synaptotagmin facilitates a Ca2+-independent stage of synaptic vesicle priming in vivo

Synaptotagmin I, a synaptic vesicle protein required for efficient synaptic transmission, contains a highly conserved polylysine motif necessary for function. Using Drosophila, we examined in which step of the synaptic vesicle cycle this motif functions. Polylysine motif mutants exhibited an apparent decreased Ca2+ affinity of release, and, at low Ca2+, an increased failure rate, increased facilitation, and increased augmentation, indicative of a decreased release probability. Disruption of Ca2+ binding, however, cannot account for all of the deficits in the mutants; rather, the decreased release probability is probably due to a disruption in the coupling of synaptotagmin to the release machinery. Mutants exhibited a major slowing of recovery from synaptic depression, which suggests that membrane trafficking before fusion is disrupted. The disrupted process is not endocytosis because the rate of FM 1-43 uptake was unchanged in the mutants, and the polylysine motif mutant synaptotagmin was able to rescue the synaptic vesicle depletion normally found in syt(null) mutants. Thus, the polylysine motif functions after endocytosis and before fusion. Finally, mutation of the polylysine motif inhibits the Ca2+-independent ability of synaptotagmin to accelerate SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor)-mediated fusion. Together, our results demonstrate that the polylysine motif is required for efficient Ca2+-independent docking and/or priming of synaptic vesicles in vivo.     

Phylogeography and sympatric differentiation of the Arctic charr Salvelinus alpinus (L.) complex in Siberia as revealed by mtDNA sequence analysis

Sequence variation in the mtDNA control region of Arctic charr Salvelinus alpinus and Dolly Varden Salvelinus malma from 56 Siberian and North American populations was analysed to assess their phylogeographic relationships and the origins of sympatric forms. Phylogenetic trees confirm the integrity of phylogroups reported in previous mtDNA studies except that the Siberian group does not separate as a single cluster. Haplotype network analysis indicates the proximity of Siberian and Atlantic haplotypes. These are considered as one Eurasian group represented by the Atlantic, east Siberian (interior Siberia including Transbaikalia, Taimyr) and Eurosiberian (Finland, Spitsbergen, Taimyr) sub-groups. Salvelinus alpinus with presumably introgressed Bering group (malma) haplotypes were found along eastern Siberian coasts up to the Olenek Bay and the Lena Delta region, where they overlap with the Eurasian group and in the easternmost interior region. It is proposed that Siberia was colonized by S. alpinus in two stages: from the west by the Eurasian group and later from the east by the Bering group. The high diversity of Eurasian group haplotypes in Siberia indicates its earlier colonization by S. alpinus as compared with the European Alps. This colonization was rapid, proceeded from a diverse gene pool, and was followed by differential survival of ancestral mtDNA lineages in different basins and regions, and local mutational events in isolated populations. The results presented here support a northern origin of Transbaikalian S. alpinus , the dispersion of S. alpinus to the Lake Baikal Basin from the Lena Basin, segregation of S. alpinus between Lena tributaries and their restricted migration over the divides between sub-basins. These results also support sympatric origin of intralacustrine forms of S. alpinus .     

Drosophila synaptotagmin I null mutants survive to early adulthood

Summary: Synaptotagmin is a synaptic vesicle protein required for efficient neurotransmitter release, yet its exact role in the synaptic vesicle cycle is unclear. Drosophila presents an ideal organism for studies aimed at determining the in vivo functions of proteins. However, synaptotagmin studies have been limited by the early (embryonic or first instar) lethality previously reported for Drosophila synaptotagmin I null (syt^null) mutants. Here we report a new culturing technique that enhances survival of severely uncoordinated mutants thereby permitting Drosophila syt^null mutants to survive through early adulthood. We examined synapses in syt^null third instar larvae by electrophysiology and found that they exhibit severely decreased and asynchronous evoked neurotransmitter release, as well as an increased rate of spontaneous neurotransmitter release, as previously seen in first instar syt^null larvae. The ability to examine severe synaptotagmin mutants as third instar larvae, a stage where electrophysiological and morphological analyses are more easily accomplished, will facilitate structure/function studies. genesis 31:30–36, 2001. © 2001 Wiley‐Liss, Inc.     

Bacterial mutagenicity and carcinogenic potential of some azapyrene derivatives

The mutagenic and carcinogenic activities of 5 azapyrenes, which are suspected of being environmental pollutants, were assessed using the Salmonella assay and the anchorage-independent survival assay. The compounds tested were: 1-azapyrene, 2-azapyrene, 4-azapyrene, 1-aza-2-hydroxypyrene, and 2-aza-1-hydroxypyrene. The compounds were mutagenic and some were also carcinogenic.     

Quantitative mRNA analysis of eight bovine 5-HT receptor subtypes in brain, abomasum, and intestine by real-time RT-PCR

Serotoninergic pathways are involved in economically important bovine gastrointestinal (GI) motility disorders such as displaced abomasum and cecal dilatation/dislocation. The existing research tools to investigate the role of serotoninergic pathways in such disorders in ruminants comprise functional pharmacological methods, e.g., in vitro contractility studies in tissue baths, and electromyographical recordings in vivo. However, no tools for quantification of bovine serotonin receptor [5-hydroxytryptamine receptor (5-HTR)] expression were available so far. This study aimed to develop real-time RT-PCR assays for quantitative mRNA analysis of bovine 5-HTR subtypes. Because the bovine 5-HTR coding sequences (CDSs) were completely unknown, multiple species (human, mouse, and rat) alignment of complete CDS was used for primer design in highly homologous regions. LightCycler real-time RT-PCR assays (partial CDS) for the following bovine 5-HTR subtypes were developed and validated: 5-HTR1A, 5-HTR1B, 5-HTR1D, 5-HTR1F, 5-HTR2A, 5-HTR2B, 5-HTR2C, and 5-HTR4. Intra- and inter-assay coefficients of variation (CV) for the eight established assays were small, ranging from 0.49% to 2.46%. As a first physiological application, 5-HTR mRNA expression levels were measured in brain, abomasum, and intestine of 10 healthy, lactating dairy cows. The 5-HTR expression was quantified by normalization to the housekeeping gene glyceraldehyde-phosphate-dehydrogenase (GAPDH). The 5-HTR subtype expression levels ranged from 0.001% (5-HTR2C in intestine) to 1% 5-HTR/GAPDH (5-HTR1B and 5-HTR4 in intestine). There were high variations of 5-HTR subtype mRNA expression within tissues across receptor subtypes and within receptor subtypes across tissues. In conclusion, accurate real-time RT-PCR assays for quantitative analysis of bovine 5-HTR subtype gene expression were developed and validated.     

Postpartum reproductive function: association with energy,metabolic and endocrine status in high yielding dairy cows

This study evaluated the effect of metabolic, endocrine and energy status on onset of ovarian cycle, days open (DO), and conception at first service in 90 multiparous Holstein cows, housed at a research farm. Dry matter intake, milk yield and body weight were measured daily from Week 2 antepartum (a.p.) to Week 20 postpartum (p.p.). Milk composition was determined four times per week and milk acetone was measured weekly. Blood samples for the determination of glucose, non-esterified fatty acids, cholesterol, creatinine, albumin, urea, beta-hydroxybutyrate, leptin, insulin, insulin-like growth factor-1, growth hormone, 3,5,3'-triiodothyronine (T(3)), and thyroxine (T(4)) were taken 2 weeks a.p., in Weeks 1-16, and 20 p.p. between 7:30 and 9:00 h. The onset of ovarian cycle was specified by weekly gynecological examination and by skim milk progesterone determination by radioimmunoassay (twice per week). Energy balance (EB) traits were calculated and expressed as accumulated negative EB from calving to EB equilibrium, EB nadir (EBN), rate of EB recovery after EBN (EBR), and time from calving to EBN and to EB equilibrium, respectively. The onset of ovarian cycle p.p. was not related to EB. However, a low degree of EBN and a fast EBR were associated with fewer DO, and EB at first service was positively related to conception. High plasma levels of T(3) and T(4) p.p. were associated with an early start of ovarian cycle, and high concentrations of glucose and cholesterol with a short calving to conception interval. Conception at first service was positively related to EB at first service and progesterone concentration 10-13 days after first service. In conclusion, thyroid hormones may play an important role in resumption of ovarian cyclicity p.p., and a good energy status enhances the chance of conception at first service and shortens DO.