Autoradiography of Serotonin 5-HT1A Receptor-Activated G Proteins in Guinea Pig Brain Sections by Agonist-Stimulated [35S]GTPγS Binding

Abstract: G protein activation mediated by serotonin 5-HT_1A and 5-HT_1B/D receptors in guinea pig brain was investigated by using quantitative autoradiography of agonist-stimulated [³⁵S]GTPγS binding to brain sections. [³⁵S]GTPγS binding was stimulated by the mixed 5-HT_1A/5-HT_1B/D agonist L694247 in brain structures enriched in 5-HT_1A binding sites, i.e., hippocampus (+140 ± 14%), dorsal raphe (+70 ± 8%), lateral septum (+52 ± 12%), cingulate (+36 ± 8%), and entorhinal cortex (+34 ± 5%). L694247 caused little or no stimulation of [³⁵S]GTPγS binding in brain regions with high densities of 5-HT_1B/D binding sites (e.g., substantia nigra, striatum, central gray, and dorsal subiculum). The [³⁵S]GTPγS binding response was antagonized by WAY100635 (10 µM) and methiothepin (10 µM). In contrast, the 5-HT_1B inverse agonist SB224289 (10 µM) did not affect the L694247-mediated [³⁵S]GTPγS binding response, and the mixed 5-HT_1B/D antagonist GR127935 (10 µM) yielded a partial blockade. The distribution pattern of the [³⁵S]GTPγS binding response and the antagonist profile suggest the L694247-mediated response in guinea pig brain to be mediated by 5-HT_1A receptors. In addition to L694247, 8-hydroxy-2-(di-n-propylamino)tetralin, and flesinoxan also stimulated [³⁵S]GTPγS binding; their maximal responses varied between 46 and 52% compared with L694247, irrespective of the brain structure being considered. Sumatriptan, rizatriptan, and zolmitriptan (10 µM) stimulated [³⁵S]GTPγS binding in the hippocampus by 20–50%. Naratriptan, CP122638, and dihydroergotamine stimulated [³⁵S]GTPγS binding to a similar level as L694247 in hippocampus, lateral septum, and dorsal raphe. It appears that under the present experimental conditions, G protein activation through 5-HT_1A but not 5-HT_1B/D receptors can be measured in guinea pig brain sections.     

Analysis of Assembly and Budding of Lujo Virus

The recently identified arenavirus Lujo virus (LUJV) causes fatal hemorrhagic fever in humans. We analyzed its mechanism of viral release driven by matrix protein Z and the cell surface glycoprotein precursor GPC. The L domains in Z are required for efficient virus-like particle release, but Tsg101, ALIX/AIP1, and Vps4A/B are unnecessary for budding. LUJV GPC is cleaved by site 1 protease (S1P) at the RKLM motif, and treatment with the S1P inhibitor PF-429242 reduced LUJV production.     

Ascorbate oxidase from Cucurbita maxima

Ascorbate oxidase is present in homogenates of the flesh of Cucurbita maxima fruits. Its activity is independent of ascorbate concentration over th     

Utility of DNA barcoding in native Oreochromis species

The importance of Oreochromis in worldwide aquaculture and regional fisheries motivates the study of their genetic diversity in their native range. In this article, all mitochondrial cytochrome c oxidase subunit I gene (COI) sequences of Oreochromis species are retrieved from Barcode of Life Data system to quantify the available DNA barcoding information from wild individuals collected within the native ranges of the respective species. It is found that 70% of the known species in the genus still lack a COI barcode, and only 15% of the available sequences are from within the respective native ranges. Many of the available sequences have been produced from specimens acquired from aquaculture and introduced, naturalized populations, making the assessment of variation within the original native range challenging. Analyses of the wild-collected fraction of available sequences indicated the presence of cryptic lineages within Nile tilapia Oreochromis niloticus and O. schwebischi, the occurrence of potential introgressive hybridization between O. niloticus and blue tilapia O. aureus, and potential ancestral polymorphism between Karonga tilapia O. karongae and black tilapia O. placidus. This article also reports a case of misidentification of O. mweruensis as longfin tilapia O. macrochir. These results stress the importance of improving the knowledge of genetic variation within the native ranges of Oreochromis species for better-informed conservation of these natural resources.     

The role of an active transport mechanism in glycerol accumulation during osmoregulation by Zygosaccharomyces rouxii

Summary At water activities (a _w) of 0.998 (no osmoticum) and 0.960 a _w(NaCl), the affinity (K _m) of glycerol transport by Zygosaccharomyces rouxii was 25.6 and 6.4 mmol/l respectively. The maximum uptake rate (V _max) was ca. 2.3 mol/g/min at both a _w's. However, at an a _wof 0.960 using polyethylene glycol (PEG) 400 the K _mand V _max for glycerol transport increased to 61.1 mmol/l and 32.2 mol/g per minute respectively. This suggests that different glycerol transport mechanisms operate during stress by the two osmotica. The addition of uncouplers (2,4-dinitrophenol or carbonylcyanide-m-chlorophenylhydrazine) resulted in the outflow of accumulated [^14C]glycerol from Z. rouxii after on osmotic upshock indicating that an active transport mechanism was operative. The transport mechanism was specific for glycerol since other polyols (mannitol, meso-erythritol and arabitol) had no effect on the uptake rate. During upshock from 0.998 to 0.960 a _w(NaCl), a transient increases in the rate of [¹⁴C]glycerol uptake was observed. However, if PEG 400 was used as osmoticum, the rate of glycerol uptake failed to increase.Offprint requests to: P. J. van Zyl     

Loss of binding antibodies against rabies in a vaccinated dog population in Flores Island,Indonesia

Effective parenteral vaccines are available to control rabies in dogs. While such vaccines are successfully used worldwide, the period between vaccine boosters required to guarantee protection of the population against rabies varies between vaccines and populations. In Flores Island, Indonesia, internationally and locally produced rabies vaccines are used during annual vaccination campaigns of predominantly free-roaming owned domestic dogs. The study objective was to identify the duration of the presence and factors associated with the loss of adequate level of binding antibodies (≥0.5 EU/ml) following rabies vaccination in a domestic dog population on Flores Island. A total of 171 dogs that developed an antibody titre higher or equal to 0.5 EU/ml 30 days after vaccination (D30), were repeatedly sampled at day 90, 180, 270, and 360 after vaccination. On the day of vaccination (D0), an interview was performed with dog owners to collect information on dog characteristics (age, sex, body condition score (BCS)), history of rabies vaccination, kind of daily food, frequency of feeding, and origin of the dog. Serum samples were collected and the level of antibodies was quantitatively assessed using ELISA tests. Dogs were categorized as having an adequate level of binding antibodies (≥0.5 EU/ml) or inadequate level of binding antibodies (<0.5 EU/ml) at each time points examined. A total of 115, 72, 23, and 31 dogs were sampled at D90, D180, D270, and D360, respectively, with the highest proportion of antibodies ≥ 0.5 EU/ml (58%, 95% CI, 49–67%) at D90, which reduced gradually until D360 (35%, 95% CI, 19–52%). Multivariable logistic regression models showed that loss of adequate level of binding antibodies is significantly associated with dogs having no history of vaccination or vaccination applied more than 12 months before D0, being less than 12 months of age, and having a poor BCS. These results highlight the importance of BCS regarding the immune response duration and provide insights into frequency of vaccination campaigns required for the internationally available vaccine used on Flores Island. For dogs without vaccination history or vaccination being applied more than 12 months before D0, a booster is recommended within 3 months (a largest drop of antibodies was detected within the first 90 days) after the first vaccination to guarantee measurable protection of the population that lasts at least for one year.     

An ion-insensitive cAMP biosensor for long term quantitative ratiometric fluorescence resonance energy transfer (FRET) measurements under variable physiological conditions

Ratiometric measurements with FRET-based biosensors in living cells using a single fluorescence excitation wavelength are often affected by a significant ion sensitivity and the aggregation behavior of the FRET pair. This is an important problem for quantitative approaches. Here we report on the influence of physiological ion concentration changes on quantitative ratiometric measurements by comparing different FRET pairs for a cAMP-detecting biosensor. We exchanged the enhanced CFP/enhanced YFP FRET pair of an established Epac1-based biosensor by the fluorophores mCerulean/mCitrine. In the case of enhanced CFP/enhanced YFP, we showed that changes in proton, and (to a lesser extent) chloride ion concentrations result in incorrect ratiometric FRET signals, which may exceed the dynamic range of the biosensor. Calcium ions have no direct, but an indirect pH-driven effect by mobilizing protons. These ion dependences were greatly eliminated when mCerulean/mCitrine fluorophores were used. For such advanced FRET pairs the biosensor is less sensitive to changes in ion concentration and allows consistent cAMP concentration measurements under different physiological conditions, as occur in metabolically active cells. In addition, we verified that the described FRET pair exchange increased the dynamic range of the FRET efficiency response. The time window for stable experimental conditions was also prolonged by a faster biosensor expression rate in transfected cells and a greatly reduced tendency to aggregate, which reduces cytotoxicity. These properties were verified in functional tests in single cells co-expressing the biosensor and the 5-HT(1A) receptor.     

Complete nucleotide sequence of pSCV50, the virulence plasmid of Salmonella enterica serovar Choleraesuis SC-B67

We carried out comparative analysis on the sequences of two 50-kb virulence plasmids of Salmonella enterica serovar Choleraesuis strains SC-B67 (pSCV50) and RF-1 (pKDSC50). The two plasmids share over 99% sequence similarity. Ninety-two nucleotide variations at 42 sites were detected between the two plasmids; pSCV50 contains 24 nucleotide substitutions, 6 deletions, and 62 insertions, compared to pKDSC50. Two regions in pSCV50 appeared to be more susceptible to changes: one is the non-virulence-associated transfer region (27.5-33.0 K) and the other a function-unknown region (9.0-10.5 K). We re-annotated pSCV50 using more advanced tools and the up-to-date databases and corrected the inaccurate annotation in pKDSC50. The results indicate that virulence-related genes on the 50-kb plasmid are under negative selection, suggesting that they play important roles in the expression of virulence during the process of infection, while other genes in this plasmid tend to evolve neutrally.