Titanium and implantology: a review in dentistry.

Implant dentistry has become a popular restorative option in clinical practice. Titanium and titanium alloys (TTA) are the gold standard for endo-osseus dental implants production, thanks to their biocompatibility, resistance to corrosion and mechanical properties. The characteristics of the TTA implant surface seem to be particularly relevant in the early phase of osseointegration. Furthermore, the microstructure of implant surface can largely influence the bone remodelling at the level of the bone-implant surface. Recently, research has stated on the long-term of both survival and success rates of osseointegrated implants and mainly on biomechanical aspects, such as load distribution and biochemical and histological processes at the bone-implant interface. This short review reports recent knowledge on chemical and mechanical properties, biological aspects, innovations in preventing peri-implantitis, describing clinical applications and recent improvements of TTA dental implants. In addition, it highlights current knowledge about a new implant coating that has been demonstrated to reduce the number of initially adhering bacteria and peri-implantitis.

Irisin alleviates renal injury caused by sepsis via the NF-κB signaling pathway.

OBJECTIVE: Renal injury caused by sepsis is a difficult point in the field of critical care medicine today, which seriously endangers the health of patients. The aim of our paper was to study the role of irisin in the inflammation and apoptosis of renal injury caused by sepsis and its potential mechanism of action. MATERIALS AND METHODS: Lipopolysaccharide (LPS) was utilized to establish an acute kidney injury model. HK-2 cells were divided into 3 groups: control group, LPS group, LPS+irisin group. The expression of TNF-α, IL-1ß, Bcl-2, and Bax were detected using Western blot. Commercial enzyme-linked immunosorbent assay (ELISA) kits were used to detect the levels of TNF-α, IL-6, and IL-1ß in the cell supernatant. The LDH content was detected to observe cell damage. TUNEL staining and flow cytometry were to investigate the apoptosis in three groups. The viability of HK-2 cells was detected using Cell Counting Kit-8 (CCK-8) assay. RESULTS: After HK-2 cells were treated with LPS, the LDH content in the cell supernatant was greatly increased, and the expression of TNF-α, IL-6, and IL-1ß was also significantly increased. However, after treatment with irisin, LDH content and expression of inflammatory factors were significantly suppressed. Similarly, LPS treatment greatly elevated the levels of TNF-α, IL-1ß, Bax, p65 and IκKα, as well as inhibited the expression of Bcl-2 and IκB-α. However, irisin treatment reversed these situations. In addition, the number of TUNEL-positive cells and the apoptotic rate were also greatly decreased in LPS+irisin group compared with those in LPS group. CONCLUSIONS: Irisin could inhibit inflammation and apoptosis of HK-2 cells treated with LPS via the NF-κB pathway.

Design, evaluation and initial imaging results of a PET insert based on strip-line readout for simultaneous PET/MRI.

We present the development of a PET insert system for potential simultaneous PET/MR imaging using a 9.4 T small animal MRI scanner to test our system. The detectors of the system adopt a strip-line based multiplexing readout method for SiPM signals. In this readout, multiple SiPM outputs in a row share a common strip-line. The position information about a hit SiPM is encoded in the propagation time difference of the signals arriving at the two ends of the strip-line. The use of strip-lines allows us to place the data acquisition electronics remotely from the detector module to greatly simplify the design of the detector module and minimize the mutual electromagnetic interference. The prototype is comprised of 14 detector modules, each of which consists of an 8x4 LYSO scintillator array (each LYSO crystal is 3x3x10 mm3) coupled to two units of Hamamatsu MPPC arrays (4x4, 3.2 mm pitch) that are mounted on a strip-line board. On the strip-line board, outputs of the 32 SiPMs are routed to 2 strip-lines so that 16 SiPM signals share a strip-line. The detector modules are installed inside a plastic cylindrical supporting structure with an inner and outer diameter of 60 mm and 115 mm, respectively, to fit inside a Bruker BioSpec 9.4 Tesla MR scanner. The axial field of view of the prototype is 25.4 mm. The strip-lines were extended by using 5-meter cables to a sampling data acquisition (DAQ) board placed outside the magnet. The detectors were not shielded in the interest of investigating how they may affect and be affected by the MRI. Experimental tests were conducted to evaluate detection performance, and phantom and animal imaging were carried out to assess the spatial resolution and the MR compatibility of the PET insert. Initial results are encouraging and demonstrate that the prototype insert PET can potentially be used for PET/MR imaging if appropriate shielding will be implemented for minimizing the mutual interference between the PET and MRI systems.

MicroRNA-599 regulates the development of Parkinson's disease through mediating LRRK2 expression.

OBJECTIVE: This study investigates whether microRNA-599 can inhibit the progression of Parkinson's disease (PD) by regulating the LRRK2 expression. We aim to search for a new therapeutic target for PD. MATERIALS AND METHODS: A mouse model of PD was first established. A relative amount of TH+ neurons in the mouse brain was quantified by immunohistochemistry. The expression levels of microRNA-599 and LRRK2 in mouse brain tissues were determined by the quantitative Real Time-Polymerase Chain Reaction (qRT-PCR) and Western blot. Cell model of PD was constructed by MPP+ treatment in SH-SY5Y cells. The expression levels of microRNA-599 and LRRK2 in MPP+-induced SH-SY5Y cells were examined as well. We verified the binding condition between microRNA-599 and LRRK2 through dual-luciferase reporter gene assay. The viability and apoptosis in MPP+-induced SH-SY5Y cells overexpressing microRNA-599 were determined by cell counting kit-8 (CCK-8) assay and flow cytometry, respectively. RESULTS: Compared with normal mice, TH+ neurons were fewer in the brain tissue of PD mice. MicroRNA-599 expression was lower, while LRRK2 expression was higher in brain tissues of PD mice relative to controls. Meanwhile, in vitro expression of microRNA-599 was downregulated and LRRK2 expression was upregulated in MPP+-induced SH-SY5Y cells. Dual-luciferase reporter gene assay verified the binding condition between microRNA-599 and LRRK2. The microRNA-599 overexpression downregulated the LRRK2 expression in SH-SY5Y cells, and conversely, the microRNA-599 knockdown upregulated the LRRK2 expression. Of note, the microRNA-599 overexpression protected MPP+-induced viability decrease and apoptosis acceleration in SH-SY5Y cells. CONCLUSIONS: MicroRNA-599 is lowly expressed in both in vivo and in vitro PD model. MicroRNA-599 inhibits the development of PD through regulating the LRRK2 expression.

The important role of manual aspiration through the guiding catheter during repeated solitaire mechanical thrombectomy in acute ischemic stroke.

OBJECTIVE: Acute ischemic stroke (AIS) is an important global health problem. Intravenous (IV) thrombolysis with recombinant tissue plasminogen activator (rt-PA) is the standard treatment. However, only a small number of patients benefit from it because of strict application restrictions. Increasing evidence has demonstrated that mechanical thrombectomy is an effective and safe therapy for AIS. PATIENTS AND METHODS: We present 14 cases of successful recanalization with Solitaire devices for AIS patients after stroke onset. During stent retrieval, continuous manual aspiration was applied through the guiding catheter, and several large pieces of thrombus were aspirated into the catheter along with the clot, which was adhered to the stent. Clinical outcomes were assessed by the NIHSS at discharge and the mRS on follow-up at 90 days. RESULTS: All 14 patients with AIS occlusions were treated with Solitaire stents during the study period. The successful recanalization rate was 100%. On discharge, all patients (100%) had improved (NIHSS of ≥ 10 points). At 90 days, 12 patients (86%) had a good functional outcome with mRS of ≤  2. CONCLUSIONS: We recommend the use of manual aspiration through a guiding catheter as an alternative technique when a specialized aspiration device is not available, to facilitate a fast, complete, and safe thrombus retrieval by the Solitaire system.

[Down-regulation of miR-146b-5p promotes malignant transformation of fusion cells after co-culture of macrophages with glioma stem cells in vitro].

Objective: To observe mutual interactions between macrophages(Mφ) and glioma stem cells (GSCs)in dual-color tracing model in vitro, to identify the biological characteristics of fusion cells in multiple levels, and to analysis the relevant molecular mechanisms. Methods: Red fluorescent protein(RFP) gene was stably transfected into human GSCs cell line SU4. Mφ cells were obtained from Balb/c nude mice with enhanced green fluorescent protein (EGFP) expression. Then two cells were co-cultured in dual-color tracing platform. RFP/EGFP double positive cells with high proliferation ability were mono-cloned. The fusion cells were verified by Western blot, fluorescence in situ hybridization, immunocytochemistry and chromosome karyotype analysis.The biological characteristics of fusion cells were further analyzed, together with relevant molecular changes. Results: RFP / EGFP double positive cells were obtained through in vitro co-culture. RFP and EGFP coexpression were proved at transcriptional and translational levels in the fusion cells. They also co-expressed GSCs marker Nestin and Mφ marker CD68, and karyotype analysis showed two types of characteristic chromosomes, which confirmed that the fusion cells originated from spontaneous fusion between SU4-RFP and Mφ.Fusion cell proliferation rate and invasion ability were higher than SU4-RFP, which were relevant with down-regulation of miR-146b-5p and activation of STAT3. Fusion cells transfected with miR-146b-5p showed a higher apoptosis rate(18.83%) and lower tumor formation(4/5). Conclusion: Mφ could fuse with GSCs spontaneously in local tumor micro-environment. The proliferation and invasion abilities of fusion cells were higher than their parent cells, which were relevant with down-regulation of miR-146b-5p and activation of STAT3. It revealed the possible mechanisms of malignant progression of gliomas.

Seawater Incursion Events in a Cretaceous Paleo-lake Revealed by Specific Marine Biological Markers.

Many large paleo-lakes in North China were formed after the Triassic Era. Seawater incursion events (SWIEs) in these lakes have been extensively discussed in the literature, yet lack reliable methodology and solid evidence, which are essential for reconstructing and confirming SWIEs. The present study employs specific marine biological markers (24-n-propyl and 24-isopropyl cholestanes) to trace SWIEs in a dated core taken from the Songliao Basin (SLB). Two SWIEs were identified. The first SWIE from 91.37 to 89.00 Ma, was continuous and variable but not strong, while the second SWIE from 84.72 to 83.72 Ma was episodic and strong. SWIEs caused high total organic carbon (TOC) and negative δ(13)Corg values in the sediments, which were interpreted as an indication of high productivity in the lake, due to the enhancement of nutrient supplies as well as high levels of aqueous CO2, due to the mixing of alkaline seawater and acidic lake water. The SWIEs in SLB were controlled by regional tectonic activity and eustatic variation. Movement direction changes of the Izanagi/Kula Plate in 90 Ma and 84 Ma created faults and triggered SWIEs. A high sea level, from 90 to 84 Ma, also facilitated the occurrence of SWIEs in SLB.

Nucleotide sequence and polymorphism of MHC class II DQB exon 2 alleles in Chinese yakow (Bos grunniens × Bos taurus).

The objective of this study is to describe the gene characteristics of the bovine lymphocyte antigen (BoLA)-DQB exon 2 locus in Chinese yakow (Bos grunniens × Bos taurus) and to compare it with previously reported patterns in other bovidae species to investigate genetic factors for disease resistance. The exon 2 of the MHC class II DQB gene was cloned and sequenced. It was revealed by sequence analyses that there are 36 DQB exon 2 alleles among 44 Chinese yakow. These alleles exhibited a high degree of nucleotide and amino acid polymorphism with most amino acid variations occurring at positions forming the peptide-binding sites (PBS). The DQB loci were analysed for patterns of synonymous (dS ) and nonsynonymous (dN ) substitution. The Chinese yakow was observed to be under strong positive selection in the DQB exon 2 peptide-binding sites (dN = 0.147, P < 0.01). It appears that this variability among Chinese yakow confers the ability to mount immune responses to a wide variety of peptides or pathogens.

Molecular characteristics of the MHC-DRA genes from yak (Bos grunniens) and Chinese yakow (Bos grunniens × Bos taurus).

Two full-length cDNAs (762 bp) of the DRA gene from yak and Chinese yakow were isolated and analysed to identify structural and functional variations. The sequences for DRA in yak (Bogr-DRA) and Chinese yakow (Bogr × BoLA-DRA) were essentially identical to those for cattle (99%) and buffalo (97%). Except for two substitutions in the amino acids comprising the domain for signal peptide (SP) in yak, the additional residues were highly conserved across the species investigated. Peptide-binding site (PBS) of Bogr-DRA and Bogr × BoLA-DRA was highly reserved in the α1 domain among all species investigated. The lack of mutation in Bogr-DRA is consistent with the conception that the gene is highly conserved among all mammalian species. The very high conservation of the DRA gene among ruminants, including yak, may be due to its recent evolutionary detachment.

First Report of Chilli veinal mottle virus Infecting Tomato (Solanum lycopersicum) in China.

Chilli veinal mottle virus (ChiVMV), a potyvirus, is widespread over the world. In China, it was first reported in chili pepper (Capsicum annuum) in Hainan Province (south China) in 2006 (2). Subsequently, it was reported in tobacco (Nicotiana tabacum) in Yunnan Province (southwest China) in 2011 (1). Sichuan Province is one of the largest vegetable producing areas of China. In May 2012, tomatoes with leaves displaying virus-infected symptoms like mottling, mosaic, narrowing, or curling were observed in several fields of Chengdu, eastern Sichuan Province, southwest China. Of the 20 fields we investigated, four fields with 90% tomato plants were infected. During 2012 and 2013, six samples were collected from symptomatic tomato leaves based on different symptoms and locations. All six samples were assayed by western blotting using polyclonal antisera (Cucumber mosaic virus [CMV], Tobacco mosaic virus [TMV]) obtained from Agdia (Elkhart) and one antiserum to ChiVMV obtained from Yunnan Academy of Agricultural Science (China). Two samples from Pengzhou and one sample from Shuangliu exhibiting mosaic leaves were positive for TMV, one sample from Pixian exhibiting narrowing leaves was positive for CMV, and the other two samples from Shuangliu exhibiting mottle and leaf distortion were positive for ChiVMV. Total RNAs was extracted from all six samples and healthy tomato leaves using Trizol reagent (Invitrogen), First-strand cDNA synthesis primed with oligo(dT) by SuperScript III Reverse Transcriptase (Invitrogen). RT-PCR was performed using primer pairs ChiVMV-CP F (5'-GCAGGAGAGAGTGTTGATGCTG-3') and ChiVMV-CP R (5'-(T)16AACGCCAACTATTG-3'), which were designed to direct the amplification of the entire capsid protein (CP) gene and 3' untranslated region (3'-UTR) of ChiVMV (GenBank Accession No. KC711055). The expected 1,166-bp DNA fragment was amplified from the two tomato samples from Shuangliu that were positive for ChiVMV in the western blot tests, but not from the others. The obtained fragments were purified and cloned into the PMD18-T vector (TaKaRa) and sequenced. The sequencing results showed that the two ChiVMV isolates from tomato in Shuangliu were identical (KF738253). Nucleotide BLAST analysis revealed that this ChiVMV isolate shared ~84 to 99% nucleotide identities with other ChiVMV isolates available in GenBank (KC711055 to KF220408). To fulfill Koch's postulates, we isolated this virus by three cycle single lesion isolation in N. tabacum, and mechanically inoculated it onto tomato leaves. The same mottle and leaf distortion symptoms in systemic leaves were observed. Subsequent RT-PCR, fragment clone, and sequence determination tests were repeated and the results were the same. All the evidence from these tests revealed that the two tomato plants were infected by ChiVMV. To our knowledge, this is the first report of ChiVMV naturally infecting tomato in China. It shows that ChiVMV is spreading in China and is naturally infecting a new solanaceous crop in the southwest area, and the spread of the virus may affect tomato crop yields in China. Thus, it is very important to seek an effective way to control this virus. References: (1) M. Ding et al. Plant Dis. 95:357, 2011. (2) J. Wang et al. Plant Dis. 90:377, 2006.

First Report of Sweet potato chlorotic fleck virus Infecting Sweet Potato in Sichuan Province, China.

Sweet potato chlorotic fleck virus (SPCFV) is one of several viruses naturally infecting sweet potato (Ipomoea batatas L.), and it has recently been classified as a new member of the genus Carlavirus (family Flexiviridae) (1). However, SPCFV is distantly related to typical carlaviruses, as most of its putative gene products share amino acid sequence identities of <40% with those of typical carlaviruses (1). China is the largest sweet potato producing country in the world. So far, SPCFV has been reported in eastern China, such as Jiangsu, Anhui, Henan (3), and Guangdong (2) provinces, but no reports exist in western China. Sichuan Province, located in southwestern China, is the largest sweet potato producing area in the country. There are big differences between the environment and climate conditions between Sichuan and eastern China. During 2012, a survey was constructed to determine the genetic diversity and distribution of sweet potato viruses in Sichuan. Forty-seven sweet potato samples exhibiting virus-like symptoms were collected from four different geographic areas of the province. Western blotting using the antisera obtained from the International Potato Center showed that two samples were positive for SPCFV, whereas with reverse transcription (RT)-PCR, only one isolate of SPCFV was obtained from a sample exhibiting symptoms of chlorosis, leaf distortion, and vein clearing. Serological detection indicated that the plant was co-infected with SPCFV, Sweet potato feathery mottle virus (SPFMV), and Sweet potato virus G (SPVG). Total RNA was extracted from symptomatic leaves using Trizol reagent (Invitrogen) according to the manufacturer's protocol, and RT-PCR was performed by using primer pairs SPCFV-CP F (5'-ATGGCGGCGAAGGAGGCTGATA-3') and SPCFV-CP R (5'-TCACTTGCACTTCCCATTAC-3') corresponding to the entire coat protein (CP) gene of SPCFV. Expected DNA fragments of 900 bp were obtained from the symptomatic plant but not from control plants. The obtained fragments were purified and cloned into the PMD19-T vector (TaKaRa). Recombinant plasmids were then transformed into competent cells of Escherichia coli strain DH5α. Nucleotide BLAST analysis revealed that the 900-bp fragment (GenBank Accession No. KC414676) shared 87 to 91% nucleotide identities with other SPCFV isolates available in the GenBank database. To our knowledge, this is the first report of the co-infection between SPCFV and other sweet potato viruses including SPFMV and SPVG in China, and this is the first molecular report of SPCFV in Sichuan, western China. It shows that SPCFV is spreading to a new ecological area of China, and the spread of the virus may affect sweet potato crop yields in western China. Some measures must be carried out quickly to control the virus. References: (1) V. Aritua et al. Arch. Virol. 152:813, 2007. (2) V. Aritua et al. Plant Dis. 93:87, 2009. (3) Q. Wang et al. Crop. Prot. 29:110, 2010.

Preparation, characterization and performance of a novel PVDF/PMMA/TPU blend hollow fiber membrane for wastewater treatment.

Polyvinylidene fluoride (PVDF)/polymethylmethacrylate (PMMA)/thermoplastic polyurethane (TPU) blend hollow fiber membranes were successfully prepared by the wet-spinning method with the loading of PMMA and TPU in a range of polymer concentrations varying from 0 to 20 wt% and at a total polymer concentration of 16 wt%. The influence of the addition of PMMA and TPU on the morphologies and the properties of such prepared membranes was investigated through FTIR-ATR, SEM, viscosity measurements, UF experiments and mechanical strength tests. Based on the experimental results, the compatibility of the PVDF, PMMA and TPU blend was best under the conditions of the PVDF-rich phase. The elongation at break of the membrane increased to a maximum of 146% with increase in the TPU concentration to 20 wt% in dope solution. The addition of PMMA increased the water permeation flux from 120 to 195 L/(m(2) h) initially. The flux then decreased when PMMA concentration was increased to over 10 wt%. The membranes obtained at optimized blending ratio were applied to the dyeing process wastewater filtration. During continuous filtration for 8 h, the flux was stabilized at about 20 L/(m(2) h) at 0.1 MPa. The reduction in COD(Cr), turbidity and color were about 63, 84 and 63% respectively.

Isolation, sequence identification and expression profile of three novel genes Rab2A, Rab3A and Rab7A from Black-boned sheep (Ovis aries).

Complete coding sequences of three Black-boned sheep (Ovis aries) genes Rab2A, Rab3A and Rab7A were amplified using reverse transcription polymerase chain reaction (RT-PCR) based on the conserved sequence information of cattle or other mammals known to be highly homologous to sheep ESTs. The Black-boned sheep Rab2A gene encodes a protein of 226 amino acids which contains the conserved putative RabL2 domain and is highly homologous to the Rab2A proteins of seven other species--cattle (96%), human (83%), Sumatran orangutan (82%), rat (81%), mouse (80%), African clawed frog (72%) and zebrafish (71%). The Black-boned sheep Rab3A gene encodes a protein of 220 amino acids that contains the conserved putative Rab3 domain and is very similar to the Rab3A proteins of four species--cattle (99%), African clawed frog (99%), Western clawed frog (98%) and zebrafish (95%). And the Black-boned sheep Rab7A gene encodes a protein of 207 amino acids that contains the conserved putative Rab7 domain and has high homology with the Rab7A proteins of six other species--human (99%), dog (99%), Sumatran orangutan (99%), zebrafish (97%), rabbit (97%) and African clawed frog (96%). Analysis of the phylogenetic tree has demonstrated that the Black-boned sheep Rab2A, Rab3A and Rab7A proteins share a common ancestor and the tissue expression analysis has shown that the corresponding genes are expressed in a range of tissues including leg muscle, kidney, skin, longissimus dorsi muscle, spleen, heart and liver. Our experiment is the first to provide the primary foundation for a further insight into these three sheep genes.

Phylogenetic analysis of 16S rRNA gene sequences reveals rumen bacterial diversity in Yaks (Bos grunniens).

Six matured male Yaks (Bos grunniens) with a mean live weight of 450 +/- 23 kg (mean +/- SD), were housed indoors in metabolism cages and fed pelleted lucerne (Medicago sativum). After an adjustment period of 24 days of feeding the diet, samples of rumen content were obtained for analysis of the bacteria in the liquor. The diversity of rumen bacteria was investigated by constructing a 16S rRNA gene clone library using the general bacterial primers F27 and R1492. A total of 130 clones, comprising nearly full length sequences (approx. 1.5 kb) were sequenced and submitted to BLAST and phylogenetic analysis. Using the criterion that similarity of 97% or greater with the sequences of cultivated bacteria, 16 clones were identified as Butyrivibrio fibrisolvens, Pseudobutyrivibrio ruminis, Ruminococcus flavefaciens, Succiniclasticum ruminis, Selenomonas ruminantium and Prevotella ruminicola, respectively. A further 10 clones shared similarity ranging from 90 to 97% with cultivated bacteria but the similarity in sequences for the remaining 104 clones were less than 90% of those of cultivated bacteria. Using a phylogenetic analysis it was found that the majority of the clones identified (63.8%) were located in the Low G + C Subdivision, with most of the remainder (35.4% of clones) located in the Cytophaga-Flexibacter-Bacteroides phylum and one clone (0.8%) was identified as a Proteobacteria. It was apparent that Yaks have a large and diverse range of bacteria in the rumen content which differ from those of cattle and other ruminants.

Pigmentation in Black-boned sheep (Ovis aries): association with polymorphism of the Tyrosinase gene.

Measurements were made in Black-boned (n = 40) and normal (n = 23) sheep (Ovis aries) from a flock in Nanping County of Yunnan Province, China, as well as a group (n = 21) of Romney Marsh sheep (O. aries) with the view to explaining the basis of the dark pigmentation occurring in the Black-boned animals. Plasma colour was significantly darker (P < 0.01) in Black-boned sheep than in their normal flock mates, which in turn had significantly darker plasma (P < 0.01) than the Romney Marsh sheep. Similar significant (P < 0.01) differences were measured for plasma tyrosinase activity and both groups of sheep from Nanping County had similar plasma concentrations of glutathione which were significantly smaller (P < 0.01) than for the Romney Marsh sheep.A partial fragment of 750 bp of exon 1 of the gene encoding tyrosinase was constructed and found to contain two silent mutation sites (G192C and C462T) but there was no effect on amino acid sequences of tyrosinase. Using restriction fragment length polymorphism analyses two allelic variants of site G192C were identified giving rise to the genotypes GG, GC and CC; the frequencies of allele G being 0.914, 0.824 and 0.286 in the Black-boned sheep, their flock mates and the Romney Marsh sheep respectively. Plasma tyrosinase activity was similar for genotypes GG and GC and for both genotypes significantly higher (P < 0.05) than for genotype CC. The sheep from Nanping County displayed only the GG and GC genotypes and had predominantly black or black and white coat colour whereas the Romney Marsh sheep were of either genotype GC or CC and exhibited only white coat colouration. It is not appears that the dark pigmentation of the Black-boned sheep arises because of polymorphisms in the exon 1 of tyrosinase gene. However, this result could explain the differences between Black-boned and Romney Marsh sheep but not for differences between Black-boned and Nanping Normal sheep. Moreover, this result has provided evidence of genetic markers in the form of polymorphisms of the tyrosinase gene which may help to find the black traits causing mutations. There would be merit in further studies using histochemical and molecular techniques to elucidate the causes of the dark pigmentation in these Black-boned sheep.

Quantitative analysis of oxytocin and vasopressin messenger ribonucleic acids in single magnocellular neurons isolated from supraoptic nucleus of rat hypothalamus.

Oxytocin (OT) and vasopressin (VP) are peptide hormones that are derived from genes predominantly expressed in distinct magnocellular neurons in the paraventricular (PVN) and supraoptic (SON) nuclei of the hypothalamus. Recent evidence suggests that some magnocellular neurons coexpress both peptides. Our qualitative RT-PCR experiments on single cells show that the majority of magnocellular neurons coexpress both peptide messenger RNAs (mRNAs) in varying amounts. Using a competitive RT-PCR method combined with a standard calibration curve, we quantitatively determined OT and VP mRNA in single magnocellular neurons from the normal female rat SON, with a detection sensitivity of less than 30 mRNA molecules/cell. We defined the phenotypes of the single magnocellular neurons according to their ratios of these two peptide mRNAs. Using this approach, we identified three major phenotypes: oxytocin neurons, where the average OT to VP mRNA ratio is about 256; vasopressin neurons, where the average VP to OT mRNA ratio is about 182; and one oxytocin/vasopressin coexisting neuron, where the OT/VP mRNA ratio is 2. Thus, there is some OT and VP mRNA coexpression in virtually all of the magnocellular neurons in supraoptic nuclei of hypothalamus. However, clear phenotypes are identifiable by considering quantitative as opposed to qualitative differences.

Analysis of synaptotagmin I-IV messenger RNA expression and developmental regulation in the rat hypothalamus and pituitary.

Synaptotagmins are a large family of synaptic vesicle membrane proteins, that appear to be involved in neurotransmitter secretion from small secretory vesicles. We have quantitatively analysed the messenger RNA levels of synaptotagmin I-IV isoforms in adult hypothalamic and pituitary tissues in order to determine which of these isoforms dominate in these tissues which mainly secrete peptides from large dense core vesicles. We also studied the expression of these isoforms during prenatal (E15, and E17) and postnatal (P1, P7, P14 and P21) rat hypothalamic development. In order to assay small individual samples (e.g., pituitary and embryonic tissues), we employed quantitative reverse transcription-polymerase chain reaction methods. Our results show that synaptotagmin I messenger RNA is the most abundant isoform in all tissues, and is about 5.4- or 38-fold higher in hypothalamus than in neurointermediate and anterior pituitary lobe, respectively. Synaptotagmin II, which is very abundant in cerebellum, is relatively low in hypothalamus (5% of cerebellum) and virtually absent from the pituitary. Synaptotagmin III is about 10 times greater in the neural tissues versus the pituitary, and synaptotagmin IV was the least abundant isoform in all the tissues. Developmental analyses of the synaptotagmin isoforms in rat hypothalamus shows that all isoforms are at low levels during embryonic stages and increase postnatally. Synaptotagmin I and II have similar patterns and rise to maximum (adult) levels around P14, whereas synaptotagmin III and IV reach their maximum levels considerably earlier, at P1. These data show that synaptotagmin I is the dominant isoform in both predominantly peptide secreting systems (e.g., in pituitary tissues) and in neurotransmitter secreting systems (e.g., in cerebellum). While the developmental expression patterns of synaptotagmin I and II parallels the temporal development of synaptogenesis in the nervous system, the early maximal expression of synaptotagmin III and IV suggests that these isoforms may have other functions during early postnatal development.

Domoic acid is a potent neurotoxin to neonatal rats.

Domoic acid induces a time-dependent neuroexcitotoxic effect in neonatal rats characterized by hyperactivity, stereotypic scratching, convulsions, and death with observable behaviors occurring at exposures 40 times lower by body weight in neonates than reported in adults. Low doses of domoic acid (0.1 mg/kg) induced c-fos in the central nervous system which was inhibited in part by 2-amino-5-phosphonovaleric acid, an NMDA receptor antagonist. Domoic acid caused no evidence of structural alteration in the brain of neonates as assessed by Nissel staining and cupric silver histochemistry. Domoic acid induced reproducible behavioral effects at doses as low as 0.05 mg/kg and induced seizures doses as low as 0.2 mg/kg. Determination of serum domoic acid levels after 60 min exposure indicated that serum levels of domoic acid in the neonates corresponded closely to the serum levels that induce similar symptoms in adult rats and mice. We conclude that neonatal rats are highly sensitive to the neuroexcitatory and lethal effects of domoic acid and that the increased sensitivity results from higher than expected serum levels of domoic acid. These findings are consistent with other findings that reduced serum clearance of domoic acid is a predisposing factor to domoic acid toxicity.

Glutamate receptors and calcium entry mechanisms for domoic acid in hippocampal neurons.

Domoic acid (50 nM) elevates cytosolic free calcium ([Ca2+]i) levels in 49% of the hippocampal pyramidal neurons isolated from postnatal day one (PND1) rats. This effect was prevented by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; an antagonist of non-N-methyl-D-asparate (NMDA) receptors, but not 2-amino-5-phosphonovaleric acid (AP-5; an antagonist of NMDA receptors). Domoic acid given at 5 microM also elevated [Ca2+]i levels in a second population (36%) of neurons in which the effect was only partially inhibited by 100 microM CNQX. Nimodipine given at 300 nM prevented the elevation in [Ca2+]i caused by 50 nM and 5 microM domoic acid, indicating that domoic acid induced Ca2+ entry through type L voltage dependent calcium channels. These results provide evidence for at least two domoic acid-sensitive non-NMDA receptor subtypes in primary cultures of neonatal hippocampal pyramidal cells and indicate that voltage-dependent calcium channels are a primary calcium entry mechanism for domoic acid action.

Maitotoxin-elevated cytosolic free calcium in GH4C1 rat pituitary cells nimodipine-sensitive and -insensitive mechanisms.

Maitotoxin includes an extracellular Ca2+-dependent membrane depolarization predominantly via activation of L-type voltage-dependent Ca2+ channels (L-VDCC) in GH4C1 rat pituitary cells. In contract to studies employing intracellular dyes, electrophysiological studies have indicated that maitotoxin activates voltage-independent conductances. In the present study, we used fura-2 calcium digital analysis to investigate the actions of very low concentrations of maitotoxin on cytosolic free calcium ([Ca2+]i) in GH4C1 cells in an effort to distinguish different calcium entry mechanisms. Maitotoxin at concentrations as low as 0.01 ng/mL elevated [Ca2+]i 35 +/- 3% and induced membrane depolarization. The concentration dependency for maitotoxin-elevated [Ca2+]i was biphasic with the first phase maximal at 0.05 to 0.5 ng/mL and the minimum EC50 of the second phase about 2.0 ng/mL. Nimodipine (100 nM), a dihydropyridine antagonist of L-VDCC, prevented the [Ca+2]i increase and depolarization induced by up to 0.1 ng/mL maitotoxin, but not at higher concentration (0.5 ng/mL) of maitotoxin. This indicates that lower concentrations (0.1 ng/mL) of maitotoxin require L-VDCC, whereas higher concentrations (>-0.5 ng/mL) of maitotoxin may require additional ionic mechanisms. Maitotoxin (0.5 ng/mL) induced 45Ca2+ uptake and depolarization in Ltk-cells which lack VDCC. Reducing extracellular Cl- from 123 to 5.8 microM increased the magnitude of membrane depolarization by maitotoxin (0.5 ng/mL), which suggests that a Cl- conductance participated in depolarization induced by higher maitotoxin concentrations. Taken together, our results indicate that maitotoxin activates at least two ionic mechanisms. At lower concentrations of maitotoxin, the primary ionic mechanism requires the activation of L-VDCC; however, at higher maitotoxin concentrations, additional ionic mechanisms are involve in the entry of extracellular Ca2+. This latter mechanism may represent the voltage-independent pathway evident under voltage clamp conditions.