Quantifying small numbers of antibodies with a 'near-universal' protein-DNA chimera

We present general means to greatly increase the sensitivity of antibody-based assays. Augmentation relies on a 'tadpole' protein-DNA chimera whose protein moiety binds most classes of mammalian antibodies but not avian immunoglobulin Y (IgY). We used this tadpole in affinity capture assays followed by real-time PCR to quantify numerous molecules, including prostate-specific antigen (PSA) in human serum, with great sensitivity and accuracy.     

Genome-wide association study in RPGRIP1(-/-) dogs identifies a modifier locus that determines the onset of retinal degeneration

Cone-rod dystrophy (CRD) is a form of inherited retinal degeneration (RD) causing blindness in man as well as in several breeds of dog. Previously, a 44 bp insertion in RPGRIP1 (retinitis pigmentosa GTPase regulator interacting protein-1) was associated with a recessive early-onset CRD (cone-rod dystrophy 1, cord1) in a Miniature longhaired dachshund (MLHD) research colony. Yet in the MLHD pet population, extensive range of the onset age has been observed among RD cases, with some RPGRIP1(-/-) dogs lacking obvious clinical signs. Phenotypic variation has been known in human homologous diseases, including retinitis pigmentosa and Leber congenital amaurosis, indicating possible involvement of modifiers. To explore additional genetic loci associated with the phenotypic variation observed in MLHDs, a genome-wide association study was carried out using Canine SNP20 arrays in 83 RPGRIP1(-/-) MLHDs with variable ages of onset or no clinical abnormality. Using these samples, comparison of 31 early-onset RD cases against 49 controls (15 late-onset RD and 34 normal dogs combined) identified a strong association (P = 5.05 × 10(-13)) at a single locus on canine chromosome 15. At this locus, the majority of early-onset RD cases but few of the controls were homozygous for a 1.49 Mb interval containing ~11 genes. We conclude that homozygosity at both RPGRIP1 and the newly mapped second locus is necessary to develop early-onset RD, whereas RPGRIP1(-/-) alone leads to late-onset RD or no apparent clinical phenotype. This study establishes a unique model of canine RD requiring homozygous mutations at two distinct genetic loci for the manifestation of early-onset RD.     

Rapid identification of marine bioluminescent bacteria by amplified 16S ribosomal RNA gene restriction analysis

To rapidly identify natural isolates of marine bioluminescent bacteria, we developed amplified ribosomal DNA restriction analysis (ARDRA) methods. ARDRA, which is based on the restriction patterns of 16S rRNA gene digested with five enzymes (EcoRI, DdeI, HhaI, HinfI, RsaI), clearly distinguished the 14 species of marine bioluminescent bacteria currently known, which belong to the genera Vibrio, Photobacterium, and Shewanella. When we applied ARDRA to 129 natural isolates from two cruises in Sagami Bay, Japan, 127 were grouped into six ARDRA types with distinctive restriction patterns; these isolates represented the bioluminescent species, P. angustum, P. leiognathi, P. phosphoreum, S. woodyi, V. fischeri, and V. harveyi. The other two isolates showing unexpected ARDRA patterns turned out to have 16S rRNA gene sequences similar to P. leiognathi and P. phosphoreum. Nevertheless, ARDRA provides a simple and fairly robust means for rapid identification of the natural isolates of marine bioluminescent bacteria, and is therefore useful in studying their diversity.     

Advances toward new antidepressants beyond SSRIs: 1-aryloxy-3-piperidinylpropan-2-ols with dual 5-HT1A receptor antagonism/SSRI activities. Part 5

A series of 1-aryloxy-3-piperidinylpropan-2-ols possessing potent dual 5-HT1A receptor antagonism and serotonin reuptake inhibition was discovered. 1-(1H-Indol-4-yloxy)-3-(4-benzo[b]thiophen-2-ylpiperidinyl)propan-2-ols exhibited selective and high affinities at the 5-HT1A receptor and serotonin reuptake site in vitro. In vivo evaluation of this series of compounds demonstrated elevated extracellular serotonin levels from the basal and quick recovery of neuron firing that was presumably suppressed by the initial acute activation of 5-HT1A somatodendritic autoreceptors.     

Screening system for <Emphasis Type="SmallCaps">d</Emphasis>-Asp-containing proteins using <Emphasis Type="SmallCaps">d</Emphasis>-aspartyl endopeptidase and two-dimensional gel electrophoresis

d-Asp-containing proteins have been implicated in many aging-related diseases. To clarify the role of d-Asp-containing proteins in such diseases, we developed a screening system for these proteins using a d-aspartyl endopeptidase that specifically cleaves the proteins at the C-terminus. The digested proteins were detected by means of two-dimensional gel electrophoresis and identified using nano-liquid chromatography/tandem mass spectrometry. We were able to detect myelin basic protein, a known d-Asp-containing protein, in the brain tissues of mice; this indicates that our system is effective for screening d-Asp-containing proteins.     

A novel fractionation method of the rough ER integral membrane proteins; Resident proteins versus exported proteins?

We treated the high salt‐washed canine pancreatic rough ER (KRM) with 0.18% Triton X‐100, separated the extract from the residual membrane (0.18%Tx KRM), and processed the extract with SM‐2 beads to recover membrane proteins in proteoliposomes. To focus on integral membrane proteins, KRM, 0.18%Tx KRM and proteoliposomes were subjected to sodium carbonate treatment, and analyzed by 2‐D gel electrophoresis. Consequently we found that a distinct group of integral membrane protein of KRM preferentially extracted from the membrane and recovered in proteoliposomes did exist, while majority of KRM integral membrane proteins were fractionated in 0.18%Tx KRM, which retained the basic structure and functions of KRM. Protein identification showed that the former group was enriched with proteins exported from the ER and the latter group comprised mostly of ER resident proteins. This result will potentially affect the prevailing view of the ER membrane structure as well as protein sorting from the ER.     

Characterization of Wistar–Kyoto rats showing hyperadiponectinemia

AimsWistar–Kyoto rats (HA-WKY) kept in the author's laboratory showed higher levels of serum adiponectin (approximately 4-fold) compared with Wistar–Kyoto/Izm rats (WKY/Izm), a WKY standard strain, at 6 weeks old. In a preliminary study, HA-WKY but not WKY/Izm showed hyperinsulinemia and severe hypercholesterolemia when fed a high-fat diet. This study analyzed the differences between HA-WKY and WKY/Izm to investigate the causes of hyperadiponectinemia.Main methodsSix-week-old male HA-WKY and WKY/Izm were used for an analysis of adiponectin-related factors.Key findingsThe main intermediates in the adiponectin signaling pathway, AMP-activated protein kinase and peroxisome proliferator-activated receptor α, were activated at similar levels in liver and skeletal muscle between HA-WKY and WKY/Izm, although HA-WKY had not only higher adiponectin concentrations but also extremely high levels of high-molecular weight (HMW, polymer) adiponectin, which is the active form. The main difference between HA-WKY and WKY/Izm was the existence of adiponectin, mainly middle-molecular weight (MMW, hexamer) and HMW adiponectin multimers, in skeletal muscle extracts from WKY/Izm but not HA-WKY. Skeletal muscle in WKY/Izm expressed much higher amounts of T-cadherin, a receptor for MMW and HMW adiponectin multimers, than that in HA-WKY. In contrast, there was no significant difference in the expression level of adiponectin receptor 2 for trimer, MMW, and HMW adiponectin multimers.SignificanceThe results suggested that the existence of adiponectin in WKY/Izm skeletal muscle was due to the binding of serum adiponectin. The difference in serum adiponectin concentrations between HA-WKY and WKY/Izm could come from the difference in adiponectin binding to skeletal muscle.     

Analgesic effects of D-amino acids in four inbred strains of mice

1. Prominent strain differences of mice were found in analgesic effects of D-amino acids. 2. In C57BL/6CrSlc and C3H/HeSlc mice, pain threshold, which was determined by using a hot-plate method, increased to 140-175% of the control after the systemic treatment of all three D-amino acids employed, such as D-phenylalanine, -leucine and -methionine, whereas in DBA/2CrSlc or BALB/cCrSlc mice, out of three only one D-amino acid, D-phenylalanine or -leucine, produced significant increase of pain threshold. 3. This lack of ability to perceive analgesic effects of specific amino acids observed in the latter two strains suggests that there probably exist different analgesia-inducing mechanisms for each of three D-amino acids in mice and the latter two strains lack two of them.