Separation and concentration of delta 17-6-keto-PGF1 alpha using monoclonal antibody to omega 3-olefin structure of trienoic prostanoids.

A monoclonal antibody against cis-3-hexen-1-ol was prepared and used to separate and/or concentrate delta 17-6-keto-prostaglandin F1 alpha (PGF1 alpha) in the human sera. cis-3-Hexen-1-ol was conjugated with the human serum albumin (HSA) according to the N-succinimidylester method and hyperimmunized to BALB/c mouse. The monoclonal antibodies were obtained from hybridoma clones established by a fusion between SP2/0-Ag14-k13 mouse myeloma cells and splenocytes of a mouse. A monoclonal antibody, named 4G9-12B, recognized the epitope characteristic for omega 3-olefin structure. The 4G9-12B antibody became more specific for delta 17-6-keto-PGF1 alpha than 6-keto-PGF1 alpha by applying inhibition ELISA using amino-residue coating plates. Using the prepared immunoaffinity columns of this antibody, delta 17-6-keto-PGF1 alpha was clearly detected in 6 pg/ml of the human blood sera by GC/MS analysis. These results suggest that the monoclonal antibody to the partial structure of trienoic prostanoid, omega 3-olefin unit, and that its immunoaffinity columns are useful in separating and concentrating delta 17-6-keto-PGF1 alpha in the human blood or urine.     

Asialoglycoprotein receptor deficiency in mice lacking the major receptor subunit. Its obligate requirement for the stable expression of oligomeric receptor

The asialoglycoprotein receptor is an abundant hetero-oligomeric endocytic receptor that is predominantly expressed on the sinusoidal surface of the hepatocytes. A number of physiological and pathophysiological functions have been ascribed to this hepatic lectin (HL), the removal of desialylated serum glycoproteins and apoptotic cells, clearance of lipoproteins, and the sites of entry for hepatotropic viruses. The assembly of two homologous subunits, HL-1 and HL-2, is required to form functional, high affinity receptors on the cell surface. However, the importance of the individual subunits for receptor transport to the cell surface is controversial. We have previously generated HL-2-deficient mice and showed that the expression of HL-1 was significantly reduced, and the functional activity as the asialoglycoprotein receptor was virtually eliminated. However, we failed to detect phenotypic abnormalities. To explore the significance of the major HL-1 subunit for receptor expression and function in vivo, we have disrupted the HL-1 gene in mice. Homozygous HL-1-deficient animals are superficially normal. HL-2 expression in the liver is virtually abrogated, indicating that HL-1 is strictly required for the stable expression of HL-2. Although these mice are almost unable to clear asialo-orosomucoid, a high affinity ligand for asialoglycoprotein receptor, they do not accumulate desialylated glycoproteins or lipoproteins in the plasma.     

Swapping of the beta-hairpin region between Sp1 and GLI zinc fingers: significant role of the beta-hairpin region in DNA binding properties of C2H2-type zinc finger peptides

The recent design strategy of zinc finger peptides has mainly focused on the alpha-helix region, which plays a direct role in DNA recognition. On the other hand, the study of non-DNA-contacting regions is extremely scarce. By swapping the beta-hairpin regions between the Sp1 and GLI zinc fingers, in this study, we investigated how the beta-hairpin region of the C(2)H(2)-type zinc finger peptides contributes to the DNA binding properties. Surprisingly, the Sp1 mutant with the GLI-type beta-hairpin had a higher DNA binding affinity than that of the wild-type Sp1. The result of the DNase I footprinting analyses also showed the change in the DNA binding pattern. In contrast, the GLI zinc finger completely lost DNA binding ability as a result of exchanging the beta-hairpin region. These results strongly indicate that the beta-hairpin region appears to function as a scaffold and has an important effect on the DNA binding properties of the C(2)H(2)-type zinc finger peptides.     

Phloretin as an Antagonist of Prostaglandin F2α Receptor in Cultured Rat Astrocytes

Abstract: The effect of phloretin on prostaglandin (PG) F_2α-induced phosphoinositide hydrolysis and elevation of intracellular Ca²⁺ concentration was examined in cultured rat astrocytes. Phloretin inhibited PGF_2α (1 μ M )-induced phosphoinositide hydrolysis in a concentration-dependent manner with an IC₅₀ value of 16 μ M . The inhibitory action of phloretin was specific for PGs. The addition of increasing concentrations of phloretin caused progressive shifts of the dose-response curves of PGF_2α to the right. In digitoninpermeabilized astrocytes, phloretin (100 μ M ) inhibited the stimulation induced by PGF_2α (1 μ M ) plus GTPγS (50 μ M ) without affecting that induced by GTPγS alone. PGF_2α at 1 μ M transiently increased astrocytic intracellular Ca²⁺ concentration in 39% of the cells tested. The response was completely blocked by 100 μ M phloretin and the calcium response recovered again after washing out phloretin. These results suggest that phloretin is an antagonist of PGF_2α receptor linked to phospholipase C in astrocytes.     

Outbreak of gastroenteritis caused by the pandemic Vibrio parahaemolyticus O3:K6 in Mexico

During 2003 and during late September of 2004, more than 1230 cases of gastroenteritis were reported in the south of Sinaloa State, north-western Mexico. All cases were attributed to the consumption of raw or undercooked shrimp collected at the Huizache-Caimanero lagunary system. Vibrio parahaemolyticus was identified by standard biochemical methods, and many strains were positive for PCR amplifications of the tlh and tdh genes and negative for the trh gene. A representative strain belonged to the O3:K6 serogroup. This is the first outbreak of gastroenteritis caused by the pandemic strains of O3:K6 V. parahaemolyticus in México.     

Bone Inner Structure Suggests Increasing Aquatic Adaptations in Desmostylia (Mammalia,Afrotheria)

Background

The paleoecology of desmostylians has been discussed controversially with a general consensus that desmostylians were aquatic or semi-aquatic to some extent. Bone microanatomy can be used as a powerful tool to infer habitat preference of extinct animals. However, bone microanatomical studies of desmostylians are extremely scarce.

Methodology/Principal Findings

We analyzed the histology and microanatomy of several desmostylians using thin-sections and CT scans of ribs, humeri, femora and vertebrae. Comparisons with extant mammals allowed us to better understand the mode of life and evolutionary history of these taxa. Desmostylian ribs and long bones generally lack a medullary cavity. This trait has been interpreted as an aquatic adaptation among amniotes. Behemotops and Paleoparadoxia show osteosclerosis (i.e. increase in bone compactness), and Ashoroa pachyosteosclerosis (i.e. combined increase in bone volume and compactness). Conversely, Desmostylus differs from these desmostylians in displaying an osteoporotic-like pattern.

Conclusions/Significance

In living taxa, bone mass increase provides hydrostatic buoyancy and body trim control suitable for poorly efficient swimmers, while wholly spongy bones are associated with hydrodynamic buoyancy control in active swimmers. Our study suggests that all desmostylians had achieved an essentially, if not exclusively, aquatic lifestyle. Behemotops, Paleoparadoxia and Ashoroa are interpreted as shallow water swimmers, either hovering slowly at a preferred depth, or walking on the bottom, and Desmostylus as a more active swimmer with a peculiar habitat and feeding strategy within Desmostylia. Therefore, desmostylians are, with cetaceans, the second mammal group showing a shift from bone mass increase to a spongy inner organization of bones in their evolutionary history.     

Electron-microscopic studies on the threshold value of calcium concentration for the release of storage granules and the acceleration of their degradation in the rat parathyroid gland

Summary To determine both a threshold value of calcium concentration (CC) for the release of storage granules and that for the acceleration of degradation of these granules, the rat parathyroid glands were perfused in situ with HEPES-Ringer solutions containing different concentration of Ca²⁺ for 10 min. With perfusates containing 0.83–1.21 mM Ca²⁺ (equivalent to 8–11 mg/dl serum calcium), the number of type-I storage granules (large core) [NSG-I] and that of type-II storage granules (small core) [NSG-II] remained unchanged. With perfusates containing 0.83 mM Ca²⁺ (7.5 mg/dl) or less, however, both NSG-I and NSG-II decreased remarkably and the former was larger than the latter. On the contrary, with perfusates containing 1.27 mM Ca²⁺ (11.5 mg/dl) or more, NSG-II increased and the ratio of NSG-I to NSG-II was changed reversely. We concluded that a thereshold value of CC required for the release of storage granules may be present between 0.88 and 0.83 mM Ca²⁺ (8 and 7.5 mg/dl) and that a threshold value of CC for accelerating the transformation of type-I granules into type-II, the degradation of storage granules, may be situated at about 1.27 mM Ca²⁺ (11.5 mg/dl). Additionally, it was suggested that both prosecretory and storage granules are not only formed at the innermost Golgi cisterna but also at the trans-Golgi network.     

Restriction map of CELO virus DNA

A restriction map of chicken embryo lethal orphan (CELO) virus DNA was reported with ten restriction endonucleases (XbaI, XhoI, SalI, HindIII, EcoRI, BglI, KpnI, BamHI, PstI and SstI). CELO virus DNA was estimated by comparing CELO virus DNA fragments with marker DNA fragments to have a molecular weight of 29.3·10⁶.