An improved method based on the Porter-Silber reaction for determining 17-hydroxycorticosteroids in urine

This paper describes a highly specific method for determining urinary 17-hydroxycorticosteroids, which has been developed by (i) changing the composition of the Porter-Silber reagent and (ii) removing contaminants interfering with the color reaction by addition of sodium bisulfite to β-glucuronidase-hydrolyzed urine before extraction with solvent. For a reference method the Norymberski-Riondel (J. K. Norymberski and A. Riondel 1970, Biochem. J. 120, 493–498) gas chromatography (glc) was used: Correlation coefficient between the present method and GLC = 0.988, deviation from the theoretical regression LINE = 6.8%, and coefficient of SIMILARITY = 0.56. These results are much better than those obtained by I. Ernest, B. Håkansson, J. Lehmann, and B. Sjögren (1964, Acta Endocrinol. 46, 552–562) for the original Porter-Silber method in comparison with the chromatographic measurement of grouped and individual steroids.     

Adenovirus infection induces HuR relocalization to facilitate virus replication

HuR is an RNA-binding protein of the embryonic lethal abnormal vision (ELAV) family, which binds to the AU-rich element (ARE) in the 3′-untranslated region (UTR) of certain mRNAs and is involved in the nucleo-cytoplasmic export and stabilization of ARE-mRNAs. The cytoplasmic relocalization of ARE-mRNAs with several proteins such as HuR and pp32 increases in cells transformed by the adenovirus oncogene product E4orf6. Additionally, these ARE-mRNAs were stabilized and acquired the potential to transform cells. Although, the relocalization of HuR and the stabilization of ARE-mRNAs are crucial for cell transformation, evidence regarding the relationship of HuR and ARE-mRNAs with adenovirus replication is lacking. In this report, we demonstrate that adenovirus infection induces the relocation of HuR to the cytoplasm of host cells. Analysis using the luciferase-ARE fusion gene and the tetracycline (tet)-off system revealed that the process of stabilizing ARE-mRNAs is activated in adenovirus-infected cells. Heat shock treatment or knockdown-mediated depletion of HuR reduced adenovirus production. Furthermore, expression of ARE-including viral IVa2 mRNA, decreased in HuR-depleted infected cells. These results indicate that HuR plays an important role in adenovirus replication, at least in part, by up-regulating IVa2 mRNA expression and that ARE-mRNA stabilization is required for both transformation and virus replication.     

MK-8776, a novel Chk1 inhibitor,exhibits an improved radiosensitizing effect compared to UCN-01 by exacerbating radiation-induced aberrant mitosis

Checkpoint kinase 1 (Chk1) is an evolutionarily conserved serine/threonine kinase that plays an important role in G₂/M checkpoint signaling. Here, we evaluate the radiosensitizing effects of a novel selective Chk1 inhibitor MK-8776, comparing its efficacy with a first-generation Chk1 inhibitor UCN-01, and attempt to elucidate the mechanism of radiosensitization. In a clonogenic survival assay, MK-8776 demonstrated a more pronounced radiosensitizing effect than UCN-01, with lower cytotoxicity. Importantly, radiosensitization by MK-8776 can be achieved at doses as low as 2.5 Gy, which is a clinically applicable irradiation dose. MK-8776, but not UCN-01, exacerbated mitotic catastrophe (MC) and centrosome abnormalities, without affecting repair kinetics of DNA double strand breaks. Furthermore, live-cell imaging revealed that MK-8776 significantly abrogated the radiation-induced G₂/M checkpoint, prolonged the mitotic phase, and enhanced aberrant mitosis. This suggests that Chk1 inhibition by MK-8776 activates a spindle assembly checkpoint and increases mitotic defects in irradiated EMT6 cells. In conclusion, we have shown that, at minimally toxic concentrations, MK-8776 enhances radiation-induced cell death through the enhancement of aberrant mitosis and MC, without affecting DNA damage repair.     

Full sequence analysis of the original Sapporo virus

In this study, the full-length genome sequence of the prototype of sapovirus, namely Sapporo virus (SV82), was identified. Sapporo virus RNA was extracted from a fecal sample, amplified by RT-PCR and the PCR products sequenced directly and analyzed. Sequence analysis showed that Sapporo virus consists of 7433 nucleotides and has three open reading frames. The Sapporo strain shows 91.7% nucleotide sequence identity to the Manchester virus. Phylogenic analysis has also revealed the closeness of Sapporo virus to other sapovirus/genogroup I strains. Basic information on the evolutionary history of sapovirus analysis is provided here.     

The clinical and laboratory manifestations profile of antiphospholipid syndrome among Saudi Arabia population: Examining the applicability of Sapporo criteria

Antiphospholipid syndrome is a organized autoimmune disease presented with vascular thrombosis and pregnancy morbidity. The Sapporo classification criteria of APS were revised in 2006 and are used as the main diagnosis guideline, which validity as standard measurements is still in debate. This study observe the clinical and laboratory indices of APS among Saudi patients. This is a retrospective study hospital-based population. The clinical and Laboratory manifestations of diagnosed APS patients from electronical medical records identifies by ICD-9 code 795.79 in the King Saud University Medical City, Riyadh, Saudi Arabia, between 1990 and 2012. We selected patients with ICD-9 code 795.79 as. Sapporo criteria applied to all patients, then divided into cases fulfilled criteria and cases failed the criteria. To notice the difference in clinical and laboratory indices and comorbidities between the two groups, the T-test was performed and Logistic regression for the fulfilled criteria and clinical indices of vascular thrombosis, DVT/PE, recurrent, and pregnancy morbidity. A total of 72 (90%) females and 8 (10%) males, with the female-to-male ratio 9:1. The mean (±SD) age at diagnosis was 28.1 (±8.7) years (range 11–63 years). There were 22 patients (27.5%) attained the revised criteria (APS confirmed) and no significant difference between the two groups was observed (p > 0.2). However, we found Sapporo confirmed APS cases had significantly higher percentage of serological manifestation presence than clinically diagnosed APS cases. Though there is no statistically significance, Sapporo confirmed APS cases had advanced odds of undergoing vascular thrombosis (OR = 1.61, 95%CI) and DVT/PE (OR = 1.53, 95%CI) and lesser odds of undergoing recurrent DVT/PE (OR = 0.67, 95%CI) and pregnancy morbidity (OR = 0.63, 95%CI) than the clinically diagnosed APS cases. Over 70% of the study population with diagnosed APS did not accomplish the revised Sapporo criteria due to negative laboratory manifestations, which reflects heterogeneous but not degreed disease severity profiles.     

Synthesis of oxytocin derivatives lipidated via a carbonate or carbamate linkage as a long-acting therapeutic agent for social impairment-like behaviors

In the course of our studies of hydrophobic oxytocin (OT) analogues, we newly synthesized lipidated OT (LOT-4a-c and LOT-5a-c), in which a long alkyl chain (C14-C16) is conjugated via a carbonate or carbamate linkage at the Tyr-2 phenolic hydroxy group and a palmitoyl group at the terminal amino group of Cys-1. These LOTs did not activate OT and vasopressin receptors. Among the LOTs, however, LOT-4c, having a C16-chain via a carbonate linkage at the phenolic hydroxyl group of the Tyr-2, showed very long-lasting action for the recovery of impaired social behavior in CD38 knockout mice, a rodent model of autistic phenotypes, whereas the effect of OT itself rapidly diminished. These results indicate that LOT-4c may serve as a potential prodrug in mice.     

Oligonucleotide conformations. (5) NMR and relaxation studies on GpU and UpG at neutral pH.

The average conformation of GpU and UpG in neutral aqueous solutions has been investigated by proton chemical shifts and coupling measurements as well as T1 relaxation time experiments. The proportion of the N and S pseudorotational conformers of the ribose ring has been derived from the vicinal coupling constants. The relaxation data provide information about the syn--anti equilibrium of the orientation of the base about the glycosidic bond. This orientation is predominantly syn for the Guo base in both dinucleoside phosphates, that of Urd is anti in the case of GpU and shows an almost equivalent syn and anti character for UpG.     

A keratin of fetal skin is reexpressed in human keratinocytes transformed by SV40 virus or treated with the tumor promoter TPA

SV40-transformation as well as treatment with tumor promoters produce alterations in morphology, differentiation and keratinization of human keratinocytes. Two cell lines of SV40-transformed keratinocytes and primary cultures of keratinocytes treated with the tumor promoter 12-O-tetradecanoyl phorbol-13-acetate (TPA) were found to contain an additional protein of 52.5 kD molecular weight (MW). This protein was identified by its reactivity with the monoclonal antibody TROMA-I as being keratin no. 8, a keratin normally present only in simple epithelia. Since this keratin is present in fetal epidermis but disappears gradually when fetal skin becomes multilayered after week 13 of development (Moll et al., Differentiation 23 (1982) 170. [23]), it suggests that SV40 virus and TPA are able to induce in human keratinocytes the reexpression of fetal characters.     

Protein-bound SH groups in frozen-thawed egg cells of the sea urchin

Unfertilized egg cells of the sea urchin St. intermedius could survive slow freezing to ?15 °C for a short period of time, but at the same freezing temperature extracellular freezing became fatal within a few hours. Such freezing injury resulted in “black” or “white” cytolysis in frozen-thawed cells. “Black” cytolysis took place in the process of both freezing and thawing, while “white” cytolysis occurred only on thawing. Rapid rewarming consistently produced “white” cytolysis in extracellularly frozen cells. The observed behavior of the injured cells during freeze-thawing appeared favorable for the explanation of freezing injury by the SH-SS hypothesis. Protein-bound SH groups were quantitatively determined in both whole cell and cortex with plasma membrane before and after freeze-thawing. However, no significant change in the SH value was observed between freeze-thaw cytolysed materials and unfrozen ones.