Functional nucleotides of U5 LTR determining substrate specificity of prototype foamy virus integrase

In order to study functional nucleotides in prototype foamy virus (PFV) DNA on specific recognition by PFV integrase (IN), we designed chimeric U5 long terminal repeat (LTR) DNA substrates by exchanging comparative sequences between human immunodeficiency virus type-1 (HIV-1) and PFV U5 LTRs, and investigated the 3'-end processing reactivity using HIV-1 and PFV INs, respectively. HIV-1 IN recognized the nucleotides present in the fifth and sixth positions at the 3'-end of the substrates more specifically than any other nucleotides in the viral DNA. However, PFV IN recognized the eighth and ninth nucleotides as distinctively as the fifth and sixth nucleotides in the reactions. In addition, none of the nucleotides present in the twelfth, sixteenth, seventeenth, eighteenth, nineteenth, and twentieth positions were not differentially recognized by HIV-1 and PFV INs, respectively. Therefore, our results suggest that the functional nucleotides that are specifically recognized by its own IN in the PFV U5 LTR are different from those in the HIV-1 U5 LTR in aspects of the positions and nucleotide sequences. Furthermore, it is proposed that the functional nucleotides related to the specific recognition by retroviral INs are present inside ten nucleotides from the 3'-end of the U5 LTR.     

Comparison of Expression of Inflammatory Cytokines in the Spinal Cord Between Young Adult and Aged Beagle Dogs

Aging is an inevitable process that occurs in the whole body system accompanying with many functional and morphological changes. Inflammation is known as one of age-related factors, and inflammatory changes could enhance mortality risk. In this study, we compared immunoreactivities of inflammatory cytokines, such as interleukin (IL)-2 (a pro-inflammatory cytokine), its receptor (IL-2R), IL-4 (an anti-inflammatory cytokine), and its receptor (IL-4R) in the cervical and lumbar spinal cord of young adult (2–3 years old) and aged (10–12 years old) beagle dogs using immunohistochemistry and western blotting. IL-2 and IL-2R-immunoreactive nerve cells were found throughout the gray matter of the cervical and lumbar spinal cord of young adult and aged dogs. In the spinal cord neurons of the aged dog, immunoreactivity and protein levels were apparently increased compared with those in the young adult dog. Change patterns of IL-4- and IL-4R-immunoreactive cells and their protein levels were also similar to those in IL-2 and IL-2R; however, IL-4 and IL-4R immunoreactivity in the periphery of the neuronal cytoplasm in the aged dog was much stronger than that in the young adult dog. These results indicate that the increase of inflammatory cytokines and their receptors in the aged spinal cord might be related to maintaining a balance of inflammatory reaction in the spinal cord during normal aging.     

Effect of Root Zone Temperature on the Mineral Composition of Xylem Sap and Plasma Membrane K+-Mg++-ATPase Activity of Grafted-Cucumber and -Figleaf Gourd Root Systems

Cucumber (Cucumis sativus L.) seedlings were grafted onto cucumber-(CG) or figleaf gourd- (FG, Cucurbita ficifolia Bouché)seedlings in order to determine the effect of solution temperature(12, 22, and 32°C) on the mineral composition of xylem sapand the plasma membrane K⁺-Mg⁺⁺-ATPase activities of the roots.Low solution temperature (12°C) lowered the concentrationof NO^–₃ and H₂PO^–₄ in xylem sap of CG plants butnot of FG plants. Concentrations of K⁺, Ca⁺⁺ and Mg⁺⁺ in xylemsap were less affected than anions by solution temperature.The plasma membrane of FG plants grown in 12°C solutiontemperature showed the highest K⁺- Mg⁺⁺-ATPase activity at allATP concentrations up to 3 mM and at low reaction temperatureup to 12°C, indicating resistance of figleaf gourd to lowroot temperature. (Received December 27, 1994; Accepted March 10, 1995)     

Effect of phosphorus deficiency on aluminium-induced citrate exudation in soybean (Glycine max)

Aluminium (Al) toxicity or phosphorus (P) deficiency can induce exudation of organic acids from the roots of some plants, which is believed to be a tolerance mechanism against Al toxicity or P deficiency. In the present study, the effect of P deficiency on Al-induced citrate exudation was investigated in three soybean varieties differing in low-P tolerance. P starvation alone failed to induce secretion of organic acids from all three soybean varieties. However, P deficiency altered Al-induced citrate exudation over time, showing a complex interaction. Short × term P starvation (4 days) produced up to 50% increase in Al-induced citrate secretion, while longer-term (10 days) starvation reduced Al-induced citrate secretion to trace amounts. However, after a further 1 day in complete nutrient solution for recovery, Al-induced citrate exudation from the recovered roots was approximately 6 times higher than that from the continuously P-starved plants, but still approximately 3.6 times lower than that from the P-sufficient control. With increasing P or Al supply, Al-induced citrate exudation increased, while Al accumulation in soybean roots decreased in parallel with the decrease of P supply. The photosynthetic rate, stomatal conductance and transpiration were decreased by P deficiency, whereas the intracellular CO₂ concentration was increased. These findings indicate that P nutrition has a significant effect on Al-induced citrate exudation and Al accumulation in soybean root apices.     

Outlier Analysis Defines Zinc Finger Gene Family DNA Methylation in Tumors and Saliva of Head and Neck Cancer Patients

Head and Neck Squamous Cell Carcinoma (HNSCC) is the fifth most common cancer, annually affecting over half a million people worldwide. Presently, there are no accepted biomarkers for clinical detection and surveillance of HNSCC. In this work, a comprehensive genome-wide analysis of epigenetic alterations in primary HNSCC tumors was employed in conjunction with cancer-specific outlier statistics to define novel biomarker genes which are differentially methylated in HNSCC. The 37 identified biomarker candidates were top-scoring outlier genes with prominent differential methylation in tumors, but with no signal in normal tissues. These putative candidates were validated in independent HNSCC cohorts from our institution and TCGA (The Cancer Genome Atlas). Using the top candidates, ZNF14, ZNF160, and ZNF420, an assay was developed for detection of HNSCC cancer in primary tissue and saliva samples with 100% specificity when compared to normal control samples. Given the high detection specificity, the analysis of ZNF DNA methylation in combination with other DNA methylation biomarkers may be useful in the clinical setting for HNSCC detection and surveillance, particularly in high-risk patients. Several additional candidates identified through this work can be further investigated toward future development of a multi-gene panel of biomarkers for the surveillance and detection of HNSCC.     

Clinical Correlates of Mass Effect in Autosomal Dominant Polycystic Kidney Disease

Mass effect from polycystic kidney and liver enlargement can result in significant clinical complications and symptoms in autosomal dominant polycystic kidney disease (ADPKD). In this single-center study, we examined the correlation of height-adjusted total liver volume (htTLV) and total kidney volume (htTKV) by CT imaging with hepatic complications (n = 461) and abdominal symptoms (n = 253) in patients with ADPKD. “Mass-effect” complications were assessed by review of medical records and abdominal symptoms, by a standardized research questionnaire. Overall, 91.8% of patients had 4 or more liver cysts on CT scans. Polycystic liver disease (PLD) was classified as none or mild (htTLV < 1,600 mL/m); moderate (1,600 ≤ htTLV <3,200 mL/m); and severe (htTLV ≥ 3,200 mL/m). The prevalence of moderate and severe PLD in our patient cohort was 11.7% (n = 54/461) and 4.8% (n = 22/461), respectively, with a female predominance in both the moderate (61.1%) and severe (95.5%) PLD groups. Pressure-related complications such as leg edema (20.4%), ascites (16.6%), and hernia (3.6%) were common, and patients with moderate to severe PLD exhibited a 6-fold increased risk (compared to no or mild PLD) for these complications in multivariate analysis. Similarly, abdominal symptoms including back pain (58.8%), flank pain (53.1%), abdominal fullness (46.5%), and dyspnea/chest-discomfort (44.3%) were very common, and patients with moderate to severe PLD exhibited a 5-fold increased risk for these symptoms. Moderate to severe PLD is a common and clinically important problem in ~16% of patients with ADPKD who may benefit from referral to specialized centers for further management.     

Adiponectin Provides Additional Information to Conventional Cardiovascular Risk Factors for Assessing the Risk of Atherosclerosis in Both Genders

Background

This study evaluated the relation between adiponectin and atherosclerosis in both genders, and investigated whether adiponectin provides useful additional information for assessing the risk of atherosclerosis.

Methods

We measured serum adiponectin levels and other cardiovascular risk factors in 1033 subjects (454 men, 579 women) from the Korean Genomic Rural Cohort study. Carotid intima–media-thickness (CIMT) was used as measure of atherosclerosis. Odds ratios (ORs) with 95% confidence intervals (95% CI) were calculated using multiple logistic regression, and receiver operating characteristic curves (ROC), the category-free net reclassification improvement (NRI) and integrated discrimination improvement (IDI) were calculated.

Results

After adjustment for conventional cardiovascular risk factors, such as age, waist circumference, smoking history, low-density and high-density lipoprotein cholesterol, triglycerides, systolic blood pressure and insulin resistance, the ORs (95%CI) of the third tertile adiponectin group were 0.42 (0.25–0.72) in men and 0.47 (0.29–0.75) in women. The area under the curve (AUC) on the ROC analysis increased significantly by 0.025 in men and 0.022 in women when adiponectin was added to the logistic model of conventional cardiovascular risk factors (AUC in men: 0.655 to 0.680, p = 0.038; AUC in women: 0.654 to 0.676, p = 0.041). The NRI was 0.32 (95%CI: 0.13–0.50, p<0.001), and the IDI was 0.03 (95%CI: 0.01–0.04, p<0.001) for men. For women, the category-free NRI was 0.18 (95%CI: 0.02–0.34, p = 0.031) and the IDI was 0.003 (95%CI: −0.002–0.008, p = 0.189).

Conclusion

Adiponectin and atherosclerosis were significantly related in both genders, and these relationships were independent of conventional cardiovascular risk factors. Furthermore, adiponectin provided additional information to conventional cardiovascular risk factors regarding the risk of atherosclerosis.     

Nitric oxide synthase and calcium-binding protein-containing neurons in the hamster visual cortex

The distribution and morphology of neurons containing neuronal nitric oxide synthase (NOS), and calcium-binding proteins calbindin D28K and calretinin in the hamster visual cortex were compared by immunocytochemistry. Staining for NOS, calbindin D28K and calretinin was seen both in the specific layers and in the selective cell types. The densest concentration of anti-NOS-immunoreactive (IR) neurons was found in layer VI. Most of the calbindin D28K-IR neurons were located in layers II/III and V while the calretinin-IR neurons were predominantly located in layers II/III. The labeled neurons varied in morphology. The large majority of NOS-IR neurons were round or oval cells with many dendrites coursing in all directions. The majority of the calbindin D28K-IR neurons were stellate and round or oval cells with multipolar dendrites. The majority of the calretinin-IR neurons were vertical fusiform cells with long processes traveling perpendicular to the pial surface. Our study showed that 14.7% and 27.5% of the NOS-IR cells in the hamster visual cortex contained calbindin D28K or calretinin, respectively. These results indicate that NOS, calbindin and calretinin are located in specific layers and specific cell types and the vast majority of NOS-containing neurons are limited to neurons that do not express calbindin D28K or calretinin.     

14-3-3epsilon inhibits MK5-mediated cell migration by disrupting F-actin polymerization

The signal pathway by which 14-3-3epsilon inhibits cell migration induced by MAPK-activated protein kinase 5 (MK5) was investigated in cultured HeLa cells. Both in vivo and in vitro analyses have revealed that 14-3-3epsilon interacts with MK5. 14-3-3epsilon bound to MK5 inhibits the phosphorylation of HSP27, a known substrate of MK5. Disturbance of actin cytoskeleton organization by 14-3-3epsilon was shown in transfected cells transiently expressing 14-3-3epsilon as well as established cells stably expressing 14-3-3epsilon. Moreover, overexpression of 14-3-3epsilon resulted in the inhibition of cell migration induced by MK5 overexpression or TNFalpha treatment. Our results suggest that 14-3-3epsilon bound to MK5 inhibits cell migration by inhibiting the phosphorylation of HSP27 whose phosphorylation regulates F-actin polymerization, actin cytoskeleton organization and subsequent actinfilament dynamics.