Identification of Heat Shock Protein 60 as a Regulator of Neutral Sphingomyelinase 2 and Its Role in Dopamine Uptake

Activation of sphingomyelinase (SMase) by extracellular stimuli is the major pathway for cellular production of ceramide, a bioactive lipid mediator acting through sphingomyelin (SM) hydrolysis. Previously, we reported the existence of six forms of neutral pH–optimum and Mg²⁺-dependent SMase (N-SMase) in the membrane fractions of bovine brain. Here, we focus on N-SMase ε from salt-extracted membranes. After extensive purification by 12,780-fold with a yield of 1.3%, this enzyme was eventually characterized as N-SMase2. The major single band of 60-kDa molecular mass in the active fractions of the final purification step was identified as heat shock protein 60 (Hsp60) by matrix-assisted laser desorption/ionization time-of-flight mass spectrometric analysis. Proximity ligation assay and immunoprecipitation study showed that Hsp60 interacted with N-SMase2, prompting us to examine the effect of Hsp60 on N-SMase2 and ceramide production. Interestingly, Hsp60 siRNA treatment significantly increased the protein level of N-SMase2 in N-SMase2-overexpressed HEK293 cells. Furthermore, transfection of Hsp60 siRNA into PC12 cells effectively increased both N-SMase activity and ceramide production and increased dopamine re-uptake with paralleled increase. Taken together, these results show that Hsp60 may serve as a negative regulator in N-SMase2-induced dopamine re-uptake by decreasing the protein level of N-SMase2.     

Trends in the Rates of Peripartum Hysterectomy and Uterine Artery Embolization

The objective of this study was to determine the trends in national rates of peripartum hysterectomy (PH) and uterine arterial embolization (UAE) in Korea. We used data collected by the Health Insurance Review & Assessment Service of Korea and analyzed data from patients who gave birth during the period from 2005 to 2008. There were 1785,178 deliveries during the study period, including 2636 cases of PH (1.48 per 1000 deliveries). The PH rate in 2005 was 1.57 per 1000 deliveries and in 2008 it was 1.33 per 1000 deliveries. UAE was performed in 161 women (incidence, 0.38 per 1000 deliveries) and 447 women (incidence, 0.98 per 1000 deliveries) in 2005 and 2008, respectively. In Korea, the rate of PH decreased slightly, while the rate of UAE rate increased dramatically during the period from 2005 to 2008. Further studies are needed to evaluate the effects of UAE on the rate of PH performed.     

Clinicopathological Correlation and Prognostic Significance of Protein Kinase Cα Overexpression in Human Gastric Carcinoma

Objectives

This study investigated the PKCα protein expression in gastric carcinoma, and correlated it with clinicopathological parameters. The prognostic significance of PKCα protein expression in gastric carcinoma was analyzed.

Methods

Quantitative real-time PCR test was applied to compare the PKCα mRNA expression in tumorous and nontumorous tissues of gastric carcinoma in ten randomly selected cases. Then PKCα protein expression was evaluated in 215 cases of gastric carcinoma using immunohistochemical method. The immunoreactivity was scored semiquantitatively as: 0 = absent; 1 = weak; 2 = moderate; and 3 = strong. All cases were further classified into two groups, namely PKCα overexpression group with score 2 or 3, and non-overexpression group with score 0 or 1. The PKCα protein expression was correlated with clinicopathological parameters. Survival analysis was performed to determine the prognostic significance of PKCα protein expression in patients with gastric carcinoma.

Results

PKCα mRNA expression was upregulated in all ten cases of gastric carcinoma via quantitative real-time PCR test. In immunohistochemical study, eighty-eight out of 215 cases (41%) of gastric carcinoma revealed PKCα protein overexpression, which was statistically correlated with age (P = 0.0073), histologic type (P<0.0001), tumor differentiation (P = 0.0110), depth of invasion (P = 0.0003), angiolymphatic invasion (P = 0.0373), pathologic stage (P = 0.0047), and distant metastasis (P = 0.0048). We found no significant difference in overall and disease free survival rates between PKCα overexpression and non-overexpression groups (P = 0.0680 and 0.0587). However, PKCα protein overexpression emerged as a significant independent prognostic factor in multivariate Cox regression analysis (hazard ratio 0.632, P = 0.0415).

Conclusions

PKCα protein is upregulated in gastric carcinoma. PKCα protein expression is statistically correlated with age, histologic type, tumor differentiation, depth of invasion, angiolymphatic invasion, pathologic stage, and distant metastasis. The PKCα protein overexpression in patients with gastric carcinoma is a significant independent prognostic factor in multivariate Cox regression analysis.     

A Novel Cell-Penetrating Peptide Derived from Human Eosinophil Cationic Protein

Cell-penetrating peptides (CPPs) are short peptides which can carry various types of molecules into cells; however, although most CPPs rapidly penetrate cells in vitro, their in vivo tissue-targeting specificities are low. Herein, we describe cell-binding, internalization, and targeting characteristics of a newly identified 10-residue CPP, denoted ECP^32–41, derived from the core heparin-binding motif of human eosinophil cationic protein (ECP). Besides traditional emphasis on positively charged residues, the presence of cysteine and tryptophan residues was demonstrated to be essential for internalization. ECP^32–41 entered Beas-2B and wild-type CHO-K1 cells, but not CHO cells lacking of cell-surface glycosaminoglycans (GAGs), indicating that binding of ECP^32–41 to cell-surface GAGs was required for internalization. When cells were cultured with GAGs or pre-treated with GAG-digesting enzymes, significant decreases in ECP^32–41 internalization were observed, suggesting that cell-surface GAGs, especially heparan sulfate proteoglycans were necessary for ECP^32–41 attachment and penetration. Furthermore, treatment with pharmacological agents identified two forms of energy-dependent endocytosis, lipid-raft endocytosis and macropinocytosis, as the major ECP^32–41 internalization routes. ECP^32–41 was demonstrated to transport various cargoes including fluorescent chemical, fluorescent protein, and peptidomimetic drug into cultured Beas-2B cells in vitro, and targeted broncho-epithelial and intestinal villi tissues in vivo. Hence this CPP has the potential to serve as a novel vehicle for intracellular delivery of biomolecules or medicines, especially for the treatment of pulmonary or gastrointestinal diseases.     

A cis-Acting Element in Retroviral Genomic RNA Links Gag-Pol Ribosomal Frameshifting to Selective Viral RNA Encapsidation

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Highlights? A retroviral RNA packaging element (GRPE) overlaps the Gag-Pol ribosomal frameshift site ? Without two stem loops in GRPE, genomic RNA encapsidation is decreased >50-fold ? Downregulating the translation termination factor eRF1 produces defective virus particles ? GPRE links ribosome frameshifting and effective retroviral packaging     

Plant Trait-Species Abundance Relationships Vary with Environmental Properties in Subtropical Forests in Eastern China

Understanding how plant trait-species abundance relationships change with a range of single and multivariate environmental properties is crucial for explaining species abundance and rarity. In this study, the abundance of 94 woody plant species was examined and related to 15 plant leaf and wood traits at both local and landscape scales involving 31 plots in subtropical forests in eastern China. Further, plant trait-species abundance relationships were related to a range of single and multivariate (PCA axes) environmental properties such as air humidity, soil moisture content, soil temperature, soil pH, and soil organic matter, nitrogen (N) and phosphorus (P) contents. At the landscape scale, plant maximum height, and twig and stem wood densities were positively correlated, whereas mean leaf area (MLA), leaf N concentration (LN), and total leaf area per twig size (TLA) were negatively correlated with species abundance. At the plot scale, plant maximum height, leaf and twig dry matter contents, twig and stem wood densities were positively correlated, but MLA, specific leaf area, LN, leaf P concentration and TLA were negatively correlated with species abundance. Plant trait-species abundance relationships shifted over the range of seven single environmental properties and along multivariate environmental axes in a similar way. In conclusion, strong relationships between plant traits and species abundance existed among and within communities. Significant shifts in plant trait-species abundance relationships in a range of environmental properties suggest strong environmental filtering processes that influence species abundance and rarity in the studied subtropical forests.     

Quantification of Blood Flow in Internal Cerebral Artery by Optical Flow Method on Digital Subtraction Angiography in Comparison with Time-Of-Flight Magnetic Resonance Angiography

Objective

This study compared data on the blood flow velocity in the internal carotid artery, which was obtained using the optical flow method (OFM) with digital subtraction angiography (DSA) and the time-of-flight (TOF) technique using magnetic resonance angiography (MRA).

Materials and Methods

Images were obtained from 12 cerebrovascular patients who underwent both brain DSA and MRA imaging. The OFM was applied on the DSA images to determine the average blood flow velocity. The calculated results were compared with the values obtained from the TOF-MRA data. A linear fit was performed on the data and Bland-Altman plots were analyzed.

Results

The blood flow velocity was closely associated with vascular diseases. Color-coding of the OFM measurements were superimposed on to the DSA images, which quantitatively illustrated the relative flow in the vessels. The average blood flow velocity was calculated using OFM and DSA, which demonstrated a high correlation with the MRA measurements in the anterior-posterior (AP) view (R = 0.71). In contrast, the average blood flow velocity was low in the lateral view (R = 0.28). The consistency between the high and low blood velocity in the AP view was better compared to the lateral view. The blood flow velocity distribution in the AP view was statistically closer to the MRA measurement compared to the lateral view.

Conclusions

This study evaluated the correlation of blood flow measured using DSA and TOF-MRA in a small heterogeneous group of patients with cerebrovascular lesions. OFM with DSA imaging reveals hemodynamic information and TOF-MRA.     

Plasma Adiponectin Levels Correlate Positively with an Increasing Number of Components of Frailty in Male Elders

Objective

Frailty is an important geriatric syndrome. Adiponectin is an important adipokine that regulates energy homeostasis. The aim of this study is to investigate the relationship between plasma adiponectin levels and frailty in elders.

Methods

The demographic data, body weight, metabolic and inflammatory parameters, including plasma glucose, total cholesterol, triglyceride, tumor necrosis factor alpha (TNF-α), c-reactive protein (CRP) and adiponectin levels, were assessed. The frailty score was assessed using the Fried Frailty Index (FFI).

Results

The mean (SD) age of the 168 participants [83 (49.4%) men and 85 (50.6%) women] was 76.86 (6.10) years. Judged by the FFI score, 42 (25%) elders were robust, 92 (54.7%) were pre-frail, and 34 (20.3%) were frail. The mean body mass index was 25.19 (3.42) kg/m². The log-transformed mean (SD) plasma adiponectin (µg/mL) level was 1.00 (0.26). The log-transformed mean plasma adiponectin (µg/mL) levels were 0.93 (0.23) in the robust elders, 1.00 (0.27) in the pre-frail elders, and 1.10 (0.22) in the frail elders, and the differences between these values were statistically significant (p  = 0.012). Further analysis showed that plasma adiponectin levels rose progressively with an increasing number of components of frailty in all participants as a whole (p for trend  = 0.024) and males (p for trend  = 0.037), but not in females (p for trend  = 0.223).

Conclusion

Plasma adiponectin levels correlate positively with an increasing number of components of frailty in male elders. The difference between the sexes suggests that certain sex-specific mechanisms may exist to affect the association between adiponectin levels and frailty.     

Intestinal Methanobrevibacter smithii but not total bacteria is related to diet‐induced weight gain in rats

It is increasingly understood that gastrointestinal (GI) methanogens, including Methanobrevibacter smithii, influence host metabolism.

Objective:

Therefore, we compared M. smithii colonization and weight gain in a rat model under different dietary conditions.

Design and Methods:

Sprague‐Dawley rats were inoculated with M. smithii or vehicle (N = 10/group), fed normal chow until day 112 postinoculation, high‐fat chow until day 182, then normal chow until day 253. Thereafter, five rats from each group were fed high‐fat and normal chow until euthanasia.

Results:

Both groups exhibited M. smithii colonization, which increased following inoculation only for the first 9 days. Change to high‐fat chow correlated with significant increases in weight (P < 0.00001) and stool M. smithii (P < 0.01) in all rats, with stool M. smithi decreasing on return to normal chow. Rats switched back to high‐fat on day 253 further increased weight (P < 0.001) and stool M. smithii (P = 0.039). Euthanasia revealed all animals had higher M. smithii, but not total bacteria, in the small intestine than in the colon. Rats switched back to high‐fat chow had higher M. smithii levels in the duodenum, ileum, and cecum than those fed normal chow; total bacteria did not differ in any bowel segment. Rats which gained more weight had more bowel segments colonized, and the lowest weight recorded was in a rat on high‐fat chow which had minimal M. smithii colonization.

Conclusions:

We conclude that M. smithii colonization occurs in the small bowel as well as in the colon, and that the level and extent of M. smithii colonization is predictive of degree of weight gain in this animal model.