[The GTP-Bound form of Rab3D Promotes Lipid Droplet Growth in Adipocyte].

Rab GTPases are major regulators of membrane trafficking. Proteome profiling reveals dozens of Rab proteins associated with lipid droplets (LDs), but few of them have been functionally validated. Cell death activator CIDE-3 protein mediates LD fusion and growth. It is highly enriched at LD-LD contact sites. Here, we investigated the role of Rab3D in lipid storage in adipocyte. We verified that the adipose levels of Rab3D transcript were higher than that of other Rab3 family members; the differences were most pronounced in white adipose tissue. Moreover, we showed that Rab3D promotes LD growth in 3T3-L1 preadipocytes in a dose dependent manner, independently of the process of CIDE-3-mediated LD fusion. Finally, we confirmed that the GTP-bound form of Rab3D is its LD promoting form; it translocates from other vesicles to LDs during adipocyte differentiation. By contrast, the Rab3D-GDP form is retained in the cytoplasm and has no effect on LD sizes. Presented results provide evidence for the role of Rab3D in controlling formation of large LDs in adipocytes. We conclude that Rab3D is a novel LD regulator with characteristics differing from these of previously identified LD associated Rab proteins, such as Rab18 and Rab8a.

[A case of craniopharyngioma presenting as cavernous sinus space occupying].

SummaryA 37-year-old female patient has the symptoms of recurrent headache for 2 years and worse for 1 month. The skull CT and MRI show a space-occupying lesion in the right of the cavernous sinus region. The patient underwent the resection of the tumor by the nasal endoscopy. The pathological biopsy showed the craniopharyngioma. This paper reports a case of craniopharyngioma in the cavernous sinus region and reviews the literature in order to increase the understanding of the disease and reduce misdiagnosis and missed diagnosis.

The function of hyperpolarization-activated cyclic nucleotide-gated channel in diabetic cystopathy.

OBJECTIVE: We aimed at investigating changes in the expression and physiological function of hyperpolarization-activated cyclic nucleotide-gated (HCN) channels in interstitial cells of Cajal (ICC) in diabetic state. MATERIALS AND METHODS: Twenty adult female Sprague-Dawley (SD) rats were randomly assigned to control and Zucker diabetic fatty (ZDF) group. The protein and mRNA expression of HCN isoforms and C-kit in the rat bladders were detected using Western blotting and reverse transcription-polymerase chain reaction (RT-PCR). The bladder contraction was evaluated using a bladder smooth muscle strip test. Whole cell patch-clamp techniques were used to detect the activity of HCN channels. Immunofluorescent staining was used to the positive expression of HCN and C-kit in ICC. RESULTS: cAMP, as HCN channel-specific stimulant, could increase the frequency and amplitude of spontaneous contractions in both group, while cAMP inducing contraction of ZDF rats, was still significantly lower compared with the control group. Acute bladder ICCs were isolated by collagenase digestion. Classic Ih current pattern was recorded on ICCs while Ih current amplitude of ICCs from ZDF diabetic rats was significantly lower than the control group. The expression and mRNA of HCN1-4 isoforms in ZDF diabetic rats were both significantly lower compared with the control group. Meanwhile, the number of c-kit positive cells in ZDF diabetic rats showed no significant differences compared with controls. The morphological structure of ICC in the bladder of ZDF rats was relatively loose and the number of their cell process was apparently decreased. CONCLUSIONS: The structure of ICCs in ZDF rats was relatively loose, their connection to each other was also diminished. The expression of HCN was down-regulated and its response to cAMP was also decreased. HCN channels in bladder ICCs might regulate detrusor contraction. Changes in HCN expression and activity in bladder ICCs might be one of the most important mechanisms of diabetic cystopathy.

Screening and functional analysis of micro-RNAs that regulate the expression of the tumor suppression TP53 gene.

OBJECTIVE: With gradual increase of cancer incidence and mortality rates, the regulatory mechanism of cancer has become a hotspot. It has been known that the expression of TP53 is closely associated with the occurrence of cancer. The microRNA (miRNA)-mediated regulation of the expression of numerous proto-oncogenes has been reported. This study aimed to identify miRNAs that regulate the expression of TP53 gene. MATERIALS AND METHODS: The sequence of TP53 gene was downloaded from NCBI and analyzed with TargetScan software to predict potential miRNAs that regulate TP53 expression. miR-122 was selected as the most potential miRNA for regulating TP53. miR-122 mimics and inhibitor were synthesized and transfected into Hela cells. The expression of TP53 mRNA was measured by qRT-PCR. The cell proliferation, migration, and invasion ability were assessed by CCK-8, scratch wounding, and transwell invasion assay, respectively. RESULTS: Cells transfected with miR-122 mimics exhibited significantly lower TP53 expression (p < 0.05), but significantly increased cell proliferation and migration compared with blank control group (p < 0.05). Notably, cells in miR-122 mimics and control groups had similar invasion ability (p > 0.05). However, cells in miR-122 inhibitor group exhibited significantly higher TP53 expression (p < 0.05), but significantly reduced cell proliferation and invasion ability. The migration ability of cells in miR-122 inhibitor group was similar to cells in control group (p > 0.05). CONCLUSIONS: The selected miR-122 effectively inhibited the expression of TP53 gene in Hela cells, and enhanced their proliferation, migration, and invasion.

Clinical transition for adolescents with developmental disabilities in Hong Kong: a pilot study.

INTRODUCTION: Children with developmental disabilities usually move from the paediatric to adult health service after the age of 18 years. This clinical transition is fragmented in Hong Kong. There are no local data for adolescents with developmental disabilities and their families about the issues they face during the clinical transition. This pilot study aimed to explore and collect information from adolescents with developmental disabilities and their caregivers about their transition from paediatric to adult health care services in Hong Kong. METHODS: This exploratory survey was carried out in two special schools in Hong Kong. Convenient samples of adolescents with developmental disabilities and their parents were taken. The questionnaire was administered by interviewers in Cantonese. Descriptive statistics were used to analyse the answers to closed-ended questions. Responses to open-ended questions were summarised. RESULTS: In this study, 22 parents (mean age ± standard deviation: 49.9 ± 10.0 years) and 13 adolescents (19.6 ± 1.0 years) completed the face-to-face questionnaire. The main diagnoses of the adolescents were cerebral palsy (59%) and cognitive impairment (55%). Of the study parents, 77% were reluctant to transition. For the 10 families who did move to adult care, 60% of the parents were not satisfied with the services. The main reasons were reluctant to change and dissatisfaction with the adult medical service. The participants emphasised their need for a structured clinical transition service to support them during this challenging time. CONCLUSIONS: This study is the first in Hong Kong to present preliminary data on adolescents with developmental disabilities and their families during transition from paediatric to adult medical care. Further studies are required to understand the needs of this population group during clinical transition.

Effect of exogenous l-arginine and ageing on NO and ET-1 in penile tissue of rat.

The aim of this study was to investigate the effect and significance of l-arginine and ageing on nitric oxide (NO)-cyclic guanosine monophosphate (cGMP) pathway and ET-1 in penile tissue of rats. The different months old rats were divided into control group and experiment group randomly, the content of NO, cGMP, the changes of activity of Nitrous Oxide Systems (NOS) and the content of endothelin-1(ET-1) in penile tissue were determined. Along with ageing, NO and the activity of NOS in penile tissue increased at first and then decreased (P < 0.001). The content of cGMP reduced obviously (P < 0.001), the content of ET-1 had a tendency to increase, and the ratio of ET-1/NO increased significantly (P < 0.001 or P < 0.01). After feeding rats with l-arginine for some time, both the activity of NOS and the content of cGMP increased significantly in penile tissue (P < 0.001), while there was no obvious change in the content of ET-1. Our study shows that whether the smooth muscle cells relax or contract might be decided by the content of cGMP and value of ET-1/NO in penile tissue. l-arginine had significant effect on increasing the activity of NOS and the content of NO and cGMP, indicating that l-arginine has potential clinical value to be used in treating ED.

Different cervical cancer screening approaches in a Chinese multicentre study.

To evaluate alternative cervical cancer screening methods, digital colposcopy and collection of cervical exfoliated cells for liquid-based cytology (LBC) and hybrid capture 2 (HC2) testing were performed among 2562 women aged 15-59 years in three study sites in the People's Republic of China (rural Shanxi province, Shenyang city in Liaoning province and Shenzhen city in Guangdong province). Visual inspection with acetic acid (VIA) was also evaluated independently from colposcopy. A total of 74 cases of histologically confirmed cervical intraepithelial neoplasia grade 2 or worse (CIN2+) were identified, and 16 CIN2+ cases were imputed among unbiopsied women to correct for verification bias. Corrected sensitivity for CIN2+ was 37% for VIA, 54% for colposcopy, 87% for LBC with a threshold of atypical cells of undetermined significance (LBC>or=ASCUS), 90% for HC2, 84% for LBC using HC2 to triage ASCUS and 96% for positivity to LBC>or=ASCUS or HC2. For VIA, sensitivity was much lower among women >or=40 years (12%) than those aged or=ASCUS or HC2, up to 94% for LBC using HC2 to triage ASCUS. In conclusion, LBC, HC2 and their combinations performed well, whereas VIA missed a majority of CIN2+, particularly in older women. Digital colposcopy performed better than VIA, but still missed nearly half of CIN2+ in this study.

INSIG2 gene polymorphism is not associated with obesity in Caucasian, Afro-Caribbean and Indian subjects.

A common polymorphism, rs7566605, 10 kb upstream of the insulin-induced gene 2 transcription start site has been associated with obesity in Caucasian and African-American populations, with the hypothesis that an alteration in gene expression results in elevated plasma triglyceride levels. The goal of this study was to verify the findings in a cohort of 2721 healthy Caucasian men (second Northwick Park Heart Study), and a separate study of 747 type 2 diabetic patients from Caucasian, Afro-Caribbean and Indian groups (University College London Diabetes and Cardiovascular Disease Study). The rs7566605 single-nucleotide polymorphism (SNP) was not related to plasma triglyceride levels in either study, and we found no association with body mass index or obesity in either cohort, despite having the power to detect the previously reported effect. This suggests that, at the least, the true size of the effect on obesity of this SNP is likely to be considerably less than reported previously.

Human papillomavirus infection in Shenyang City, People's Republic of China: A population-based study.

To investigate the prevalence of, and risk factors for, cervical infection with human papillomavirus (HPV) in Shenyang City, People's Republic of China, we interviewed and obtained cervical cell samples from 685 women aged 15-59 years enumerated from local population lists. Human papillomavirus DNA was detected in cervical cell samples using a GP5+/6+-based PCR assay for 44 HPV types. Human papillomavirus prevalence was 16.8% overall and 13.6% among women without cervical abnormalities (16.6% and 12.4%, respectively, age-standardised to the world standard population), with no significant trends in HPV prevalence by age group. Of the 32 types identified, high-risk HPV types predominated in all age groups, HPV16 being the most common (3.4% of all women), followed by HPV52 (2.5%) and 58 (1.9%). Multiple-type infections accounted for 31.3% of all infected women. Not being married, reporting multiple sexual partners and husband's extramarital sexual relationships were all significantly associated with being HPV-positive. The disclosure of a relatively high HPV prevalence in Shenyang, in comparison with other worldwide populations, raises important questions concerning the prevention of cervical cancer in China, especially given the promising efficacy of prophylactic HPV vaccines.

Enhanced collagen synthesis in cultured skin fibroblasts from insulin-dependent diabetic patients with nephropathy.

Excessive production and deposition of extracellular matrix proteins are characteristic features of diabetic nephropathy. This study tests the hypothesis that cells from diabetic patients who develop nephropathy have a disturbance in collagen metabolism compared with cells from diabetic patients without complications. Kinetics of overall collagen metabolism and total protein synthesis were examined in serially passaged, subconfluent, quiescent skin fibroblasts cultured in either normal (5 mM) or high (25 mM) glucose concentrations from 14 insulin-dependent diabetic (IDDM) patients with nephropathy; 14 IDDM patients without nephropathy matched for age, diabetes duration, and body mass index; and 14 healthy subjects. Fibroblasts were incubated in the presence of 2 microCi/ml [3H]proline, and after labeling the incorporation of [3H]proline into total protein, collagen (collagenase-sensitive material), and noncollagen proteins (collagenase-resistant material) was determined at different time points. Collagen degradation was determined in pulse-chase experiments by following the residual collagen-bound radioactivity after incubation for 8 h with 10 microCi/ml [3H]proline. In high glucose concentrations (25 mM), overall collagen synthesis (measured as [3H]proline incorporation into extracellular and intracellular collagenase-sensitive material) was significantly greater in the patients with nephropathy (mean +/- SEM after a 24-h labeling period: 7189 +/- 671 dpm/10(6) cells) than in the patients without (4341 +/- 267 dpm/10(6) cells; P < 0.01) or healthy control subjects (3836 +/- 234 dpm/10(6) cells; P < 0.01). No significant differences were observed in noncollagen protein production or in collagen degradation rates among the three groups of subjects. In the presence of normal glucose concentrations (5 mM), collagen synthesis was lower in all groups studied, but the differences between IDDM patients with nephropathy and those without remained unaltered. These results suggest that long-term cultured fibroblasts derived from diabetic patients with nephropathy exhibit an abnormality in collagen metabolism. Cells from long-standing diabetic patients without nephropathy have normal collagen metabolism. The increased collagen synthesis is likely to be intrinsic to those diabetic patients susceptible to nephropathy and may play an important role in the sclerotic processes that occur in the kidneys, arteries, and heart.

Application of a bi-directional associative memory (BAM) network in computer assisted learning in chemistry.

A computer assisted learning software based on a bi-directional associative memory (BAM) network was developed. The software was implemented to assist students in associating the names of the elements in the periodic table with their chemical symbols. The use of the BAM facilitates the analysis and interpretation of students' responses. The software package can be modified easily as an educational tool for other disciplines.

Molecular cloning of the common carp (Cyprinus carpio) rhodopsin cDNA.

A recombinant phage clone containing a 1584 nucleotides rhodopsin cDNA was screened from a carp retinal cDNA library. The inserted DNA consisting of a single open reading frame of 1062 nucleotides at positions 72 to 1133 encodes a 354 amino acid polypeptide. The deduced amino acid sequence of carp rhodopsin showed 95.7, 85.5 and 74.4% identity with that of goldfish, sand goby and lamprey, respectively. The sites of palmitoylation, glycosylation, disulfide bond formation and Schiff base formation in the putative rhodopsin are all conserved.

Intracellular pH and Na+/H+ antiport activity of cultured skin fibroblasts from diabetics.

Increased leucocyte Na+/H+ antiport activity has previously been demonstrated in both hypertensive subjects and Type 1 diabetic patients with nephropathy and may indicate a predisposition to hypertension in such diabetic patients. We have studied intracellular pH and Na+/H+ antiport activity in cultured skin fibroblasts from diabetic patients with and without nephropathy, together with non-diabetic controls to assess if such differences persisted in cultured cells. Fibroblasts from diabetic patients with nephropathy were significantly more alkaline [median (range): 6.90 (6.82 to 7.07)] compared to both normoalbuminuric diabetic patients [6.81 (6.75 to 6.89)] or normal controls [6.82 (6.77 to 6.93)] (P < 0.001 for both). This was associated with a raised Na+/H+ antiport activity in cells from patients with nephropathy when intracellular pH (pHi) was clamped to pH 6.5, without any differences in the maximal transport capacity of the antiport at pHi 6.2. Using both intracellular pH and Na+/H+ antiport activity at pHi 6.5, patients with nephropathy were separated from uncomplicated subjects with a sensitivity of 92% and a specificity of 100%. In conclusion, the raised Na+/H+ antiport activity in cells from patients with diabetic nephropathy persists despite passaging in vitro, thus indicating a heritable component, and results mainly from an increased apparent affinity of the antiport for intracellular H+.

Na+/H+ antiport activity and cell growth in cultured skin fibroblasts of IDDM patients with nephropathy.

IDDM patients with incipient and overt nephropathy have been found to exhibit an overactivity of RBC sodium-lithium countertransport. To explore the physiological relevance of this finding, we measured the activity of Na+/H+ antiport in serially passaged cultured skin fibroblasts from IDDM patients with and without nephropathy and from normal, nondiabetic control subjects. Na+/H+ antiport activity (measured as the rate of amiloride-sensitive Na+ influx at pHi = 6.4, extracellular pH = 8.0, and [Na+] = 1 mM) was elevated significantly in IDDM patients with nephropathy compared with IDDM patients without nephropathy and nondiabetic control subjects (13.35 +/- 3.8 vs. 8.54 +/- 2.0 vs. 7.33 +/- 2.3 nmol Na+.mg protein-1.min-1; P less than 0.006 and P less than 0.001, respectively). A kinetic analysis of Na+/H+ antiport activity showed that the raised activity in IDDM patients with nephropathy was caused by an increased Vmax for extracellular Na+. Km values were similar in the three groups. pH-stimulated amiloride-sensitive Na+ influx also was higher under baseline conditions and after serum stimulation in cells from IDDM patients with nephropathy. pHi values were significantly higher, both during active proliferation and after 10-min exposure to serum, in cells from IDDM patients with nephropathy, compared with IDDM patients without nephropathy and nondiabetic control subjects. Serum-stimulated incorporation of [3H]thymidine into DNA was greater in IDDM patients with nephropathy than in the other two groups (35.7 +/- 18.9- vs. 17.4 +/- 7.5- vs. 11.9 +/- 8.7-fold stimulation above baseline; P less than 0.01 for both.(ABSTRACT TRUNCATED AT 250 WORDS)

Increased sodium-lithium countertransport activity in red cells of patients with insulin-dependent diabetes and nephropathy.

Susceptibility to diabetic nephropathy may be related to a predisposition to arterial hypertension. We have studied the activity of sodium-lithium countertransport in red cells, a marker of risk for essential hypertension, in white European adults with insulin-dependent diabetes and diabetic nephropathy, a matched group of patients with diabetes without renal disease, and nondiabetic patients with renal disease. Measures of metabolic control and concentrations of plasma free insulin and growth hormone were similar in the two diabetic groups. The degree of impairment in renal function was similar in the diabetic and nondiabetic patients with renal disease. Body-mass index and plasma potassium concentrations were similar in all three groups. Diastolic blood pressure was elevated to a similar degree in the two groups with renal disease, as compared with that in the diabetic patients without renal disease. The rates of sodium-lithium countertransport in red cells were significantly higher in the diabetic patients with renal disease (mean +/- SD, 0.55 +/- 0.19 mmol of lithium per liter of red cells per hour) than in the diabetic patients without renal disease (0.33 +/- 0.16; P less than 0.005) and in the nondiabetic patients with renal disease (0.31 +/- 0.14; P less than 0.001). Predisposition to hypertension, as indicated by elevated sodium-lithium countertransport activity in red cells, may serve as a marker for the risk of renal disease in patients with insulin-dependent diabetes.

Biofragmentable bowel anastomosis ring: comparative efficacy studies in dogs.

Three studies were conducted in a total of 178 dogs to determine the efficacy of a biofragmentable bowel anastomosis ring (BAR) composed of polyglycolic acid and 12.5% barium sulfate. Wound strength and healing of BAR, suture, and staple colonic anastomoses were compared for intervals of up to 1 year. The effect of systemic steroid treatment and BAR size on anastomotic wound strength and healing was also studied. The BARs fragmented at a mean time of 15.06 days postoperatively and were passed in the feces without injury. Wound strength was determined by measurements of the pressure required to burst the anastomosed colonic segment and measurements of the tension required to break 10 mm wide longitudinal strips of the anastomosed segment. The studies demonstrated that wound strength had progressed to a point where continued mechanical support (with sutures or staples) was no longer required by 14 days in both nontreated and steroid-treated dogs. Gross healing evaluations at 21 days and beyond showed no differences due to anastomotic method. Microscopic evaluations suggested that residual granulation tissue was less at the sites of BAR asastomoses than at sites of suture or staple anastomoses at the 1-year interval, suggesting that healing may be better with BAR than with standard methods of colon anastomosis.

Age dependent lipid and protein changes in individual bovine lenses.

Successive fiber fractions isolated from individual bovine lenses were fractionated to examine changes in lens proteins and membrane lipids as a function of age. In calf lens of about 1.2 gm wet weight, cholesterol (C) is maintained at a level of 3.3 microgram/mg dry weight in the outer cortical 30% of the lens. In the inner cortex, a C content of 2.4 micrograms/mg was found that decreased somewhat to 2.1 micrograms/mg in the inner nuclear 20% of the lens. The almost linear decrease in phospholipid (P) content from 11.6 in the cortex to 1.7 micrograms/mg in the nucleus resulted in a cortex to nucleus increase in C/P ratio from 0.5 to about 2.0 (mol/mol). Compared to calf lenses, a low C level of 2.4 micrograms/mg was observed in the outer cortex of cow lenses (approximately 3.0 gm wet weight). No significant difference in C level was found between the calf and cow lenses either in the inner cortical or nuclear regions. The P level was reduced to 6 and 1.2 micrograms/mg in the outer cortex and nucleus of the cow lens, respectively. The low nuclear P content is responsible for the observed high C/P value of 3.6. The lower lipid content found in the cortex of older lens suggests an age dependent decrease in the amount of available membrane lipid to envelope the newly formed fibers. A cortex to nucleus increase in the amount of urea-soluble (US) protein fraction from about 6 to 14% of total fiber mass was observed with the calf lens. In the cow lenses, the nuclear US fraction accounts for almost 30% of the fiber.(ABSTRACT TRUNCATED AT 250 WORDS)

Age-dependent changes in the distribution and concentration of human lens cholesterol and phospholipids.

Analyses of total lipid in individual lenses 1.8-63 years of age indicate that both the cholesterol and the phospholipid concentrations have reached a high level of 10 and 14 micrograms/mg lens dry weight, respectively, after the first ten years of growth. Thereafter, the rate of phospholipid accumulation was greatly reduced to a value of 0.05 microgram/mg per year while that of cholesterol reduced to 0.19. Analyses of the distribution of lipid in successive lens fiber layers indicate that both the cholesterol and phospholipid levels increase in the entire lens between the age of 1.8 and 9 years. Older lenses showed a continuous increase in the accumulation of cholesterol in the deep cortical fibers, while little or no increase in phospholipid concentration was observed. These results indicate that the accumulation of lipids is greater than that of lens dry mass (protein) during the first decade of lens growth. Since more than 90% of lenticular lipids are associated with fiber cell membranes, these data suggest a gradual change in the differentiation of the newly formed secondary fibers from the epithelium during this period. Analyses of the phospholipid composition of the successive fiber fractions indicate that the major phospholipids of phosphatidyl ethanolamine (PE), phosphatidylserine (PS) and sphingomyelin maintained a uniform distribution in the 1.8- and 5-year-old lenses. While no change was observed with the cortical fibers, older lenses showed a gradual loss of PE and PS in the nuclear fiber up to 63 years of age. By the late teen years, nuclear PS can no longer be detected, while high levels of PE are maintained in lens nucleus. The disappearance of nuclear PE begins in the teen years and is completed by the age of 40. The decrease in PE and PS resulted in a continuous increase in the cholesterol/phospholipid ratio, a measure of membrane rigidity in the nuclear fiber in lenses 20 years of age and older. This decrease is also responsible for the exceedingly high rigidity of the nuclear fibers of lenses 60 years of age and older. Possible lamellar cholesterol organization in the lens fiber membrane is discussed.