Acute and subchronic co-administrations to cadmium,diazinon and selenium induce apparent osteoporotic symptoms in adult male rats

Cadmium (Cd) and diazinon (DZN) are known to be environmental risk factors for various bone diseases including osteoporosis. Selenium (Se), an essential constituent of many antioxidant enzymes, has in higher concentrations negative effects on the bone. The present study was aimed to investigate possible changes in femoral bone of adult male rats after their acute and subchronic exposures to Cd, DZN and Se. A total of 30 male Wistar rats were randomized into three experimental groups. The rats in the group A (4-months-old) were injected intraperitoneally with a mixture of 2 mg CdCl₂ kg^?1, 20 mg DZN kg^?1 and 2 mg Na₂SeO₃ kg^?1 body weight and killed 36 h after xenobiotics had been injected. In the group B, young males (1-month-old) were administered with a combination of 30 mg CdCl₂ L^?1, 40 mg DZN L^?1 and 5 mg Na₂SeO₃ L^?1 in their drinking water, for 90 days. Ten 4-months-old males without toxicant supplementation served as a control group (C). After treatment period, detailed histological analysis of femoral bone was performed in each group. Our results revealed apparent osteoporotic symptoms (resorption lacunae, osteoporotic fractures) in rats from groups A and B. Moreover, histomorphometrical evaluation showed reduced bone vascularization (constricted primary osteons’ vascular canals and Haversian canals) and weakness mechanical properties of bones (smaller size of the secondary osteons) in these rats in comparison with those of the control group. Our study demonstrates for the first time that acute and subchronic co-administrations to Cd, DZN and Se induce evident manifestation characteristics of osteoporosis in male rats.     

The effect of T-2 toxin on bone microstructure in rabbits

T-2 toxin is the most toxic of the trichothecene mycotoxins. Its effect on bone microstructure is still unknown. This study focuses on acute effects of the T-2 toxin on compact and trabecular bone tissues structure of rabbits after a single intramuscular administration. Experimental E group (n?=?4) consisted of animals which were intramuscularly injected with T-2 toxin at dose 0.08 mg.kg^?1 body weight 72 h before slaughter. Group C (n?=?4) without T-2 toxin application served as a control. An absence of primary vascular longitudinal bone tissue near endosteal surfaces, its deposition on periosteal surfaces and a lower density of secondary osteons in the middle part of the substantia compacta were observed in both females and males injected with T-2 toxin. On the contrary, morphometrical analysis of the compact bone showed no demonstrable alternations in the sizes of primary osteons’ vascular canals, Haversian canals or secondary osteons between rabbits from E and C groups. Also, no significant effects of the T-2 toxin on trabecular bone morphometry and cortical bone thickness were observed between rabbits of either sex. The single intramuscular application of T-2 toxin at the dose used in our study affects only qualitative histological characteristics of the compact bone in rabbits.     

All-trans retinoic-acid inhibits heterodimeric bone morphogenetic protein 2/7-stimulated osteoclastogenesis,and resorption activity

Background

Bone regenerative heterodimeric bone morphogenetic protein 2/7 (BMP2/7) enhances but all-trans retinoic acid (ATRA) inhibits osteoclastogenesis. However, the effect of ATRA on physiological and/or BMP2/7-induced osteoclastogenesis in still unclear. In this study, we aimed to test the effect of combined treatment of BMP2/7 and ATRA on osteoclastogenesis, and resorption activity.

Results

All-trans retinoic acid (1 µM)?±?BMP2/7 (5 or 50 ng/ml) was added in murine pre-osteoclasts cell line RAW264.7 or mouse bone marrow derived macrophages (BMM) cultures. Osteoclast marker gene expression, osteoclastogenesis, and resorption activity were analyzed. BMP2/7 robustly enhanced osteoclast maker gene expression, osteoclastogenesis, and resorption activity. Interestingly, ATRA completely inhibited osteoclast formation in presence or absence of BMP2/7. Pan-antagonist of retinoic acid receptors (RARs) and antagonist of RARα, β or γ failed to reverse the inhibitory effect of ATRA on osteoclastogenesis. ATRA strongly inhibited Rank and Nfatc1 expression.

Conclusions

All-trans retinoic acid inhibits BMP2/7-induced osteoclastogenesis, and resorption activity possibly via RANKL–RANK pathway. Our findings from previous and current study suggest that combination of ATRA and BMP2/7 could be a novel approach to treat hyperactive osteoclast-induced bone loss such as in inflammation-induced severe osteoporosis and bone loss caused by cancer metastasis to bone.

     

The role of ACE2, angiotensin-(1–7) and Mas1 receptor axis in glucocorticoid-induced intrauterine growth restriction

Background

Plasma and urine levels of the potent vasodilator Ang-(1–7) are elevated in mid and late pregnancy and are correlated with elevated placental angiogenesis, fetal blood flow, and rapid fetal growth. We hypothesized that Ang-(1–7), its receptor (Mas1) and the enzymes involved in Ang-(1–7) production (ACE2 and Membrane metallo-endopeptidase; MME) are down regulated in response to glucocorticoid administration contributing to IUGR.

Methods

Pregnant female Sprague–Dawley rats were injected with dexamethasone (DEX; 0.4 mg/kg/day) starting from 14 day gestation (dg) till sacrifice at 19 or 21 dg while control groups were injected with saline (n?=?6/group). The gene and protein expression of ACE2, MME, Ang-(1–7) and Mas1 receptor in the placental labyrinth (LZ) and basal zones (BZ) were studied.

Results

DEX administration caused a reduction in LZ weight at 19 and 21 dg (p?<?0.001). IUGR, as shown by decreased fetal weights, was evident in DEX treated rats at 21 dg (p?<?0.01). ACE2 gene expression was elevated in the LZ of control placentas at 21 dg (p?<?0.01) compared to 19 dg and DEX prevented this rise at both gene (p?<?0.01) and protein levels (p?<?0.05). In addition, Ang-(1–7) protein expression in LZ was significantly reduced in DEX treated rats at 21 dg (p?<?0.05). On the other hand, Mas1 and MME were upregulated in LZ at 21 dg in both groups (p?<?0.05 and p?<?0.001, respectively).

Conclusion

The results of this study indicate that a reduced expression of ACE2 and Ang-(1–7) in the placenta by DEX treatment may be responsible for IUGR and consequent disease programming later in life.

     

Experimental research into the potential therapeutic effect of GYY4137 on Ovariectomy-induced osteoporosis

Background

Evidence has shown that endogenous H₂S plays an important role in the physiological and pathophysiological processes of many organs. The study aimed to explore whether exogenous H₂S has a potential therapeutic effect on a rat ovariectomy-induced model of osteoporosis.

Methods

The OVX osteoporosis model was established in female Sprague-Dawley rats by full bilateral ovariectomy. The rats were randomly divided into four groups, with the two experimental groups receiving an intraperitoneal injection of GYY4137 or sodium alendronate. The level of H₂S in the plasma was determined and common laboratory indicators to diagnose osteoporosis, such as alkaline phosphatase (ALP) activity and the levels of osteocalcin (OCN), calcitonin, parathyroid hormone and leptin were measured. The bone mineral density (BMD) of the 4th and 5th lumbar vertebrae was measured using dual-energy X-ray absorptiometry. The maximum stress of femoral fracture was obtained through a three-point bending test of the femur.

Results

The OVX osteoporosis model was successfully established. GYY4137 was injected to increase the level of H₂S in the plasma in one group, designated OVX-GYY during the observation period (p?<?0.05). At 12 weeks, the BMD value of the fourth lumbar vertebra in the OVX-GYY group had increased (p?<?0.05). The BMD femur value in the OVX-vehicle group had decreased (p?<?0.05). Bilateral ovariectomy leads to biochemical disorders related to bone metabolism and hormone levels in rat plasma (all p?<?0.05). Ovariectomy also reduced blood calcium, blood phosphate and calcitonin, and increased parathyroid hormone and leptin. The opposite results were obtained for the groups with alendronate sodium or GYY4137 treatment (all p?<?0.05).

Conclusions

Through the slow release of H₂S, GYY4137 did an excellent job of simulating endogenous neuroendocrine gaseous signaling molecules. Exogenous H₂S had a regulatory effect on osteoporosis in ovariectomized rats, showing potential value for the treatment of human postmenopausal osteoporosis.

     

<Emphasis Type="Italic">Chlamydomonas fla</Emphasis> mutants reveal a link between deflagellation and intraflagellar transport

Background

Cilia and flagella are often lost in anticipation of mitosis or in response to stress. There are two ways that a cell can lose its flagella: resorption or deflagellation. Deflagellation involves active severing of the axoneme at the base of the flagellum; this process is defective in Chlamydomonas fa mutants. In contrast, resorption has been thought to occur as a consequence of constitutive disassembly at the tip in the absence of continued assembly, which requires intraflagellar transport (IFT). Chlamydomonas fla mutants are unable to build and maintain flagella due to defects in IFT.

Results

fla10 cells, which are defective in kinesin-II, the anterograde IFT motor, resorb their flagella at the restrictive temperature (33°C), as previously reported. We find that in standard media containing ~300 microM calcium, fla10 cells lose flagella by deflagellation at 33°C. This temperature-induced deflagellation of a fla mutant is not predicted by the IFT-based model for flagellar length control. Other fla mutants behave similarly, losing their flagella by deflagellation instead of resorption, if adequate calcium is available. These data suggest a new model whereby flagellar resorption involves active disassembly at the base of the flagellum via a mechanism with components in common with the severing machinery of deflagellation. As predicted by this model, we discovered that deflagellation stimuli induce resorption if deflagellation is blocked either by mutation in a FA gene or by lack of calcium. Further support for this model comes from our discovery that fla10-fa double mutants resorb their flagella more slowly than fla10 mutants.

Conclusions

Deflagellation of the fla10 mutant at the restrictive temperature is indicative of an active disassembly signal, which can manifest as either resorption or deflagellation. We propose that when IFT is halted by either an inactivating mutation or a cellular signal, active flagellar disassembly is initiated. This active disassembly is distinct from the constitutive disassembly which plays a role in flagellar length control.

     

Novel mutation G324C in <Emphasis Type="Italic">WNT1</Emphasis> mapped in a large Pakistani family with severe recessively inherited <Emphasis Type="Italic">Osteogenesis Imperfecta</Emphasis>

Introduction

Osteogenesis imperfecta (OI) is a clinically and genetically heterogeneous disease with skeletal fragility and variable extra-skeletal manifestations. To date several point mutations in 18 different genes causing different types of OI have been identified. Mutations in WNT1 compromise activity of the osteoblasts leading to disturbed bone mass accrual, fragility fractures and progressive skeletal abnormalities. The present study was conducted to determine the underlying genetic cause of an autosomal recessive skeletal dysplasia in a large consanguineous family from Chinute, Pakistan.

Materials and methods

Blood was collected from 24 individuals of affected family along with clinical data. Homozygosity mapping was performed to confirm consanguinity. SNPs were identified, followed by whole exome and Sanger sequencing. In silico characterization of WNT1 mutation was performed using multiple platforms.

Results

Nine affected family members exhibited severe bone deformities, recurrent fractures, short stature and low bone mineral density. SNP array data revealed homozygous segments >?1 Mb in length accounting for 2.1–12.7% of the genome in affected individuals and their siblings and a single 6,344,821 bp homozygous region in all affected individuals on chromosome 12q12-q13. This region includes two potential OI candidate genes WNT1 and VDR. We did whole-exome sequencing for both genes in two patients and identified a novel damaging missense mutation in exon 4 of WNT1: c.1168G?>?T (NM_005430) resulting in p.G324C. Sanger sequencing confirmed segregation of mutation with the disease in family.

Conclusion

We report a novel mutation responsible for OI and our investigation expands the spectrum of disease-causing WNT1 mutations and the resulting OI phenotypes.

     

Characterization and prognosis of estrogen receptor-positive/progesterone receptor-negative male breast cancer: a population-based study

Background

The aim of this study was to explore the characteristics and prognostic information of estrogen receptor-positive/progesterone receptor-negative (ER+/PR?) male breast cancer.

Methods

Using the US National Cancer Institute’s Surveillance, Epidemiology, and End Results database, we compared the demographics, clinical characteristics, and outcome of estrogen receptor-positive/progesterone receptor-positive (ER+/PR+) patients with ER+/PR? male breast cancer patients from 1990 to 2010. Two thousand three hundred twenty-two patients with ER+/PR+ tumors and 355 patients with ER+/PR? tumors were included in our study.

Results

ER+/PR? patients were younger (P?=?0.008) and more likely to be African American (P?<?0.001) while presented with higher histological grade (P?<?0.001), larger tumor size (P?=?0.010), and more invasion to the lymph nodes (P?=?0.034) and distant sites (P?<?0.001), thus later stage (P?=?0.001). Despite higher chance of receiving chemotherapy (51.0% vs 36.5%, P?<?0.001), ER+/PR? patients experienced significantly worse breast cancer-specific survival (BSCC) (P?<?0.001) and shorter overall survival (OS) (P?=?0.003). Multivariate Cox model confirmed that tumor size, lymph node invasion, metastasis, and surgery were independent prognostic factors of both BSCC and OS for ER+/PR? male breast cancer. Age at diagnosis and chemotherapy were significantly associated with OS but not with BSCC.

Conclusion

ER+/PR? male breast cancer was more aggressive and experienced shorter survival than ER+/PR+ patients. The prognosis was mainly associated with tumor size, lymph node invasion, metastasis, and surgery.

     

Role of perfusion parameters on DCE-MRI and ADC values on DWMRI for invasive ductal carcinoma at 3.0 Tesla

Background

The value of apparent diffusion coefficient (ADC) values and quantitative parameters (Ktrans, Kep, Ve) in detecting prognostic factor at 3.0 Tesla remains unclear, especially in predicting prognosis of breast cancer.

Methods

A total of 151 patients with IDC underwent breast DCE-MRI and DWI-MRI at 3.0 Tesla following surgery. The ADC values were acquired with b values of 0 and 1000?s/mm². The relationship between ADC values or DCE-MRI quantitative parameters and size, histologic grade (HG), lymph node metastasis (LNM), ER, PR, and Ki67 was evaluated. The predictive values of ADC, Ktrans, Kep, and Ve to prognosis of IDC were assessed.

Results

ADC value was positively related to size (P?=?0.04) and HER2 (P?=?0.046) expression and negatively related to ER (P?=?0.012) and PR (P?<?0.001) expression. Ktrans value has positive correlation with size (P?<?0.001), HG (P?<?0.001), LNM (P?<?0.001), HER2 (P?=?0.007), and Ki67 (P?<?0.001) expression and negative correlation with ER (P?<?0.001) and PR (P?<?0.001) expression. Kep value was positively related to size (P?<?0.001) and negatively related to ER (P?<?0.001) and PR (P?<?0.001) expression. Ve value was negatively related to HER2 expression (P?=?0.004). The Cox hazard ratio (HR) of ADC, Ktrans, Kep, and Ve values on survival was 5.26 (P?=?0.093), 1.081 (P?=?0.002), 1.006 (P?=?0.941), and 0.883 (P?=?0.926), respectively.

Conclusions

Ktrans value was a best predictive indicator of HG, LNM, ER, PR, and Ki67 expression, and ADC value was the best predictive indicator of HER2. Preoperative use of the 3.0 Tesla could provide important information to determine the optimal treatment plan.

     

Effect of ATP and Bax on the apoptosis of <Emphasis Type="Italic">Eimeria tenella</Emphasis> host cells

Background

Eimeria tenella (E. tenella) is a species of Eimeria that causes haemorrhagic caecal coccidiosis, resulting in major economic losses in the global poultry industry. After E. tenella infection, the amount of ATP and Bax in host cells showed highly significant changes. Therefore, it is necessary to investigate the effects of ATP and Bax on the apoptosis of E. tenella host cells.

Results

The ATP-treated group and the V5-treated group had higher E. tenella infection rates than the untreated group at 24, 48, 72, 96, and 120 h after infection with E. tenella. The results of flow cytometry showed that compared with the control group, the mitochondrial permeability transition pore (MPTP) opening in the untreated group was highly significantly increased (P?<?0.01) at 4, 24, 48, 72, 96, and 120 h. Moreover, results from Hoechst-Annexin V-PI staining and flow cytometry showed that the rates of early apoptosis, late apoptosis, and necrosis in the untreated group were significantly lower (P?<?0.05) or highly significantly lower (P?<?0.01) than those of the control group at 4 h, while the rates of early apoptosis, late apoptosis, and necrosis in the untreated group were higher at varying degrees than those in the control group at 24–120 h (P?<?0.05 or P?<?0.01). After treatment with ATP and Bax inhibitors, the rates of early apoptosis, late apoptosis, and necrosis, in addition to the MPTP opening in both the ATP-treated and V5-treated groups, were significantly lower (P?<?0.05) or highly significantly lower (P?<?0.01) than those in the untreated group.

Conclusions

ATP and Bax play important roles in regulating the apoptosis of E. tenella host cells.

     

Automated computed tomographic scoring of lung disease in adults with primary ciliary dyskinesia

Background

The present study aimed to develop an automated computed tomography (CT) score based on the CT quantification of high-attenuating lung structures, in order to provide a quantitative assessment of lung structural abnormalities in patients with Primary Ciliary Dyskinesia (PCD).

Methods

Adult (≥18?years) PCD patients who underwent both chest CT and spirometry within a 6-month period were retrospectively included. Commercially available lung segmentation software was used to isolate the lungs from the mediastinum and chest wall and obtain histograms of lung density. CT-density scores were calculated using fixed and adapted thresholds based on various combinations of histogram characteristics, such as mean lung density (MLD), skewness, and standard deviation (SD). Additionally, visual scoring using the Bhalla score was performed by 2 independent radiologists. Correlations between CT scores, forced expiratory volume in 1?s (FEV₁) and forced vital capacity (FVC) were evaluated.

Results

Sixty-two adult patients with PCD were included. Of all histogram characteristics, those showing good positive or negative correlations to both FEV₁ and FVC were SD (R?=???0.63 and???0.67; p?<?0.001) and Skewness (R?=?0.67 and 0.67; p?<?0.001). Among all evaluated thresholds, the CT-density score based on MLD?+?1SD provided the best negative correlation with both FEV₁ (R?=???0.68; p?<?0.001) and FVC (R?=???0.71; p?<?0.001), close to the correlations of the visual score (R?=???0.60; p?<?0.001 for FEV₁ and R?=???0.62; p?<?0.001, for FVC).

Conclusions

Automated CT scoring of lung structural abnormalities lung in primary ciliary dyskinesia is feasible and may prove useful for evaluation of disease severity in the clinic and in clinical trials.

     

<Emphasis Type="Italic">Streptococcus pyogenes</Emphasis>-purpura fulminans as an invasive form of group A streptococcal infection

Background

Streptococcus pyogenes is an uncommon pathogen of purpura fulminans, and the pathogenesis of S. pyogenes-purpura fulminans remains unclear because of paucity of cases. We reported a pediatric case of S. pyogenes-purpura fulminans with literature review of the disease.

Case presentation

A 3-year-old boy showed limping, lethargy and acral gangrene within 24 h. A diagnosis of S. pyogenes-purpura fulminans was made for bacterial isolation from throat and peripheral blood. Intensive therapy led to a survival with amputation of the left distal metatarsal bone, and normal development. The isolated M12 carried no mutation of csrS/R or rgg. Thrombophilia or immunodeficiency was excluded.

Discussion

Twelve-reported cases (9 pediatric and 3 elderly) of S. pyogenes-purpura fulminans started with shock and coagulopathy. Five patients age <?8 years had no underlying disease and survived. One youngest and two immunocompromised patients died.

Conclusion

Streptococcus pyogenes-acute infectious purpura fulminans is a distinctive rare form of aggressive GAS infections.

     

Gastrointestinal specific anxiety in irritable bowel syndrome: validation of the Japanese version of the visceral sensitivity index for university students

Objective

The visceral sensitivity index (VSI) is a useful self-report measure of the gastrointestinal symptom-specific anxiety (GSA) of patients with irritable bowel syndrome (IBS). Previous research has shown that worsening GSA in IBS patients is related to the severity of GI symptoms, suggesting that GSA is an important endpoint for intervention. However, there is currently no Japanese version of the VSI. We therefore translated the VSI into Japanese (VSI-J) and verified its reliability and validity.

Material and methods

Participants were 349 university students aged 18 and 19 years and recruited from an academic class. We analyzed data from the VSI-J, Anxiety Sensitivity Index (ASI), Hospital Anxiety and Depression scale (HAD), and Irritable Bowel Syndrome Severity Index (IBS-SI). The internal consistency, stability, and factor structure of the VSI-J and its associations with anxiety, depression and severity measures were investigated.

Results

The factor structure of the VSI-J is unidimensional and similar to that of the original VSI (Cronbach’s α?=?0.93). Construct validity was demonstrated by significant correlations with ASI (r?=?0.43, p?<?0.0001), HAD-ANX (r?=?0.19, p?=?0.0003), and IBS-SI scores (r?=?0.45, p?<?0.0001). Furthermore, the VSI-J was a significant predictor of severity scores on the IBS-SI and demonstrated good discriminant (p?<?0.0001) and incremental (p?<?0.0001) validity.

Conclusion

These findings suggest that the VSI-J is a reliable and valid measure of visceral sensitivity.

     

Expression of hypoxia-inducible factor-1α and vascular density in mammary adenomas and adenocarcinomas in bitches

Background

The study aimed at examining hypoxia-inducible factor (HIF)1α expression in adenocarcinomas and adenomas in bitches in regard to tumour malignancy grade, proliferation, apoptosis and vascularisation. Therefore, paraffin sections of 15 adenomas and 64 adenocarcinomas sampled from 79 dogs aged 6 to 16 years were analysed.

Results

A significantly higher HIF-1α expression was noted in adenocarcinomas in comparison to adenomas (P?<?0.0004). Moreover, HIF-1α expression in adenocarcinomas correlated positively with tumour malignancy grade (r?=?0.59, P?<?0.05), Ki-67 antigen expression (r?=?0.43; P?<?0.0005), TUNEL-positive cells (r?=?0.62, P?<?0001) and tumour vascularity measured by quantification of vessels characterized by the expression of von Willebrand Factor (r?=?0.57, P?<?0.05).

Conclusion

Results of this study indicate a similar biological role of HIF-1α in dogs and in humans, which may confirm suitability of the animal model in investigations on progression of tumours in humans.

     

Production of <Emphasis Type="Italic">trans</Emphasis>-10,<Emphasis Type="Italic">cis</Emphasis>-12-conjugated linoleic acid using permeabilized whole-cell biocatalyst of <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis>

Objective

To improve the production of trans-10,cis-12-conjugated linoleic acid (t10,c12-CLA) from linoleic acid in recombinant Yarrowia lipolytica.

Results

Cells of the yeast were permeabilized by freeze/thawing. The optimal conditions for t10,c12-CLA production by the permeabilized cells were at 28 °C, pH 7, 200 rpm with 1.5 g sodium acetate l^?1, 100 g wet cells l^?1, and 25 g LA l^?1. Under these conditions, the permeabilized cells produced 15.6 g t10,c12-CLA l^?1 after 40 h, with a conversion yield of 62 %. The permeabilized cells could be used repeatedly for three cycles, with the t10,c12-CLA extracellular production remaining above 10 g l^?1.

Conclusion

Synthesis of t10,c12-CLA was achieved using a novel method, and the production reported in this work is the highest value reported to date.

     

4-Vinylphenol production from glucose using recombinant <Emphasis Type="Italic">Streptomyces mobaraense</Emphasis> expressing a tyrosine ammonia lyase from <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis>

Objectives

To find a novel host for the production of 4-vinylphenol (4VPh) by screening Streptomyces species.

Results

The conversion of p-coumaric acid (pHCA) to 4VPh in Streptomyces mobaraense was evaluated using a medium containing pHCA. S. mobaraense readily assimilated pHCA after 24 h of cultivation to produce 4VPh. A phenolic acid decarboxylase, derived from S. mobaraense (SmPAD), was purified following heterologous expression in Escherichia coli. SmPAD was evaluated under various conditions, and the enzyme’s k_cat/K_m value was 0.54 mM ^?1 s^?1. Using intergenetic conjugation, a gene from Rhodobacter sphaeroides encoding a tyrosine ammonia lyase, which catalyzes the conversion of l-tyrosine to p-coumaric acid, was introduced into S. mobaraense. The resulting S. mobaraense transformant produced 273 mg 4VPh l^?1 from 10 g glucose l^?1.

Conclusion

A novel strain suitable for the production of 4VPh and potentially other aromatic compounds was isolated.

     

Production of a therapeutic protein by fusing it with two fragments of the carboxyl-terminal peptide of human chorionic gonadotropin β-subunit in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

Objective

To produce a therapeutic protein (endostatin) by fusion with two fragments of the carboxyl-terminal peptide (CTP) of the human chorionic gonadotropin β-subunit in Pichia pastoris.

Results

Two CTP sequences were fused to the C-terminal of human endostatin, and the fusion protein (endo-CTP) was expressed by P. pastoris. Endo-CTP inhibited proliferation of endothelial cells with an IC₅₀ of 7 μg ml^?1, and 30 % of cells were annexin V-positive after treatment with 20 μg endo-CTP ml^?1 for 48 h. Migration of endothelial cells was inhibited by endo-CTP in a concentration-dependent manner. The half-life of endo-CTP in Sprague–Dawley rats was much longer than that of its commercial counterpart (Endostar).

Conclusion

A long-acting endostatin can be produced using CTP technology.

     

Metabolic engineering of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> for linalool production

Objectives

To engineer the yeast Saccharomyces cerevisiae for the heterologous production of linalool.

Results

Expression of linalool synthase gene from Lavandula angustifolia enabled heterologous production of linalool in S. cerevisiae. Downregulation of ERG9 gene, that encodes squalene synthase, by replacing its native promoter with the repressible MET3 promoter in the presence of methionine resulted in accumulation of 78 µg linalool l^?1 in the culture medium. This was more than twice that produced by the control strain. The highest linalool titer was obtained by combined repression of ERG9 and overexpression of tHMG1. The yeast strain harboring both modifications produced 95 μg linalool l^?1.

Conclusions

Although overexpression of tHMG1 and downregulation of ERG9 enhanced linalool titers threefold in the engineered yeast strain, alleviating linalool toxicity is necessary for further improvement of linalool biosynthesis in yeast.

     

Urine metabolites are associated with glomerular lesions in type 2 diabetes

Introduction

Little is known about the association of urine metabolites with structural lesions in persons with diabetes.

Objectives

We examined the relationship between 12 urine metabolites and kidney structure in American Indians with type 2 diabetes.

Methods

Data were from a 6-year clinical trial that assessed renoprotective efficacy of losartan, and included a kidney biopsy at the end of the treatment period. Metabolites were measured in urine samples collected within a median of 6.5 months before the research biopsy. Associations of the creatinine-adjusted urine metabolites with kidney structural variables were examined by Pearson’s correlations and multivariable linear regression after adjustment for age, sex, diabetes duration, hemoglobin A_1c, mean arterial pressure, glomerular filtration rate (iothalamate), and losartan treatment.

Results

Participants (n?=?62, mean age 45?±?10 years) had mean?±?standard deviation glomerular filtration rate of 137?±?50 ml/min and median (interquartile range) urine albumin:creatinine ratio of 34 (14–85) mg/g near the time of the biopsy. Urine aconitic and glycolic acids correlated positively with glomerular filtration surface density (partial r?=?0.29, P?=?0.030 and r?=?0.50, P?<?0.001) and total filtration surface per glomerulus (partial r?=?0.32, P?=?0.019 and r?=?0.43, P?=?0.001). 2-ethyl 3-OH propionate correlated positively with the percentage of fenestrated endothelium (partial r?=?0.32, P?=?0.019). Citric acid correlated negatively with mesangial fractional volume (partial r=-0.36, P?=?0.007), and homovanillic acid correlated negatively with podocyte foot process width (partial r=-0.31, P?=?0.022).

Conclusions

Alterations of urine metabolites may associate with early glomerular lesions in diabetic kidney disease.