Exploiting the antivirulence efficacy of an ajoene-ciprofloxacin combination against Pseudomonas aeruginosa biofilm associated murine acute pyelonephritis

The study investigated the in vitro, ex vivo and in vivo efficacy of ajoene and ciprofloxacin (CIP) alone and in combination against Pseudomonas aeruginosa biofilms and biofilm-associated murine acute pyelonephritis. The ajoene–CIP combination exhibited significant greater (p < 0.05) antimotility and biofilm inhibitory effects than those obtained when they were applied individually. The combined action of the agents resulted in a significant increase in serum sensitivity and phagocytic uptake and killing of P. aeruginosa (p < 0.001) compared to the untreated control. Mice groups treated with an ajoene (25 mg kg^?1) and CIP (30 mg kg^?1 or 15 mg kg^?1) combination showed a significantly (p < 0.001) reduced bacterial load in the kidney and bladder as compared to that of infected controls and mice treated with solo agents on the fifth day post-infection. The decreased levels of biomarkers and photomicrographs of the kidney tissue of the treated mice showed a reduced severity of damage. Hence, the study highlights the antivirulent and therapeutic efficacy of the ajoene-CIP combination at the minimal dosage of CIP.     

Methyl farnesoate induced ovarian maturation in the spider crab,Libinia emarginata

Summary

To overcome the problem of getting crustaceans to reproduce in captivity, eyestalk ablation or X-organ sinus gland removal is commonly utilized in commercially important species such as shrimp. We have investigated the effect of unilateral and bilateral eyestalk ablation on methyl farnesoate (MF) production by mandibular organs (MOs) and on ovarian maturation in female spider crabs Libinia emarginata, a useful model since these animals are in a terminal molt and are devoid of a functional Y-organ. Non-reproductive, over-wintering female L. emarginata were induced to be reproductive by feeding and increasing the holding temperature to stimulate the endocrine system. In addition, we removed X-organ sinus glands by eyestalk ablation either unilaterally (UEA) or bilaterally (BEA) to further stimulate MF synthesis by MOs. Endogenous MF in the hemolymph was extracted and quantified by means of HPLC and in some cases by GC/MS. Oocyte growth and egg quality were studied simultaneously to determine how they were related to MF levels found during vitellogenesis. The initial MF concentration in unablated controls was low, 0.31 ng/ml of hemolymph, and this increased (p<0.05) to about 1 ng/ml by 2 weeks, remaining at about that level for the remainder of the experiment. Eyestalk ablation significantly stimulated MF concentrations by week 1 to nearly 2 and 3.5ng/ml in the UEA (p <0.01) and BEA (p <0.001) animals, respectively. Oocytes appeared to respond to increased MF levels, as ovarian maturation was initiated from the point at which MF increased (p <0.05). Thereafter, the rate of oocyte growth was directly correlated with the extent of elevation of MF. The gonado-somatic index [(GSI) = gonad weight/body weight × 100] of controls at the start was about 1.5 and increased to 6.5 by week 4. Mature oocytes were reached at a GSI around 7. Oocyte maturation was accomplished at week 2 in BEA, week 3 in UEA, and later than week 4 in controls. After maturation, oocytes started to degrade in some ablated animals, particularly in the bilaterally ablated ones where the highest MF concentrations were observed. These data indicate that MF elevations are required for stimulating ovarian maturation in Crustacea. MF appears to accelerate gonad development during the vitellogenic process, but may be deleterious at high concentrations. These results have a significant and important application and implications for aquaculture.     

Melatonin does not affect the black pigment migration in the crab Neohelice granulata

N-acetyl-5-methoxytryptamine or melatonin is a multifunctional molecule. The main physiological function, at least in vertebrates, is to transduce to the animal the photoperiodic information and regulate rhythmic parameters. But studies have also observed the action of this molecule on pigment migration in ectothermic vertebrates. Thus the aim of this paper was to investigate in vivo and in vitro the influence of melatonin on the pigment migration in melanophores of the crab Neohelice granulate. Injections of melatonin (2 × 10⁻⁹ moles · crab⁻¹) at 07:00 h or 19:00 h did not affect (p > 0.05) the circadian pigment migration of the melanophores in constant darkness. Additionally no significant pigment migration (p > 0.05) was verified in normal and eyestalkless crabs injected with melatonin (10⁻¹⁰–10⁻⁷ moles · crab⁻¹) during the day or night. In the ^{in vitro} assay, the response of melanophores to the pigment-dispersing hormone in eyestalkless crabs injected with melatonin (2 × 10⁻⁹ moles · crab⁻¹) 1 and 12 hours before the observations did not differ (p > 0.05) from the control group (injected with physiological solution). These results suggest that melatonin does not act as a signaling factor for pigment dispersion or aggregation in the melanophores of N. Granulate.     

Seasonal variation in in vitro immune activity of milk leukocytes in elite and non-elite crossbred cows of Indian sub-tropical semi-arid climate

Immunity of mammary gland in terms of in vitro activity of milk leukocytes has been evaluated during hot-humid, summer, and winter season in elite (n = 10) and non-elite (n = 10) crossbred cows. Milk samples were collected from all the cows throughout the year at 15-day interval. Milk somatic cell counts (SCC) and differential leukocyte counts (DLC) were evaluated microscopically. Milk neutrophils, macrophages, and lymphocytes were isolated and cultured in vitro. In vitro PI of milk neutrophils and macrophages was evaluated by colorimetric NBT (nitro-blue tetrazolium) reductive assay. Mitogen-induced milk lymphocyte blastogenic response was measured by colorimetric MTT (thiazolyl blue tetrazolium bromide) assay. Milk SCC was found to be significantly (p < 0.01) higher in elite cows compared to non-elite cows irrespective of season. There was significant (p < 0.05) increase in milk SCC during hot-humid season compared to winter season in both the group of the cows. There was no significant difference between group and season in terms of DLC. In vitro phagocytic index of elite cows was significantly (p < 0.01) higher than non-elite cows. The phagocytic index was significantly (p < 0.01) decreased in summer and hot-humid season compared to winter season in both the group of animals. Macrophages isolated from elite cows having significantly (p < 0.01) lower phagocytic index than non-elite cows which significantly (p < 0.01) decreased during summer and hot-humid season compared to winter. In vitro milk lymphocyte proliferative response was significantly (p < 0.01) lower in elite cows. Activity of B-lymphocytes decreased significantly (p < 0.01) during summer and hot-humid season than winter, but activity of T-lymphocytes remains unaltered during different seasons. In conclusion, the mammary immunity in terms of in vitro activity of milk leukocytes is compromised during summer and hot-humid season in elite crossbred cows; therefore, better care and management should be taken in high-yielding cows during summer and hot-humid season to minimize intramammary infections.     

In vitro effect of methyl farnesoate on the vitellogenin content of ovary and hepatopancreas,in the crayfish Cherax quadricarinatus

Vitellogenin levels were determined in pieces of either ovary or hepatopancreas taken from females of the crayfish Cherax quadricarinatus during both early pre-reproductive and reproductive periods, and exposed in vitro to 0.15, 1.5, or 15?µM of the juvenoid methyl farnesoate (MF). A significant accumulation of vitellogenin was seen in ovaries treated with 15?µM of MF during the early pre-reproductive period. Besides, protein synthesis, measured as tritiated leucine incorporation, was correspondingly increased in ovaries taken during both pre- and post-reproductive periods and exposed to 15?µM of MF. On the other hand, no stimulating effect of MF on the vitellogenin content of the hepatopancreas was seen in any period. These results, taken together, support the hypothesis of the endogenous vitellogenin synthesis stimulated by MF during the early ovarian maturation. However, this hormone was not able to stimulate vitellogenin synthesis in the ovary during the reproductive period.     

IGF1 stimulates differentiation of primary follicles and their growth in ovarian explants of zebrafish (<Emphasis Type="Italic">Danio rerio)</Emphasis> cultured <Emphasis Type="Italic">in vitro</Emphasis>

The present study is an attempt to elucidate the involvement of insulin-like growth factor (IGF1) in the differentiation and growth of primary follicles in ovarian explant cultures of zebrafish. Ovaries from adult females were cultured in triplicate sets/treatment group for 15 days at 22°C in the laboratory. Culture medium was supplemented with either insulin (1 ng/mL) or IGF1 (1 ng/mL) or insulin + IGF1 (Experiment 1) or 0.1 or 1.0 or 10 ng/mL of IGF1 (Experiment 2). Ovaries cultured in medium alone served as controls and those fixed at the beginning of the culture as initial controls. Experiments were repeated. On the 16th day ovarian explants were fixed in Bouin’s fluid and processed for paraffin embedding, sections (3 µm) were cut and stained with hematoxylin-eosin. Follicles were classified into 6 stages and atretic follicles (AF). Previtellogenic, vitellogenic and total follicle number was calculated. At the start of the culture, ovaries contained all stages of growing and degenerating follicles. In in vitro cultured control ovaries, vitellogenic follicles underwent atresia, while, primary follicles remained unaffected. Insulin or insulin + IGF1 treated ovaries did not differ significantly while IGF1 exposed ovarian explants had greater (P < 0.05) number of primary follicles compared to controls. IGF1 also caused an increase in the number and growth of primary follicles in a dose dependent manner although; cultures were not supplemented with gonadotrophic hormones. Results suggest that locally derived intra-ovarian IGF1 may have a role in the differentiation and growth of primary follicles in zebrafish ovary.     

Methyl jasmonate promotes anthocyanins’ production in Prunus salicina × Prunus persica in vitro shoot cultures

In vitro shoot cultures of Prunus salicina × Prunus persica, “Citation®” rootstock, were treated with 50-μM methyl jasmonate (MJ) or 100-μM abscisic acid (ABA); in MJ-treated shoots, total anthocyanins increased significantly (1.88 mg/g fresh weight) relative to controls (0.43 mg/g fresh weight). Cyanidin 3-glucoside was the most abundant anthocyanin in both MJ-treated and control explants. The addition of ABA to the culture medium did not elicit anthocyanins’ accumulation.     

Effects of different processing methods of flaxseed on ruminal degradability and in vitro post-ruminal nutrient disappearance

The aim of the study was to determine the effects of different heat-processing methods of flaxseed on the in situ effective dry matter degradability (EDMD) and the in situ effective crude protein degradability (ECPD). The treatments included roasting, steep roasting, rolled roasting, rolled steep roasting, microwave irradiation and extrusion. Three rumen-fistulated sheep were used for in situ incubations. Furthermore, the effects of heat-processing methods on post-ruminal in vitro nutrient disappearance and total tract disappearance were measured by a three-step in vitro technique. The seeds were roasted and extruded at 140°C to 145°C. One lot of roasted seeds was gradually cooled for about 1 h (roasting) and another lot was held in temperature isolated barrels for 45 min (steep roasting). Moreover, roasted and steep roasted flaxseed was rolled in a roller mill. The lowest and highest EDMD was observed for unheated and extruded flaxseed, respectively (p < 0.05). The highest ECPD was observed for extruded flaxseed (p < 0.05). Roasting and microwave irradiation reduced ECPD of flaxseed (p < 0.05). In vitro post-ruminal disappearance of crude nutrients including fibre fractions was highest for rolled-roasted and rolled steep-roasted flaxseed (p < 0.05). The lowest and highest total tract disappearance rates of crude nutrients and fibre fractions were estimated for unheated and extruded flaxseed, respectively (p < 0.05). The post-ruminal disappearance of crude nutrients was also increased by roasting, in which rolling enhanced this effect. In conclusion, all investigated heat treatments had significant effects on in situ and in vitro degradability of nutrients. As well, rolling of roasted flaxseed enhanced the respective effects. Therefore, different methods of heat processing can be used to modify the feed value of flaxseed for specific purposes.     

Predatory activity and passage of six nematophagous fungi species in gastrointestinal tract of trichostrongylide-infected sheep*

With the development of anthelmintic resistance of parasitic nematodes, screening the potential of predatory fungi candidates by in vitro and in vivo assay is necessary for biocontrol of parasitic nematodes in sheep. Fifteen native isolates of fungi species of six predators were evaluated through in vitro and in vivo experiment to evaluate the capacity of passing through the gastrointestinal tract of sheep. The results of the in vitro experiment showed that the reduction percentages of the third-stage larvae (L3) of trichostrongylides in faeces were 97.02–98.49% for five isolates of Arthrobotrys (Monacrosporium) sinense; 83.47–99.12% for three isolates of Arthrobotrys oviformis; 80.00–97.41% for four isolates of Arthrobotrys conoides; and 99.18%, 77.56%, and 75.72% for one isolate of Arthrobotrys microscaphoides, Arthrobotrys salina, and Dactylellina leptospora, respectively. In the in vivo assay, the results exhibited that the larval recoveries in faeces were significantly decreased (p?D. leptospora. After fungal treatment (within 24?h) of one isolate of A. salina, although the efficacy against L3 was 43.83%, tested fungus was detected in the faeces. The remaining isolates, except one isolate of D. leptospora and one isolate of A. conoides, were positive for either L3 reduction or fungal survival in faeces. The present study showed that nematophagous fungi could survive in the passage through the alimentary tract of sheep and should be potential candidates as a feed supplement.     

Characterization of progeny of Coleus blumei following an in vitro selection for salt tolerance

An in vitro selection method was developed for Coleus blumei to enhance salt tolerance of this amenity species. Leaf disc explants were incubated on a Murashige & Skoog medium containing benzylaminopurine, 2 mg l^-1, and napthalene acetic acid, 1 mg l^-1, which initiated both callus and plantlets from the explants. A large number of explants were incubated on this differentiating medium containing 90 mM NaCl, which inhibited over 90% of plantlet formation. Surviving plantlets. were grown to maturity, when apical cuttings were taken and propagated. Plants were also allowed to flower and set seed. Cuttings from the selected regenerated plants showed consistently better growth in the presence of NaCl than unselected cuttings. Seed progeny of selected plants also showed more vigorous growth in the presence and absence of NaCl than progeny from unselected plants. The in vitro selection was compared with the results of an earlier in vivo selection to assess the contribution from tissue culture derived somaclonal variation. Progeny from the in vitro selection showed a higher level of tolerance than progeny from the in vivo selection.     

Effect of copper on callus growth and gene expression of in vitro-cultured pith explants of Nicotiana glauca

Abstract

Copper is a vital component of electron transfer reactions mediated by proteins such as superoxide dismutase, cytochrome c oxidase and plastocyanin, but its concentrations in the cells needs to be maintained at low levels. In fact, the same ability of this essential metal ion to transfer electrons can also make it toxic to cells when present in excess. In vitro cultured explants of Nicotiana have been extensively used as a model to analyse metal-DNA interactions. In this report, we examined the effect of copper (1, 10 and 100 μM CuSO₄) on callus growth and protein synthesis of in vitro-cultured pith explants of Nicotiana glauca. In addition, a N. glauca cDNA library from Cu-treated (100 μM CuSO₄) pith explants cultured in vitro for 24 h was analysed by mRNA differential screening. The copper treatments inhibited callus growth of pith explants. The extent of inhibition was directly correlated to metal concentration. One and 10 μM CuSO₄ induced a notable increase of proteins synthesis relative to control explants. By contrast, 100 μM CuSO₄ inhibited protein synthesis relative to control extracts. The SDS-PAGE fluorography of pith proteins revealed, in Cu-treated extracts qualitative and/or quantitative differences in the synthesis of some polypeptides compared with control explants. Copper-modulated patterns of gene expression were also analysed by mRNA differential screening. The N. glauca genes isolated from Cu-treated pith explants shared common identities with other genes known to be elicited by diverse stresses, including pathogenesis and abiotic stress. In particular, the cDNAs were homologues to genes encoding cell wall proteins (i.e., extensin, and arabinogalactan-protein) and pathogenesis-related proteins (i.e., osmotin, endochitinase and a member of the Systemic Acquired Resistance gene family). In addition, an MD-2-related lipid-recognition (ML) domain protein and the enzyme S-adenosyl-L-homocysteine (AdoHcy) hydrolase appeared involved in the response to copper stress. In animal cells, AdoHcy hydrolase is a copper binding protein in vivo, which suggests that, also in plant tissues, this enzyme may play an important role in regulating the levels and intracellular distribution of copper.     

The influence of habitat, season and tidal regime in the activity of the intertidal crab Neohelice (=Chasmagnathus) granulata

The activity pattern of intertidal crabs is influenced by factors that usually change rhythmically following tidal and/or diel cycles, and is often associated with the use of refuges. The movement activity of the burrowing crab Neohelice granulata was compared among three populations from SW Atlantic coastal areas where they face different tidal regimes, water salinities, substrata and biological factors. At each site, we examined the seasonal activity of the crabs (individuals collected in pitfall traps) in two types of habitat: mudflat and salt marsh. The working hypothesis is that the activity would vary according to the diverse environmental conditions encountered at geographical and local scales. Crab activity varied between sites and seasons showing to be more intense when habitats were covered by water. The most active groups were large males, followed by large non-ovigerous females. Ovigerous females were almost inactive. Most crabs were near or inside burrows at low tides in Mar Chiquita and Bahía Blanca, but they were active at both low and high tides in San Antonio during spring and summer. N. granulata were active in a wide range of temperatures: from 10 to 37 °C at low tides and at temperatures as low as 2 °C when covered by water. Differences of activity between mudflat and salt marsh varied among sites depending on flooding frequencies. Movement activity of N. granulata varied both in space and in time; crabs move under very different abiotic conditions (e.g., low or high tide, daylight or night, low and high temperature) and their movement may also be prevented or elicited by biotic conditions like burrow complexity, food quality and predation pressure. The wide set of conditions under which N. granulata can be active may explain why this is the only semiterrestrial crab inhabiting latitudes higher than 40°S in South America.     

Monitoring of polymorphonuclear leukocyte functions in diabetes mellitus— a comparative study of conventional radiometric function tests and low-light imaging systems

In this study neutrophil (PMN) phagocytic capacity was investigated using a conventional radiometric ingestion assay (IN) in comparison with PMN respiratory burst activity assessed by luminol-enhanced chemiluminescence (LCL) in response to phorbolesters and LCL induction during phagocytosis of opsonized Staphylococous aureus (STLCL) in diabetes mellitus and healthy controls. PMN ingestion was measured with ³H-thymidine-labelled S. aureus in a kinetic radiometric assay. LCL and STLCL were assessed in a parallel detecting microtitre-plate luminometer (MTP-Reader). PMN of diabetic subjects showed a highly significant reduction of peak LCL in response to PMA as well as during phagocytosis of S. aureus (STLCL) compared to non-diabetic controls (p<0.001 respectively). PMN ingestion in diabetic patients (51.8±4.6%) was significantly reduced compared to controls (78.3±6.2%) (p<0.01). The in vitro data displayed impaired PMN oxidative burst activity at glucose concentrations ? 13.8mmol/L, whereas PMN IN was significantly reduced at glucose levels ?27.75mmol/L. The control group showed a positive correlation of peak LCL response and IN (p<0.05) but not of STCL and IN; in diabetic patients this was also true, but did not reach statistical significance. The data obtained in this study clearly demonstrated impaired PMN respiratory burst activity and markedly reduced phagocytic PMN functions in diabetic patients ex vivo and in vitro as measured by LCL and by ingestion of ³H-thymidine-labelled S. aureus suggesting inhibitory effects of elevated glucose concentrations on various PMN-functions, which might be of clinical importance concerning altered host defence.     

The effects of copper additives on the quantity and cell viability of adherent Staphylococcus epidermidis in silicone implants

This in vitro study evaluated the antibacterial effect of copper additives in silicone implants. Specimens of a standard silicone material used in breast augmentation and modified copper-loaded silicone specimens were prepared and incubated in a Staphylococcus epidermidis suspension (2 h, 37°C). After the quantification of adhering staphylococci using a biofluorescence assay (Resazurin), the viability of the adhering bacterial cells was quantified by live or dead cell labeling in combination with fluorescence microscopy. In the Resazurin fluorometric quantification, a higher amount of adhering S. epidermidis cells was detected on pure silicone (4612 [2319/7540] relative fluorescence units [rfu]) than on silicone with copper additives (2701 [2158/4153] rfu). Additionally, a significantly higher amount of adhering bacterial cells (5.07% [2.03%/8.93%]) was found for pure silicone than for silicone with copper additives (1.72% [1.26%/2.32%]); (p < 0.001). Calculations from live or dead staining showed that the percentage of dead S. epidermidis cells adhered on pure silicone (52.1%) was significantly lower than on silicone with copper additives (79.7%); (p < 0.001). In vitro, silicone material with copper additives showed antibacterial effects against S. epidermidis. Copper-loaded silicone may prevent bacterial colonization, resulting in lower infection rates of silicone implants.     

Structure of Carbohydrate Moiety in Asterosaponin A. Isolation of Three New Disaccharides

ABSTRACT

This study aimed to investigate the role of serine/threonine kinase PkaE in Streptomyces coelicolor A3(2). Liquid chromatography tandem mass spectrometry was performed for comparative phosphoproteome and proteome analyses of S. coelicolor A3(2), followed by an in vitro phosphorylation assay. Actinorhodin production in the pkaE deletion mutant was lower than that in wild-type S. coelicolor A3(2), and the spores of the pkaE deletion mutant were damaged. Furthermore, phosphoproteome analysis revealed that 6 proteins were significantly differentially hypophosphorylated in pkaE deletion mutant (p < 0.05, fold-change ≤ 0.66), including BldG and FtsZ. In addition, the in vitro phosphorylation assay revealed that PkaE phosphorylated FtsZ. Comparative proteome analysis revealed 362 differentially expressed proteins (p < 0.05) and six downregulated proteins in the pkaE deletion mutant involved in actinorhodin biosynthesis. Gene ontology enrichment analysis revealed that PkaE participates in various biological and cellular processes. Hence, S. coelicolor PkaE participates in actinorhodin biosynthesis and morphogenesis.     

Influence of supplemental endoglucanase or xylanase on volatile fatty acid production from ruminant feed by ruminal in vitro cultures

Abstract

This study employed two commercial enzyme preparations to examine the effects of endoglucanase, xylanase or their combination on in vitro volatile fatty acid (VFA) production by ruminal microbial populations. Batch ruminal cultures were established with one of various feedstuffs or with a fescue hay-based diet and ruminal fluid from a heifer fed a 40% forage:60% concentrate diet. Addition of xylanase at 135 xylanase units (XU) per ml increased total VFA production from the fescue hay-based diet (44.3 vs. 57.2 mM, p < 0.05) without changing the acetate to propionate (A:P) ratio. Addition of endoglucanase at 2, 3, 4, and 5 carboxymethyl cellulase units (CMCU) per ml increased total VFA production from the fescue hay-based diet on average by 36% (p < 0.05). Addition of 3, 4 and 5 CMCU/ml also decreased (p < 0.05) the A:P ratio. The combined addition of xylanase (135 XU/ml) and endoglucanase (5 CMCU/ml) increased total VFA production from the fescue hay-based diet (40.9 vs. 61.5 mM, p < 0.05) and reduced the A:P ratio (3.4 vs. 1.5, p < 0.05). The effects of endoglucanase and xylanase supplementation on in vitro VFA production varied across the various substrates used. However, endoglucanase supplementation consistently reduced the A:P ratio with all substrates tested. The effects of the enzyme combination were generally greater than either enzyme alone. We conclude that endoglucanase and xylanase activities differ in their ability to affect ruminal VFA production, and endoglucanase but not xylanase, may improve fermentation efficiency by reducing the A:P ratio.     

Effect of methoprene and 20-hydroxyecdysone on vitellogenin production in cultured fat bodies and backless explants from unfed female Dermacentor variabilis

The effect of 20-hydroxyecdysone (20HE) and the juvenile hormone analogue methoprene (JHA) on vitellogenin (Vg) production in fat body organ cultures and backless explants of unfed female Dermacentor variabilis was measured. An indirect double antibody enzyme linked immunosorbent assay (ELISA) was developed using a monoclonal antibody that recognized a 98 kDa subunit of Vg and a Vg specific polyclonal antibody made against vitellin (Vn). Peak Vg titers in culture medium from fat body cultures treated with 0.1 &mgr;M 20HE or 1 &mgr;M 20HE were 24 ng/ml and 20 ng/ml respectively. In culture medium from backless explants treated with 0.1 &mgr;M 20HE or 1 &mgr;M 20HE, peak Vg titers were 36 ng/ml and 26 ng/ml, respectively. JHA produced only a slight increase in Vg titers that was statistically different from Vg titers produced by 20HE but was not statistically different from hormone-free controls. These results support the conclusion that Vg production in fat body trophocytes of D. variabilis is regulated by 20HE.     

β-hCG promotes epithelial ovarian cancer metastasis through ERK/MMP2 signaling pathway

Epithelial ovarian cancer (EOC) is the most lethal gynecologic malignancy, with typically extensive intraperitoneal implantation leading to poor prognosis. Our previous study preliminarily demonstrated β-hCG can promote tumorigenesis in immortalized nontumorigenic ovarian epithelial cells. In this study, the roles and mechanisms of β-hCG in regulating EOC proliferation and metastasis were thoroughly explored. First, histologically, β-hCG was aberrantly overexpressed in human EOC metastatic tissues, and significantly correlated with FIGO stage, tumor size, differentiation, histologic grade and high grade serous ovarian carcinoma (HGSOC) (P < 0.05). However, serologically, β-hCG expression showed no significant difference between EOC and nonmalignant ovarian patients. Second, β-hCG was confirmed to have no significant effects on EOC proliferation in vitro and in vivo, while β-hCG upregulation was proven to promote migration and invasion ability in ES-2 and OVCAR-3 cells in vitro (P < 0.05), and β-hCG downregulation in SKOV3 cells had the opposite effect. Moreover, more invadopodia protrusions, mitochondria accumulations and cytoskeletal rearrangements were observed in β-hCG-overexpressing ES-2 cells, while β-hCG-depleted SKOV3 cells produced the opposite effect. Furthermore, β-hCG was confirmed to clearly facilitate intraperitoneal metastasis in nude mouse orthotopic ovarian xenograft models. Importantly, these effects of β-hCG were mediated by activation of the ERK/MMP2 signaling pathway, independently of luteinizing hormone/chorionic gonadotropin receptor (LHCGR) presence, and inhibition the pathway with the p-ERK1/2 inhibitor SCH772984 significantly impaired the tumor-promoting effects induced by β-hCG. Collectively, these data provide new insight into the roles and mechanisms of β-hCG in regulating EOC metastasis through ERK/MMP2 signaling pathway and may become a new target for therapeutic intervention.     

Effects of homocysteine on adipocyte differentiation and CD36 gene expression in 3T3-L1 adipocytes

The aim of this study was to investigate the effects of homocysteine (Hcy), a risk factor for cardiovascular diseases, hypertension, stroke and obesity, on expression of CD36 that regulates uptake of oxidized low-density lipoprotein (Ox-LDL) by adipocytes and differentiation of 3T3-L1 cells to adipocytes. Cell viability was determined using MTT assay, and density of triglycerides were measured with Oil Red O staining. The expression levels of CD36 were analyzed using SYBR green assay by quantitative RT-PCR. Our results showed that the addition of Hcy inhibited differentiation of 3T3-L1 preadipocytes in a dose-dependent manner without a significant cell toxicity (p < 0.05). Percentage CD36 gene expression increased in the Hcy treatment groups, but not statistically significantly (p > 0.05) compared to differentiated adipocytes. Hcy reduced adipocyte differentiation, but had no effect on the expression level of CD36 in vitro conditions. The effect of Hcy on uptake and clearance of Ox-LDL by adipose tissue now needs to be investigated in vivo.