Ultrastructure of isolated mouse ovarian follicles cultured <Emphasis Type="Italic">in vitro</Emphasis>

Background  

In vitro maturation of ovarian follicles, in combination with cryopreservation, might be a valuable method for preserving and/or restoring fertility in mammals with impaired reproductive function. Several culture systems capable of sustaining mammalian follicle growth in vitro have been developed and many studies exist on factors influencing the development of in vitro grown oocytes. However, a very few reports concern the ultrastructural morphology of in vitro grown follicles.     

<Emphasis Type="Italic">In vitro</Emphasis> bioassay as a predictor of <Emphasis Type="Italic">in vivo</Emphasis> response

Background  

There is a substantial discrepancy between in vitro and in vivo experiments. The purpose of the present work was development of a theoretical framework to enable improved prediction of in vivo response from in vitro bioassay results.     

Clinical grade mesenchymal stem cells transdifferentiated under xenofree conditions alleviates motor deficiencies in a rat model of Parkinson's disease

Bone marrow derived mesenchymal stem cells (BMMSCs) is a valid, definitive candidate for repair of damaged tissues in degenerative disorders in general and neurological diseases in particular. We have standardized the processing conditions for proliferation of BMMSCs using xenofree medium and checked their in vitro and in vivo neurogenic potential.

Method

The proliferative potential of BMMSCs was analyzed using xenofree media and functionality checked by transplantation into Parkinson's disease (PD) animal models. In vitro neuronal differentiation was investigated by neuronal induction media supplemented with growth factors. Differentiated cells were characterized at cellular and molecular levels. In vitro functionality estimated by dopamine secretion.

Results

A pure population of BMMSCs showing an 8–10 fold expansion was obtained using xenofree media. On differentiation to neuronal lineage, they exhibited neuronal morphology. Detectable levels of dopamine (1.93 ng/ml) were secreted into the culture media of differentiated cells. There was a significant behavioural improvement in PD models 3 months post transplantation.

Conclusion

Our study demonstrates that BMMSCs can be transdifferentiated efficiently into functional dopaminergic neurons both in vitro and in vivo. This holds immense clinical potential as a replacement therapy for PD and other neurodegenerative diseases.     

Human liver-derived cells stably modified for regulated proinsulin secretion function as bioimplants in vivo

Background

Insulin deficiency is currently treated with pharmacological insulin secretagogues, insulin injections or islet transplants. Secondary failure of pharmacological agents is common; insulin injections often fail to achieve euglycemic control; and islet transplants are rare. Non‐β cells capable of regulated insulin secretion in vivo could be a functional cure for diabetes. Hepatocytes are good candidates, being naturally glucose‐responsive, protein‐secreting cells, while the liver is positioned to receive direct nutrient signals that regulate insulin production.

Methods

Human liver‐derived Chang cells were modified with a plasmid construct in which a bifunctional promoter comprising carbohydrate response elements and the human metallothionein IIA promoter controlled human proinsulin cDNA expression. Secretory responses of stable cell clones were characterized in vitro and in vivo by proinsulin radioimmunoassay.

Results

Transfected Chang cells secreted 5–8 pmol proinsulin/10⁶ cells per 24 h in continuous passage for at least a year in response to 5–25 mM glucose and 10–90 µM zinc in vitro. Glucose and zinc synergistically increased proinsulin production by up to 30‐fold. Non‐glucose secretagogues were also active. Glucose transporter 2 (GLUT2) and glucokinase cDNA co‐transfection enhanced glucose responsiveness. Intraperitoneally implanted Chang cells secreted proinsulin in scid and Balb/c mice. Serum proinsulin levels were further increased 1.3‐fold (p<0.05) after glucose and 1.4‐ to 1.6‐fold (p<0.005) after zinc administration in vivo.

Conclusions

These results are the first to demonstrate stable proinsulin production in a human liver‐derived cell line with activity in vitro and in vivo and provide a basis for engineering hepatocytes as in vivo bioimplants for future diabetes treatment. Copyright © 2002 John Wiley & Sons, Ltd.

     

<Emphasis Type="Italic">In vitro</Emphasis> gene regulatory networks predict <Emphasis Type="Italic">in vivo</Emphasis> function of liver

Background  

Evolution of toxicity testing is predicated upon using in vitro cell based systems to rapidly screen and predict how a chemical might cause toxicity to an organ in vivo. However, the degree to which we can extend in vitro results to in vivo activity and possible mechanisms of action remains to be fully addressed.     

Equol an isoflavonoid: potential for improved prostate health, <Emphasis Type="Italic">in vitro</Emphasis> and <Emphasis Type="Italic">in vivo</Emphasis>evidence

Background  

To determine: in vitro binding affinity of equol for 5alpha-dihydrotestosterone (5alpha-DHT), in vitro effects of equol treatment in human prostate cancer (LNCap) cells, and in vivo effects of equol on rat prostate weight and circulating levels of sex steroid hormones.     

Identification of pancreatic cancer invasion-related proteins by proteomic analysis

Background  

Markers of pancreatic cancer invasion were investigated in two clonal populations of the cell line, MiaPaCa-2, Clone #3 (high invasion) and Clone #8 (low invasion) using proteomic profiling of an in vitro model of pancreatic cancer.     

A comparative study of protein synthesis in <Emphasis Type="Italic">in vitro</Emphasis> systems: from the prokaryotic reconstituted to the eukaryotic extract-based

Background  

Cell-free protein synthesis is not only a rapid and high throughput technology to obtain proteins from their genes, but also provides an in vitro platform to study protein translation and folding. A detailed comparison of in vitro protein synthesis in different cell-free systems may provide insights to their biological differences and guidelines for their applications.     

Crude extracts of bacterially expressed dsRNA can be used to protect plants against virus infections

Background  

Double-stranded RNA (dsRNA) is a potent initiator of gene silencing in a diverse group of organisms that includes plants, Caenorhabditis elegans, Drosophila and mammals. We have previously shown and patented that mechanical inoculation of in vitro-transcribed dsRNA derived from viral sequences specifically prevents virus infection in plants. The approach required the in vitro synthesis of large amounts of RNA involving high cost and considerable labour.     

Listeria monocytogenes mediated CFTR transgene transfer to mammalian cells

Background

Several approaches for gene therapy of cystic fibrosis using viral and non‐viral vectors are currently being undertaken. Nevertheless, the present data suggest that vectors currently being used will either have to be further modified or, alternatively, novel vector systems need to be developed. Recently, bacteria have been proven as suitable vehicles for DNA transfer to a wide variety of eukaryotic cells. In this study, we assessed the ability of the facultative intracellular pathogen Listeria monocytogenes to deliver a cDNA encoding the human cystic fibrosis transmembrane conductance regulator (CFTR) to CHO‐K1 cells, since these cells have been extensively used for heterologous CFTR expression.

Methods

An established in vitro gene transfer system based on antibiotic‐mediated lysis of intracellular L. monocytogenes was exploited to transfer eukaryotic expression plasmids. Transient as well as stable CFTR transgene expression was analyzed by microscopical and biochemical methods; functionality was tested by whole‐cell patch‐clamp recordings.

Results

L. monocytogenes mediated gene transfer to CHO‐K1 cells was facilitated by an improved transfection protocol. In addition, the use of the isogenic mutant L. monocytogenes hlyW491A, engineered to produce a hemolysin variant with low toxigenic activity, greatly enhanced the efficiency of gene transfer. This strain allowed the transfer of functional CFTR to CHO‐K1 cells.

Conclusions

This is the first demonstration of L. monoyctogenes mediated CFTR transgene transfer. The successful in vitro transfer suggests that L. monocytogenes might be a potential vector for cystic fibrosis gene therapy or alternative applications and deserves further investigation in vitro as well as in vivo. Copyright © 2002 John Wiley & Sons, Ltd.

     

<Emphasis Type="Italic">In vitro</Emphasis> regeneration of the medicinal woody plant <Emphasis Type="Italic">Phellodendron amurense</Emphasis> Rupr. through excised leaves

Shoot organogenesis and plant establishment has been achieved for Phellodendron amurense Rupr. from excised leaf explants. Young leaf explants were collected from in vitro established shoot cultures and used for the induction of direct shoot regeneration, callus and subsequent differentiation into shoots on MS medium. Direct shoot regeneration was achieved by culturing 1 cm² sections of about 10-day-old leaves on MS medium enriched with 4.4 M BAP and 1.0 M NAA after 4 weeks of culture. The leaf explants produced callus from their cut margins within 3 weeks of incubation on medium supplemented with 2.0 M TDZ and 4.0 M 2,4-D or 4.0 M NAA. The maximum number of adventitious shoots was regenerated from the leaf-derived callus within 4 weeks of culture on MS medium containing 1.5 M BAP and 1.0 M NAA. The highest rate of shoot multiplication was achieved at the third subculture, and more than 65 shoots were produced per callus clump. For rooting, the in vitro proliferated and elongated shoots were excised into 2–4 cm long microcuttings, which were planted individually on a root-induction MS medium containing 2.0 M IBA. Within 3 weeks of transfer to the rooting medium, all the cultured microcuttings produced 2–6 roots. The in vitro regenerated plantlets were transferred to Kanuma soil, and the survival rate ex vitro was 90%.     

Chromatin remodeling agent trichostatin A: a key-factor in the hepatic differentiation of human mesenchymal stem cells derived of adult bone marrow

Background  

The capability of human mesenchymal stem cells (hMSC) derived of adult bone marrow to undergo in vitro hepatic differentiation was investigated.     

<Emphasis Type="Italic">Brucella abortus ure2</Emphasis> region contains an acid-activated urea transporter and a nickel transport system

Background  

Urease is a virulence factor that plays a role in the resistance of Brucella to low pH conditions, both in vivo and in vitro. Brucella contains two separate urease gene clusters, ure1 and ure2. Although only ure1 codes for an active urease, ure2 is also transcribed, but its contribution to Brucella biology is unknown.     

Identification and characterization of rhizosphere fungal strain MF‐91 antagonistic to rice blast and sheath blight pathogens

Aim

To examine the inhibition effects of rhizosphere fungal strain MF‐91 on the rice blast pathogen Magnaporthe grisea and sheath blight pathogen Rhizoctonia solani.

Methods and Results

Rhizosphere fungal strain MF‐91 and its metabolites suppressed the in vitro mycelial growth of R. solani. The inhibitory effect of the metabolites was affected by incubation temperature, lighting time, initial pH and incubation time of rhizosphere fungal strain MF‐91. The in vitro mycelial growth of M. grisea was insignificantly inhibited by rhizosphere fungal strain MF‐91 and its metabolites. The metabolites of rhizosphere fungal strain MF‐91 significantly inhibited the conidial germination and appressorium formation of M. grisea. Moreover, the metabolites reduced the disease index of rice sheath blight by 35·02% in a greenhouse and 57·81% in a field as well as reduced the disease index of rice blast by 66·07% in a field. Rhizosphere fungal strain MF‐91 was identified as Chaetomium aureum based on the morphological observation, the analysis of 18S ribosomal DNA internal transcribed spacer sequence and its physiological characteristics, such as the optimal medium, temperature and initial pH for mycelial growth and sporulation production.

Conclusions

Rhizosphere fungus C. aureum is effective in the biocontrolling of rice blast pathogen M. grisea and sheath blight pathogen R. solani both in in vitro and in vivo conditions.

Significance and Impact of the Study

This study is the first to show that rhizosphere fungus C. aureum is a potential fungicide against rice blast and sheath blight pathogens.     

Physiological tonicity improves human chondrogenic marker expression through nuclear factor of activated T-cells 5 <Emphasis Type="Italic">in vitro</Emphasis>

Introduction  

Chondrocytes experience a hypertonic environment compared with plasma (280 mOsm) due to the high fixed negative charge density of cartilage. Standard isolation of chondrocytes removes their hypertonic matrix, exposing them to nonphysiological conditions. During in vitro expansion, chondrocytes quickly lose their specialized phenotype, making them inappropriate for cell-based regenerative strategies. We aimed to elucidate the effects of tonicity during isolation and in vitro expansion on chondrocyte phenotype.     

Effects of bone morphogenic protein 4 (BMP4) and its inhibitor,Noggin, on <Emphasis Type="Italic">in vitro</Emphasis>maturation and culture of bovine preimplantation embryos

Background  

BMP4 is a member of the transforming growth factor beta (TGFbeta) superfamily and Noggin is a potent BMP inhibitor that exerts its function by binding to BMPs preventing interactions with its receptors. The aim of this work was to investigate the role of BMP4 and Noggin, on oocytes in vitro maturation (m experiments) and embryos in vitro development (c experiments) of bovine.     

Effect of ethanol on the response of the rat urinary bladder to in vitro ischemia: Protective effect of α-lipoic acid

Purpose: Ethanol exposure has been used to demonstrate the increase of oxidative stress to a variety of tissues. We studied the effect of ethanol on the response of isolated strips of rat bladder to In vitro hypoxia in the absence of glucose (In vitro ischemia). Secondly, we determined if -lipoic acid (LA) could alter the response to ethanol + In vitro ischemia.Methods: Sixty-four rats were used for the these experiments. Each rat was anesthetized and its urinary bladder excised. The bladder body was cut into two longitudinal strips and each strip mounted in individual baths filled with oxygenated Tyrodes solution containing glucose at 37 C. Ethanol (0.3%, 1%, or 3%) was placed in the first six baths (two strips at each concentration). The last two baths did not receive ethanol. Each strip was incubated for 1 h and then stimulated with field stimulation at 2, 8, and 32 Hz. Each strip was stimulated with 10 M carbachol, washed three times with fresh oxygenated buffer and ethanol re-added to their respective baths. Each strip was then stimulated with 120 mM KCl and washed three times as before. Strips were then subjected to 1 h In vitro ischemia (incubation in the absence of glucose with Tyrodes equilibrated with nitrogen instead of oxygen). During the ischemic period, each strip was stimulated for 5 s every 10 min by 32 Hz FS to simulate hyperreflexia. At the end of the hour, the tissues were incubated for an additional hour in the presence of oxygen + glucose and subjected to a second series of stimulations as before. At all times, ethanol was maintained in baths 1–6. In set 2, 1% ethanol was added to the first six baths. LA was added to every other bath, and the experiments performed as mentioned earlier.Results: (a) Ethanol at 0.3% or 1% had no effect on the contractile responses prior to exposure to In vitro ischemia; 3% was inhibitory. (b) In vitro ischemia mediated a significant decrease in the contractile responses to all forms of stimulation except for carbachol. (c) Ethanol mediated a dose-response enhancement of the contractile dysfunctions caused by In vitro ischemia. (d) LA completely reversed the effects of ethanol on contractile responses following In vitro ischemia except for carbachol.Conclusions: The results demonstrate that direct exposure to ethanol significantly enhanced contractile dysfunctions mediated by In vitro ischemia followed by re-oxygenation and that the presence of LA significantly inhibits this effect of ethanol. (Mol Cell Biochem 271: 133–138, 2005)This material is based upon work supported in part by the Office of Research and Development, Department of Veterans Affairs, and NIH RO-1-DK067114