Methods for Morphological and Biochemical Analysis of Invasion in vitro

Author(s):  
M. Mareel ◽  
E. Bruyneel ◽  
G. De Bruyne ◽  
C. Dragonetti
Keyword(s):  
2012 ◽  
Vol 18 (S2) ◽  
pp. 110-111
Author(s):  
L. Rothschild ◽  
F. Mwaura ◽  
J. Kabaru ◽  
N. Lobo ◽  
K. Moulton ◽  
...  

Extended abstract of a paper presented at Microscopy and Microanalysis 2012 in Phoenix, Arizona, USA, July 29 – August 2, 2012.


Author(s):  
Storm N. S. Reid ◽  
Joung-Hyun Park ◽  
Yunsook Kim ◽  
Yi Sub Kwak ◽  
Byeong Hwan Jeon

Exogenous lactate administration has more recently been investigated for its various prophylactic effects. Lactate derived from potential functional foods, such as fermented oyster extract (FO), may emerge as a practical and effective method of consuming exogenous lactate. The current study endeavored to ascertain whether the lactate derived from FO may act on muscle cell biology, and to what extent this may translate into physical fitness improvements. We examined the effects of FO in vitro and in vivo, on mouse C2C12 cells and exercise performance indicators in mice, respectively. In vitro, biochemical analysis was carried out to determine the effects of FO on lactate content and muscle cell energy metabolism, including adenosine triphosphate (ATP) activity. Western blot analysis was also utilized to measure the protein expression of total adenosine monophosphate-activated protein kinase (AMPK), p-AMPK (Thr172), lactate dehydrogenase (LDH), succinate dehydrogenase (SDHA) and peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α) in response to FO administration. Three experimental groups were formed: a positive control (PC) treated with 1% horse serum, FO10 treated with 10 μg/mL and FO50 treated with 50 μg/mL. In vivo, the effects of FO supplementation on exercise endurance were measured using the Rota-rod test, and Western blot analysis measured myosin heavy-chain 2 (MYH2) to assess skeletal muscle growth, alongside p-AMPK, total-AMPK, PGC-1α, cytochrome C and UCP3 protein expression. Biochemical analysis was also performed on muscle tissue to measure the changes in concentration of liver lactate, lactate dehydrogenase (LDH), glycogen and citrate. Five groups (n = 10/per group) consisted of a control group (CON), exercise group (Ex), positive control treated with Ex and 500 mg/kg Taurine (Ex-Tau), Ex and 100 mg/kg FO supplementation (Ex-FO100) and Ex and 200 mg/kg FO supplementation (Ex-FO200) orally administered over the 4-week experimental period.FO50 significantly increased PGC-1α expression (p < 0.001), whereas both FO10 and FO50 increased the expression of p-AMPK (p < 0.001), in C2C12 muscle cells, showing increased signaling important for mitochondrial metabolism and biogenesis. Muscle lactate levels were also significantly increased following FO10 (p < 0.05) and FO50 (p < 0.001). In vivo, muscle protein expression of p-AMPK (p < 0.05) and PGC-1α were increased, corroborating our in vitro results. Cytochrome C also significantly increased following FO200 intake. These results suggest that the effects of FO supplementation may manifest in a dose-response manner. FO administration, in vitro, and supplementation, in vivo, both demonstrate a potential for improvements in mitochondrial metabolism and biogenesis, and even for potentiating the adaptive effects of endurance exercise. Mechanistically, lactate may be an important molecule in explaining the aforementioned positive effects of FO.


2019 ◽  
Vol 44 (5) ◽  
pp. 630-634
Author(s):  
Fazıla Atakan Erkal ◽  
Güzin Aykal ◽  
Hayriye Melek Yalçınkaya ◽  
Nihal Aksoy ◽  
Murat Özdemir

Abstract Objective Vast majority of laboratory errors occurs in preanalytical phase and in vitro hemolysis is the most common among preanalytical errors. Automated serum index measurement is being used in routine biochemical analysis in Antalya Public Health Care Laboratory, since June 2014. Our aim in this study is to reveal the impact of serum index usage on rejected samples and rejected test rates due to hemolysis. Materials and methods Hemolysis, icterus and lipemia (HIL) spectral interference reagent and program have been used in our laboratory since June 2014. In the current study, the number of samples and tests that were rejected due to hemolysis in June–August 2014 were compared with those rejected in the same period of 2013. Results In 2014, the sample rejection rate was 2.53% and the rejected test rate was 0.48%. In 2013, the sample rejection rate was 0.56% and the rejected test rate was 0.55%. When compared two periods, statistically significant increase in rejected sample number due to hemolysis in 2014 is result of, visually undetectable hemolyzed samples previously can be identified by HIL method (p<0.05). Conclusion Usage of hemolysis index program in automated systems for detecting hemolysis was evaluated as a method which is standardized, semi-quantitative, with high reproducibility and allows test based rejection.


1981 ◽  
Vol 241 (3) ◽  
pp. F203-F218 ◽  
Author(s):  
H. R. Jacobson

Although each of the major experimental techniques applied to the study of renal physiology has provided its fair share of new information, the technique of in vitro microperfusion of nephron segments is notable for two major contributions. First, it has supplied a more direct and controlled means of studying epithelial transport processes, some of which already have helped us to understand certain aspects of kidney function and others of which have yet to find their application in unraveling the mysteries of the kidney. Second, in the process of delineating these transport characteristics, it has served to emphasize the epithelial specialization present in the kidney, providing functional counterparts to the already recognized anatomic heterogeneity present in the kidney. In this second role microperfusion has spawned the application of biochemical analysis of the hormonal responses of various nephron segments and contributed to the impetus for work in culturing the various cell types present in each nephron segment. This review outlines the functional characteristics of the 11 major segments of the nephron, incorporating what has been learned from some of the biochemical work on hormone response and correlating the latter with transport events.


Parasitology ◽  
1972 ◽  
Vol 65 (2) ◽  
pp. 265-271 ◽  
Author(s):  
P. I. Trigg ◽  
W. E. Gutteridge

When ring-stage parasites are grown to the late trophozoite stagein vitro, the incorporation of [3H]adenosine into parasite DNA and RNA is in the ratio of ~ 1:1·5 and not 1:4 as expected from direct biochemical analysis of the parasite. A method is described by which large quantities of infected blood can be grownin vitrofrom the ring to the trophozoite stage thus allowing direct biochemical analysis of parasites grownin vitro. The results of direct biochemical analysis indicated that parasites grownin vitrohave similar DNA and protein contents to those grownin vitrobut that RNA contents are much lessin vitro. Quantitative histochemical studies also indicated a deficiency in RNA in parasites grownin vitro. It is concluded from the evidence of three distinct methods that RNA synthesis is defective during the first asexual cyclein vitro. In the second cycle all three macromolecular biosyntheses studied are reduced when compared to the first so that the actual rate of RNA synthesis is further reduced when compared to thatin vitro. It is suggested that the successive decrease in RNA synthesis parallels the reduction in multiplication obtained on subculture of the parasite.


1995 ◽  
Vol 16 (2) ◽  
pp. 140-147 ◽  
Author(s):  
Gloria V. Callard ◽  
Joan C. Jorgensen ◽  
J. Michael Redding

2006 ◽  
Vol 387 (4) ◽  
pp. 451-460 ◽  
Author(s):  
Christina Sieber ◽  
Frank Plöger ◽  
Raphaela Schwappacher ◽  
Rolf Bechtold ◽  
Michael Hanke ◽  
...  

Abstract Growth and differentiation factor 5 (GDF-5) is a homodimeric protein stabilized by a single disulfide bridge between cysteine 465 in the respective monomers, as well as by three intramolecular cysteine bridges within each subunit. A mature recombinant human GDF-5 variant with cysteine 465 replaced by alanine (rhGDF-5 C465A) was expressed in E. coli, purified to homogeneity, and chemically renatured. Biochemical analysis showed that this procedure eliminated the sole interchain disulfide bond. Surprisingly, the monomeric variant of rhGDF-5 is as potent in vitro as the dimeric form. This could be confirmed by alkaline phosphatase assays and Smad reporter gene activation. Furthermore, dimeric and monomeric rhGDF-5 show comparable binding to their specific type I receptor, BRIb. Studies on living cells showed that both the dimeric and monomeric rhGDF-5 induce homomeric BRIb and heteromeric BRIb/BRII oligomers. Our results suggest that rhGDF-5 C465A has the same biological activity as rhGDF-5 with respect to binding to, oligomerization of and signaling through the BMP receptor type Ib.


2006 ◽  
Vol 188 (18) ◽  
pp. 6669-6679 ◽  
Author(s):  
Brigitte Saint-Joanis ◽  
Caroline Demangel ◽  
Mary Jackson ◽  
Priscille Brodin ◽  
Laurent Marsollier ◽  
...  

ABSTRACT The twin arginine translocation (Tat) system is used by many bacteria to export fully folded proteins containing cofactors. Here, we show genetically that this system is essential for Mycobacterium tuberculosis, as the tatAC operon and tatB genes could be inactivated only in partially diploid strains. Using comparative genomics, the rv2525c gene of M. tuberculosis was identified as encoding a histidine-rich protein, with a twin arginine signal peptide, and orthologous genes were shown to be present in several but not all actinobacterial species. Conservation of this gene by Mycobacterium leprae, which has undergone reductive evolution, suggested an important role for rv2525c. An rv2525c knockout mutant was constructed, and biochemical analysis indicated that the mature Rv2525c protein is secreted. Upon exposure to antituberculous drugs, rv2525c expression is significantly up-regulated together with those of other genes involved in cell wall biogenesis. Phenotypic comparison of the mutant with the parental strain revealed an increase in susceptibility to some β-lactam antibiotics and, despite slower growth in vitro, enhanced virulence in both cellular and murine models of tuberculosis. The Tat system thus contributes in multiple ways to survival of the tubercle bacillus.


Sign in / Sign up

Export Citation Format

Share Document