Adult 175 kDa collagenase antigen ofSetaria cerviin immunoprophylaxis againstBrugia malayiin jirds

2004 ◽  
Vol 78 (4) ◽  
pp. 347-352 ◽  
Author(s):  
Y. Srivastava ◽  
S. Rathaur ◽  
Y.P. Bhandari ◽  
M.V.R. Reddy ◽  
B.C. Harinath
Keyword(s):  
Meriones Unguiculatus ◽  
Host Cells ◽  
Specific Substrate ◽  
Collagenase Activity ◽  
Setaria Cervi ◽  
Protein Substrates ◽  
Infective Larvae ◽  
Optimum Ph

AbstractA 175 kDa antigen fraction with collagenase activity was isolated and purified from somatic extracts of adultSetaria cervifemales using column chromatography involving consecutive steps of DEAE-Sepharose CL6B and Sephadex G-100. The optimum pH for 175 kDa collagenase was found to be pH 7.0. Sensitivities to a variety of inhibitors and activators indicated that the 175 kDa coIlagenolytic enzyme was metalloserine in nature. The enzyme hydrolysed a variety of protein substrates such as haemoglobin, casein, azocasein (general substrates) and collagen, FALGPA (furanoyl-acryloyl-leu-gly-pro-ala), the specific substrate of collagenase. The enzyme showed 57% inhibition by jird anti-somatic collagenase antibodies and reacted insignificantly with normal jird sera. Further analysis was undertaken on the immunoprophylactic potential of 175 kDa collagenase in inducing immunity againstBrugia malayi(a human filarial parasite) in jirds (Meriones unguiculatus)in vitroandin situ. Immune sera of jirds raised against this antigen promoted partial adherence of peritoneal exudate cells toB. malayimicrofilariae (mf) and infective larvae (L3)in vitroand induced partial cytotoxicity to the parasites within 48 h. The anti-S. cervi175 kDa antigen serum was more effective in inducing cytotoxicity toB. malayiL3, than mf. In the microchambers implanted inside immune jirds, host cells could migrate and adhere to the mf and infective larvae thereby killing them partially within 48 h.

SUSCEPTIBILITY OF PROTEINS USED IN CALF MILK REPLACERS TO HYDROLYSIS BY VARIOUS PROTEOLYTIC ENZYMES

1980 ◽  
Vol 60 (4) ◽  
pp. 907-914 ◽  
Author(s):  
K. J. JENKINS ◽  
S. MAHADEVAN ◽  
D. B. EMMONS
Keyword(s):  
Trichloroacetic Acid ◽  
Proteolytic Enzymes ◽  
Milk Proteins ◽  
In Vitro Study ◽  
Enzyme Treatment ◽  
Protein Hydrolysis ◽  
Protein Substrates ◽  
Optimum Ph ◽  
Milk Replacers

An in vitro study was conducted to assess the hydrolytic susceptibility of various milk and non-milk proteins (soybean, rapeseed, fish) used in calf milk replacers to endogenous and commercial proteolytic enzymes. Extent of protein hydrolysis (%) was calculated from the reduced amount of protein precipitated by 10% trichloroacetic acid following enzyme treatment. All of the enzymes tested hydrolyzed the milk proteins more extensively than the non-milk proteins both at their optimum pH, and at the pH (6.1) of calf abomasal contents immediately after feeding. At both optimum pH and pH 6.1, the highest average hydrolysis value for all protein substrates was obtained with pronase followed by papain, trypsin, pancreatin, chymotrypsin, Mucor miehei rennet andchymosin (calf rennet). All substrates were hydrolyzed extensively by pepsin at pH 2.0 but, as expected, very little hydrolysis occurred with this enzyme atpH6.1.

Intron-containing β-tubulin transcripts in Cryptosporidium parvum cultured in vitro

Microbiology ◽  
2004 ◽  
Vol 150 (5) ◽  
pp. 1191-1195 ◽  
Author(s):  
Xiaomin Cai ◽  
Cheryl A. Lancto ◽  
Mitchell S. Abrahamsen ◽  
Guan Zhu
Keyword(s):  
Life Cycle ◽  
Cryptosporidium Parvum ◽  
Late Life ◽  
Host Cells ◽  
Intron Splicing ◽  
Transcript Processing ◽  
Life Cycle Stages ◽  
Tubulin Mrna

The genome of Cryptosporidium parvum contains a relatively small number of introns, which includes the β-tubulin gene with only a single intron. Recently, it was observed that the intron was not removed from some of the β-tubulin transcripts in the late life cycle stages cultured in vitro. Although normally spliced β-tubulin mRNA was detected in all parasite intracellular stages by RT-PCR (e.g. HCT-8 or Caco-2 cells infected with C. parvum for 12–72 h), at 48–72 h post-infection unprocessed β-tubulin transcripts containing intact introns started to appear in parasite mRNA within infected host cells. The intron-containing transcripts could be detected by fluorescence in situ hybridization (FISH) using an intron-specific probe. The intron-containing β-tubulin transcripts appeared unique to the in vitro-cultured C. parvum, since they were not detected in parasite-infected calves at 72 h. As yet, it is unclear whether the late life cycle stages of C. parvum are partially deficient in intron-splicing or the intron-splicing processes have merely slowed, both of which would allow the detection of intron-containing transcripts. Another possible explanation is that the decay in transcript processing might simply be due to the onset of parasite death. Nonetheless, the appearance of intron-containing transcripts coincides with the arrest of C. parvum development in vitro. This unusual observation prompts speculation that the abnormal intron-splicing of β-tubulin transcripts may be one of the factors preventing complete development of this parasite in vitro. Furthermore, the presence of both processed and unprocessed introns in β-tubulin transcripts in vitro may provide a venue for studying overall mechanisms for intron-splicing in this parasite.

scholarly journals The absorption of oral morroniside in rats: In vivo, in situ and in vitro studies

2019 ◽  
Vol 69 (2) ◽  
pp. 287-296 ◽  
Author(s):  
Shan Xiong ◽  
Jinglai Li ◽  
Yanling Mu ◽  
Zhenqing Zhang
Keyword(s):  
Iridoid Glycosides ◽  
Specific Substrate ◽  
Sprague Dawley ◽  
Glycoprotein P ◽  
Cornus Officinalis ◽  
Sd Rats ◽  
Basolateral Side

Abstract Morroniside is one of the most important iridoid glycosides from Cornus officinalis Sieb. et Zucc. In the present study, the pharmacokinetics and bioavailability studies of morroniside were conducted on Sprague-Dawley (SD) rats. A rat in situ intestinal perfusion model was used to characterize the absorption of morroniside. Caco-2 cells were used to examine the transport mechanisms of morroniside. The pharmacokinetic study of morroniside exhibited linear dose-proportional pharmacokinetic characteristics and low bioavailability (4.3 %) in SD rats. Its average Peff value for transport across the small intestinal segments changed from (3.09 ± 2.03) × 10−6 to (4.53 ± 0.94) × 10−6 cm s−1. In Caco-2 cells, the Papp values ranged from (1.61 ± 0.53) × 10−9 to (1.19 ± 0.22) × 10−7 cm s−1 for the apical to basolateral side and the Pratio values at three concentrations were all lower than 1.2. Morroniside showed poor absorption and it might not be a specific substrate of P-glycoprotein (P-gp).

scholarly journals Relative Expression Levels Rather Than Specific Activity Plays the Major Role in Determining In Vivo AKT Isoform Substrate Specificity

2011 ◽  
Vol 2011 ◽  
pp. 1-18 ◽  
Author(s):  
Rachel S. Lee ◽  
Colin M. House ◽  
Briony E. Cristiano ◽  
Ross D. Hannan ◽  
Richard B. Pearson ◽  
...  
Keyword(s):  
Substrate Specificity ◽  
Specific Activity ◽  
Dominant Role ◽  
Protein Substrates ◽  
Cellular Processes ◽  
Disease States ◽  
The Individual

The AKT protooncogene mediates many cellular processes involved in normal development and disease states such as cancer. The three structurally similar isoforms: AKT1, AKT2, and AKT3 exhibit both functional redundancy and isoform-specific functions; however the basis for their differential signalling remains unclear. Here we show that in vitro, purified AKT3 is ∼47-fold more active than AKT1 at phosphorylating peptide and protein substrates. Despite these marked variations in specific activity between the individual isoforms, a comprehensive analysis of phosphorylation of validated AKT substrates indicated only subtle differences in signalling via individual isoforms in vivo. Therefore, we hypothesise, at least in this model system, that relative tissue/cellular abundance, rather than specific activity, plays the dominant role in determining AKT substrate specificity in situ.

The uptake in vitro of dyes, monosaccharides and amino acids by the filarial worm Brugia pahangi

Parasitology ◽  
1979 ◽  
Vol 78 (3) ◽  
pp. 343-354 ◽  
Author(s):  
S. N. Chen ◽  
R. E. Howells
Keyword(s):  
Trypan Blue ◽  
Calf Serum ◽  
Fluorescein Isothiocyanate ◽  
Meriones Unguiculatus ◽  
Scintillation Counting ◽  
Brugia Pahangi ◽  
Infective Larvae ◽  
Filarial Worm ◽  
Aspiculuris Tetraptera

SUMMARYThe uptake in vitro of various substances by Brugia pahangi was investigated using infective larvae obtained from Aedes aegypti and worms removed from Meriones unguiculatus at 2, 3, 10, 20 and 90 days post-infection. Worms incubated in growth medium 199 containing 1% Trypan blue possessed demonstrable dye in the oral orifice, the anterior oesophageal lumen and the external openings of the vulva and the cloaca or anus but the dye was not found in the gut lumen even after incubation for 24 h. No uptake of ferritin particles into the intestine of the worms was found and no fluorescence could be demonstrated in the gut lumen of worms incubated in medium containing 50% (v/v) fluorescein isothiocyanate-conjugated calf serum for up to 24 h. Trypan blue uptake by the gut of Aspiculuris tetraptera was clearly observed after incubation for several hours. The uptake of D-glucose and L-leucine by B. pahangi was demonstrated using autoradiographic and scintillation counting techniques and incorporation into worm tissues was detected. Glucose was found to be readily incorporated in the apical, glycogen-rich areas of the myocytes of worms of all ages studied and in the uterine epithelium of the adult female. In contrast, a lower incorporation of D-glucose was found in the eggs, embryos and vas deferens and especially in the gut. The incorporation of L-leucine occurred throughout the tissue of the worms during a 30 mm incubation. Labelling was also located over the surface of the cuticle of the worms, when incubated for a period of 15 to 60 mm in L-[3H]leucine. Scintillation counting techniques demonstrated that there was no uptake of 14C-labelled L-glucose or sucrose by B. pahangi. The data presented on the uptake in vitro of nutrients or other compounds by infective larvae and adult stages of B. pahangi did not demonstrate an intestinal route of uptake but indicated that the transcuticular route of uptake may be employed.

scholarly journals Efficacy ofClonostachys roseaandDuddingtonia flagransin Reducing theHaemonchus contortusInfective Larvae

2015 ◽  
Vol 2015 ◽  
pp. 1-5 ◽  
Author(s):  
Manoel Eduardo da Silva ◽  
Fabio Ribeiro Braga ◽  
Pedro Mendoza de Gives ◽  
Miguel Angel Mercado Uriostegui ◽  
Manuela Reyes ◽  
...  
Keyword(s):  
Aqueous Suspension ◽  
Duddingtonia Flagrans ◽  
Gastrointestinal Helminths ◽  
Clonostachys Rosea ◽  
Free Living ◽  
Infective Larvae ◽  
Group 4 ◽  
Group 1

The biocontrol is proven effective in reducing in vitro and in situ free-living stages of major gastrointestinal helminths, allowing progress in reducing losses by parasitism, maximizing production, and productivity. This study aimed at evaluating the predatory activity of fungal isolates ofDuddingtonia flagransandClonostachys roseaspecies and its association on infective larvae (L3) ofH. contortusin microplots formed by grasses and maintained in a protected environment. All groups were added with 10 mL of an aqueous suspension with 618H. contortusL3approximately. Group 1 was used as control and only received the infective larvae. Groups 2 and 3 receivedD. flagranschlamydospores andC. roseaconidia at doses of 5 × 106. Group 4 received the combination of 5 × 106D. flagranschlamydospores + 5 × 106C. roseaconidia.D. flagransandC. roseashowed nematicidal effectiveness reducing by 91.5 and 88.9%, respectively, the population ofH. contortusL3. However, when used in combination efficiency decreased to 74.5% predation ofH. contortusL3. These results demonstrate the need for further studies to determine the existence of additive effects, synergistic or antagonistic, between these species.

Setaria cervi: in vitro released collagenases and their inhibition by Wuchereria bancrofti infected sera

2003 ◽  
Vol 77 (1) ◽  
pp. 77-81 ◽  
Author(s):  
R.N. Singh ◽  
S. Rathaur
Keyword(s):  
Specific Activity ◽  
Wuchereria Bancrofti ◽  
Thiol Group ◽  
Collagenase Activity ◽  
Setaria Cervi ◽  
Adult Females ◽  
Human Sera ◽  
Molecular Masses ◽  
Comparable Activity

AbstractIn vitro released products of adult Setaria cervi females, microfilariae and extracts showed considerable amounts of collagenase activity. On the basis of per mg protein released in vitro, the products of both microfilariae and adult females exhibited comparable activity but this was much higher than that of extract of microfilariae and adult females. Two collagenase enzymes with molecular masses of 50 kDa and 70 kDa were separated using DEAE-sepharose CL6B and Sephadex G-100 column chromatography. The 50 kDa and 70 kDa collagenase exhibited pH optima of 5.2 and 7.0, respectivly. Considering specific activity, the 50 kDa enzyme was found to contribute about ten times more collagenase activity as compared to the 70 kDa enzyme. An inhibition study revealed obvious differences between them. Thiol group inhibitors such as N-ethylmaleimide and leupeptin inhibited the 50 kDa enzyme but this was strongly activated by dithiothreitol, a thiol group stabilizer. Alternatively, the 70 kDa enzyme showed a sensitivity to a metal chelator and a serine group inhibitor indicating its metalloserine protease nature. The antifilarial drug diethylcarbamazine did not demonstrate any inhibition under in vitro conditions. Both enzymes were significantly inhibited by antibody IgG separated from Wuchereria bancrofti infected human sera, showing a possible immunoprotective role.

Correlated Studies of Cellular Interactions Using TEM, Freeze Etching, and SEM

1974 ◽  
Vol 32 ◽  
pp. 320-321
Author(s):  
J. P. Revel
Keyword(s):  
Developmental Stages ◽  
Three Dimensional ◽  
Cell Movement ◽  
Cellular Interactions ◽  
Serial Sections ◽  
Cell Sheets ◽  
Freeze Etching ◽  
Early Developmental

Movement of individual cells or of cell sheets and complex patterns of folding play a prominent role in the early developmental stages of the embryo. Our understanding of these processes is based on three- dimensional reconstructions laboriously prepared from serial sections, and from autoradiographic and other studies. Many concepts have also evolved from extrapolation of investigations of cell movement carried out in vitro. The scanning electron microscope now allows us to examine some of these events in situ. It is possible to prepare dissections of embryos and even of tissues of adult animals which reveal existing relationships between various structures more readily than used to be possible vithout an SEM.

In vitro and in vivo polysaccharides assembly: ultrastructural and cytochemical study of growing plant cell wall components.

1978 ◽  
Vol 36 (2) ◽  
pp. 434-435
Author(s):  
D. Reis ◽  
B. Vian ◽  
J. C. Roland
Keyword(s):  
Cell Wall ◽  
Self Assembly ◽  
Plant Cell Wall ◽  
Higher Plants ◽  
Three Dimensional ◽  
Cytochemical Study ◽  
Wall Components

Wall morphogenesis in higher plants is a problem still open to controversy. Until now the possibility of a transmembrane control and the involvement of microtubules were mostly envisaged. Self-assembly processes have been observed in the case of walls of Chlamydomonas and bacteria. Spontaneous gelling interactions between xanthan and galactomannan from Ceratonia have been analyzed very recently. The present work provides indications that some processes of spontaneous aggregation could occur in higher plants during the formation and expansion of cell wall.Observations were performed on hypocotyl of mung bean (Phaseolus aureus) for which growth characteristics and wall composition have been previously defined.In situ, the walls of actively growing cells (primary walls) show an ordered three-dimensional organization (fig. 1). The wall is typically polylamellate with multifibrillar layers alternately transverse and longitudinal. Between these layers intermediate strata exist in which the orientation of microfibrils progressively rotates. Thus a progressive change in the morphogenetic activity occurs.

In Situ Localization of PPAR Gamma and Uncoupling Protein in Mouse Embryo Sections Using Digoxigenin-Labeled Riboprobes

1996 ◽  
Vol 54 ◽  
pp. 32-33
Author(s):  
C. Jennermann ◽  
S. A. Kliewer ◽  
D. C. Morris
Keyword(s):  
Alkaline Phosphatase ◽  
Room Temperature ◽  
Uncoupling Protein ◽  
Ppar Gamma ◽  
Nuclear Hormone Receptor ◽  
Full Length ◽  
Northern Analysis ◽  
Peroxisome Proliferator

Peroxisome proliferator-activated receptor gamma (PPARg) is a member of the nuclear hormone receptor superfamily and has been shown in vitro to regulate genes involved in lipid metabolism and adipocyte differentiation. By Northern analysis, we and other researchers have shown that expression of this receptor predominates in adipose tissue in adult mice, and appears first in whole-embryo mRNA at 13.5 days postconception. In situ hybridization was used to find out in which developing tissues PPARg is specifically expressed.Digoxigenin-labeled riboprobes were generated using the Genius™ 4 RNA Labeling Kit from Boehringer Mannheim. Full length PPAR gamma, obtained by PCR from mouse liver cDNA, was inserted into pBluescript SK and used as template for the transcription reaction. Probes of average size 200 base pairs were made by partial alkaline hydrolysis of the full length transcripts. The in situ hybridization assays were performed as described previously with some modifications. Frozen sections (10 μm thick) of day 18 mouse embryos were cut, fixed with 4% paraformaldehyde and acetylated with 0.25% acetic anhydride in 1.0M triethanolamine buffer. The sections were incubated for 2 hours at room temperature in pre-hybridization buffer, and were then hybridized with a probe concentration of 200μg per ml at 70° C, overnight in a humidified chamber. Following stringent washes in SSC buffers, the immunological detection steps were performed at room temperature. The alkaline phosphatase labeled, anti-digoxigenin antibody and detection buffers were purchased from Boehringer Mannheim. The sections were treated with a blocking buffer for one hour and incubated with antibody solution at a 1:5000 dilution for 2 hours, both at room temperature. Colored precipitate was formed by exposure to the alkaline phosphatase substrate nitrobluetetrazoliumchloride/ bromo-chloroindlylphosphate.

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