The location and mechanism of hyperosmotic fluid secretion in the rectum of the saline-water mosquito larvae Aedes taeniorhynchus

1977 ◽  
Vol 66 (1) ◽  
pp. 111-126
Author(s):  
T. J. Bradley ◽  
J. E. Phillips
Keyword(s):  
Saline Water ◽  
Fluid Secretion ◽  
Opposite Polarity ◽  
Posterior Segment ◽  
Transport Processes ◽  
Cell Border ◽  
Experimental Conditions ◽  
Ultrastructural Studies ◽  
Aedes Taeniorhynchus

1. Ligation between the anterior and posterior segments of the rectum in vitro was used to demonstrate that the posterior rectum is the site of hyperosmotic secretion to the lumen side. Observations were consistent with a reabsorptive function for the anterior rectum. These results support predictions from ultrastructural studies of these two segments. 2. The initial potential of the rectal lumen, relative to the haemocoel side, was of opposite polarity in the anterior (−10 mV) and posterior (+ 10 mV) segments and these values decreased to −2 and +6 mV respectively in ligated recta which had secreted for 2 h. 3. A comparison of these potential difference measurements with concentration differences developed across the rectal epithelium under the same experimental conditions indicates that Na+, K+, Mg2+, and Cl- are all actively transported by the posterior segment to the lumen side. 4. The influence of different haemolymph concentrations of Na+, K+, and Cl- on the potential differences across the basal cell border and across the whole rectal epithelium are reported. Based on this and previous data, we propose a model for the organization of transport processes within the single celltype present in the posterior rectal epithelium.

The effect of external salinity on drinking rate and rectal secretion in the larvae of the saline-water mosquito Aedes taeniorhynchus

1977 ◽  
Vol 66 (1) ◽  
pp. 97-110
Author(s):  
T. J. Bradley ◽  
J. E. Phillips
Keyword(s):  
Sea Water ◽  
External Medium ◽  
Narrow Range ◽  
Saline Water ◽  
Fluid Secretion ◽  
Osmotic Concentration ◽  
Drinking Rate ◽  
Aedes Taeniorhynchus ◽  
External Salinity ◽  
Kinetics Of

1. The drinking rate of the saline-water mosquito larva Aedes taeniorhyncus (100 nl.mg-1.h-1) is unaffected by the salinity of the external medium, but is directly proportional to the surface area of the animal. 2. Haemolymph Na+, Mg2+, K+, Cl-, SO42- and osmotic concentrations were measured in larvae adapted to 10%, 100% and 200% seawater and were found to be regulated within a narrow range. 3. With the exception of potassium, ionic concentrations in rectal secretion were found to increase with increasing concentrations of the sea water in which larvae were reared. 4. The osmotic concentration of rectal secretion was unaffected by changes in haemolymph osmotic concentration but did rise when sodium or chloride concentrations of the haemolymph were increased. High levels of these ions also stimulated the rate of fluid secretion. 5. Transport of chloride and sodium by the rectum exhibits the kinetics of allosteric rather than classical enzymes.

Fluid secretion and microvillar ultrastructure in mosquito malpighian tubules

1989 ◽  
Vol 257 (5) ◽  
pp. R1096-R1102
Author(s):  
T. J. Bradley ◽  
C. Snyder
Keyword(s):  
Fluid Transport ◽  
Fluid Secretion ◽  
Blood Feeding ◽  
Malpighian Tubules ◽  
Primary Cells ◽  
Ultrastructural Examination ◽  
Aedes Taeniorhynchus ◽  
Physiological Limit ◽  
Secretion Rates

The Malpighian tubules of fourth instar larvae, pupae, and female adults of the mosquito Aedes taeniorhynchus were examined with regard to in vitro fluid secretion rate and the ultrastructural features of the microvillar border of the primary cells. In vitro fluid secretion rates were determined after stimulation with 5-hydroxytryptamine. While larval tubules are capable of rapid fluid secretion, the tubules of pupae exhibit very low rates of secretion, indistinguishable from 0 nl/h. The capacity to secrete fluid returns after the pupal-adult molt and is further enhanced after blood feeding. Similar results were obtained in tubules stimulated in vitro with dibutyryl adenosine 3',5'-cyclic monophosphate. Ultrastructural examination of the microvillar border of the primary cells of the Malpighian tubules revealed that the period of reduced secretion capacity in the pupal tubules is correlated with a marked reduction in microvillar volume, microvillar surface area, and mitochondrial content in the microvillar border. The results suggest that microvilli of a certain size and containing extensions of mitochondria are required for rapid fluid transport. The absence of these conditions in pupal tubules cannot be overcome by in vitro stimulation with known secretagogues and therefore represents a physiological limit on transport performance in the pupal tubules of mosquitoes.

The secretion of hyperosmotic fluid by the rectum of a saline-water mosquito larva, Aedes taeniorhynchus

1975 ◽  
Vol 63 (2) ◽  
pp. 331-342 ◽  
Author(s):  
T. J. Bradley ◽  
J. E. Phillips
Keyword(s):  
Sea Water ◽  
Saline Water ◽  
Average Rate ◽  
Ionic Composition ◽  
Fluid Secretion ◽  
Osmotic Concentration ◽  
Aedes Taeniorhynchus ◽  
Osmotic Balance ◽  
Anal Papillae

1. Fourth-instar larvae of the mosquito A. taeniorhynchus (Wiedemann), when living in sea water, drink at a rate of 100 nl h(−1) larva(−1) and maintain ionic and osmotic levels in the haemolymph at about one-third those of the external medium. 2. Hyperosmotic urine is produced in the rectum by secretion of fluid having an osmotic concentration and ionic composition similar to that of sea water, with the exception that potassium levels are elevated 18-fold in the secretion. The average rate of fluid secretion observed was 19 nl h-1) larva(−1) with a maximum of 92 nl h(−1) larva(−1). 3. The concentration and volume of rectal secretion may be too low to account completely for osmotic balance. The possible role of anal papillae is discussed in this regard.

Probing the ultrastructure of the mitotic and meiotic spindle in eggs: An expeditious approach based on semi-thin (0.25 μm) serial sections

1983 ◽  
Vol 41 ◽  
pp. 552-555
Author(s):  
Conly L. Rieder ◽  
S. Bowser ◽  
R. Nowogrodzki ◽  
K. Ross ◽  
G. Sluder
Keyword(s):  
Small Sample Size ◽  
Small Sample ◽  
Meiotic Spindle ◽  
Serial Sections ◽  
Mitotic Apparatus ◽  
Thin Sections ◽  
Cell Cycles ◽  
Ultrastructural Studies

Eggs have long been a favorite material for studying the mechanism of karyokinesis in-vivo and in-vitro. They can be obtained in great numbers and, when fertilized, divide synchronously over many cell cycles. However, they are not considered to be a practical system for ultrastructural studies on the mitotic apparatus (MA) for several reasons, the most obvious of which is that sectioning them is a formidable task: over 1000 ultra-thin sections need to be cut from a single 80-100 μm diameter egg and of these sections only a small percentage will contain the area or structure of interest. Thus it is difficult and time consuming to obtain reliable ultrastructural data concerning the MA of eggs; and when it is obtained it is necessarily based on a small sample size.We have recently developed a procedure which will facilitate many studies concerned with the ultrastructure of the MA in eggs. It is based on the availability of biological HVEM's and on the observation that 0.25 μm thick serial sections can be screened at high resolution for content (after mounting on slot grids and staining with uranyl and lead) by phase contrast light microscopy (LM; Figs 1-2).

Ultrastructural studies on the interaction of haemophilus influenzae with human endothelial cells in vitro

1990 ◽  
Vol 48 (3) ◽  
pp. 636-637
Author(s):  
D.J.P. Ferguson ◽  
M. Virji ◽  
H. Kayhty ◽  
E.R. Moxon
Keyword(s):  
Endothelial Cells ◽  
Haemophilus Influenzae ◽  
Intercellular Junctions ◽  
Blood Stream ◽  
Ultrastructural Studies ◽  
Endothelial Barriers ◽  
Serotype B ◽  
Meningitis In Children ◽  
Respiratory Epithelial

Haemophilus influenzae is a human pathogen which causes meningitis in children. Systemic H. influenzae infection is largely confined to encapsulated serotype b organisms and is a major cause of meningitis in the U.K. and elsewhere. However, the pathogenesis of the disease is still poorly understood. Studies in the infant rat model, in which intranasal challenge results in bacteraemia, have shown that H. influenzae enters submucosal tissues and disseminates to the blood stream within minutes. The rapidity of these events suggests that H. influenzae penetrates both respiratory epithelial and endothelial barriers with great efficiency. It is not known whether the bacteria penetrate via the intercellular junctions, are translocated within the cells or carried across the cellular barrier in 'trojan horse' fashion within phagocytes. In the present studies, we have challenged cultured human umbilical cord_vein endothelial cells (HUVECs) with both capsulated (b+) and capsule-deficient (b-) isogenic variants of one strain of H. influenzae in order to investigate the interaction between the bacteria and HUVEC and the effect of the capsule.

Observations of Frozen-Hydrated Microtubules

1990 ◽  
Vol 48 (1) ◽  
pp. 504-505
Author(s):  
D. Chrétien ◽  
D. Job ◽  
R.H. Wade
Keyword(s):  
Fourier Transforms ◽  
Structural Complexity ◽  
Image Contrast ◽  
Phase Behaviour ◽  
Experimental Conditions ◽  
Thin Sections ◽  
Surface Lattice ◽  
Cilia And Flagella ◽  
Outstanding Question

Microtubules are filamentary structures found in the cytoplasm of eukaryotic cells, where, together with actin and intermediate filaments, they form the components of the cytoskeleton. They have many functions and show various levels of structural complexity as witnessed by the singlet, doublet and triplet structures involved in the architecture of centrioles, basal bodies, cilia and flagella. The accepted microtubule model consists of a 25 nm diameter hollow tube with a wall made up of 13 paraxial protofilaments (pf). Each pf is a string of aligned tubulin dimers. Some results have suggested that the pfs follow a superhelix. To understand how microtubules function in the cell an accurate model of the surface lattice is one of the requirements. For example the 9x2 architecture of the axoneme will depend on the organisation of its component microtubules. We should also note that microtubules with different numbers of pfs have been observed in thin sections of cellular and of in-vitro material. An outstanding question is how does the surface lattice adjust to these different pf numbers?We have been using cryo-electron microscopy of frozen-hydrated samples to study in-vitro assembled microtubules. The experimental conditions are described in detail in this reference. The results obtained in conjunction with thin sections of similar specimens and with axoneme outer doublet fragments have already allowed us to characterise the image contrast of 13, 14 and 15 pf microtubules on the basis of the measured image widths, of the the image contrast symmetry and of the amplitude and phase behaviour along the equator in the computed Fourier transforms. The contrast variations along individual microtubule images can be interpreted in terms of the geometry of the microtubule surface lattice. We can extend these results and make some reasonable predictions about the probable surface lattices in the case of other pf numbers, see Table 1. Figure 1 shows observed images with which these predictions can be compared.

Superoxide, Xanthine Oxidase and Platelet Reactions: Further Studies on Mechanisms by which Oxidants Influence Platelets

1981 ◽  
Vol 45 (03) ◽  
pp. 290-293 ◽  
Author(s):  
Peter H Levine ◽  
Danielle G Sladdin ◽  
Norman I Krinsky
Keyword(s):  
Superoxide Dismutase ◽  
Cytochrome C ◽  
Xanthine Oxidase ◽  
Direct Effect ◽  
Platelet Function ◽  
Experimental Conditions ◽  
Release Reaction

SummaryIn the course of studying the effects on platelets of the oxidant species superoxide (O- 2), Of was generated by the interaction of xanthine oxidase plus xanthine. Surprisingly, gel-filtered platelets, when exposed to xanthine oxidase in the absence of xanthine substrate, were found to generate superoxide (O- 2), as determined by the reduction of added cytochrome c and by the inhibition of this reduction in the presence of superoxide dismutase.In addition to generating Of, the xanthine oxidase-treated platelets display both aggregation and evidence of the release reaction. This xanthine oxidase induced aggreagtion is not inhibited by the addition of either superoxide dismutase or cytochrome c, suggesting that it is due to either a further metabolite of O- 2, or that O- 2 itself exerts no important direct effect on platelet function under these experimental conditions. The ability of Of to modulate platelet reactions in vivo or in vitro remains in doubt, and xanthine oxidase is an unsuitable source of O- 2 in platelet studies because of its own effects on platelets.

Desmopressin (DDAVP) Enhances Platelet Adhesion to the Extracellular Matrix of Cultured Human Endothelial Cells through Increased Expression of Tissue Factor

1997 ◽  
Vol 77 (05) ◽  
pp. 0975-0980 ◽  
Author(s):  
Angel Gálvez ◽  
Goretti Gómez-Ortiz ◽  
Maribel Díaz-Ricart ◽  
Ginés Escolar ◽  
Rogelio González-Sarmiento ◽  
...  
Keyword(s):  
Extracellular Matrix ◽  
Endothelial Cells ◽  
Tissue Factor ◽  
Platelet Adhesion ◽  
Umbilical Vein ◽  
Procoagulant Activity ◽  
Experimental Conditions ◽  
Chromogenic Assay

SummaryThe effect of desmopressin (DDAVP) on thrombogenicity, expression of tissue factor and procoagulant activity (PCA) of extracellular matrix (ECM) generated by human umbilical vein endothelial cells cultures (HUVEC), was studied under different experimental conditions. HUVEC were incubated with DDAVP (1, 5 and 30 ng/ml) and then detached from their ECM. The reactivity towards platelets of this ECM was tested in a perfusion system. Coverslips covered with DD A VP-treated ECMs were inserted in a parallel-plate chamber and exposed to normal blood anticoagulated with low molecular weight heparin (Fragmin®, 20 U/ml). Perfusions were run for 5 min at a shear rate of 800 s1. Deposition of platelets on ECMs was significantly increased with respect to control ECMs when DDAVP was used at 5 and 30 ng/ml (p <0.05 and p <0.01 respectively). The increase in platelet deposition was prevented by incubation of ECMs with an antibody against human tissue factor prior to perfusion. Immunofluorescence studies positively detected tissue factor antigen on DDAVP derived ECMs. A chromogenic assay performed under standardized conditions revealed a statistically significant increase in the procoagulant activity of the ECMs produced by ECs incubated with 30 ng/ml DDAVP (p <0.01 vs. control samples). Northern blot analysis revealed increased levels of tissue factor mRNA in extracts from ECs exposed to DDAVP. Our data indicate that DDAVP in vitro enhances platelet adhesion to the ECMs through increased expression of tissue factor. A similar increase in the expression of tissue factor might contribute to the in vivo hemostatic effect of DDAVP.

Applicability of Instability Index for In vitro Protein Stability Prediction

2019 ◽  
Vol 26 (5) ◽  
pp. 339-347 ◽  
Author(s):  
Dilani G. Gamage ◽  
Ajith Gunaratne ◽  
Gopal R. Periyannan ◽  
Timothy G. Russell
Keyword(s):  
Protein Stability ◽  
Caulobacter Crescentus ◽  
Effect Of Temperature ◽  
Instability Index ◽  
Dipeptide Composition ◽  
Experimental Conditions ◽  
The Stability ◽  
Vitro Protein

Background: The dipeptide composition-based Instability Index (II) is one of the protein primary structure-dependent methods available for in vivo protein stability predictions. As per this method, proteins with II value below 40 are stable proteins. Intracellular protein stability principles guided the original development of the II method. However, the use of the II method for in vitro protein stability predictions raises questions about the validity of applying the II method under experimental conditions that are different from the in vivo setting. Objective: The aim of this study is to experimentally test the validity of the use of II as an in vitro protein stability predictor. Methods: A representative protein CCM (CCM - Caulobacter crescentus metalloprotein) that rapidly degrades under in vitro conditions was used to probe the dipeptide sequence-dependent degradation properties of CCM by generating CCM mutants to represent stable and unstable II values. A comparative degradation analysis was carried out under in vitro conditions using wildtype CCM, CCM mutants and two other candidate proteins: metallo-β-lactamase L1 and α -S1- casein representing stable, borderline stable/unstable, and unstable proteins as per the II predictions. The effect of temperature and a protein stabilizing agent on CCM degradation was also tested. Results: Data support the dipeptide composition-dependent protein stability/instability in wt-CCM and mutants as predicted by the II method under in vitro conditions. However, the II failed to accurately represent the stability of other tested proteins. Data indicate the influence of protein environmental factors on the autoproteolysis of proteins. Conclusion: Broader application of the II method for the prediction of protein stability under in vitro conditions is questionable as the stability of the protein may be dependent not only on the intrinsic nature of the protein but also on the conditions of the protein milieu.

scholarly journals Non-invasive skin sampling of tryptophan/kynurenine ratio in vitro towards a skin cancer biomarker

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Skaidre Jankovskaja ◽  
Johan Engblom ◽  
Melinda Rezeli ◽  
György Marko-Varga ◽  
Tautgirdas Ruzgas ◽  
...  
Keyword(s):  
Skin Cancer ◽  
Relative Increase ◽  
Cancer Biomarker ◽  
Cancer Diagnostics ◽  
Sampling Time ◽  
Skin Permeability ◽  
Experimental Conditions ◽  
Non Invasive

AbstractThe tryptophan to kynurenine ratio (Trp/Kyn) has been proposed as a cancer biomarker. Non-invasive topical sampling of Trp/Kyn can therefore serve as a promising concept for skin cancer diagnostics. By performing in vitro pig skin permeability studies, we conclude that non-invasive topical sampling of Trp and Kyn is feasible. We explore the influence of different experimental conditions, which are relevant for the clinical in vivo setting, such as pH variations, sampling time, and microbial degradation of Trp and Kyn. The permeabilities of Trp and Kyn are overall similar. However, the permeated Trp/Kyn ratio is generally higher than unity due to endogenous Trp, which should be taken into account to obtain a non-biased Trp/Kyn ratio accurately reflecting systemic concentrations. Additionally, prolonged sampling time is associated with bacterial Trp and Kyn degradation and should be considered in a clinical setting. Finally, the experimental results are supported by the four permeation pathways model, predicting that the hydrophilic Trp and Kyn molecules mainly permeate through lipid defects (i.e., the porous pathway). However, the hydrophobic indole ring of Trp is suggested to result in a small but noticeable relative increase of Trp diffusion via pathways across the SC lipid lamellae, while the shunt pathway is proposed to slightly favor permeation of Kyn relative to Trp.

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