scholarly journals Proteins of the kidney microvillar membrane. Immunoelectrophoretic analysis of the membrane hydrolases: identification and resolution of the detergent- and proteinase-solubilized forms

1979 ◽  
Vol 179 (2) ◽  
pp. 397-405 ◽  
Author(s):  
A G Booth ◽  
L M Hubbard ◽  
A J Kenny
Keyword(s):  
Neutral Endopeptidase ◽  
Specific Staining ◽  
Hydrophobic Domain ◽  
Aminopeptidase M ◽  
Crossed Immunoelectrophoresis ◽  
Detergent Treatment ◽  
Staining Methods ◽  
Aminopeptidase A ◽  
The Difference ◽  
Charge Shift

Antibodies raised in rabbits to detergent-solubilized pig kidney microvillar proteins have been used to investigate the membrane hydrolases by crossed immunoelectrophoresis. Eight enzymes were detected by specific staining methods: aminopeptidase M, dipeptidylpeptidase IV, neutral endopeptidase, aminopeptidase A, carboxypeptidase P, gamma-glutamyltransferase, trehalase and phosphodiesterase I. The mobility of all these enzymes, with the exception of trehalase and neutral endopeptidase, was increased by treatment of the detergent-solubilized preparation with papain. The difference between the detergent and proteinase forms of these enzymes is attributed to the removal of a small, non-antigenic peptide to which detergent is bound in significant quantities. This interpretation was further supported by experiments in which the microvillus fraction was labelled with an intramembrane photolabelling reagent, 1-azido-4-[125I]iodobenzene. After photolysis, the radioactivity in the membrane could be solubilized by detergent treatment but not by papain treatment. Radioautography after crossed charge-shift immunoelectrophoresis showed a good correlation between charge-shift (signifying the presence of detergent bound to a hydrophobic domain) and the presence of the label.

scholarly journals Intestinal parasites in cancer patients in the South of Brazil

2017 ◽  
Vol 78 (3) ◽  
pp. 574-578 ◽  
Author(s):  
S. Jeske ◽  
T. F. Bianchi ◽  
M. Q. Moura ◽  
B. Baccega ◽  
N. B. Pinto ◽  
...  
Keyword(s):  
Cancer Patients ◽  
Intestinal Parasites ◽  
Strongyloides Stercoralis ◽  
Clinic Visit ◽  
Parasitic Infections ◽  
The South ◽  
Intestinal Protozoa ◽  
Specific Staining ◽  
Staining Methods ◽  
South Of Brazil

Abstract Intestinal parasitic infections in immunocompromised patients can lead to serious complications when not diagnosed and treated early. This study aimed to investigate the frequency of intestinal parasites in cancer patients undergoing chemotherapy in the South of Brazil. Three fecal samples collected from each patient (73 individuals) were processed by Ritchie and Faust techniques and submitted to specific staining methods for intestinal protozoa. A 61.6% parasite and/or commensal positivity was found. Helminths identified were Ascaris lumbricoides (33.3%), Taenia spp. (6.6%), Strongyloides stercoralis (4.4%) and Trichuris trichiura (2.2%). Among protozoans, Giardia lamblia (26.6%), Cryptosporidium spp. (13.3%) and Cystoisospora belli (4.4%) were identified. The presence of Entamoeba coli, Endolimax nana and Entamoeba hartmanni was also recorded. The results obtained warn of the importance of fecal parasitological diagnosis and the use of specific staining methods for the detection of intestinal parasites in cancer patients. These exams should be regularly requested at the patient’s first clinic visit, given the high prevalence found in this study and the possible severity of such conditions for these individuals.

Bradykinin metabolism in rat hearts with left-ventricular hypertrophy following myocardial infarction

2003 ◽  
Vol 81 (7) ◽  
pp. 740-746 ◽  
Author(s):  
Marie-Josée Dumoulin ◽  
Albert Adam ◽  
Jean-Lucien Rouleau ◽  
Hugues Gosselin ◽  
Daniel Lamontagne
Keyword(s):  
Myocardial Infarction ◽  
Left Ventricular Hypertrophy ◽  
Ventricular Hypertrophy ◽  
Neutral Endopeptidase ◽  
Left Ventricular ◽  
Coronary Artery Ligation ◽  
Artery Ligation ◽  
Relative Contribution ◽  
The Difference ◽  
Level Of Significance

The aim of the present study was to assess the contribution of angiotensin I converting enzyme (ACE) and neutral endopeptidase (NEP) in the coronary degradation of bradykinin (BK) after left-ventricular hypertrophy following myocardial infarction (MI) in rats. Myocardial infarction was induced by left descendant coronary artery ligation, and the contribution of ACE and NEP in the degradation of exogenous BK after a single passage through the coronary bed was assessed at 2, 5, and 36 days post-MI. BK degradation rate (Vmax/Km) was found to be significantly lower in hearts at 36 days (3.30 ± 0.28 min–1) compared with 2 days (4.39 ± 0.32 min–1) for noninfarcted hearts, but this reduction was just above the statistical level of significance for post-MI hearts. In infarcted hearts, Vmax/Km was increased significantly 5 days post-MI (4.91 ± 0.28 min–1) compared with the 2 and 36 day-groups (3.43 ± 0.20 and 2.78 ± 0.16 min–1, respectively). The difference between noninfarcted and MI was significant only 2 days post-MI. Treatment with the vasopeptidase inhibitor, omapatrilat, showed that the relative contribution of ACE and NEP combined increased over time in infarcted hearts and became significantly higher 36 versus 2 days post-MI. Finally, the treatment with an ACE inhibitor (enalaprilat) and a NEP inhibitor (retrothiorphan) in the 36-day infarcted and noninfarcted hearts showed that the relative contribution of ACE in infarcted hearts was comparable with that of noninfarcted hearts, whereas the relative contribution of NEP was increased significantly in infarcted hearts. In conclusion, experimental MI in rats induces complex changes in the metabolism of exogenous BK. The changes resulted in an increased relative contribution of NEP 36 days after infarction.Key words: bradykinin, ACE, NEP, myocardial infarction.

The Difference Between Platelet and Plasma FXIII Used to Study the Mechanism of Platelet Microvesicle Formation

1993 ◽  
Vol 70 (04) ◽  
pp. 681-686 ◽  
Author(s):  
Pål André Holme ◽  
Frank Brosstad ◽  
Nils Olav Solum
Keyword(s):  
Polyclonal Antibodies ◽  
Calcium Ionophore ◽  
Calcium Ionophore A23187 ◽  
Ionophore A23187 ◽  
B Subunit ◽  
Mechanical Disruption ◽  
Crossed Immunoelectrophoresis ◽  
The Difference ◽  
The Complement System ◽  
Filtration Technique

SummaryThe formation of microvesicles from platelets was induced either by activation of the complement system by a monoclonal antibody to CD9, or by incubation of platelets with the calcium ionophore A23187. A filter technique to isolate the microvesicles without plasma contamination is described. The microvesicles contained FXIIIa2 from the platelet cytoplasm which shows that these particles contain significant amounts of intracellular material. This was shown by the use of crossed immunoelectrophoresis with rabbit antibodies to total human platelet proteins in the second dimension gel and polyclonal antibodies against the a- and b-subunit of FXIII in the intermediate gel. The FXIIIa2 in the microvesicle was found to be functional as an enzyme. To prove this, it was shown that FXIII in its immunoprecipitate arc could catalyze the incorporation of monodansylcadaverine into casein as identified by fluorescence of this arc in ultraviolet light. The observation that the plasma form of FXIII (FXIIIa2b2) was absent from the microvesicles collected by the filtration technique, whereas it was present in platelet fragments obtained by mechanical disruption by ultrasonication, indicates that the activation-dependent microvesicles are formed by a true budding process with the inclusion of intracellular, but not extracellular material.

scholarly journals Apoptosis in Candida biofilms exposed to amphotericin B

2010 ◽  
Vol 59 (2) ◽  
pp. 149-157 ◽  
Author(s):  
Rawya S. Al-Dhaheri ◽  
L. Julia Douglas
Keyword(s):  
Amphotericin B ◽  
Antifungal Agents ◽  
Candida Parapsilosis ◽  
Candida Krusei ◽  
Clinical Use ◽  
Caspase Activity ◽  
Persister Cells ◽  
Specific Staining ◽  
General Inhibitor ◽  
Staining Methods

Candida biofilms are resistant to a range of antifungal agents in current clinical use. The basis of this drug resistance is not clear, but in some cases it could be due to the presence of a small number of drug-tolerant or persister cells. In this study, specific staining methods were used to investigate the existence of persisters and apoptosis in Candida biofilms subjected to different concentrations of amphotericin B. Fluorescein diacetate staining revealed the presence of persisters in biofilms of one of two strains of Candida albicans tested, and in biofilms of Candida krusei and Candida parapsilosis. Caspase activity, indicative of apoptosis, was detected with SR-FLICA and (aspartyl)2-rhodamine 110 fluorochrome-based staining reagents in all of these biofilms. The general inhibitor of mammalian caspases, Z-VAD-FMK, when used at a low concentration (2.5 μM), increased the viability of drug-treated biofilms up to 11.5-fold (P <0.001 %). Seven specific caspase inhibitors had different effects on C. albicans biofilm viability, but inhibitors of caspases-1, −9, −5, −3 and −2 all significantly increased cell survival (40-fold, 8-fold, 3.5-fold, 1.9-fold and 1.7-fold, respectively). However, histone deacetylase (HDA) inhibitors enhanced the activity of amphotericin B for biofilms of all three Candida species. Sodium butyrate and sodium valproate, for example, when added concurrently with amphotericin B, completely eliminated biofilm populations of C. albicans. Overall, our results demonstrate an apoptotic process in amphotericin-treated biofilms of three Candida species. They also indicate that HDA inhibitors can enhance the action of the drug and in some cases even eradicate persister subpopulations, suggesting that histone acetylation might activate apoptosis in these cells.

scholarly journals Analysis of Serum Proteins and Enzymes Level in Human Subjects with Osteoarthritis

2011 ◽  
Vol 30 (1) ◽  
pp. 15-24 ◽  
Author(s):  
Areeba Ahmad ◽  
Mohd Irshad ◽  
Waseem Ahmad ◽  
Abdul Khan ◽  
Riaz Ahmad
Keyword(s):  
Serum Proteins ◽  
Human Subjects ◽  
Significant Decline ◽  
Specific Staining ◽  
Native Page ◽  
Software Analysis ◽  
Major Activity ◽  
Ldh Isoenzymes ◽  
Sds Page ◽  
The Difference

Analysis of Serum Proteins and Enzymes Level in Human Subjects with OsteoarthritisThe aim of the present study was to assess the serum proteins and enzymes level using polyacrylamide gel electrophoretic (PAGE) profiles in human subjects with osteoarthritis (OA). Forty-one subjects with confirmed OA were selected for the present study. Sera were collected from these individuals and loaded in equal amounts on native and denaturing PAGE separately. Software analysis of these profiles was done using Scion Imaging (Beta release-4, Scion Corporation) and GelPro (Media Cybernetics, USA) programs. To visualize esterases (Est) and lactate dehydrogenase (LDH) isoenzymes in the sera of these patients substrate specific staining was performed. Differences in the values of control and OA subjects were tested statistically. Software analysis of native-PAGE profiles revealed the presence of nineteen peptides in control and twenty one in OA subjects respectively. Two extra peptides were present in the β-globulins region of OA subjects. Significant decline from 42.77% to 34.72% in albumin levels (hypoalbuminemia) was observed in OA subjects with total albumin to globulin ratio 0.58. In SDS-PAGE, the difference in control and OA subjects was observed among eight peptides with molecular weight 25, 22 and 20 kDa (absent in OA) and five novel peptides 270, 125, 30, 21.36 and 18.4 kDa (absent in controls), while albumin retains the major activity. For enzymes, Est follow a relative order, BchEst (42.86%)> ArylEst (16.24%)>AchEst (6.85%) in OA subjects with the expression of a new BchEst isoform in 4.78% and two isoforms of ArylEst at 2.13 and 1.61% concentrations respectively. Significantly declined albumin esterase-like activity (AlbEst) was observed (34%) (P<0.05) in diseased subjects compared with controls (47%). Significant increase in LDH-5 and decline in LDH-1 and -2 isoenzymes were also observed in the sera of OA subjects. However, the overall rank of LDH isoenzymes was similar in control and OA subjects. Our results demonstrate noticeable differences in the sera PAGE profiles and enzymes activity in control and OA subjects and provide evidence to select serum for its use in the search for suitable biochemical markers in osteoarthritis.

THE DYSFIBRINOGEN OF CHILDHOOD NEPHROSIS

1987 ◽  
Author(s):  
T Abshire ◽  
L Fink ◽  
J Christian ◽  
J O'Connell ◽  
W Hathaway
Keyword(s):  
Western Blot ◽  
Degradation Products ◽  
Polyacrylamide Gel Electrophoresis ◽  
Electrophoretic Pattern ◽  
Enzyme Linked Immunosorbent Assay ◽  
Thrombin Time ◽  
Fetal Plasma ◽  
Crossed Immunoelectrophoresis ◽  
Adult Plasma ◽  
The Difference

An abnormal fibrinogen (Fib) related to increased sialic acid (SA) has been described in adults with liver disease. This dysfibrinogen (Dysfib) seems most like fetal Fib. A review of 11 patients with nephrosis revealed an unexplained prolonged thrombin time (TT) and otherwise normal coagulation studies. Based on these observations, we sought to answer whether the prolonged TT defined a Dysfib and if this abnormal Fib was similar to fetal Fib. Pooled adult, fetal plasma, and the plasma of 3 patients with nephrosis were studied with TT and reptilase times (RT). Fib was measured by functional (Fib-act) and immunologic (Fib-ag) assays. An enzyme linked immunosorbent assay (ELISA) was established using antifibrinogen as the first antibody and either peroxidase conjugated Fib or a lectin (Limulus Polyphemus) specific for SA as the second antibody. The optical density was recorded per μgm Fib for both conjugated antifibrinogen or lectin and the ratio compared in order to estimate SA reactivity. Patient 3 was also studied by: 1) crossed immunoelectrophoresis (CIE) employing lectin in the first dimension and 2) polyacrylamide gel electrophoresis (PAGE) with transfer to nitrocellulose paper using Western Blot technique.Results of the CIE showed patient 3 and fetal plasma were similar in electrophoretic pattern and different from adult plasma. The PAGE with Western Blot revealed a similar pattern of Fib for patient 3, fetal and adult plasma. We conclude that the prolonged TT and RT, the greater amount of Fib-ag when compared to Fib-act in patients 1-3 and fetal plasma and the absence of evidence for Fib degradation products, support the diagnosis of Dysfib. The similarity of the CIE for patient 3 and fetal plasma and the difference between ELISA lectin/Fib ratio of patients 1-3 and fetal compared with adult plasma suggest that the Dysfib of nephrosis may be similar to fetal Fib.

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