Testicular function after torsion of the spermatic cord.
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Animals↗
Biomedical subjects
A J Visser
Publications and source records attributed to A J Visser.
Formation of quasi-regular compact structure of poly(methacrylic acid) upon an interaction with alpha-chymotrypsin.
Structure and dynamic properties of free poly(methacrylic acid) (PMA) and PMA complexed with alpha-chymotrypsin (CT) were studied using the time resolved fluorescence anisotropy technique. We have found that the interaction of PMA with CT induces the formation of a quasi-regular structure of PMA. At a CT/PMA weight ratio of 4:1 the interaction with CT leads to formation of approximately four equal segments of polyelectrolyte, each binding one CT molecule and characterized by an independent rotational mobility. Increase of the CT/PMA weight ratio above 8:1 gives rise to the overall rotation of the whole enzyme-polyelectrolyte complex. In water-ethanol mixtures the mobility of PMA segments containing CT decreases and the structure of the complex becomes even more rigid due to enhancement of the electrostatic interaction between CT and PMA. Formation of the compact and quasi-regular structure of the complex is perhaps the main reason behind the enhancement of enzyme stability and suppression of enzyme aggregation in water-organic cosolvent mixtures.
Animals↗
Fluorescence correlation spectroscopy of flavins and flavoenzymes: photochemical and photophysical aspects.
Fluorescence Correlation Spectroscopy (FCS) was used to investigate the excited-state properties of flavins and flavoproteins in solution at the single molecule level. Flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD) and lipoamide dehydrogenase served as model systems in which the flavin cofactor is either free in solution (FMN, FAD) or enclosed in a protein environment as prosthetic group (lipoamide dehydrogenase). Parameters such as excitation light intensity, detection time and chromophore concentration were varied in order to optimize the autocorrelation traces. Only in experiments with very low light intensity ( < 10 kW/cm2), FMN and FAD displayed fluorescence properties equivalent to those found with conventional fluorescence detection methods. Due to the high triplet quantum yield of FMN, the system very soon starts to build up a population of non-fluorescent molecules, which is reflected in an apparent particle number far too low for the concentration used. Intramolecular photoreduction and subsequent photobleaching may well explain these observations. The effect of photoreduction was clearly shown by titration of FMN with ascorbic acid. While titration of FMN with the quenching agent potassium iodide at higher concentrations ( > 50 mM of I-) resulted in quenched flavin fluorescence as expected, low concentrations of potassium iodide led to a net enhancement of the de-excitation rate from the triplet state, thereby improving the fluorescence signal. FCS experiments on FAD exhibited an improved photostability of FAD as compared to FMN: As a result of stacking of the adenine and flavin moieties, FAD has a considerably lower triplet quantum yield. Correlation curves of lipoamide dehydrogenase yielded correct values for the diffusion time and number of molecules at low excitation intensities. However, experiments at higher light intensities revealed a process which can be explained by photophysical relaxation or photochemical destruction of the enzyme. As the time constant of the process induced at higher light intensities resembles the diffusion time constant of free flavin, photodestruction with the concomitant release of the cofactor offers a reasonable explanation.
Ascorbic Acid↗
Exploring the conformational equilibrium of E. coli thioredoxin reductase: characterization of two catalytically important states by ultrafast flavin fluorescence spectroscopy.
The conformational dynamics of wild-type Escherichia coli thioredoxin reductase (TrxR) and the mutant enzyme C138S were studied by ultrafast time-resolved fluorescence of the flavin cofactor in combination with circular dichroism (both in the flavin fingerprint and far-UV regions) and steady-state fluorescence and absorption spectroscopy. The spectroscopic data show two conformational states of the enzyme (named FO and FR), of which the physical characteristics differ considerably. Ultrafast fluorescence lifetime measurements make it possible to distinguish between the two different populations: Dominant picosecond lifetimes of approximately 1 ps (contribution 75%) and 7 ps (8%) are associated with the FO species in TrxR C138S. Long-lived fluorescence with two time constants in the range of 0.2-1 ns (total contribution 17%) originates from enzyme molecules in the FR conformation. The near absence of fast lifetime components in oxidized wild-type TrxR supports the idea of this enzyme being predominantly in the FR conformation. The emission spectrum of the FO conformation is blue-shifted with respect to that of the FR conformation. Because of the large difference in fluorescence characteristics, fluorescence measurements on time scales longer than 100 ps are fully determined by the fraction of enzyme molecules in the FR conformation. Binding of the thiol reagent phenyl mercuric acetate to wild-type enzyme and TrxR C138S stabilizes the enzymes in the FR conformation. Specific binding of the NADPH-analog, AADP(+), to the FR conformation resulted in dynamic fluorescence quenching in support of the multiple quenching sites model. Raising the temperature from 277K-323K resulted in a moderate shift to the FR conformation for TrxR C138S. High concentrations of the cosolvent glycerol triggered the domain rotation from the FO to the FR conformation.
Escherichia coli↗
Supramaximal stimuli do not evoke a maximal contraction in urinary bladder smooth muscle fibers.
BACKGROUND: Smooth muscle fibers can be stimulated with an electrical field, high potassium or carbachol. We studied the effect of combined, supramaximal stimulation on the isometric force and the maximum shortening velocity of the pig urinary bladder. MATERIALS AND METHODS: After determining the dose response curve of each stimulation type, we stimulated 8 fibers with cumulative addition of supramaximal stimuli. RESULTS: The isometric force elicited with either potassium, carbachol or electrical field stimulation alone was the same for each stimulus. After addition of a second or third different supramaximal stimulus, the force further increased to a value that was on average 40% higher. CONCLUSIONS: Carbachol, high potassium or electrical field stimulation work through different stimulation pathways. Maximum stimulation with one of the stimuli does not result in a maximum isometric force development and maximum shortening velocity.
Animals↗
Intracellular electrical activity in human urinary bladder smooth muscle: the effect of high sucrose medium.
INTRODUCTION: The primary key to pharmacotherapy of bladder instability is in the excitation-contraction coupling of detrusor smooth muscle cells. To study this process, simultaneous recordings of mechanical and electrical activity are required. However, recording of mechanical activity induces movement, which may affect the quality of intracellular recordings. MATERIALS AND METHODS: We therefore compared the electrical activity of human detrusor smooth muscle cells in normal Krebs' solution and in a hypertonic solution, which immobilizes the tissue, enabling us to study the effect of movement on the membrane potential. Carbachol and KCl were applied to induce contractions. RESULTS: Sucrose in the medium made the tissue rigid and abolished its movement, while the electrical response was not affected. When compared with recordings in normal Krebs' solution, the average resting membrane potential was not altered. However, the membrane potential was more stable, with far less spike-shaped potentials. The spike-shaped potential amplitude was larger, while the duration was decreased. CONCLUSIONS: Impairing the ability of tissue movement resulted in changes in the electrophysiological properties of detrusor smooth muscle cells. The results suggest that stretch has an effect on L-type Ca2+ channels.
Action Potentials↗
Early diagnosis of prostate cancer in the Western Cape.
BACKGROUND: Early stage prostate cancer does not cause symptoms, and even metastatic disease may exist for years without causing symptoms or signs. Whereas early stage prostate cancer can be cured with radical prostatectomy or radiotherapy, the prognosis of patients with locally advanced or metastatic cancer is significantly poorer. OBJECTIVES: In view of the high incidence of advanced and therefore incurable prostate cancer seen at the oncology clinic of the Department of Urology, Tygerberg Hospital, we started a prostate clinic with the aim of detecting early stage prostate cancer which is potentially curable. A secondary objective was to investigate the question whether there is a higher incidence of prostate cancer among black African men. PATIENTS AND METHODS: Men aged 50-70 years were invited by means of media communications (newspaper and radio) to attend our prostate clinic for a free physical examination, including a digital rectal examination (DRE) and serum prostate specific antigen (PSA) assay. If the DRE was clinically suspicious of malignancy and/or the serum PSA was > 4 ng/ml, the patient was appropriately counselled and referred for transrectal ultrasound (TRUS)-guided sextant prostate biopsy. RESULTS: In the period June 1997-September 1999 a total of 1,056 men attended the prostate clinic. Biopsies were indicated in 160 cases, and were obtained in 114 (71.3%, i.e. 10.8% of the entire cohort). Prostate cancer was detected on first biopsy in 3.5% of the entire group of men (in 35.9% of those with a clinically abnormal DRE, in 41.3% of those with a serum PSA > 4 ng/ml and in 88.6% of those with an abnormal DRE and serum PSA > 4 ng/ml. In the 37 men with prostate cancer, the clinical tumour stage was T1-2 in 83.8% and T3-4 in 16.2%. In the group of patients with clinical stage T1-2 tumours, the treatment was watchful waiting in 62.5% of cases, radiotherapy in 20.8% and radical prostatectomy in 16.7%. Analysis of the data according to race showed that in the group of 47 black men there was a higher percentage of clinically abnormal DRE, PSA > 4.0 ng/ml and biopsies showing malignancy, and a higher overall prostate cancer detection rate (8.5%). CONCLUSIONS: Our prostate cancer detection rate of 3.5% is slightly lower than that reported in larger studies (4.7%), which may be due to the fact that prostate biopsy was performed in only 71% of those who had an indication for biopsy. In the men diagnosed with clinically localised prostate cancer, potentially curative treatment was given in only 37.5% of cases. This compares unfavourably with the historical cohort of men seen at our oncology clinic, where 53% received potentially curative treatment, and a large European study where potentially curative treatment was given in 89% of cases. Our finding that black men had a higher percentage of clinically abnormal DRE, PSA > 4.0 ng/ml and biopsies showing malignancy and a higher overall detection rate of prostate cancer should be interpreted with caution, since black men comprised only 4.5% of our overall study cohort.
Aged↗
Serum prostate-specific antigen as surrogate for the histological diagnosis of prostate cancer.
INTRODUCTION: To determine whether there is a cut-off value of serum prostate-specific antigen (PSA) which can be used confidently to make the diagnosis of prostate cancer, thereby obviating the need for biopsy. PATIENTS AND METHODS: During the period October 1991 to March 1998 the Department of Chemical Pathology at Tygerberg Hospital performed a total of 6,733 serum PSA assays on 3,960 patients. The histopathological and clinical diagnoses of these patients were obtained from records in the departments of Anatomical Pathology, Urological Oncology and Radiation Oncology. The serum PSA levels were correlated with the histopathology reports, using different PSA cut-off values ranging from 5 to 500 ng/ml, to calculate the sensitivity, specificity, and positive and negative predictive values of each cut-off value of PSA in predicting the presence of prostate cancer. RESULTS: In total, 3,837 (57%) of the 6,733 serum PSA assays were < or = 4 ng/ml, 1,045 (15.5%) of the assays were > or = 50 ng/ml, and 798 (11.9%) were > or = 100 ng/ml. Of the total of 3,960 individual patients, 531 (13.4%) had a serum PSA > or = 50 ng/ml and 423 (10.7%) had a PSA > or = 100 ng/ml. A serum PSA of > or = 30 ng/ml had a positive predictive value (PPV) of 90% at a specificity of 87% and sensitivity of 78%, while a PSA > or = 60 ng/ml had a PPV of 98% at a specificity of 98% and sensitivity of 65% for the presence of prostate cancer. The PPV reached 99% at a PSA > or = 100 ng/ml and 100% at a PSA > or = 500 ng/ml, with a specificity of 99% and 100%, but sensitivity of only 53% and 19%, respectively. CONCLUSIONS: A serum PSA > or = 60 ng/ml has a PPV of 98% for the presence of adenocarcinoma of the prostate, and may be used as a surrogate for histological diagnosis where facilities for obtaining prostatic biopsies are not readily available, thus decreasing costs and patient morbidity.
Aged↗
Vesicosuspension for female stress incontinence and cystocele using pubic bone anchors.
OBJECTIVE: The purpose of this study was to evaluate the results of vesicosuspension for female stress incontinence and cystocele using fixation with pubic bone anchors in a cohort of patients operated on by a single surgeon. METHODS: Using a standard questionnaire, an independent female interviewer not employed by the surgeon's practice contacted 100 consecutive patients with stress incontinence and/or cystocele who had undergone vesicosuspension using suprapubic fixation with bone anchors between October 1996 and July 1997. The patients' responses were recorded on a computerised database and analysed. All procedures were performed by a single surgeon (A.J.V.). The duration of the operation was normally 45 minutes, the patient was mobilised on day 3 postoperatively, and the catheter removed on day 7. RESULTS: The mean age of the 100 women was 50.4 years (range 26-84 years) and the mean follow-up was 11 months (range 1.4-19 months). Previous operations for incontinence had been performed in 44 patients. Preoperatively 56 patients had to wear protective pads, using an average of 4.4 pads per day, and 36 had to change underwear because of urine leakage. Postoperatively 14 patients had to wear protective pads, using an average of 3.5 pads per day, while 11 had to change their underwear because of urine leakage. Only 3 patients used a catheter to empty the bladder, 2 used medication for incontinence, and none had had a subsequent operation for urine leakage. Postoperatively 16 patients reported having problems with pain in the pelvis, and 6 had pain during intercourse, but there were no cases of osteitis pubis. Subjective improvement reported by the patients was 93% on average, and overall patient satisfaction with the procedure was 8.6 on a scale of 0-10. In total, 89 patients said they would be prepared to undergo the operation again, while 92 would recommend it to a friend. CONCLUSION: The use of pubic bone anchors for colposuspension is safe and reliable, with results comparable to those of other methods, and the added advantages of faster mobilisation and few short-term complications.
Adult↗
Simultaneous recording of mechanical and intracellular electrical activity in guinea-pig urinary bladder smooth muscle: a comparison with human detrusor contraction.
OBJECTIVES: To study the relationship between the electrical and mechanical activity of guinea-pig detrusor muscle and compare this with the results obtained in human tissue. The guinea pig is the most used model to study the electrical basis of human bladder contraction. METHODS: We simultaneously recorded the mechanical and intracellular electrical activity in muscle strips. To study the effect of tissue movement on the membrane potential, the medium was made hypertonic with sucrose. Carbachol and KCl were applied to the bath to induce contractions. RESULTS: Carbachol resulted in a force response without a consistent change in the membrane electrical activity. KCl induced depolarization of the membrane associated with force development. Sucrose in the medium greatly impaired the ability to contract, without affecting the electrical activity. Compared with recordings in normal Krebs' solution, the resting membrane potential was not altered. In both media, spike-shaped potentials with variable amplitudes and shapes were recorded. These events were minimally affected by sucrose. CONCLUSIONS: In contrast to the response to KCl, the overall mechanical response of guinea-pig detrusor strips to muscarinic receptor stimulation did not correlate with the electrical activity of a single cell. Sucrose had only a minimal effect on the electrical activity, demonstrating that the electrical responses we measured were not affected by movement. Our intracellular recordings in guinea-pig tissue differed from the results obtained by other groups, but show great resemblance with those we recorded in human urinary bladder smooth muscle strips.
Animals↗
Fluorescence dynamics of green fluorescent protein in AOT reversed micelles.
We have used the enhanced green fluorescent protein (EGFP) to investigate the properties of surfactant-entrapped water pools in organic solvents (reversed micelles) with steady-state and time-resolved fluorescence methods. The surfactant used was sodium bis(2-ethylhexyl)sulfosuccinate (AOT) and the organic solvents were isooctane and (the more viscous) dodecane, respectively. The water content of the water pools could be controlled through the parameter w0, which is the water-to-surfactant molar ratio. With steady-state fluorescence, it was observed that subtle fluorescence changes could be noted in reversed micelles of different water contents. EGFP can be used as a pH-indicator of the water droplets in reversed micelles. Time-resolved fluorescence methods also revealed subtle changes in fluorescence decay times when the results in bulk water were compared with those in reversed micelles. The average fluorescence lifetimes of EGFP scaled with the relative fluorescence intensities. Time-resolved fluorescence anisotropy of EGFP in aqueous solution and reversed micelles yielded single rotational correlation times. Geometrical considerations could assign the observed correlation times to dehydrated protein at low w0 and internal EGFP rotation within the droplet at the highest w0.
Alkanes↗
Oxidation of unsaturated phospholipids in membrane bilayer mixtures is accompanied by membrane fluidity changes.
Steady-state and time-resolved fluorescence spectroscopy has been used to obtain information on oxidation processes and associated dynamical and structural changes in model membrane bilayers made from single unilamellar vesicles (SUV's) of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) mixed with increasing amounts of 1-stearoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (SAPC). The highly unsaturated arachidonoyl chain containing four double bonds is prone to oxidation. Lipid oxidation was initiated chemically by a proper oxidant and could be followed on line via the fluorescence changes of an incorporated fluorescent lipophilic fatty acid: 4,4-difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-s-indacene-3-undecanoic acid (BP-C11). The oxidation rate increases with an increasing amount of SAPC. Size measurements of different SUV's incorporated with a trace amount of a phosphatidylcholine analogue of BP-C11 using fluorescence correlation spectroscopy have demonstrated that an increase of lipid unsaturation results in smaller sized SUV's and therefore to a larger curvature of the outer bilayer leaflet. This suggests that the lipid-lipid spacing has increased and that the unsaturated fatty acyl chains are better accessible for the oxidant. Oxidation results in some characteristic physical changes in membrane dynamics and structure, as indicated by the use of specific fluorescence probes. Fluorescence measurements of both dipyrenyl- and diphenylhexatriene-labelled PC introduced in non-oxidised and oxidised DOPC-SAPC membranes clearly show that the microfluidity (local fluidity at the very site of the probes) significantly decreases when the oxidised SAPC content increases in the lipid mixture. A similar effect is observed from the lateral diffusion experiments using monopyrenyl PC in the same membrane systems: the lateral diffusion is distinctly slower in oxidised membranes.
Diffusion↗
Structural dynamics of green fluorescent protein alone and fused with a single chain Fv protein.
Structural information on intracellular fusions of the green fluorescent protein (GFP) of the jellyfish Aequorea victoria with endogenous proteins is required as they are increasingly used in cell biology and biochemistry. We have investigated the dynamic properties of GFP alone and fused to a single chain antibody raised against lipopolysaccharide of the outer cell wall of gram-negative bacteria (abbreviated as scFv-GFP). The scFv moiety was functional as was proven in binding assays, which involved the use of both fluorescence correlation spectroscopy observing the binding of scFv-GFP to gram-negative bacteria and a surface plasmon resonance cell containing adsorbed lipopolysaccharide antigen. The rotational motion of scFv-GFP has been investigated with time-resolved fluorescence anisotropy. However, the rotational correlation time of scFv-GFP is too short to account for globular rotation of the whole protein. This result can only be explained by assuming a fast hinge motion between the two fused proteins. A modeled structure of scFv-GFP supports this observation.
Animals↗
Conformational stability of human interferon-gamma on association with and dissociation from liposomes.
The integrity of a therapeutic protein has to be safeguarded when formulated in delivery systems such as liposomes. In this study, we investigated the conformational stability of recombinant human interferon gamma (hIFNgamma) on association with and after dissociation from liposomal bilayers using circular dichroism (CD) and steady-state fluorescence spectroscopy as well as time-resolved fluorescence methodology. We used hIFNgamma adsorption to and desorption from empty liposomes as a model for hIFNgamma-containing liposomes prepared via the film hydration method. CD studies indicated that no changes in the secondary and tertiary protein structure occur during and after interaction of hIFNgamma with the liposomes. Steady-state fluorescence emission spectra of untreated and liposome-desorbed hIFNgamma revealed that the environment of the sole Trp residue was not affected by the adsorption/desorption process. The Trp-36 residue remained fully quenchable by acrylamide after desorption of hIFNgamma from the liposomes. Time-resolved fluorescence studies were conducted to probe the local environment and the mobility of Trp-36 before, during, and after interaction of hIFNgamma with the liposomal membrane. Differences in rotational correlation time between free and liposomal hIFNgamma were attributed to immobilization of the protein on adsorption to the liposome bilayer. Disparities were detected between the average lifetimes of liposome-adsorbed hIFNgamma and hIFNgamma-liposomes, indicating that subtle changes in the Trp-36 environment took place during preparation of the liposomes via the film hydration method compared with the adsorption of hIFNgamma to the liposome surface. The results of this study indicate that association of hIFNgamma with negatively charged liposomes results in minimal changes in the secondary and tertiary structure of the protein. We conclude that all techniques used point to a full retention or restoration of the protein conformation after desorption from the liposomes.
Acrylamide↗
The role of intracellular and extracellular calcium in mechanical and intracellular electrical activity of human urinary bladder smooth muscle.
We studied the role of extracellular and intracellular Ca2+ in human detrusor smooth muscle contraction. Simultaneous recordings of mechanical and intracellular electrical activity were made in three different Ca2+ concentrations: normal Krebs' solution (100%), 10% of the standard Ca2+ concentration and a solution in which Ca2+ was omitted from the medium (0%). Spontaneous contractions and KCl or CCh induced contractions were studied. Ryanodine and caffeine were used to manipulate the intracellular Ca2+ stores. The present results show that only a very small amount of Ca2+ in the extracellular space is sufficient to support spontaneous and induced contractions. Spike-shaped potentials and long lasting depolarisations were recorded in all three solutions. However, the prevalence of long lasting depolarisations increased when the extracellular Ca2+ concentration was reduced. The amplitude of the spike-shaped potentials and long lasting depolarisations appeared to be negatively affected by diminishing the extracellular Ca2+ concentration. Additionally, the duration of the long lasting depolarisations was reduced in 0% Ca2+. The contraction upon KCl stimulation was primarily depending on the extracellular Ca2+. Upon muscarinic receptor stimulation, a combined activation of Ca2+ mobilisation from intracellular and extracellular stores may occur; the ratio of contribution of these two sources changes in accordance with the requirements of the conditions.
Acetylcholine↗
Purification and ligand exchange protocols for antenna proteins from bioluminescent bacteria.
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Chromatography, Gel↗
In vivo fluorescence correlation microscopy (FCM) reveals accumulation and immobilization of Nod factors in root hair cell walls.
Fluorescence correlation microscopy (FCM) is a new single-molecule detection technique based on the confocal principle to quantify molecular diffusion and concentration of fluorescent molecules (particles) with sub-micron resolution. In this study, FCM is applied to examine the diffusional behaviour of fluorescent Nod factor analogues on living Vicia sativa root hairs. Three recently described Nod factors with a fluorescent acyl chain (Goedhart et al. Biochemistry 1999, 38, 10898-10907) were used. Plasmolysis of fluorescently labelled root hairs showed that the Nod factors are predominantly located in the cell wall, as hardly any fluorescence could be detected in the plasma membrane. After Nod factor-induced root hair deformation, the new outgrowth was not labelled, indicating a lack of migration of Nod factors to the newly synthesized cell wall. In agreement, FCM showed a > 1,000-fold reduction of molecular mobility of the fluorescence Nod factors upon binding to the cell wall. In addition, FCM demonstrated that Nod factors, when exogenously applied in aqueous solution at 10 nM, markedly concentrate in the cell wall of root hairs (up to 50-fold). The feasibility of applying FCM for the study of living plant cells as well as the implications of our results for the perception of Nod factors are discussed.
Arabidopsis↗
Simultaneous recording of mechanical and intracellular electrical activity in human urinary bladder smooth muscle.
OBJECTIVE: To elucidate the role of the membrane potential in human detrusor smooth muscle contraction, by simultaneously recording mechanical and intracellular electrical activity in muscle strips. Materials and methods The agonists acetylcholine and carbachol were applied to induce a contraction on muscarinic receptor stimulation; to block the response, atropine was added to the bath. The Ca2+ necessary for activating the contractile machinery can be recruited via two pathways: release from intracellular stores or influx from the extracellular matrix. High potassium was applied to induce Ca2+ influx through voltage-sensitive Ca2+ channels. RESULTS: There were significant changes in the force when agonist, antagonist and high potassium was administered. However, there were significant changes in membrane potential only when KCl was applied to the bath and not with muscarinic agonist or antagonist application. Activity in the form of spike potentials did not change significantly on applying any of the test substances. CONCLUSION: The present results indicate that the Ca2+ mobilized on M3 receptor stimulation originates primarily from intracellular stores, with no systematic changes in membrane potential. Atropine only caused a relaxation in muscle previously contracted by M3-receptor agonist stimulation; it had no effect on relaxed muscle strips.
Adult↗