A novel method for assessing effects of hydrostatic fluid pressure on intracellular calcium: a study with bovine articular chondrocytes

Chondrocytes in articular cartilage are exposed to hydrostatic pressure and distortional stress during weight bearing and joint loading. Because these stresses occur simultaneously in articular cartilage, the mechanism of mechanosignal transduction due to hydrostatic pressure alone in chondrocytes is not clear. In this study, we attempted to characterize the change in intracellular calcium concentration ([Ca²⁺]_i) in response to the application of hydrostatic fluid pressure (HFP) to cultured bovine articular chondrocytes isolated from defined surface (SZ) and middle zones (MZ) by using a fluorescent indicator (X-rhod-1 AM), a novel custom-made pressure-proof optical chamber, and laser confocal microscopy. Critical methodology implemented in this experiment involved application of high levels of HFP to the cells and the use of a novel imaging apparatus to measure the peak [Ca²⁺]_i in individual cells. The peak [Ca²⁺]_i in MZ cells cultured for 5 days showed a significant twofold increase after the application of HFP at constant 0.5 MPa for 5 min. The peak [Ca²⁺]_i in SZ cells was lower (43%) than that of MZ cells. The peak was suppressed with an inhibitor of dantrolene, gadolinium, or a calcium ion-free buffer, but not with verapamil. This study indicated that the increase in [Ca²⁺]_i in chondrocytes to HFP is dependent on the zonal origin. HFP stimulates calcium mobilization and stretch-activated channels. real-time optical measurement; intracellular calcium; mechanosignal transduction     

Methodological considerations for conducting tumor surveys of fishes

The usefulness of tumor surveys in indicating the availability of carcinogenic contaminants in ecosystems hinges largely on decisions made during the planning process. Species used as indicator organisms for monitoring programs are best selected from those known to be susceptible to epizootics of neoplasms. Lesions which can be quickly located and preserved in the field and for which a data base exists to help interpretation include liver neoplasms and external neoplasms. Since tumor frequencies increase with age and may change from season to season, studies should hold these variables constant among locations. During the design of the study investigators should determine the number of fish they need to examine in order to achieve the degree of resolution desired. Finally the field crew must be familiar with fish anatomy and necropsy techniques as well as storage and handling of the chosen preservative.     

A review and evaluation of study design considerations for site-specifically assessing the health of fish populations

Since there are not any well developed procedures for site-specifically evaluating the health of fish populations, most field surveillance programs have been restricted to collecting information on chemistry, toxicity, bioaccumulation, biochemical alterations orin situ benthic community structure. Identification of the mechanism and significance of contaminant effects on fish populations depends on the ability to identify changes and to distinguish changes in survival, food availability or food conversion efficiency, from coincidental changes associated with alterations in habitat or natural variability. Preliminary identification of the characteristics of adult fish can be used to cost-effectively focus financial resources on the alterations which are of relevance to interpretation of impacts and identification of causal factors. Proper interpretation requires that appropriate attention be given to monitoring level and strategy, selection of species and timing of sampling, sample size requirements and choice of reference site.     

Methodological considerations in the study of population density and social pathology

A previous study of the effects of overcrowding on social behavior is discussed and replicated in this article. Consideration of two major methodological problems — the construction of the class and ethnicity indices and the measurement of social class — leads to reanalysis of the data and to results differing in several significant respects from the original results. Although these new results do not provide a definitive answer to the question of the effects of population density on social pathologies, they do show that studies of the problem are subject to several, possibly severe, measurement problems. Until these are more adequately dealt with, conclusions regarding the relation between density and human behavior must be both cautious and tentative.     

Methodological considerations on the study of sexual dimorphism in past human populations

The degree of sexual dimorphism in human populations is influenced by stress, social role and by labour division. However, studies on ethnographic populations provided contradictory results. Unfortunately, most of these studies were based on stature only, which, as we could observe in a survey on pre-protohistoric circum-Medirerranean samples, is a poor indicator of functionally related dimorphism. A number of skeletal measurements were examined: skull, stature, transverse trunk diameters, long bones length, circumference and section, in order to assess their usefulness as indicators of functionally related dimorphism. The best indicators were represented by section and circumference of the long bones of the limbs, followed by cross-shoulder breadth (biclavicular length), stature and limb bone length, facial measurements, cranial measurements and sacral breadth. From the methodological point of view, it was found that:

1. It is better to calculate the index of dimorphism for each trait or set of traits within each sample. Then a weighted average of all the available samples is taken. The index derived from pooling a number of samples does not make biological sense. In pooled samples the distinction of between versus within sample differences is obscured;
2. It is better to combine an index which is based on the difference between averages and one which takes variability into account, because variability can also be an aspect of sexual dimorphism;
3. It is better to apply some allometric correction to the measurements used. For instance, the log transformation produces clearer trends of differential dimorphism among the various traits.

     

Pleiotropic functions of brain proteinases: Methodological considerations and search for caspase substrates

Analysis of the literature and our own data suggest that the so-called “apoptotic” proteinases play important roles in brain function. However, mechanisms of their involvement in normal neuronal plasticity remain obscure. One of the main reasons for this is broad substrate specificity of proteinases; the number of potential substrates of each can reach several thousands. Obviously, a real approach to study functions of “apoptotic” proteinases, caspase-3 in particular, is to identify their intracellular substrates. It is the nature of a substrate that defines the direction of signal transduction or metabolic changes; therefore, identification of molecular partners of particular proteases should be the key study, not just measuring its activity or respective protein or mRNA expression. This approach will allow studying regulatory mechanisms not only for proteinases, but also for other pleiotropic enzymes usually possessing broad substrate specificity.     

Methodological considerations for the human platelet 5-HT2A receptor binding kinetic assay

Analysis of an extensive database of human platelet 5-HT2A receptor binding assays has been conducted in order to identify factors that may affect the assay results. Despite anecdotal reports that storage of frozen platelet pellets may affect 5-HT2A binding affinity and capacity, no quantitative study has been reported in the literature. Analysis of binding data for 373 frozen samples with a storage time up to three years is presented in this paper. It is shown that prolonged storage significantly decreases binding. The loss of binding capacity begins in the first six month of storage. Bmax declines by half after 17 month. The impact of storage time on the binding affinity is much smaller. There is only about 20% increase in the value of affinity K(D) during the half-life of Bmax. Differences in sample storage time may partly explain discrepancies in results between different research groups. Nonspecific binding due to binding to filter material diminishes accuracy and reliability of the binding assays as a result of a decrease in the ratio of specific to nonspecific ratio. A data analysis based on our suggested mathematical model shows that this effect depends on tissue concentration in test tube and becomes pronounced when the concentration is below 0.1 mg protein/ml (at 0.2 nM of ligand). Above 0.1 mg protein/ml, percentage of specific to total binding exceeds 65%, which is an acceptable level for the ratio. The majority of the binding studies reported in the literature employed a tissue concentration more than 0.5 mg/ml, well above the minimal limit sufficient for a reliable assay. However, development of microassays to conserve precious tissue must take the limit into consideration.     

Reactions involving carbamyl phosphate in the presence of precipitated calcium phosphate with formation of pyrophosphate: A model for primitive energy-conservation pathways

The formation of carbamyl phosphate (CAP) in dilute solutions of cyanate (NCO^–) and orthophosphate (Pi) was measured both in the absence and in the presence of a precipitated matrix of calcium phosphate (Pi.Ca). The second-order rate constant and the free energy of CAP synthesis were not modified by the presence of the solid matrix, indicating that synthesis occurs in the homogeneous Pi-containing solution. The elimination reaction of CAP to form NCO^– and Pi followed first-order kinetics and the rate constant was the same whether or not calcium phosphate was present. Elimination was not complete, and the steady level of remaining CAP was that expected from the free energy of synthesis. The formation of pyrophosphate (PPi) was detected in CAP-containing medium only in the presence of calcium, showing a close correlation with the amount of precipitated Pi.Ca. Phosphorolysis of CAP followed a sigmoidal time course, compatible with adsorption of CAP to the solid matrix as a prelude to transphosphorylation. Addition of 5-AMP and of short linear polyphosphates inhibited phosphorolysis of CAP. It is proposed that the presence of a solid phosphate matrix and the relative concentrations of cyano compounds, as well as those of nucleotides and inorganic polyphosphates, could have played a crucial role in the conservation of chemical energy of CAP and in its use in prebiotic phosphorylation reactions.     

Dent disease: A window into calcium and phosphate transport

This review examines calcium and phosphate transport in the kidney through the lens of the rare X‐linked genetic disorder Dent disease. Dent disease type 1 (DD1) is caused by mutations in the CLCN5 gene encoding ClC‐5, a Cl^?/H⁺ antiporter localized to early endosomes of the proximal tubule (PT). Phenotypic features commonly include low molecular weight proteinuria (LMWP), hypercalciuria, focal global sclerosis and chronic kidney disease; calcium nephrolithiasis, nephrocalcinosis and hypophosphatemic rickets are less commonly observed. Although it is not surprising that abnormal endosomal function and recycling in the PT could result in LMWP, it is less clear how ClC‐5 dysfunction disturbs calcium and phosphate metabolism. It is known that the majority of calcium and phosphate transport occurs in PT cells, and PT endocytosis is essential for calcium and phosphorus reabsorption in this nephron segment. Evidence from ClC‐5 KO models suggests that ClC‐5 mediates parathormone endocytosis from tubular fluid. In addition, ClC‐5 dysfunction alters expression of the sodium/proton exchanger NHE3 on the PT apical surface thus altering transcellular sodium movement and hence paracellular calcium reabsorption. A potential role for NHE3 dysfunction in the DD1 phenotype has never been investigated, either in DD models or in patients with DD1, even though patients with DD1 exhibit renal sodium and potassium wasting, especially when exposed to even a low dose of thiazide diuretic. Thus, insights from the rare disease DD1 may inform possible underlying mechanisms for the phenotype of hypercalciuria and idiopathic calcium stones.     

Orbital floor reconstruction using calcium phosphate cement paste: an animal study

Orbital floor defects were created in 10 New Zealand white rabbits and were reconstructed using an injectable calcium phosphate paste. These animals were euthanized at 2, 4, 8, and 12 months after implantation and were examined for biocompatibility and osteoconductivity. Grossly, implants were found to be adherent to the floor and covered with fibrous tissues. There was no sign of infection, extrusion, or migration of implant within the orbit and maxilla. The orbital floor was completely restored. Histological examination showed active new bone formation that encroached within the implant and gradually increased in density with time. Maxillary mucosa and glands were likewise reconstituted. Thin fibrovascular tissues were seen on top of and within the surface of the implant, and few to slight inflammatory cells were seen. Microradiography showed direct apposition between the new bone and the implant. These findings compare favorably with previously published reports on the biocompatibility and osteoconductivity of calcium phosphate cement. The authors believe that, together with ease of use and structural integrity, calcium phosphate paste can be useful in orbital floor reconstruction.     

Methodological and empirical considerations when assessing freshwater ecosystem service provision in a developing city context: Making the best of what we have

This study contributes to both the methodological and empirical literature by developing an integrative approach to assessing temporal and spatial change in riparian ecosystem service delivery by drawing on available and diverse data sets. These data sets act as multiple lines of evidence in supporting comparisons between data sets to test the validity of developed methods and the application of such methods. In order to synthesise these data as well as to determine the fluctuations in riparian ecosystem service provision a scoring system was developed. Methodologically, the scoring system proved informative across the majority of ecosystem services categories, showing close to 80% similarity in outcomes when comparing the scoring method to trends in long-term water quality measurements. Other benefits of the scoring system included its design simplicity, cost-effectiveness, and applicability and replicability across various urban settings. Empirically, the data sets used support the findings of the ecosystem services scoring exercise and suggests that fluctuations in ecosystem service delivery through time and across the river reaches are linked to land-use change and other human activities. Findings suggest that as water leaves an urban protected area and travels across transformed and impacted landscapes, the results are poor water quality and diminished ability of rivers to yield ecosystem services the further the river flows into the urban setting. Urbanisation and changes in land-uses in developing city contexts is therefore shown to affect potential ecosystem services benefits, necessitating increasing management interventions.     

Methodological considerations for examining the relationship between sperm morphology and motility

Sperm cells of all taxa share a common goal to reach and fertilize an ovum, yet sperm are one of the most diverse cell types in nature. While the structural diversity of these cells is well recognized, the functional significance of variation in sperm design remains elusive. An important function of spermatozoa is a need to migrate toward the ova, often over long distances in a foreign environment, which may include a complex and hostile female reproductive tract. Several comparative and experimental studies have attempted to address the link between sperm morphology and motility, yet the conclusions drawn from these studies are often inconsistent, even within the same taxa. Much of what we know about the functional significance of sperm design in internally fertilizing species has been gleaned from in vitro studies, for which experimental parameters often vary among studies. We propose that discordant results from these studies are in part due to a lack of consistency of methods, conditions that do not replicate those of the female reproductive tract, and the overuse of simple linear measures of sperm shape. Within this review, we provide a toolkit for imaging, quantifying, and analyzing sperm morphology and movement patterns for in vitro studies and discuss emerging approaches. Results from studies linking morphology to motility enhance our understanding of the evolution of adaptive sperm traits and the mechanisms that regulate fertility, thus offering new insights into methods used in assisted reproductive technologies in animal science, conservation and public health.     

Methodological challenges in assessing the environmental status of a marine ecosystem: case study of the Baltic Sea

Assessments of the environmental status of marine ecosystems are increasingly needed to inform management decisions and regulate human pressures to meet the objectives of environmental policies. This paper addresses some generic methodological challenges and related uncertainties involved in marine ecosystem assessment, using the central Baltic Sea as a case study. The objectives of good environmental status of the Baltic Sea are largely focusing on biodiversity, eutrophication and hazardous substances. In this paper, we conduct comparative evaluations of the status of these three segments, by applying different methodological approaches. Our analyses indicate that the assessment results are sensitive to a selection of indicators for ecological quality objectives that are affected by a broad spectrum of human activities and natural processes (biodiversity), less so for objectives that are influenced by a relatively narrow array of drivers (eutrophications, hazardous substances). The choice of indicator aggregation rule appeared to be of essential importance for assessment results for all three segments, whereas the hierarchical structure of indicators had only a minor influence. Trend-based assessment was shown to be a useful supplement to reference-based evaluation, being independent of the problems related to defining reference values and indicator aggregation methodologies. Results of this study will help in setting priorities for future efforts to improve environmental assessments in the Baltic Sea and elsewhere, and to ensure the transparency of the assessment procedure.