Investigation of inorganic deposits in selected organic matrices

Inorganic deposits in the wall of human and animal arteries and in experimental tumor (Morris hepatoma 7777) were examined using proton induced X-ray emission (PIXE) and PIXE in combination with proton microprobe (micro-PIXE) techniques. The sections adjacent to the irradiated ones part were submitted to histological investigations and one part of the material was additionally investigated by infrared (IR) spectroscopy. For identification of mineral deposits, the micro-PIXE method appeared the most sensitive. The mineral deposits were detected in the artery samples, even in those without visible morphological changes, as well as in tumor samples. The deposites showed different localization and composition, depending on age and type of vessel. There were also differences between human and animal arteries. IR spectroscopy revealed the presence of carbonate apatite within the artery samples from old individuals. Matching of histological observations with data obtained by micro-PIXE method allows a better correlation of morphological and analytical results.     

Heterogeneity of membrane properties in vascular muscle cells from various vascular beds

It is becoming increasingly more apparent that vascular smooth muscle cannot be treated as a homogeneous group and cannot be compared to other types of smooth muscle at least in terms of responsiveness to vasoactive agents. This heterogeneity may have as part of its basis differences in the molecular structure at the level of the plasma membrane. Such differences manifest themselves in terms of resting membrane potential (Em), response to cardiac glycosides, ionic conductances, and membrane responses to neurotransmitters. Results show that the middle cerebral artery of the cat has a higher Em and depolarizes to excess K+ with a steeper slope than other peripheral arteries of the same animal. Furthermore, cerebral arteries from different areas of the brain exhibit distinctly different resting membrane properties as well as different responses to norepinephrine. In addition, cerebral arteries exhibit a high degree of electromechanical coupling not always observed in other peripheral arteries.     

Quantitative and qualitative heterogeneity in smooth muscle myosin heavy chains

Previous studies have shown that smooth muscle myosin consists of two heavy chains (MHCs) of unequal molecular weight; however, it is not clear whether there are intermuscle, inter- and intraspecies differences in the MHCs. The purpose of these experiments was to quantitatively and qualitatively compare MHCs in different smooth muscles. Extracts of bovine aorta (BAo), dog saphenous vein (dSV) and femoral artery (dFA), and rat aorta (rAo), femoral artery (rFA), carotid artery (rCA), ileum (rGI) and uterus (rUt) were electrophoresed on 5% polyacrylamide-1% SDS gels. All tissues exhibited two MHCs with molecular weights of 207,000 (MHC1) and 204,000 (MHC2) daltons. In all cases the proportion of total MHC made up by MHC1 was greater than that by MHC2. Based on their relative proportions (MHC1:MHC2), the tissues fell into one of three groups: (1) 55:45 - rAo, rCA, dFA; (2) 60:40 - dSV, BAo, rGI; and (3) 65:35 - rUt, rFA. Group 1 differed significantly from group 3 in the proportion of each MHC. One dimensional peptide maps indicated that BAo, dSV and dFA were similar while subtle differences existed between rUt and rAo. Differences between rUt and rAo were also observed in their cross-reactivity to a monoclonal antibody to smooth muscle MHC, confirming the differences seen on peptide maps. These results indicate that there are intertissue and inter- and intraspecies differences in smooth muscle MHCs. The significance of these differences to muscle function remains to be determined.     

Sensitivity and adrenoceptor affinity in the mesenteric artery of the deoxycorticosterone acetate hypertensive rat

This study examines vascular reactivity to alpha-adrenoceptor agonists in mineralocorticoid (deoxycorticosterone acetate (DOCA-salt) hypertensive and normotensive rats. The rats were anesthetized and the mesenteric artery was excised and cut helically into strips that were mounted in a muscle bath for the measurement of isometric force development. Addition of norepinephrine, epinephrine, phenylephrine, methoxamine, or clonidine to the bath caused contractions in all arteries. Arteries from hypertensive rats were more sensitive (lower ED50 values) to each of the agonists than arteries from normotensive rats. alpha-Adrenoceptor affinity for phentolamine (Schild analysis; norepinephrine as the agonist) in hypertensive arteries was not significantly different from that in normotensive arteries. Maximal force generation to clonidine was greater in hypertensive arteries than in normotensive arteries. These results demonstrate an augmented vascular sensitivity to several alpha-adrenoceptor agonists in DOCA hypertensive rats. This change in sensitivity is independent of a change in affinity for the adrenoceptor antagonist, phentolamine. It may be that a change in receptor number or an alteration in a post-receptor activation event accounts for this enhanced adrenoceptor responsiveness in mineralocorticoid hypertension.     

The experimental animal models for assessing treatment of restenosis

Coronary restenosis after percutaneous interventions remains a major clinical problem. The assessment of therapies for the prevention of restenosis relies on the use of experimental models. This review describes the most frequently used animal models of coronary artery retenosis and the intraspecies differences among them, particularly in the extent and composition of the neointimal thickening. These differences in neointima formation should be considered in the interpretation of effective antiproliferative therapies before they are transferred into clinical trials.     

Reactivity in human cerebral artery: species variation

It is becoming obvious that the reactivity of vascular smooth muscle to vasoactive agents is not homogeneous in different arteries (cerebral vs. other arteries) from the same animal species or in cerebral arteries from different species. In this communication, the reactivity of human cerebral arteries to norepinephrine (NE), dopamine (DA), small amounts of K+ and ouabain and their mechanisms of action are compared with those in monkey and dog cerebral arteries. NE produces moderate contractions in the primate arteries, mediated by alpha 1 adrenoceptors, and slight contractions in the dog arteries, possibly mediated by alpha 2 receptors. DA relaxes human and monkey cerebral arteries but contracts the dog arteries. The primate artery relaxation mediated by dopaminergic receptors is large enough to predominate over the alpha 1 receptor-mediated contraction. Minute amounts of K+ preferentially relax cerebral arteries from humans, monkeys, and dogs, possibly activating the electrogenic Na+ pump. Ouabain, a Na+ pump inhibitor, contracts these cerebral arteries with low concentrations. Human and monkey cerebral arteries respond similarly to vasoactive agents presented so far, and appear to share the same mechanisms of action; however, the responsiveness of dog cerebral arteries differs.     

The distance between bacterial species in sequence space

Despite the revolution caused by information from macromolecular sequences, the basis of bacterial classification remains the genus and the species. How do these terms relate to the variety of bacteria that exist on earth? In this paper, the inter- and intraspecies differences in amino acid sequence of several bacterial electron transport proteins, cytochromesc, and blue copper proteins are compared. For the soil and water organisms studied, bacterial species can be classed as “tight” when there is little intraspecies variation, or “loose” when this variation is large. For this set of proteins and organisms, interspecies variation is much larger than that within a species. Examples of “tight” species arePseudomonas aeruginosa andRhodobacter sphaeroides, whilePseudomonas stutzeri andRhodopseudomonas palustris are loose species. The results are discussed in the context of the origin and age of bacterial species, and the distribution of genomes in “sequence space.” The situation is probably different for commensal or pathogenic bacteria, whose population structure and evolution are linked to the properties of another organism.     

Gender Difference in Accumulation of Calcium and Phosphorus in the Left Coronary Arteries of Thais

To examine whether there were gender differences in compositional changes of the coronary artery with aging, the authors investigated the gender difference in age-related changes of elements in the left coronary arteries of Thais by direct chemical analysis. After ordinary dissections by students at Chiang Mai University were finished, the left coronary arteries were resected from Thai subjects. The Thai subjects consisted of 69 men and 34 women. The ages of the male subjects ranged from 25 to 87?years (average age?=?62.6?±?11.4?years) and of the female subjects from 24 to 86?years (average age?=?59.4?±?14.6?years). After incinerating the arteries with nitric acid and perchloric acid, the element content was determined by inductively coupled plasma-atomic emission spectrometry. The Ca and P contents tended to increase in the left coronary arteries of men with age, but the increases were not statistically significant. In the left coronary arteries of women, the Ca and P contents increased significantly and progressively with aging. In addition, the Na content increased significantly in the left coronary arteries of both men and women with aging. The differences in the average contents of Ca and P by age group were observed between the left coronary arteries of men and women. With Student's t test, significant gender differences in the average contents of Ca and P were found in both the 40s and the 70s. The Ca and P contents of the left coronary arteries in the 40s were significantly higher in men than in women. In contrast, the Ca and P contents in the 70s were significantly higher in women than in men. These results indicated that the accumulation of Ca and P in the left coronary arteries of Thais occurred at least 10?years earlier in men than in women, but a higher accumulation of Ca and P in old age occurred in the left coronary arteries of women compared with those of men. The present study revealed that there were significant gender differences in the left coronary arteries with regard to the accumulation of Ca and P with aging. It is reasonable to presume that taking clinical findings into consideration, the gender differences in the left coronary arteries may result from hormonal and/or genetic factors rather than lifestyle factors.     

Lifelong training preserves some redox-regulated adaptive responses after an acute exercise stimulus in aged human skeletal muscle

Several redox-regulated responses to an acute exercise bout fail in aged animal skeletal muscle, including the ability to upregulate the expression of antioxidant defense enzymes and heat shock proteins (HSPs). These findings are generally derived from studies on sedentary rodent models and thus may be related to reduced physical activity and/or intraspecies differences as opposed to aging per se. This study, therefore, aimed to determine the influence of age and training status on the expression of HSPs, antioxidant enzymes, and NO synthase isoenzymes in quiescent and exercised human skeletal muscle. Muscle biopsy samples were obtained from the vastus lateralis before and 3 days after an acute high-intensity-interval exercise bout in young trained, young untrained, old trained, and old untrained subjects. Levels of HSP72, PRX5, and eNOS were significantly higher in quiescent muscle of older compared with younger subjects, irrespective of training status. 3-NT levels were elevated in muscles of the old untrained but not the old trained state, suggesting that lifelong training may reduce age-related macromolecule damage. SOD1, CAT, and HSP27 levels were not significantly different between groups. HSP27 content was upregulated in all groups studied postexercise. HSP72 content was upregulated to a greater extent in muscle of trained compared with untrained subjects postexercise, irrespective of age. In contrast to every other group, old untrained subjects failed to upregulate CAT postexercise. Aging was associated with a failure to upregulate SOD2 and a downregulation of PRX5 in muscle postexercise, irrespective of training status. In conclusion, lifelong training is unable to fully prevent the progression toward a more stressed muscular state as evidenced by increased HSP72, PRX5, and eNOS protein levels in quiescent muscle. Moreover, lifelong training preserves some (e.g., CAT) but not all (e.g., SOD2, HSP72, PRX5) of the adaptive redox-regulated responses after an acute exercise bout. Collectively, these data support many but not all of the findings from previous animal studies and suggest parallel aging effects in humans and mice at rest and after exercise that are not modulated by training status in human skeletal muscle.     

Characterization of agonist-induced vasoconstriction in mouse pulmonary artery

In recent years, transgenic mouse models have been developed to examine the underlying cellular and molecular mechanisms of lung disease and pulmonary vascular disease, such as asthma, pulmonary thromboembolic disease, and pulmonary hypertension. However, there has not been systematic characterization of the basic physiological pulmonary vascular reactivity in normal and transgenic mice. This represents an intellectual "gap", since it is important to characterize basic murine pulmonary vascular reactivity in response to various contractile and relaxant factors to which the pulmonary vasculature is exposed under physiological conditions. The present study evaluates excitation- and pharmacomechanical-contraction coupling in pulmonary arteries (PA) isolated from wild-type BALB/c mice. We demonstrate that both pharmaco- and electromechanical coupling mechanisms exist in mice PA. These arteries are also reactive to stimulation by alpha(1)-adrenergic agonists, serotonin, endothelin-1, vasopressin, and U-46619 (a thromboxane A(2) analog). We conclude that the basic vascular responsiveness of mouse PA is similar to those observed in PA of other species, including rat, pig, and human, albeit on a different scale and to varying amplitudes.     

A Comparison of Methodologies for the Study of Functional Transmitter Neurochemistry in Human Brain

A number of different approaches to the study of functional neurochemistry in human brain are discussed. The advantages and disadvantages of three main techniques are contrasted: (i) using animal tissue preparations as models of the human brain; (ii) using human peripheral tissue preparations as models of dynamic CNS processes; and (iii) studying human tissue, obtained postmortem, directly. Animal models are often readily obtained and reliable, and the high degree of inbreeding of common laboratory animals ensures that they usually yield consistent results. However, there are a number of human disorders for which animal models are either poor or unavailable, and species differences make extrapolation from the animal to the human case difficult. Human peripheral tissue models rely on a degree of homology between peripheral and CNS processes; in most cases, the evidence for such homologies derives from animal, rather than human, studies. Moreover, several examples are known where a peripheral process mimics the equivalent glial cell activity more closely than the neuronal, which can be a serious drawback for studies of neurotransmission. The use of postmortem human brain tissue presents a number of obvious difficulties, resulting from variations in the patient's age, agonal state, sex, preterminal medication, postmortem delay, etc. Human beings are genetically and nutritionally heterogeneous, so that data variability is usually greater here than when using tissue from laboratory animals. However, it is possible to control for a number of these factors, for example, by matching samples for basal metabolic rate and tissue integrity, and recently developed tissue freezing and storage techniques permit the use of within-subject experimental designs to help reduce experimental variation. A range of neurotransmitter functions are well retained in such tissue samples, so that regional variations, differential transmitter activities, drug effects, etc., can be studied in normal tissue samples, as well as in samples taken from cases of neurological and psychiatric disease. This allows, for example, changes in neuroanatomical indices to be correlated with localised alterations in a specific neurotransmitter function. A systematic approach to the analysis and matching of tissue samples is advocated. The three approaches should be considered to be complementary, especially for the study of human brain diseases.     

Vasoconstrictor responsiveness of hind body vascular beds is diminished in tail-suspended rats

It is well known that the mechanisms of occurrence of orthostatic intolerance induced by exposure to microgravity deal with multiple factors including alterations of arteries. In the previous works, the diminished contractile responsiveness of abdominal aorta and hind body medium-sized conduit arteries, mesenteric artery and femoral artery, were observed in tail-suspended rats, and the data showed that the femoral artery have subjected to the greatest changes. These results suggested that the vasoreactivity of resistance vessels might be affected by the real or simulated microgravity. Since the arterioles are the main site of peripheral resistance and of its regulation. Therefore, changes in responsiveness of arteriolar network, especially in the lower/hind body region, would be of primary importance in the genesis of postflight orthostatic intolerance. The aim of the present work was to examine whether simulated weightlessness may lead to an impairment in vasoconstrictor responsiveness in hind body vascular beds.     

Isolectins in Narcissus: complexity, inter- and intraspecies differences and developmental control

Using high resolution ion-exchange chromatography and isoelectric focusing the heterogeneity of the daffodil ( Narcissus sp.) lectin in terms of isolectin composition was analyzed. A survey of about 30 cultivars and species of Narcissus demonstrates (i) that they all contain over 50 different lectin polypeptides and (ii) that there are pronounced inter- and intraspecies differences in the isolectin patterns. Analyses of lectin preparations isolated from different tissues at different developmental stages further indicate that the isolectin composition is tissue specific and developmentally regulated. Finally, affinity chromatography experiments suggest differences in affinity for a mannose-Sepharose 4B column of different isolectins.     

Cell culture quality control by rapid isoenzymatic characterization

Summary Procedures that involve cell cultures require careful quality control to avoid inter- and intraspecies contamination. We have developed and electrophoresis technique that can be used routinely in cell culture laboratories to monitor cell line integrity. The method involves the isoenzymatic separation of nine polymorphic enzymes, three of which can be used for cell line species determinations and seven of which can be used for human cell line characterizations. Examples of how the system has been applied to both inter- and intraspecies identifications are described. The routine application of this protocol would be a valuable asset for laboratories concerned with establishing effective cell culture quality control. This work was supported by Contract N01-CP-9-1003 from the National Cancer Institute, Bethesda, MD.     

Mechanical determinants of bone form: insights from skeletal remains

Analysis of skeletal remains from humans living in the past forms an important complement to observational and experimental studies of living humans and animal models. Including earlier humans in such analyses increases the range of variation in both behavior and body size and shape that are represented, and can provide insights into the adaptive potential of the modern human skeleton. I review here a variety of studies of archaeological and paleontological remains that have investigated differences in skeletal structure from a mechanical perspective, focusing in particular on diaphyseal strength of the limb bones. Several conclusions can be drawn from these studies: 1) there has been a decline in overall skeletal strength relative to body size over the course of human evolution that has become progressively steeper in recent millennia, probably due to increased sedentism and technological advancement; 2) differences in pelvic structure and hip mechanical loadings affect femoral shape; 3) activity patterns affect overall strength and shape of both the lower and upper limb bones; and 4) responsiveness to changes in mechanical loading varies between skeletal features (e.g., articulations versus diaphyses) and by age.     

A possible balance of calcium accumulations among bone, cartilage, artery, and vein in single human individuals

To elucidate the relationships between the decrease of mineral contents in human bones and the accumulation of minerals in the other human tissues, the relative contents (RCs) of calcium were analyzed by inductively coupled plasma atomic emission spectrometry among human bones, arteries, veins, and cartilages in 27 subjects (17 men and 10 women). These were resected from subjects who died in the age range from 40 to 98 yr old. Calcanei were chosen for analysis of mineral contents in contrast with femoral, popliteal and common carotid arteries, internal jugular veins, and pubic symphysis. It was found that the RCs of calcium in calcanei were agreeable to association with those in both the pubic symphysis and the femoral artery, but they were not agreeable to association with those in the popliteal and common carotid arteries, and the internal jugular veins. This suggests that calcium released from bones is accompanied by accumulations of calcium in the artery and cartilage.     

Cell culture quality control by rapid isoenzymatic characterization

Procedures that involve cell cultures require careful quality control to avoid inter- and intraspecies contamination. We have developed an electrophoresis technique that can be used routinely in cell culture laboratories to monitor cell line integrity. The method involves the isoenzymatic separation of nine polymorphic enzymes, three of which can be used for cell line species determinations and seven of which can be used for human cell line characterizations. Examples of how the system has been applied to both inter- and intraspecies identifications are described. The routine application of this protocol would be a valuable asset for laboratories concerned with establishing effective cell culture quality control.     

Effects of Vendor and Genetic Background on the Composition of the Fecal Microbiota of Inbred Mice

The commensal gut microbiota has been implicated as a determinant in several human diseases and conditions. There is mounting evidence that the gut microbiota of laboratory mice (Mus musculus) similarly modulates the phenotype of mouse models used to study human disease and development. While differing model phenotypes have been reported using mice purchased from different vendors, the composition and uniformity of the fecal microbiota in mice of various genetic backgrounds from different vendors is unclear. Using culture-independent methods and robust statistical analysis, we demonstrate significant differences in the richness and diversity of fecal microbial populations in mice purchased from two large commercial vendors. Moreover, the abundance of many operational taxonomic units, often identified to the species level, as well as several higher taxa, differed in vendor- and strain-dependent manners. Such differences were evident in the fecal microbiota of weanling mice and persisted throughout the study, to twenty-four weeks of age. These data provide the first in-depth analysis of the developmental trajectory of the fecal microbiota in mice from different vendors, and a starting point from which researchers may be able to refine animal models affected by differences in the gut microbiota and thus possibly reduce the number of animals required to perform studies with sufficient statistical power.