The anatomical record. Part A, Discoveries in molecular, cellular, and evolutionary biology最新文献

The aim of the present study was to evaluate the possible protective effects of Nigella sativa L. (NS) against beta-cell damage from streptozotocin (STZ)-induced diabetes in rats. STZ was injected intraperitoneally at a single dose of 50 mg/kg to induce diabetes. NS (0.2 ml/kg/day, i.p.) was injected for 3 days prior to STZ administration, and these injections were continued throughout the 4-week study. Oxidative stress is believed to play a role in the pathogenesis of diabetes mellitus (DM). To assess changes in the cellular antioxidant defense system, we measured the activities of antioxidant enzymes (such as glutathione peroxidase (GSHPx), superoxide dismutase (SOD), and catalase (CAT)) in pancreatic homogenates. We also measured serum nitric oxide (NO) and erythrocyte and pancreatic tissue malondialdehyde (MDA) levels, a marker of lipid peroxidation, to determine whether there is an imbalance between oxidant and antioxidant status. Pancreatic beta-cells were examined by immunohistochemical methods. STZ induced a significant increase in lipid peroxidation and serum NO concentrations, and decreased antioxidant enzyme activity. NS treatment has been shown to provide a protective effect by decreasing lipid peroxidation and serum NO, and increasing antioxidant enzyme activity. Islet cell degeneration and weak insulin immunohistochemical staining was observed in rats with STZ-induced diabetes. Increased intensity of staining for insulin, and preservation of beta-cell numbers were apparent in the NS-treated diabetic rats. These findings suggest that NS treatment exerts a therapeutic protective effect in diabetes by decreasing oxidative stress and preserving pancreatic beta-cell integrity. Consequently, NS may be clinically useful for protecting beta-cells against oxidative stress.

We examined atomic force microscopy (AFM) and lateral force microscopy (LFM) images of human, avian, reptilian, amphibian, and piscine erythrocytes to determine whether the general pattern of erythrocyte membrane architecture has been largely conserved in the course of phylogenetic evolution or relatively minor modifications have taken place. The general pattern of the cell surface structure is indeed very similar among the phyla examined. The surface features include a number of blebs or globular structures and hole-like depressions. Such features are particularly clear in fish (Heteropneustes sp.), in which globular blebs are arranged in tiers around the depressions. The same pattern is found in the other phyla, although the sizes of the blebs and depressions vary. The depressions are approximately 340 and approximately 100 nm in diameter in chickens and fish, respectively, and are smaller in other phyla. The images of human erythrocytes presented here show holes more clearly than the images obtained by Zhang et al. (Scanning Electron Microsc., 1995; 9:981-989), who showed for the first time the highly uneven surface of these cells. The globules range in size from approximately 50-150 nm in diameter. These nanostructures have a width of approximately 333-1,000 atoms, assuming that the average dimension of an atom is 1.5 A. The size range of the holes is approximately 40-432 nm (equivalent to a width of approximately 266-2880 atoms). LFM images, which take into account the lateral component of the force, represent the variation of surface friction (roughness) on the erythrocyte surface. This is very clear in the toad images, which show well-ordered strata that have not been revealed in ordinary AFM images.

Bone morphogentic proteins (BMPs) play an important role in cardiac development. Using an in vitro explant analysis, we show that BMPs are crucial for myocardium formation. As a first approach to identify which BMP may be involved in myocardium formation in intra- and extracardiac mesenchyme in vivo, a survey of the expression patterns of BMP2, -4, -5, -6, and -7 mRNA is prepared by in situ hybridization in chicken embryonic hearts from HH5 to 44. During recruitment of mesodermal cells to the outflow tract myocardium (HH10-23), BMP2, -4, -5, and -7 mRNA are expressed in the distal myocardial border and the flanking mesenchyme. After completion, BMP2 and -4 mRNA become restricted to the mesenchyme and BMP5 and -7 mRNA to the myocardium. At the venous pole, BMP2, -5, and -7 mRNA are expressed in the distal myocardial border of the caval vein, while BMP2, -5, -6, and -7 mRNA are expressed in the distal myocardium around the pulmonary vein. BMP4 mRNA is expressed in the adjacent mesenchyme at both sides. During muscularization of the atrioventricular cushions and the tricuspid valve, the cardiomyocytes that protrude into the mesenchyme express BMP2, -4, -5, and -7 mRNA, whereas BMP6 mRNA is expressed in the cushion mesenchyme. The myocardial protrusions formed in the mesenchymal proximal outlet septum express BMP4, -5, and -7 mRNA, while BMP2 and -6 mRNA are expressed in the mesenchyme. The spatiotemporal expression patterns of these BMPs in relation to myocardium formation at the distal ends and within the heart suggest a role for BMPs in myocardium formation. During delamination of the valves, BMP4 and -6 mRNA are expressed at the ventricular side of the forming mitral valve, BMP4 mRNA at the ventricular side of the forming tricuspid valve, and BMP2, -4, and -6 mRNA at the vascular side of the forming semilunar valves.

Callitrichid primates (tamarins, marmosets) have extreme variation in the vomeronasal organ (VNO), including ontogenetic differences in the neuroepithelium and vomeronasal duct (VND) patency at birth. Such differences render the timing and extent of VNO maturation debatable in callitrichids, but no studies have used neuron-specific immunohistochemical markers to address this question. The present study compared the number of VNO epithelial cells that express immunoreactivity to neuron-specific beta-tubulin III (BT), VNO length, and VNO cross-sectional area between two species of tamarins (Leontopithecus rosalia and Saguinus geoffroyi) that differed in perinatal VND patency. Neonatal lemurs and adult marmosets and bushbabies were also examined for a comparison to species previously shown to have a relatively large amount of VNO neuroepithelium and patent VNDs. The head of each specimen was serially sectioned in the coronal plane. Based on known rostrocaudal start/stop points of the VNO, selected unstained sections were used for BT protocols and area measurement at three percentiles (25th, 50th, 75th) in each specimen. Each section was photographed and enlarged for cell counts and measurement of cross-sectional epithelial area. In each specimen, the number of BT(+) cells in the VNO was counted at each percentile and expressed as a number per mm(2). Results indicated that lemur VNOs had a dense population of BT(+) cells at birth, but the VNO was more varied in the tamarin species. S. geoffroyi had few or no BT(+) cells in VNOs of neonates, which had fused VNDs, but had an increased BT(+) population by 1 and 2 months postnatal age, when the VND was patent. Of the species with patent VNDs at birth, neonatal L. rosalia had a denser population of BT(+) cells compared to S. geoffroyi, though not to the degree seen in neonatal lemurs or adult marmosets and bushbabies. These findings show that BT immunohistochemistry is a useful comparative method for the study of VNOs in subadult primates. Since the quantity of nonsensory VNO epithelium varies substantially between species, epithelial area measurements may be misleading, and BT(+) cell counts appeared to be the best quantitative method for comparing receptor neuron numbers among primates. It is suggested that the greater BT(+) cell population in L. rosalia at all subadult stages examined reveals an earlier maturation of the neuroepithelium compared to S. geoffroyi. Further investigation should consider whether this may relate to a comparatively brief subadult ontogeny and early onset of adult behaviors in L. rosalia compared to other tamarins studied to date.

Since Rinehart and Farquhar reported the presence of agranulated cells in the anterior pituitary gland in 1953, the functions of the folliculo-stellate cell remain to be clarified. Intercellular junctions have been described in the monkey, rat, and teleost anterior pituitary glands, indicating the existence of cell-to-cell communication within the organ. We pointed to their possible role in the rapid dissemination of information through a complex interconnecting system of follicles involving gap junctions. The gap junctional/folliculo-stellate cellular network was essential in the maturation and regulation of the pituitary gland system such as the hypothalamic-pituitary-gonadal axis. It has been was shown that a network participated in the conduction of electrophysiological information over a long distance using the ion Ca(++), which propagates to other folliculo-stellate cells by signaling through gap junctions. Sixty-day-old male rats were used in this study for light microscopic immunohistochemistry of S-100 protein, type I collagen, and connexin 43, and for electron microscopy to observe the morphological relationships between the cellular networks of folliculo-stellate cells and granulated pituitary cells. Clusters of anti-S-100 protein-positive cells were clearly observed in a region of the hypophysis tentatively named the transition zone. Anti-S-100 protein-positive cells and their cytoplasmic processes were also present in the anterior lobe and assembled together to form follicular lumina. Type I collagen was clearly shown outlining the incomplete lobular or ductule-like structure making cell cords in the anterior pituitary gland. Numerous microvilli were present within the follicular lumen while around the lumina, junctional specializations including gap junctions were positive for the connexin 43 protein. A nonuniform distribution of the connexin 43-positive sites were observed. Small or dot-shaped positive sites were noted where two clusters of cells were connected; the cells were identified as S-100 cells. Double immunohistochemical staining of the connexin 43 and growth hormone (GH) or connexin 43 and luteinizing hormone (LH) was also performed, demonstrating no direct relationship between the connexin 43 and either the GH or LH cells. These findings indicate that there are two kinds of messages necessary for the hormone release in the pituitary gland. One is via the portal vein system, the other is through the gap junction-mediated networks of folliculo-stellate cells. The granulated cells directly associate with cell membrane of folliculo-stellate cells are able to discharge secretory granules through communication via gap junctions, while those granulated cells that are more distant from the folliculo-stellate cells are only able to discharge hormones via the pituitary hormone-releasing hormone from the portal vein system.

Meiosis is the special double cellular division characterized by the reduction of chromosome number of the final products and recombination of genetic information present in maternal and paternal homologous chromosomes. Early stages of meiotic prophase, leptotene and zygotene (L/Z), are functionally important since homologous chromosomes recognize, align, and pair during them. They are poorly represented in the seminiferous tubules of mammalian species, and this fact turns studies focused on these stages difficult to perform. As a consequence, the molecular bases of these important events are so far poorly known and understood in higher eukaryotes. The purpose of this work was to provide an advantageous experimental mammalian model (with a reasonable number of cells) for biochemical and molecular analysis of early meiotic prophase stages. Here, we present the results of our quantitative study on testes material of both immature and adult guinea pig specimens (Cavia porcellus). We show that their seminiferous tubules contain a comparatively high percentage of L/Z spermatocytes, as well as a very conspicuous chromosome bouquet at the L/Z transition, which points out this species as a well-suited one to address studies on such stages in mammals.

Mitochondria of human Leydig cells were reconstructed in three dimension utilizing the technique of electron microscopic tomography to obtain a better understanding of the topology of the internal membrane system and the relationship of these cristae to the inner boundary membrane (IBM). Cristae structure, in many respects, is consistent with previous tomographic studies from typical mitochondria, i.e., mitochondria from nonsteroid-producing cells. Cristae are diverse in form, with well-defined lamellar cristae interconnected to pleomorphic and tubular regions. Occasional fenestrations are present in the lamellar regions. Also consistent with other mitochondria studied by tomography, the openings of the cristae to the IBM (referred to as crista junctions) are roughly circular or elliptical and approximately 20-25 nm in diameter. Morphological contact sites between the outer mitochondrial membrane and IBM are also present. Cristae membranes in these steroid-producing mitochondria are often found in close proximity to the IBM. Unique to steroid-producing mitochondria is a form of the cristae in which multiple lamellae are in very close apposition, previously defined as the lamellar association. Tomographic reconstructions of the lamellar association reveal that these well-organized membranes also open to the IBM via crista junctions. These regions of closely apposed lamellar cristae are also interconnected and display small tubular extensions from the lamellae. The current study is the first electron microscopic tomography study of mitochondria from steroid-producing cells. The results show the cristae interconnect to form an extensive internal membrane system, which is perhaps better termed the cristae compartment. This internal membrane system is notable due to the high surface area with few small openings to the IBM. Such a morphology is more analogous to the thylakoid membrane system of chloroplasts than the long-standing view of mitochondrial cristae. The significance of the lamellar association form of the cristae is unknown.

Although mammals do not regenerate most appendages, they are able to regenerate toetips if the amputation occurs through the nail bed. The reasons for different outcomes following amputation at different levels are not understood. It is possible that cells at regenerating and nonregenerating sites migrate from fundamentally different tissues. If so, different migratory pathways could be detected. To identify putative migrating cells, microscope slides were made from both regenerating and nonregenerating toes of rats and mice on successive days after amputation. Fluorescent-labeled phalloidin, which binds polymerized f-actin, was used to identify actin filaments and fibers. Cells containing prominent actin bundles were distinguishable from those containing diffuse fibrils and those in which visible fibers were absent. Phalloidin labeling was similar in regenerating and nonregenerating digits after amputation. As early as 2 days after amputation at either proximal or distal levels, many cells of the hypodermis adjacent to the wound became labeled with phalloidin. The number and intensity of labeled hypodermal cells containing stress fiber-like bundles increased rapidly with time, and at successive times cells were seen progressively further distally. By approximately 7 days, they occupied the wound site immediately distal to bone of both regenerating and nonregenerating digits. Most dermal cells were unlabeled and endosteal and marrow cells contained only fibrillar actin. Phalloidin labeling does not support the concept of migration from different tissues in regenerating and nonregenerating amputation sites.

Mesenchymal stem cells derived from human bone marrow (hBM-MSCs) can differentiate into chondrogenic cells for the potential treatment of injured articular cartilage. To evaluate agarose gels as a supportive material for chondrogenesis of hBM-MSCs, this study examined chondrogenesis of hBM-MSCs in the agarose cultures. Pellet cultures were employed to confirm the chondrogenic potential of the hBM-MSCs that were used in agarose cultures. The hBM-MSCs were seeded in 2% agarose constructs at the initial cell-seeding densities of 3, 6, and 9 x 10(6) cells/ml while each of pellets was formed using 2.5 x 10(5) cells. Chondrogenesis of hBM-MSCs was induced by culturing cell-agarose constructs and pellets for 21 days in the presence of a defined medium containing transforming growth factor beta3 (TGF-beta3). The analysis of reverse transcription-polymerase chain reaction showed that hBM-MSCs of agarose and pellet cultures expressed the chondrogenic markers of collagen type II and aggrecan in the presence of TGF-beta3. The deposition of cartilage-specific macromolecules was detected in both agarose and pellet cultures by histological and immunohistochemical assessments. Chondrogenesis of hBM-MSCs in agarose gels directly correlated with the initial cell-seeding density, with the cell-agarose constructs of higher initial cell-seeding density exhibiting more cartilage-specific gene expressions. This study establishes a basic model for future studies on chondrogenesis of hBM-MSCs using the agarose cultures.

This work investigated the origin and development of microcirculation in the rat humeral head and the expression of vascular endothelial growth factor (VEGF) as a factor supporting the vascular growth and the development of the secondary ossification centers. Sixty rats aging 1, 3-4, 6-8, 11, and 21 days, 5 weeks, and 4 and 8 months were used. Samples of humeral head were collected for histology and immunohistochemistry for VEGF. Some animals were perfused with Mercox resin in order to obtain vascular corrosion casts (vcc) observed by scanning electron microscopy (SEM). No cartilage canals were present at birth. At 6 days postnatal, blood vessels coming from the perichondrium and the region near the capsule attachment invaded the cartilage; at 11 days postnatal, signs of calcification were present and within the third week some bone trabeculae were formed. Just before the vascular invasion of the epiphysis, a positive reaction for VEGF was localized in chondrocytes of the epiphyseal cartilage close to the capsule insertion. During the development and expansion of the secondary ossification center, VEGF expression was higher in chondrocytes but decreased when epiphysis was diffusely ossified. VEGF was expressed also by mesenchymal cells present in and around the fibrous tissue where the secondary ossification center will develop. SEM vcc confirmed that vessels penetrating into the epiphysis arose merely from the periosteal and the capsular networks, and vascular connections with the diaphyseal circulation were not evident. These observations demonstrated that VEGF production by chondrocytes begun some days after birth, supported the rapid vascular growth from the surrounding soft tissues, and was chronologically related to the development of the secondary ossification center in rat proximal humerus. Finally, the possible role of VEGF as mediator of angiogenesis and, at least indirectly, as a trigger factor also in the ossification and the bone remodeling of the secondary ossification centers has been discussed.