Intracellular events associated with ACTH action.

Fractions enriched in plasma membranes were prepared from the Y1 mouse adrenocortical tumor cell line and were characterized with respect to adenylate cyclase activity. Optimal requirements of the adenylate cyclase system for guanyl nucleotides. Mg2+, ATP, and corticotropin (ACTH) were determined. The sensitivity of the adenylate cyclase system to ACTH in plasma membrane fractions was comparable with that observed in isolated intact cells. Polycations such as poly-L-arginine and histone competitively inhibited the action of ACTH, supporting the view that the affinity of ACTH for the adenylate cyclase system is determined by the basic core of amino acids at residues 15-18. ACTH was at least one order of magnitude more potent than ACTH in stimulating adenylate cyclase activity in plasma membrane fractions.

Mutant isolates from the Y1 mouse adrenocortical tumor cell line (designated Y1DR) were previously shown to resist homologous desensitization of adenylyl cyclase via endogenous ACTH receptors or transfected beta 2-adrenergic receptors. To further localize the site of the DR mutation, we examined the agonist-binding properties of the transfected beta 2-adrenergic receptor in parental Y1 cells and the DR mutant before and after desensitization with the beta-adrenergic agonist isoproterenol. Binding studies were carried out using [125I]iodocyanopindolol as the labeled ligand and isoproterenol as the competing agonist. We found that the DR mutant has a greater number of high affinity receptors in the basal state than does the Y1 parent (70-80% vs. < or = 50%) and that treatment of membranes from parent or mutant cells with guanyl-5'-yl imidodiphosphate and NaCl converts all of the high affinity sites to low affinity sites. After desensitization with isoproterenol, only low affinity binding sites are detected in parental Y1 cells, whereas the DR mutants retain an appreciable number of high affinity receptors. These results indicate that the DR mutant may resist desensitization by affecting agonist-induced uncoupling of receptors from their guanyl nucleotide-binding regulatory G-proteins.

ACTH induces up-regulation of ACTH receptor mRNA in mouse and human adrenocortical cell lines

High voltage electron microscopy (HVEM) has been employed in various ways (whole mounts of cells stereo pair imaging, axial tomography, and serial sections for reconstruction) to elucidate three dimensional (3-D) ultrastructural data. The increased specimen thickness allows further data analysis unobtainable from ultra-thin sections. HVEM can reduce the number of sections needed in 3-D reconstructiortby approximately ten times over conventional transmission electron microscopy (CTEM). But increasing section thickness also increases wear on the diamond knife used to section. We have compared the serial sections obtained from a histo-grade diamond knife with those from an E.M. grade ultra-knife. Both sets of sections were cut 0.5 μm thick from the same block, and evaluated under the one million volt beam of the HVEM.

Ad4BP, also known as SF-1, is a steroidogenic tissue-specific transcription factor that is also essential for adrenal and gonadal development. Two mechanisms for the transcriptional regulation of the mammalian FTZ-F1 gene encoding Ad4BP in adrenocortical cells have been proposed in the previous studies: the crucial role of a cis-element, an E box for the steroidogenic cell-specific expression of mouse and rat FTZ-F1 genes, and a possible autoregulatory mechanism of the rFTZ-F1 gene by Ad4BP itself through binding to the Ad4 (or SF-1) site in the first intron. In the present study, the transcriptional regulation of the human FTZ-F1 gene in adrenocortical cells was investigated from several angles, including the above two mechanisms. Using a series of deletion analyses of the 5'-flanking region of the hFTZ-F1 gene and site-directed mutagenesis for transient transfection studies, an E box element, CACGTG at -87/-82 from the transcriptional start site, was also found to be essential for the transcription of the hFTZ-F1 gene in mouse or human adrenocortical cell lines as well as in non-steroidogenic CV-1 cells. Despite the presence of a corresponding Ad4 site, CCAAGGCC at +163/+156 in the first intron of the hFTZ-F1 gene, an autoregulatory mechanism through the Ad4 site was found to be unlikely in the hFTZ-F1 gene mainly due to site-directed mutagenesis. In addition, the forced expression of Ad4BP had little effect on hFTZ-F1 gene transcription in non-steroidogenic CV-1 cells. Such Ad4BP-independent regulation of the hFTZ-F1 gene was in striking contrast to the regulation of steroidogenic CYP genes, such as the human CYP11A gene, in which the proximal promoter activity is Ad4BP-dependent and the transactivation by Ad4BP is silenced by DAX-1. Even though the Ad4BP-dependent transcriptional regulation of the DAX-1 gene has been reported, DAX-1 did not affect the transcriptional activity of the hFTZ-F1 gene in our study. Taken together, these observations suggest that the E box is indeed required for the expression of the FTZ-F1 gene, at least in mammalian species, but may not determine the tissue-specific expression of the hFTZ-F1 gene, and that, unlike the steroidogenic CYP gene, the regulation of the hFTZ-F1 gene appears to be independent of both Ad4BP and DAX-1.

Applications of high voltage electron microscopy to botanical ultrastructure

Examination of the morphology of osteocytes within the bone supporting endosteal dental implants was performed using conventional transmission and high-voltage transmission electron microscopy (HVEM). The in vivo dog model used 72 implants inserted into the premolar region of 18 experimental animals. Forty-eight implants in 12 dogs were used as anterior abutments for fixed bridges for periods up to 12 months. The mineralized matrix of the supporting bone was either directly apposed to the implant surface or was separated from the implant by a narrow region of unmineralized matrix. Osteocytes were routinely observed to be closely associated with the bone-implant interface, as well as at a distance from the implant. Osteocytes were found to extend cellular processes directly to the implant surface through canaliculi. The osteocyte processes contained microfilaments. The three-dimensional capabilities of HVEM elucidated the nature of these cell processes at the point of exit from the osteocyte, as the processes extended through the mineralized matrix, and as the processes terminated at the implant interface. This report suggests that avenues of communication may exist between the implant and the osseous cells, providing intriguing hypotheses regarding biomechanical forces and osteogenesis at the implant interface. Furthermore, an electron-dense deposit was observed upon the inner confines of the canalicular wall, upon the outer aspects of the osteocyte lacuna, and upon the outer aspect of the bone interfacing the implant.

FGF2 elicits a strong mitogenic response in the mouse Y-1 adrenocortical tumor cell line, that includes a rapid and transient activation of the ERK-MAPK cascade and induction of the c-Fos protein. ACTH, itself a very weak mitogen, blocks the mitogenic response effect of FGF2 in the early and middle G1 phase, keeping both ERK-MAPK activation and c-Fos induction at maximal levels. Probing the mitogenic response of Y-1 cells to FGF2 with ACTH is likely to uncover reactions underlying the effects of this hormone on adrenocortical cell growth.

STEM role in failure analysis

Electron microscopy has been a valuable tool for the study of platelet biology and thrombosis for more than 70 years. Early studies using conventional transmission and scanning electron microscopy (EM) provided a foundation for our initial understanding of platelet structure and how it changes upon platelet activation. EM approaches have since been utilized to study platelets and thrombi in the context of basic, translational and clinical research, and they are instrumental in the diagnosis of multiple platelet function disorders. In this brief review, we provide a sampling of the many contributions EM based studies have made to the field, including both historical highlights and contemporary applications. We will also discuss exciting new imaging modalities based on EM and their utility for the study of platelets, hemostasis and thrombosis into the future.

It is well known that the combination of a field emission gun (FEG) and a conventional transmission electron microscope (CTEM) is extremely important for nanometer area analysis in analytical electron microscopy. However, the smaller illumination angle and reduced energy spread of FEG than those of a conventional electron gun (W hair pin filament or LaB6) give a slowly damping envelop function in phase contrast transfer function (PCTF). Thus the FEG ensures application not only to analytical microscopy but also to high resolution electron microscopy to improve the information limit.In a high voltage electron microscope (above 200 kV), high-speed vacuum pumps have to be provided below the acceleration tube to get an ultra high vacuum (UHV) around the field emission tip located at the top of the acceleration tube. However, this method is not always the best way to provide UHV because of the poor vacuum conductance caused by the electrodes inside the acceleration tube.

In a conventional transmission electron microscope (CTEM, or TEM for short) (Fig. 1.1), a thin specimen is irradiated with an electron beam of uniform current density. The acceleration voltage of routine instruments is 80–120 kV. Medium-voltage instruments work at 200–500 kV to provide a better transmission and resolution, and in high-voltage electron microscopy (HVEM) the acceleration voltage reaches 500 kV – 3 MV. Earlier books on the subject are listed as references [1.1–50]. The development of both theory and instrumentation as well as of the different application of TEM can be followed by consulting the proceedings of the International Conferences on Electron Microscopy [1.51–61].KeywordsTransmission Electron MicroIncrease Energy LossPlasmon LossUniform Current DensityScan Tunnel MicroThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

In many fields of research in science, engineering, and medicine, electron microscopy as a method for directly imaging submicroscopic structures has become increasingly important in recent decades. Electron microscopy (EM) includes several different techniques: conventional transmission electron microscopy (TEM), high-resolution electron microscopy (HREM), highvoltage electron microscopy (HVEM), scanning electron microscopy (SEM), analytical electron microscopy (AEM), emission electron microscopy (EEM), and others. In the past the central aim of using electron microscopy was structure determination, but recently it has been of growing importance for also investigating different processes, i.e., changes in materials by interaction with several influential factors (e.g., heat, electric or mechanic fields, mechanical loading). Of particular interest is the study of the micromechanical processes of deformation and fracture. Therefore, electron microscopy is a very powerful tool for materials science. The present review reports on some capabilities and limitation of the application of electron microscopy to solid polymers. In Section II the techniques of electron microscopy are briefly reviewed, followed by a section that describes the main methods of specimen preparation of solid polymeric materials. In Section IV the results of several applications of electron microscopy are discussed to reveal the morphology as well as the micromechanical deformation processes of several polymeric materials.

Whole-cell imaging of the budding yeast Saccharomyces cerevisiae by high-voltage scanning transmission electron tomography

Sturgeons are bony fish that retain structural traits typical of the more primitive Chondrostei. From an evolutionary viewpoint, sturgeons are considered relic fish. However, they show remarkable ecological plasticity and are well adapted to contemporary environmental conditions. Although development of the cardiovascular system is critical for all organs and systems, and is affected by evolutionary changes, the structure of the sturgeon heart has been mostly overlooked. This is also true for the conus arteriosus, which, as in Chondrostei, is endowed with several rows of valves and a layer of contractile myocardium. This work reports on the structure of the valves, the endocardium, and the subendocardium of the conus arteriosus of the sturgeon (Acipenser naccarii) heart. It is part of a broader study that aims to cover the entire structure of the sturgeon heart. The conus arteriosus of 15 A. naccarii hearts, ranging in age from juveniles to sexually-differentiated adults, has been studied by conventional light, transmission (TEM), and scanning electron microscopy (SEM). In addition, maceration of the soft tissues with NaOH, and actin localization by fluorescent phalloidin has been used. The conus is a tubular chamber that arises from the right ventricular side and presents two constrictions at the conus-ventricle and conus-aorta junctions. The conus is endowed with three rows of valves: one distal and two proximal. The segment of the conus located between the distal and the two proximal rows is devoid of valvular structures. The distal row has four leaflets, while the two proximal rows show the greatest variation in leaflet number, size, and shape. All leaflets have collagenous chordae tendineae arising from the free border and from the parietal side of the leaflets. The endocardium is a flat endothelium which shows a thick, irregular basement membrane. The leaflet body is formed by a loose connective tissue which blends with the subendocardium. The subendocardium is a connective tissue consisting of myofibroblasts, collagen, and elastin. It is divided into two distinct areas: one proximal, which shows little elastin and poorly organized collagen; and one distal, which is rich in elastin, with cells and extracellular fibers organized into layers that are oriented in alternative circumferential and longitudinal directions. The present report is the first systematic analysis of the structure of the sturgeon conus. Descriptions of the conus valves should recognize the existence of three valve rows only. The variability in valve morphology, and the loose structure of the leaflet tissue make it unlikely that the valves play an effective role in preventing blood backflow. In this regard, the ventricle-conus constriction may act as a sphincter. The subendocardium is an elastic coat capable of actively sustaining the tissue deformation that accompanies the heart contractile cycle. Further comparative studies are needed to provide deeper insight into the structural changes that accompany phyletic diversification.