scholarly journals Immunoelectron Microscopic Characterization of Vasopressin-Producing Neurons in the Hypothalamo-Pituitary Axis of Non-Human Primates by Use of Formaldehyde-Fixed Tissues Stored at –25 °C for Several Years

2021 ◽  
Vol 22 (17) ◽  
pp. 9180
Author(s):  
Akito Otubo ◽  
Sho Maejima ◽  
Takumi Oti ◽  
Keita Satoh ◽  
Yasumasa Ueda ◽  
...  
Keyword(s):  
Translational Research ◽  
Median Eminence ◽  
Glutamate Transporter ◽  
Microscopic Analysis ◽  
Nerve Endings ◽  
Size Difference ◽  
Posterior Pituitary ◽  
Electron Microscopic ◽  
Post Mortem Brain

Translational research often requires the testing of experimental therapies in primates, but research in non-human primates is now stringently controlled by law around the world. Tissues fixed in formaldehyde without glutaraldehyde have been thought to be inappropriate for use in electron microscopic analysis, particularly those of the brain. Here we report the immunoelectron microscopic characterization of arginine vasopressin (AVP)-producing neurons in macaque hypothalamo-pituitary axis tissues fixed by perfusion with 4% formaldehyde and stored at –25 °C for several years (4–6 years). The size difference of dense-cored vesicles between magnocellular and parvocellular AVP neurons was detectable in their cell bodies and perivascular nerve endings located, respectively, in the posterior pituitary and median eminence. Furthermore, glutamate and the vesicular glutamate transporter 2 could be colocalized with AVP in perivascular nerve endings of both the posterior pituitary and the external layer of the median eminence, suggesting that both magnocellular and parvocellular AVP neurons are glutamatergic in primates. Both ultrastructure and immunoreactivity can therefore be sufficiently preserved in macaque brain tissues stored long-term, initially for light microscopy. Taken together, these results suggest that this methodology could be applied to the human post-mortem brain and be very useful in translational research.

scholarly journals Immunoelectron microscopic characterization of vasopressin neurons in macaques by use of formaldehyde-fixed tissues stored for several years

2021 ◽  
Author(s):  
Akito Otubo ◽  
Sho Maejima ◽  
Takumi Oti ◽  
Kaita Satoh ◽  
Yasumasa Ueda ◽  
...  
Keyword(s):  
Translational Research ◽  
Median Eminence ◽  
Glutamate Transporter ◽  
Microscopic Analysis ◽  
Nerve Endings ◽  
Size Difference ◽  
Posterior Pituitary ◽  
Electron Microscopic ◽  
Vasopressin Neurons

Translational research often requires the testing of experimental therapies in primates, but research in non-human primates is now stringently controlled by law around the world. Tissues fixed in formaldehyde without glutaraldehyde have been thought to be inappropriate for use in electron microscopic analysis, particularly those of the brain. Here we report the immunoelectron microscopic characterization of arginine vasopressin (AVP)-producing neurons in macaque hypothalamo-pituitary axis tissues fixed with 4% formaldehyde and stored at −25°C for several years. The size difference of dense-cored vesicles between magnocellular and parvocellular AVP neurons was detectable in their cell bodies and perivascular nerve endings located, respectively, in the posterior pituitary and median eminence. Furthermore, glutamate and the vesicular glutamate transporter 2 were colocalized with AVP in perivascular nerve endings of both the posterior pituitary and the external layer of the median eminence, suggesting that both magnocellular and parvocellular AVP neurons are glutamatergic in primates. Both ultrastructure and immunoreactivity can therefore be sufficiently preserved in macaque brain tissues stored long-term for light microscopy. Taken together, these results suggest that this methodology could be applied to the human post-mortem brain and be very useful in translational research.

Characterization of the ryanodine receptor/channel of invertebrate muscle

1998 ◽  
Vol 274 (2) ◽  
pp. R494-R502 ◽  
Author(s):  
Kerry E. Quinn ◽  
Loriana Castellani ◽  
Karol Ondrias ◽  
Barbara E. Ehrlich
Keyword(s):  
Ryanodine Receptor ◽  
Single Channel ◽  
Microscopic Analysis ◽  
Ruthenium Red ◽  
Electron Microscopic ◽  
Release Channel ◽  
Single Channel Currents ◽  
Channel Currents ◽  
Bell Shaped Curve

Electron-microscopic analysis was used to show that invertebrate muscle has feetlike structures on the sarcoplasmic reticulum (SR) displaying the typical four-subunit appearance of the calcium (Ca2+) release channel/ryanodine receptor (RyR) observed in vertebrate skeletal muscle (K. E. Loesser, L. Castellani, and C. Franzini-Armstrong. J. Muscle Res. Cell Motil. 13: 161–173, 1992). SR vesicles from invertebrate muscle exhibited specific ryanodine binding and single channel currents that were activated by Ca2+, caffeine, and ATP and inhibited by ruthenium red. The single channel conductance of this invertebrate RyR was lower than that of the vertebrate RyR (49 and 102 pS, respectively). Activation of lobster and scallop SR Ca2+ release channel, in response to cytoplasmic Ca2+ (1 nM–10 mM), reflected a bell-shaped curve, as is found with the mammalian RyR. In contrast to a previous report (J.-H. Seok, L. Xu, N. R. Kramarcy, R. Sealock, and G. Meissner. J. Biol. Chem. 267: 15893–15901, 1992), our results show that regulation of the invertebrate and vertebrate RyRs is quite similar and suggest remarkably similar paths in these diverse organisms.

scholarly journals ISOLATION OF NERVE ENDINGS FROM THE POSTERIOR PITUITARY GLAND

1965 ◽  
Vol 25 (3) ◽  
pp. 179-193 ◽  
Author(s):  
Frank S. Labella ◽  
Madhu Sanwal
Keyword(s):  
Electron Microscopic Examination ◽  
Basement Membranes ◽  
Nerve Endings ◽  
Posterior Pituitary ◽  
Neurosecretory Granules ◽  
Electron Microscopic ◽  
Free Nerve Endings ◽  
Pituitary Glands ◽  
Posterior Pituitary Gland ◽  
Staining Techniques

Bovine posterior pituitary glands were homogenized in 10 per cent sucrose and fractionated by differential centrifugation. The following centrifugation procedure resulted in the most satisfactory separation: 1000 g for 15 minutes—nuclei, connective tissue, basement membranes with associated endothelium, giant nerve endings, and whole pituicytes; 4200 g for 15 minutes—free nerve endings, including Herring bodies; 17,000 g for 15 minutes—mitochondria; 68,000 g for 15 minutes—neurosecretory granules. Electron microscopic examination was carried out on whole tissue and on the isolated fractions. Isolated nerve endings were examined also by negative staining techniques. Isolated nerve endings retain an apparently normal complement of mitochondria, neurosecretory granules, and microvesicles ("synaptic" vesicles). The free nerve endings closely resemble those observed in sections of intact posterior pituitary tissue. Free microvesicles were not observed in any of the fractions isolated and apparently sediment at centrifugal forces higher than those employed in this study.

scholarly journals Purification and molecular characterization of the NAD+-dependent acetaldehyde/alcohol dehydrogenase from Entamoeba histolytica

1994 ◽  
Vol 303 (3) ◽  
pp. 743-748 ◽  
Author(s):  
I Bruchhaus ◽  
E Tannich
Keyword(s):  
Alcohol Dehydrogenase ◽  
Entamoeba Histolytica ◽  
Dna Sequences ◽  
Microscopic Analysis ◽  
Kinetic Studies ◽  
Protozoan Parasite ◽  
Striking Similarity ◽  
Electron Microscopic ◽  
Catalytic Functions

A bifunctional 95 kDa polypeptide (EhADH2) harbouring acetaldehyde dehydrogenase and alcohol dehydrogenase activities was purified to homogeneity from trophozoite extracts of the protozoan parasite Entamoeba histolytica. Kinetic studies revealed that the enzyme utilizes NAD+ rather than NADP+ as cofactor. Km values for acetyl-CoA, acetaldehyde and ethanol were found to be 0.015, 0.15 and 80 mM respectively in the presence of 0.2 mM NAD+. The primary structure of EhADH2 as deduced from respective amoebic DNA sequences showed striking similarity to the trifunctional AdhE protein of Escherichia coli and the bifunctional AAD protein of Clostridium acetobutylicum. Alignment with a number of aldehyde dehydrogenases and alcohol dehydrogenases from various species suggested that the two catalytic functions of EhADH2 are located on separate parts of the molecule. By cross-linking experiments and electron-microscopic analysis, native EhADH2 was found to be organized in a homopolymeric fashion consisting of more than 20 associated promoters which form rods about 50-120 nm in length.

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