Ultrastructure and enzyme histochemistry of the pancreatic islets in the horse

Summary The characteristic localization of the silver-negative A₂ cells in the central part of the pancreatic islets in the horse offers a good opportunity to study the ultrastructure and histochemistry of this type of islet cell. Electron microscopical analyses revealed that the A₂ cells contained dense spherical granules varying considerably in size. Light and dark A₂ cells were identified. The presence of numerous secretory granules of very low density was the most conspicous feature of the B cells. These cells also showed considerable differences in density. A second type of peripheral islet cell was characterized by a very high content of mitochondria and ribosomes. These small islet cells contained tiny granules and are probably identical with the A₁ cells.Negative reactions for alkaline and acid phosphatases were obtained throughout the islet tissue, while a strong glucose-6-phosphatase activity was displayed by the peripheral cells. The diphosphopyridine and triphosphopyridine nucleotide diaphorase activities were high in the peripheral cells, considerably weaker reactions being noted in the A₂ cells. On the whole there was a low succinic dehydrogenase activity in the islet tissue with a somewhat weaker enzyme staining in the A₂ than in the peripheral cells. The reactions for glucose-6-phosphate dehydrogenase and lactic dehydrogenase were also less pronounced in the A₂ cells than in the intensely reacting peripheral cells.The following abbreviations are used DPN Diphosphopyridine nucleotide - DPND Diphosphopyridine nucleotide diaphorase - DPNH Diphosphopyridine nucleotide, reduced form - G-6-PD Glucose-6-phosphate dehydrogenase - LD Lactic dehydrogenase - MTT 3,5-diphenyl-2-(4,5-dimethylthiazol-2-yl)-tetrazolium bromide - Nitro-BT 2,2-di-p-nitrophenyl-5,5-diphenyl-3,3-(3,3-dimethoxy-4,4-biphenylene)-ditetrazolium chloride - SD Succinic dehydrogenase - TPN Triphosphopyridine nucleotide - TPND Triphosphopyridine nucleotide diaphorase - TPNH Triphosphopyridine nucleotide, reduced form Supported by the Swedish Medical Research Council and the research grant A-5759 from the National Institute of Arthritis and Metabolic Diseases, United States Public Health Service.     

Effects of parathyroid hormone on cyclic AMP-formation and cytoplasmic free Ca2+ in the osteosarcoma cell line UMR 106-01

The effects of parathyroid hormone (PTH) on cytoplasmic free CA²⁺ (Ca _i ²⁺ ) and cAMP-formation were investigated in the rat osteosarcoma cell line UMR 106-01.In fura-2 loaded adherent single cells bPTH 1-34 (10 nM–1M) induced a rapid transient increase in Ca _i ²⁺ in 11% of the studied cells. In fura-2 tracings from UMR 106-01 cells in suspension, bPTH 1-34 (0.1 M) induced a transient increase in Ca _i ²⁺ in 20% of the experiments. The transient increase in Ca _i ²⁺ seen in suspensions of cells was not abolished by addition of EGTA (2.5 mM) prior to challenge with PTH, suggesting that the increase in Ca _i ²⁺ was derived from intracellular stores.A marked rapid increase in cAMP-formation was observed in all experiments with cells in suspension, also in the experiments where PTH did not affect Ca _i ²⁺ .These data show that PTH causes a release of Ca²⁺ from intracellular stores in a small percentage of osteosarcoma UMR 106-01 cells, and that PTH is capable of inducing an increase in cAMP-formation without affecting Ca _i ²⁺ in osteoblasts.     

Lysis protein T of bacteriophage T4

Summary Lysis protein T of phage T4 is required to allow the phage's lysozyme to reach the murein layer of the cell envelope and cause lysis. Using fusions of the cloned gene t with that of the Escherichia coli alkaline phosphatase or a fragment of the gene for the outer membrane protein OmpA, it was possible to identify T as an integral protein of the plasma membrane. The protein was present in the membrane as a homooligomer and was active at very low cellular concentrations. Expression of the cloned gene t was lethal without causing gross leakiness of the membrane. The functional equivalent of T in phage is protein S. An amber mutant of gene S can be complemented by gene t, although neither protein R of (the functional equivalent of T4 lysozyme) nor S possess any sequence similarity with their T4 counterparts. The murein-degrading enzymes (including that of phage P22) have in common a relatively small size (molecular masses of ca. 18 000) and a rather basic nature not exhibited by other E. coli cystosolic proteins. The results suggest that T acts as a pore that is specific for this type of enzyme.     

Export and sorting of theEscherichia coli outer membrane protein OmpA

Results of studies, mostly using the outer membrane, 325 residue protein OmpA, are reviewed which concern its translocation across the plasma membrane and incorporation into the outer membrane ofEscherichia coli. For translocation, neither a unique export signal, acting in a positive fashion within the mature part of the precursor, nor a unique conformation of the precursor is required. Rather, the mature part of a secretory protein has to be export-compatible. Export-incompatibility can be caused by a stretch of 16 (but not 8 or 12) hydrophobic residues, too low a size of the polypeptide (smaller than 75 residue precursors), net positive charge at the N-terminus, or lack of a turn potential at the same site. It is not yet clear whether binding sites for chaperonins (SecB, trigger factor, GroEL) within OmpA are importantin vivo. The mechanism of sorting of outer membrane proteins is not yet understood. The membrane part of OmpA, encompassing residues 1 to about 170, it thought to traverse the membrane eight times in antiparallel -sheet conformation. At least the structure of the last -strand (residues 160–170) is of crucial importance for membrane assembly. It must be amphiphilic or hydrophobic, these properties must extend over at least nine residues, and it must not contain a proline residue at or near its center. Membrane incorporation of OmpA involves a conformational change of the protein and it could be that the last -strand initiates folding and assembly in the outer membrane.     

Orientation Cage and Release Experiments with Migratory Wheatears (Oenanthe oenanthe) in Scandinavia and Greenland: The Importance of Visual Cues

Migratory orientation of Scandinavian and Greenland wheatears was recorded during the autumn migration periods of 1988 and 1989. Orientation cage tests were conducted under clear sunset skies, to investigate the importance of different visible sky sections on orientation performance. In addition, wheatears were released under clear starry skies and under total overcast to examine the orientation of free-flying birds. The following results were obtained:

• 1 Wheatears tested with a restricted visible sky section (90° centered around zenith) in orientation cages, showed a mean orientation towards geographic W/geomagnetic NW (Greenland) and towards geographic and magnetic WNW-NW (Sweden). These mean directions are clearly inconsistent with the expected autumn migration directions, SW-SSW in Scandinavia and SE in Greenland, as revealed by ringing recoveries for the two populations.
• 2 When the birds were allowed a much more extensive view of the sky, almost down to the horizon (above 10° elevation), Scandinavian wheatears chose headings in agreement with ringing data. Greenland birds were not significantly oriented.
• 3 Release experiments under clear starry skies resulted in mean vanishing directions in good agreement with ringing data from both sites. Greenland wheatears released under total overcast showed a similar orientation as under clear skies, indicating that a view of the stars may not be of crucial importance for selecting a seasonally accurate migratory direction.

The results suggest that an unobstructed view of the sky, including visual cues low over the horizon, is important, possibly in combination with geomagnetic cues, for the orientation of migratory naive wheatears. Furthermore, the birds showed remarkably similar orientation responses in Greenland and Scandinavia, respectively, indicating that they use basically the same orientation system, despite considerable differences in visual and geomagnetic orientation premises at the two different geographic and magnetic latitudes.     

RNA-DNA Covalent Bonds Between the RNA Primers and the DNA Products Formed by RNA Tumor Virus DNA Polymerase

Initiation of DNA synthesis by endogenous RNA primer molecules was studied with three different RNA tumor viruses. The influence of the method of virus disruption on the observed RNA-DNA bonds was ascertained. Ether disrupted virions of both murine leukemia virus (MuLV) and the B77 strain of avian sarcoma virus (B77 virus) have rC-dC and rA-dA covalent linkages between RNA primers and newly synthesized DNA. None of the 14 other possible bonds were formed. Ether-disrupted virions of avian myeloblastosis virus (AMV) have rU-dC and rA-dA linkages. In contrast, work reported herein and from other laboratories shows that Nonidet P-40 (NP-40)-disrupted virions of all three viruses have only the rA-dA junction. Studies with virus particles which were first disrupted with ether and then treated with NP-40 indicated that the detergent treatment disallowed the formation of the ribopyrimidine-dC internucleotide bond. The same transfers are found with AMV in the presence or absence of actinomycin D, where only single-stranded DNA is formed. This finding is consistent with the notion that virtually all of the significant primers have been recognized. In contrast to mature virions, transfer experiments with ether-disrupted early harvest (5 min) MuLV showed only the rC-dC bond; the rA-dA bond was absent. The short-time harvest contains a significantly higher proportion of infectious virions than 24-h harvests. Also, since the RNA from early harvest virus is appreciably more homogenous than the RNA of mature MuLV, it is concluded that the ribopyrimidine-dC linkage is the more significant initiation event from a biochemical standpoint.     

Model for the Structure of the Shape-Maintaining Layer of the Escherichia coli Cell Envelope

The surface area per repeating murein unit (i.e. per molecule of diaminopimelate) has been determined for the cell envelopes of the Escherichia coli strains K-12 and W. This area was constantly found to be 1.3 nm(2). Using this value and other previously determined properties of E. coli murein, a three-dimensional model of murein is proposed. The model specifies a monomolecular layer in which disaccharide units are each 1.03 nm long, and the polysaccharide chains, all parallel, are 1.25 nm apart. The cross-linking peptide side-chains have the same atomic coordinates and are arranged above or below the polysaccharide chains.     

The frequency response of the action of light on the membrane potential ofNitella

Summary Cells ofNitella flexilis were illuminated with light of sinusoidally modulated intensity at frequencies between 0.1 and 64 cycles/min and the frequency response of the changes of the membrane potential caused by light were measured. A band-pass characteristic was found which is known from similar investigations inAcetabularia. The slope of the frequency response was of the 1/f²-type. This slope and the band-pass characteristic lead to the conclusion that three poles and one zero are comprised in the network function of the action of light on the membrane potential. It is discussed which models regarding the mechanism of the action of light on the membrane potential account for the frequency response measured.

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Zusammenfassung Der Frequenzgang der Lichtwirkung auf das Membranpotential vonNitella.Internodienzellen der AlgeNitella flexilis wurden mit sinusförmig moduliertem Licht im Frequenzbereich von 0,1 bis 64 Schwingungen/min beleuchtet und der Frequenzgang der durch das Licht verursachten Änderungen der Membranspannung gemessen. Es trat eine Bandpaßcharakteristik auf, die jedoch nicht im Deteil untersucht wurde, da sie schon von Messungen anAcetabularia bekannt ist. Das Interesse lag auf der hochfrequenten Flanke. Es wurde gefunden, daß die Asymptote mit 1/f² abfällt. Aus der Bandpaßcharakteristik und der Flankensteilheit ist zu entnehmen, daß die Netzwerkfunktion der Lichtwirkung auf das Membranpotential im untersuchten Frequenzbereich 3 Polstellen und eine Nullstelle enthält. Es wird diskutiert, welche Schlüsse daraus über den Mechanismus der Lichtwirkung zu ziehen sind.