Hepes may stimulate cultured endothelial cells to make growth-retarding oxygen metabolites

Summary Zwitterion buffers are often used to modulate the pH of cell culture medium but their effect on cultured cells is controversial. We found that addition of 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) caused superoxide dismutase (SOD) inhibitable increases in nitroblue tetrazolium dye reduction and SOD and catalase inhibitable decreases in the growth of cultured bovine pulmonary artery endothelial cells. The findings suggest that HEPES stimulates endothelial cells to make toxic oxygen metabolites that contribute to decreased cell growth. This work was supported in part by the National Institutes of Health, Colorado and American Lung Associations, Colorado and American Heart Associations, the Council for Tobacco Research, and the Kroc, Hill, Swan and Kleberg Foundations. Dr. Bowman is a Clinician Scientist Awardee of the American Heart Association.     

Über die Entwicklung der Arbeits- und Erregungsleitungsmuskulatur des Herzens von Ratte und Meerschweinchen

Zusammenfassung Die Chemodifferenzierung der Herzmuskulatur von Ratte (215 Tiere) und Meerschweinchen (180 Tiere) wird untersucht und zur Morphodifferenzierung und elektrophysiologisch nachweisbaren Funktionsentwicklung in Beziehung gesetzt. Ferner wird die Entwicklung der Arbeitsmuskulatur mit der des Reizleitungssystems verglichen. Prinzipiell verhält sich die Herzentwicklung bei Ratte und Meerschweinchen ähnlich, doch bestehen erhebliche Unterschiede im Terminplan der Entwicklung. Bei der Ratte erfolgen wesentliche Schritte der Herzentwicklung nach der Geburt, beim Meerschweinchen ist die Entwicklung etwa zur Zeit der Geburt abgeschlossen. In der frühen Embryonalzeit ist die Herzmuskulatur reich an Glykogen, aber arm an Enzymen des oxidativen Stoffwechsels (Bernsteinsäuredehydrogenase, Cytochromoxidase) und -Hydroxibuttersäuredehydrogenase. Kapillaren fehlen. Dann — bei der Ratte etwa ab 12. Embryonaltag — beginnt der Glykogenbestand der Arbeitsmuskulatur abzunehmen. Zunächst werden die subepikardial gelegenen Schichten betroffen. Von hier schreitet die Glykogenverminderung nach innen fort. In den glykogenarm gewordenen Zonen vermehrt sich der Bestand an Atmungsfermenten und Kapillaren. Die Chemodifferenzierung in der Kammermuskulatur erfolgt grundsätzlich von außen nach innen. Parallel hierzu verläuft die Strukturentwicklung der Herzmuskelzellen und es kommt zu elektrophysiologisch nachgewiesenen Änderungen in der Permeabilität der Herzmuskelzellmembranen. Abgeschlossen ist die Entwicklung bei der Ratte nach färberisch-lichtmikroskopischen Befunden in der Mitte der 2. Lebenswoche, nach histochemischen Befunden in der 4. Lebenswoche, nach Maßgabe elektrophysiologischer Beobachtungen nach dem 31. Lebenstag. Die Vorhofmuskulatur ist mit Ausnahme frühembryonaler Stadien stets reicher an Glykogen und ärmer an Atmungsfermenten als die Kammermuskulatur. — Das Reizleitungssystem durchläuft eine eigene Entwicklung. Ursprungsgewebe für das Atrioventricularsystem ist der atrioventriculäre Muskelring. Von hieraus wachsen spezifische Fasern in die Kammermuskulatur vor. Sie unterscheiden sich histochemisch von vornherein von der Arbeitsmuskulatur.

---

Summary The present study deals with the chemodifferentiation of the cardiac muscle in rats (215 animals) and Guinea pigs (180 animals). The results obtained are compared with the morphological differentiation and the functional development, as determined by electrophysiological methods. Finally, the development of the cardiac muscle is compared with the development of the conducting system. Fundamentally the development as such is the same in rats and Guinea pigs; however, there are striking differences as far as the schedule for the development is concerned. In rats, important steps in the development of the heart take place after birth, where as in Guinea pigs the development is terminated at approximately the time of birth. In the early embryonic stages the cardiac muscle has a high content of glycogen, but it is poor in enzymes of the oxidative metabolism (succinate dehydrogenase, cytochrome oxidase) and -hydroxibutyricaciddehydrogenase. There are no capillaries. Approximately from the 12th embryonic day onwards a decrease of the glycogen content is observed in the cardiac muscle of rats. The first layers to undergo this change are the subepicardial layers. From there it progresses to the innermost layers. The activity of the respiratory enzymes and the number of the capillaries increases in the areas where the glycogen content is now relatively low. On principle the chemodifferentiation of the ventricular mucsle progresses always from the outermost to the innermost layer. The structural development of the cells of the cardiac muscle goes parallel with it; at the same time changes in the permeability of the membranes of cardiac muscle cells are demonstrable by electro-physiological methods. According to light microscopical findings the development in rats is terminated in the middle of the second week of life, according to histochemical findings in the 4th week of life, electrophysiologically after the 31st day of life. It seems to be a rule that apart from the early embryonic stages the auricular muscle is richer in glycogen and poorer in respiratory enzymes than the ventricular muscle. The conducting system has a separate pattern of development. The initial form of the atrioventricular system is the atrio-ventricular muscular ring. From there specific fibres penetrate into the ventricular musculature. Histochemically they are a priori different from the cardiac muscle.

---

---

Mit Unterstützung durch die Deutsche Forschungsgemeinschaft.

---

Stipendiatin der Humboldt-Stiftung.

---

Frau Prof. Berta Scharrer mit herzlichem Glückwunsch zum 60. Geburtstag gewidmet.     

Effects of microbial challenge on sleep in rabbits

Rabbits challenged with viable Staphylococcus aureus exhibit marked time-dependent changes in sleep patterns. To examine the generality of this observation, we monitored sleep patterns for 24 h before and for 48 h after intravenous inoculation of rabbits with Streptococcus pyogenes, Escherichia coli, or Candida albicans. All three agents produced complex time-dependent changes in sleep. Inoculation with S. pyogenes or C. albicans increased the time spent in slow-wave sleep (SWS) during h 4-20 after challenge. Electroencephalographic delta wave amplitudes (DWA) increased during h 4-8 after injection, but decreased during h 24-38 after inoculation. Altered sleep patterns were not observed when similar doses of heat-killed organisms were administered. In contrast, inoculation with E. coli produced a large increase in both SWS time and DWA for the first 2-4 h after inoculation. DWA then decreased from 6 to 32 h after inoculation. Similar effects occurred when heat-killed E. coli were administered. Rapid eye movement sleep was reduced by all three agents. These data demonstrate that altered sleep patterns occur in response to infectious challenge in rabbits, and that these changes are related to the type of infectious organism involved.     

Immediate breast reconstruction with deepithelialized TRAM flaps: techniques for improving breast reconstruction

In selected patients with lower quadrant breast masses, large breasts, and sufficient abdominal tissue, standard techniques for breast reconstruction can be modified to improve overall results. The transverse abdominal island flap can be deepithelialized and mobilized to reconstruct unilateral or bilateral defects. Furthermore, skin markings prior to mastectomy that conform to a modified Wise pattern will allow for more aesthetic positioning of eventual scars. We present a case report of a patient who underwent immediate breast reconstruction with bilateral deepithelialized lower rectus abdominis myodermal flaps.     

Effects of oxymorphazone in frogs: long lasting antinociception in vivo, and apparently irreversible binding in vitro

Oxymorphazone (at doses of 50-200 mg/kg) was found to be a relatively weak antinociceptive drug in intact frog (Rana esculenta) when acetic acid was used as pain stimulus. Frogs remained analgesic for at least 48 hrs following oxymorphazone (200 mg/kg) administration. The ligand increased the latency of wiping reflex in spinal frogs too. These effects were blocked by naloxone. In equilibrium binding studies (3H)oxymorphazone had high affinity to the opioid receptors of frog brain and spinal cord as well (apparent Kd values were 8.9 and 10.6 nM, respectively). Kinetic experiments show that only 25% of the bound (3H)oxymorphazone is readily dissociable. Preincubation of the membranes with labeled oxymorphazone results in a washing resistant inhibition of the opioid binding sites. At least 70% of the (3H)oxymorphazone specific binding is apparently irreversible after reaction at 5 nM ligand concentration, and this can be enhanced by a higher concentration of tritiated ligand.     

The pharmacophore of the human C5a anaphylatoxin.

We have determined which amino acids contribute to the pharmacophore of human C5a, a potent inflammatory mediator. A systematic mutational analysis of this 74-amino acid protein was performed and the effects on the potency of receptor binding and of C5a-induced intracellular calcium ion mobilization were measured. This analysis included the construction of hybrids between C5a and the homologous but unreactive C3a protein and site-directed mutagenesis. Ten noncontiguous amino acids from the structurally well-defined 4-helix core domain (amino acids 1-63) and the C-terminal arginine-containing tripeptide were found to contribute to the pharmacophore of human C5a. The 10 mostly charged amino acids from the core domain generally made small incremental contributions toward binding affinity, some of which were independent. Substitutions of the C-terminal amino acid Arg 74 produced the largest single effect. We also found the connection between these 2 important regions to be unconstrained.     

A differential proteomics study of cerebrospinal fluid from individuals with Niemann-Pick disease,Type C1

Niemann-Pick, type C1 (NPC1) is a fatal, neurodegenerative disease, which belongs to the family of lysosomal diseases. In NPC1, endo/lysosomal accumulation of unesterified cholesterol and sphingolipids arise from improper intracellular trafficking resulting in multi-organ dysfunction. With the proximity between the brain and cerebrospinal fluid (CSF), performing differential proteomics provides a means to shed light to changes occurring in the brain. In this study, CSF samples obtained from NPC1 individuals and unaffected controls were used for protein biomarker identification. A subset of these individuals with NPC1 are being treated with miglustat, a glycosphingolipid synthesis inhibitor. Of the 300 identified proteins, 71 proteins were altered in individuals with NPC1 compared to controls including cathepsin D, and members of the complement family. Included are a report of 10 potential markers for monitoring therapeutic treatment. We observed that pro-neuropeptide Y (NPY) was significantly increased in NPC1 individuals relative to healthy controls; however, individuals treated with miglustat displayed levels comparable to healthy controls. In further investigation, NPY levels in a NPC1 mouse model corroborated our findings. We posit that NPY could be a potential therapeutic target for NPC1 due to its multiple roles in the central nervous system such as attenuating neuroinflammation and reducing excitotoxicity.