Evidence for conversion of N-Tyr-MIF-1 into MIF-1 by a specific brain aminopeptidase

N-Tyr-MIF-1 (Tyr-Pro-Leu-Gly·NH₂), an immunoreactive neuropeptide exhibiting saturable high affinity binding in rat brain was found to be converted into MIF-1 (Pro-Leu-Gly·NH₂) by a specific brain aminopeptidase present in rat brain homogenates or cytosol, but with low activity associated with synaptosomal plasma membranes and microsomes. Conversion occurred at a rate of 16 μmol per g w/wt per h and was unaffected by puromycin but inhibited by bestatin (I₅₀, 5 × 10^?5 M). Aminopeptidases purified from cytosolic fractions of rat brain (arylamidase), mouse brain (Mn²⁺-activated aminopeptidase) or porcine kidney (leucine aminopeptidase) were inactive towards N-Tyr-MIF-1 but degraded MIF-1 with release of Leu-Gly·NH₂ as detected by RP-HPLC procedures. Morphiceptin (Tyr-Pro-Phe-Pro·NH₂), a μ opioid agonist, also acted as a substrate for the N-Tyr-MIF-1 converting enzyme with cleavage of the Tyr-Pro bond. These tetrapeptides, but not MIF-1 or its N-blocked analogs, were degraded in vitro by a metalloendopeptidase purified from kidney membranes. Since dipeptide products were not detected for crude extracts, a significant role for brain metalloendopeptidase on turnover can be excluded. Thus the results point to the presence of a specific (X-Pro-degrading) aminopeptidase in brain cytosol as an enzyme responsible for converting N-Tyr-MIF-1 and inactivating morphiceptin.     

DNA circles with cruciforms from Isospora (Toxoplasma) gondii

We have isolated a closed circular duplex DNA fraction from the unicellular parasite Isospora (Toxoplasma) gondii and examined the purified DNA by electron microscopy. A major part of this circular DNA consists of 12-micron circles containing a cruciform with 0.5-micron tails. We also found 23-micron circles with the properties expected of head-to-tail dimers of the 12-micron circles. Some of these dimers have two cruciforms with 0.4-micron tails, some have one cruciform with 0.8-micron tails. When ethidium bromide was diffused into the DNA solution, circles with tails were replaced by twisted circles without tails. Direct mixing of the DNA with high ethidium bromide concentrations (5 micrograms/ml) gave rise to highly twisted circles with tails. This proves that the tailed circles are covalently continuous and indicates that ethidium bromide blocks branch migration. The 0.5-micron tails are part of a 1.7-micron palindrome, which was visualized by spreading denatured DNA under snap-back conditions. We argue that the cruciform is not present in vivo and that the 12-micron circles may represent the mitochondrial DNA of Toxoplasma.     

Preparation of isolated liver endothelial cells and Kupffer cells in high yield by means of an enterotoxin

A new method for preparing non-parenchymal rat liver cells (NPC) is described. The liver cell suspension, prepared by perfusing the liver with collagenase, was treated with enterotoxin from Clostridium perfringens for 15 min. The enterotoxin made the parenchymal cells leaky, and these cells could be separated from the NPC by centrifugation in a solution containing Nycodenz (20%, w/v). During the centrifugation, the NPC floated, while the parenchymal cells sedimented. The yield of NPC per liver (200 g rat) was about 250 X 10(6) cells. The NPC were further separated into endothelial cells, Kupffer cells and stellate cells by centrifugal elutriation. This method was particularly useful for preparing endothelial cells in high yield (100 X 10(6) cells per liver). Intravenously injected formaldehyde-treated albumin was selectively taken up by the endothelial cells. Isolated endothelial cells in suspension as well as in surface culture maintained their ability to endocytose this ligand.     

Newly administered [3H]retinol is transferred from hepatocytes to stellate cells in liver for storage

We have recently shown that newly administered vitamin A (retinol) is initially taken up by the parenchymal cells of the liver, and subsequently (within 1-2 h) transferred to non-parenchymal liver cells (NPC) (Blomhoff et al., ref. [10]). In the present study we have separated the NPC by different methods to determine the cell type responsible for this uptake of [3H]retinol. When liver cells were prepared between 5 and 18 h after intraduodenal administration of [3H]retinol, the radioactive retinol was recovered mainly in the stellate cells. Other liver cells (i.e., hepatocytes, endothelial cells and Kupffer cells) contained only small amounts of [3H]retinol. Further, fluorescence microscopy studies indicated that stellate cells contain large quantities of retinol. Our results show that newly administered [3H]retinol, which is initially located in the hepatocytes, is transferred to the stellate cells and stored there.     

Degradation of soluble collagen by ozone or hydroxyl radicals

Collagen exposed to ozone or hydroxyl radicals was degraded in a time- and dose-dependent manner. This degradation was inhibited by free radical scavengers. Furthermore, lower levels of these oxidants did not degrade the molecule, but caused it to become susceptible to proteolytic degradation. We suggest an alternative mechanism by which oxygen-derived free radicals participate in the destruction of extracellular matrix observed during acute lung injury by oxidant gas, in addition to the commonly accepted proteinase-antiproteinase theory of lung injury.     

Identification of Mr variants of proclatin with monoclonal antibodies

Monoclonal antibodies (QB01 and 1200) prepared against human proclatin (hPRL) have helped define a variant form of the hormone. This variant is of apparently higher molecular mass (26kDa) than the predominant form of the hormone (24kDa) and its presence does not appear to be species-restricted. The demonstration of the 26 kDa form of the hPRL in fresh pituitary tissue and amniotic fluid suggests it may retain some specific function.     

Transplantation of a time-signal in honeybees

Summary It has been possible — by transplantation of brain tissue (i.e. mushroom-bodies) — to perform an interindividual transfer of a learned time-signal in honeybees. The information of the donor bees becomes determinative for the temporal activity pattern of the recipients about 3 to 4 days following transplantation.As seen from histological investigations done in parallel, the donor tissue is treated as a xenograft by the recipient's organism including disintegration and encapsulation processes. These observations give evidence for a humoral transfer of information.The results are discussed from the point of view of the analysis of the mechanism of time reception.Dedicated to Prof. Dr. H. Autrum on the occasion of his 70th birthdaySupported by the Akademie der Wissenschaften und der Literatur zu Mainz, the Stiftung Volkswagenwerk and the Deutsche ForschungsgemeinschaftAll observations done were individual per hand registrations. We want to give thanks to all people in the department who helped us to do the experiments.     

Proline excretion in Escherichia coli: A comparison of an argD + strain and a proline-excreting argD − derivative

In an attempt to deduce the physiological basis of proline excretion in argD ^– strains of Escherichia coli K12, several properties of an argD ⁺ (nonexcreting) and an argD ^– (excreting) derivative were compared. No difference was found in the transport or in the utilization of either proline or its immediate precursor, ¹-pyrroline-5-carboxylate (PCA). Furthermore, no differences were found in the physical or kinetic properties of partially purified preparations of the enzyme mediating the final step in proline biosynthesis, PCA reductase. The specific activity of PCA reductase was, however, consistently higher in crude extracts prepared from the argD ^– mutant.This work was supported by grants from the National Institutes of Allergy and Infectious Diseases (Public Health Service No. AI-10862) and The University of Connecticut Research Foundation (to C. M. B.). J. J. R. was supported by an NDEA Predoctoral Fellowship.     

Genetics of the peroxidase isoenzymes in Petunia

Summary By starch gel electrophoresis three mobility variants of a cathodic moving doublet of bands, encoded by the structural gene prxC, were detected in all organs of flowering petunias. In root tissue two of the variants showed a lower electrophoretic mobility than in other organs. During development of flower buds the PRXc enzymes showed an increase in mobility. The gene prxC was located on chromosome IV by showing linkage to the genes An3 and Dw1, by trisomic segregation, and by the construction of triply heterozygous trisomics IV. The gene order on chromosome IV is B1-An3/Dw1-prxC. It was concluded that the temporal programming difference in the expression of the alleles prxC2 and prxC3 is caused by internal site mutation. Analysis of progeny obtained by crossing of lines to the trisomic IV with genotype prxC1/C1/C2 showed differential expression of the two prxC1 alleles of the trisomic IV.