Some cytologic characteristics of the hemocytes of Limulus during clotting

The hemolymph of the horseshoe crab, Limulus (Xiphosura) polyphemus, contains a single cell type. The hemocytes are ovoid and contain many refractile granules. One-half to one minute after the onset of clotting the hemocytes swell and a hyaline cytoplasmic ring, essentially devoid of granules, appears about their circumference. During this time the granules disperse and the nucleus becomes visible. Three to five minutes following the initiation of clotting, the cell extends long pseudopodial processes. Phase contrast time-lapse cinematography reveals that the cells are extremely motile during this phase. Concomitant with these changes, many of the granules lose their refractility and one by one disappear from the cytoplasm leaving what appears to be a vacuole. Electron micrographs of native (un-clotted) hemocytes and of clots fixed in glutaraldehyde and post-fixed in osmium reveal that the membrane-bounded granules of native cells are very dense and homogeneous with no evidence of an internal structure. One-half to one minute after clotting, however, they become less dense and 250 Å microtubules spaced at ca. 500 Å intervals appear parallel to the long axis of the granule. Further degradation of the granule ensues and involves (a) change to a spherical shape, and (b) further decrease in density and better resolution of the microtubules. The microtubular component subsequently disintegrates leaving a membrane-bounded granule containing particulate material. Finally, the membrane of the granule fuses with the plasma membrane and the components of the granule are dispersed in the plasma where they presumably contribute to the formation of the gelatinous clot.     

Food dependent color patterns inThamnocephalus platyurus Packard (Branchiopoda: Anostraca); a laboratory study

Coloration of phyllopods varies from place to place and from one life stage to another. It ranges from translucent or whitish through gray, blue, green, orange, and reddish. Here, we present experimental evidence for a food- dependent color pattern inThamnocephalus platyurus Packard. The presence or absence of the synthetic pigment trans — — carotene in a baker's yeast diet was the controlling factor. All the 24 old larvae used in the experiment were whitish in color. From day 6 until the end the experiment (day 11), 100% of the shrimps under a diet with synthetic trans — — carotene (treatment 1) exhibited a characteristic color pattern which consisted of an orange color in the cercopods, and in all theracopods; the rest of the body exhibited no particular color. In comparison, 100% of the shrimps under a diet without synthetic trans — — carotene (treatment 2) were whitish throughout the body. In females from treatment 1, the ovaries and oocytes were green-bluish, while in females from treatment 2 the ovaries and oocytes were whitish. No significant differences in survival and growth were found, except that at day 9, there was a significant difference in growth, the females with the synthetic trans — — carotene group growing faster.     

Distinct distribution of immunoreactive dynorphin and leucine enkephalin in various populations of isolated adrenal cromaffin cells

The distribution of immunoreactive-dynorphin (ir-Dyn) in isolated subpopulations of bovine adrenal chromaffin cells was examined and compared with that of adrenaline (A), noradrenaline (NA) and ir-Leucine-Enkephalin (ir-Leu-Enk). Using a stepwise bovine serum albumin (BSA) gradient, various populations of catecholamine-storing cells were separated and designated as cell layers I, II and III. Cell layer I contained more NA than A; cell layer II contained slightly more A than NA whereas cell layer III was highly enriched in A. The original cell preparation contained 2.9 times more ir-Leu-Enk than ir-Dyn (4.7 and 1.6 pmoles per 10(6) cells, respectively). After separation of the cells on BSA gradient, ir-Dyn was mainly detected in cell layer I (4.0 pmoles/10(6) cells) whereas ir-Leu-Enk was concentrated in cell layer III (8.3 pmoles/10(6) cells). Both peptides were secreted in response to acetylcholine (5 x 10(-5) M), but the amount secreted was in accordance with the cell content in each peptide. After subcellular fractionation of the adrenal medulla, the neuropeptides were found in close association with catecholamines in the secretory granules. These results indicate that bovine adrenal chromaffin cells can be isolated according to their specific content in A, NA and opioid peptides and are consistent with the hypothesis of distinct biosynthetic pathways for Dyn and the Enk.     

On nine Tropodiaptomus-species (Copepoda,Calanoida) from equatorial East Africa

Of the nine species of Tropodiaptomus occurring in Equatorial East Africa, seven are redescribed and figured in great detail. Their relationships are discussed. A primary homonym is eliminated, and types are indicated wherever possible.     

Characterization of mature rat oligodendrocytes: a proteomic approach

Oligodendrocytes are glial cells responsible for the synthesis and maintenance of myelin in the central nervous system (CNS). Oligodendrocytes are vulnerable to damage occurring in a variety of neurological diseases. Understanding oligodendrocyte biology is crucial for the dissemination of de- and remyelination mechanisms. The goal of the present study is the construction of a protein database of mature rat oligodendrocytes. Post-mitotic oligodendrocytes were isolated from mature Wistar rats and subjected to immunocytochemistry. Proteins were extracted and analyzed by means of two-dimensional gel electrophoresis and two-dimensional liquid chromatography, both coupled to mass spectrometry. The combination of the gel-based and gel-free approach resulted in confident identification of a total of 200 proteins. A minority of proteins were identified in both proteomic strategies. The identified proteins represent a variety of functional groups, including novel oligodendrocyte proteins. The results of this study emphasize the power of the applied proteomic strategy to study known or to reveal new proteins and to investigate their regulation in oligodendrocytes in different disease models.     

Expression of 25 human ABC transporters in the yeast Pichia pastoris and characterization of the purified ABCC3 ATPase activity

Human ATP-binding cassette (ABC) transporters comprise a family of 48 membrane-spanning transport proteins, many of which are associated with genetic diseases or multidrug resistance of cancers. In this study, we present a comprehensive approach for the cloning, expression, and purification of human ABC transporters in the yeast Pichia pastoris. We analyzed the expression of 25 proteins and demonstrate that 11 transporters, including ABCC3, ABCB6, ABCD1, ABCG1, ABCG4, ABCG5, ABCG8, ABCE1, ABCF1, ABCF2, and ABCF3, were expressed at high levels comparable to that of ABCB1 (P-glycoprotein). As an example of the purification strategy via tandem affinity chromatography, we purified ABCC3 (MRP3) whose role in the transport of anticancer drugs, bile acids, and glucuronides has been controversial. The yield of ABCC3 was 3.5 mg/100 g of cells in six independent purifications. Purified ABCC3, activated with PC lipids, exhibited significant ATPase activity with a Vmax of 82 +/- 32 nmol min-1 mg-1. The ATPase activity was stimulated by bile acids and glucuronide conjugates, reaching 170 +/- 28 nmol min-1 mg-1, but was not stimulated by a variety of anticancer drugs. The glucuronide conjugates ethinylestradiol-3-glucuronide and 17beta-estradiol-17-glucuronide stimulated the ATPase with relatively high affinities (apparent Km values of 2 and 3 microM, respectively) in contrast to bile acids (apparent Km values of >130 microM), suggesting that glucuronides are the preferred substrates for this transporter. Overall, the availability of a purification system for the production of large quantities of active transporters presents a major step not only toward understanding the role of ABCC3 but also toward future structure-function analysis of other human ABC transporters.     

Synthesis of 3-O-methylviridicatin analogues with improved anti-TNF-alpha properties

We synthesized 3-O-methylviridicatin and several analogues of this fungal metabolite. We showed that replacement of the methoxy moiety by a thiomethyl enhanced dramatically its ability to inhibit TNF-alpha secretion. These results strongly suggest that 4-phenyl-3-methylthioquinolinone may provide the basis for the development of new anti-inflammatory agents.     

Perceptual interactions between characteristic notes smelled above aqueous solutions of odorant mixtures

Twenty-two experienced panelists rated odor intensity of aqueous solutions of citral, octen-1-ol-3, and hexanal. The panel assessed unmixed components and mixtures (9 binary and 4 ternary). In sensory sessions dedicated to mixtures (n = 6), evaluation was focused on one target odor, presented at a fixed concentration. All components had lower odor intensity on mixed presentations. In many cases, information obtained from simpler systems was not extended to complex mixtures. In a mixture, the competition between odorant molecules on qualitative aspects (dominance/suppression) imbalanced components contribution, anticipated from the quantitative distribution. Hexanal appeared to be the potentially weaker odorant in paired combinations, whereas octen-1-ol-3 had a lower relative impact on ternary systems. Suppression of the odor of octen-1-ol-3 and a concomitant increase in the odor of hexanal was common to all ternary mixtures. Reciprocal inhibition of octen-1-ol-3 and citral odors through perceptual interactions was suspected. Mutual suppression is suspected to have eased the perception of hexanal intensity.