No evidence for a peripheral effect of L-DOPA on plasma prolactin in the rat

Male and female rats with two permanently indwelling intravenous catheters were infused for 2 hours with ovine prolactin. During equilibrium conditions the effects of intravenously injected L-DOPA and benzerazide (a blocker of dopa-decarboxylase) on steady state levels of ovine prolactin were measured. A dose of 4.5 mg L-DOPA per 100 gr body weight (b.w.) caused a transient increase of plasma ovine prolactin. A dose of 0.3 mg L-DOPA/100 gr b.w. had no effect, neither in males nor in females, while benzerazide (20 mg/100 gr b.w.) had only a slight effect. The experiments suggest that L-DOPA does not affect the peripheral uptake of prolactin from the plasma.     

Proteolytic fragmentation of Helix pomatia alpha-hemocyanin: isolation of a functionally active chemically pure domain and evidence for subunit heterogeneity.

α-Hemocyanin of the vineyard snail, Helix pomatia, is a large oligomer composed of 20 subunits with a molecular weight of 360,000 ± 30,000. Limited proteolysis showed these subunits to be composed of about seven structural domains, each having one oxygen binding site (1). This paper describes the production of these structural domains by tryptic digestion of $\text{1}\text{10}$ hemocyanin molecules. The digestion pattern was followed as a function of time by examining the proteolytically obtained fragments electrophoretically in sodium dodecyl sulfate-containing polyacrylamide gels. Based on the molar ratio of each fragment present during digestion the first part of the reaction sequence for trypsinolysis could be deduced. This reaction scheme was simulated by means of an analog computer. Pseudo-first-order reaction rate constants of the various proteolytic cleavages were estimated from the computer generated time course of digestion. On the basis of these results it is postulated that Helix pomatia α-hemocyanin possesses at least two kinds of subunits which differ in their proteolytic susceptibility. These subunits occur in equimolar amounts. A functionally active domain with a molecular weight of about 50,000 has been isolated from a tryptic digest of hemocyanin subunits. This domain seems to be chemically pure, as suggested by the unique sequence of its first six amino acids, viz: Lys-Val-His-Leu-Asn-Lys.     

In vivo magnetic resonance imaging of the blue crab, Callinectes sapidus: effect of cadmium accumulation in tissues on proton relaxation properties.

Nuclear magnetic resonance imaging (MRI) has been used to visualize the internal anatomy of a living blue crab. The resolution obtained in these studies was sufficient to distinguish individual organs by the differences in their proton densities and proton relaxation properties. T1 (spin-lattice relaxation time)-weighted imaging revealed the lipid-rich nature of the hepatopancreas and gonadal tissue. To evaluate the effect of metal-induced stress on the different organs, crabs were exposed to elevated levels of cadmium in their diet, which resulted in increased concentrations of both cadmium and copper in the hepatopancreas. The spin-spin relaxation time, T2, of mobile protons in the metal-exposed tissue was significantly greater than T2 in the control tissues. These measurements suggest that the excess copper in the exposed tissues was diamagnetic [Cu(I)], since the presence of paramagnetic copper [Cu(II)] would result in a decrease of observed T2 values. We hypothesize that the increased T2 value is a reflection of increased free water in the hepatopancreas. These studies show that magnetic resonance imaging is an important nondestructive tool for the study of morphological and physiological changes that occur in marine invertebrates in response to anthropogenic and natural stresses.     

Maturation- and differentiation-dependent responsiveness of human CD4+ T helper subsets.

The human CD45R0+ (memory) CD4+ T cell population can be subdivided into a large (82%) CD27+ and a small (18%) CD27- subset. Within the CD45R0+CD27- subset, cells accumulate that have been persistently stimulated by Ag in vivo. As an apparent consequence, TLC with a differentiated functional phenotype, producing either high levels of IFN-gamma (Th1-like), high levels of IL-4 (Th2-like) or high amounts of both these cytokines (here referred to as Thx) can primarily be generated from the CD27- memory CD4+ T cell subset. In this study we examined the requirements for induction of proliferation of distinct CD4+CD45R0+ Th subsets. Immobilized CD3 mAb induced proliferation with comparable magnitude and kinetics in all types of TLC. However, interference with intracellular signaling pathways in this activation system, resulted in a strong inhibition of proliferation in TLC derived from CD27+ cells whereas, in contrast, TLC from CD27- cells were relatively resistant to elevation of [cAMP]i, inhibition of protein kinase C activation and the immunosuppressive effects of cyclosporin A. Stimulation with CD3 mAb in soluble form resulted in Il-4 secretion by Th2-like and Thx-type TLC but did not induce IFN-gamma or Il-2 secretion in any Th subset. Interestingly, Th2-like cells but not Thx-type cells were able to use endogenously produced Il-4 for proliferation. These data demonstrate a differential sensitivity of CD45R0+CD4+ Th subsets for immune activation and suppression, which correlated with their maturation stage, as reflected by CD27 membrane expression, as well as with their effector phenotype.     

Interaction of copper-metallothionein from the American lobster, Homarus americanus, with glutathione

Organisms have harnessed the unique chemistry of copper for a variety of purposes. However, that same chemistry makes this essential metal toxic at elevated concentrations. Metallothioneins (MTs), a family of small metal-binding proteins, are thought to play a crucial role in the regulation of this reactive ion. Here we report that copper-metallothioneins from the American lobster, Homarus americanus, interact with the tripeptide glutathione (gamma-Glu-Cys-Gly). Glutathione in the cytosolic fraction prepared from the digestive gland of the American lobster coelutes with copper-metallothionein during size-exclusion chromatography. The latter protein can be separated into three isoforms by anion-exchange chromatography. All three isoforms belong to the class I MTs. CuMT-I and -II are very similar, whereas CuMT-III is distinct from isoforms I and II. The interaction between glutathione and MT isoforms was examined by ultrafiltration experiments and size-exclusion HPLC. CuMT-III forms a stable 1:1 complex with glutathione, with a dissociation constant of 1 microM. CuMT-I/II makes a transient complex with glutathione, which releases copper as a copper-glutathione complex. This complex can function as the source of Cu(I) in the restoration of the oxygen-binding capacity of copper-free hemocyanin. These studies suggest that metallothionein and glutathione are intricately linked in the biochemistry of copper regulation.     

Endocytosis of lactate dehydrogenase isoenzyme M4 in rats in vivo. Experiments with enzyme labelled with O-(4-diazo-3,5-di[125I]iodobenzoyl)sucrose.

1. Pig lactate dehydrogenase isoenzyme M4 was labelled with O-(4-diazo-3,5-di[125I]iodobenzoyl)sucrose and injected intravenously into rats. Previous work has shown that this label does not influence the clearance of the enzyme (half-life about 26 min) and that it is retained within the lysosomes for several hours after endocytosis and breakdown of the protein [De Jong, Bouma & Gruber (1981) Biochem. J. 198, 45--51]. 2. The distribution of the radioactivity over a large number of tissues was determined 2 h after injection. A high percentage of the injected dose was found in liver (41%), spleen (10%) and bone including marrow (21%). 3. Autoradiography indicated uptake of the enzyme mainly by Kupffer cells of the liver, by spleen macrophages and by bone marrow macrophages. 4. Liver cells were isolated 1 h after injection of the enzyme. Kupffer cells, endothelial cells and parenchymal cells were found to endocytose the enzyme at rates corresponding to 4230, 35 and 25 ml of plasma/day per g of cell protein, respectively. 5. Previous injection of carbon particles greatly reduced the uptake of the enzyme by liver and spleen, but the uptake by bone marrow was not significantly changed.     

Three novel components of the human exosome

The yeast exosome is a complex of 3' --> 5' exoribonucleases. Sequence analysis identified putative human homologues for exosome components, although several were found only as expressed sequence tags. Here we report the cloning of full-length cDNAs, which encode putative human homologues of the Rrp40p, Rrp41p, and Rrp46p components of the exosome. Recombinant proteins were expressed and used to raise rabbit antisera. In Western blotting experiments, these decorated HeLa cell proteins of the predicted sizes. All three human proteins were enriched in the HeLa cells nucleus and nucleolus, but were also clearly detected in the cytoplasm. Size exclusion chromatography revealed that hRrp40p, hRrp41p, and hRrp46p were present in a large complex. This cofractionated with the human homologues of other exosome components, hRrp4p and PM/Scl-100. Anti-PM/Scl-positive patient sera coimmunoprecipitated hRrp40p, hRrp41p, and hRrp46p demonstrating their physical association. The immunoprecipitated complex exhibited 3' --> 5' exoribonuclease activity in vitro. hRrp41p was expressed in yeast and shown to suppress the lethality of genetic depletion of yeast Rrp41p. We conclude that hRrp40p, hRrp41p, and hRrp46p represent novel components of the human exosome complex.     

Esa1, an Arabidopsis mutant with enhanced susceptibility to a range of necrotrophic fungal pathogens, shows a distorted induction of defense responses by reactive oxygen generating compounds

An Arabidopsis thaliana mutant, esa1, that shows enhanced susceptibility to the necrotrophic pathogens Alternaria brassicicola, Botrytis cinerea and Plectosphaerella cucumerina, but has wild-type levels of resistance to the biotrophic pathogens Pseudomonas syringae pv. tomato and Peronospora parasitica. The enhanced susceptibility towards necrotrophic pathogens correlated with a delayed induction of phytoalexin accumulation and delayed induction of the plant defensin gene PDF1.2 upon inoculation with pathogens. Two reactive oxygen generating compounds, paraquat and acifluorfen, were found to cause induction of both phytoalexin accumulation and PDF1.2 expression in wild-type plants, but this induction was almost completely abolished in esa1. This finding suggests that esa1 may somehow be involved in transduction of signals generated by reactive oxygen species.