Inhibition of Dopamine Agonist-Induced Phosphoinositide Hydrolysis by Concomitant Stimulation of Cyclic AMP Formation in Brain Slices

Abstract: We examined the effects of cyclic AMP on dopamine receptor-coupled activation of phosphoinositide hydrolysis in rat striatal slices. Forskolin, dibutyryl cyclic AMP, and the protein kinase A activator Sp -cyclic adenosine monophosphothioate ( Sp -cAMPS) significantly inhibited inositol phosphate formation stimulated by the dopamine D₁ receptor agonist SKF 38393. Conversely, the protein kinase A antagonist Rp -cyclic adenosine monophosphothioate ( Rp -cAMPS) dose-dependently potentiated the SKF 38393 effect. In the presence of 200 µ M Rp -cAMPS, the dose-response curves of the dopamine D₁ receptor agonists SKF 38393 and fenoldopam were shifted to the left and maximal agonist responses were markedly increased. The agonist EC₅₀ values, however, were not significantly altered by protein kinase A inhibition. Neither Sp -cAMPS nor Rp -cAMPS significantly affected basal inositol phosphate accumulation. These findings demonstrate that dopaminergic stimulation of phosphoinositide hydrolysis is inhibited by elevations in intracellular cyclic AMP. Dopamine receptor agonists that stimulate adenylyl cyclase could suppress their activation of phosphoinositide hydrolysis by concomitantly stimulating the formation of cyclic AMP in striatal tissue. The interaction between dopamine D₁ receptor-stimulated elevations in cyclic AMP and dopaminergic stimulation of inositol phosphate formation suggests a cellular colocalization of these dopamine-coupled transduction pathways in at least some cells of the rat striatum.     

CHARACTERIZATION OF TWO CHLORELLA VULGARIS (CHLOROPHYCEAE) STRAINS ISOLATED FROM WASTEWATER OXIDATION PONDS1

We report here on the characterization and isolation of two ecotypes of Chlorella vulgaris Beyerinck that coexist in wastewater reservoirs. One ecotype (C1) contains high amounts of chlorophyll b, is capable of autotrophic growth, and can utilize only a few organic solutes for growth. The second ecotype (C2) contains low amounts of chlorophyll b, requires vitamin B₁₂, and can support its growth with a broad range of organic compounds. Of the two ecotypes, the latter showed slower growth rates when light was the sole source of energy. Cells of C2-type Chlorella attained higher photosynthetic activities than C1-type cells at saturating irradiances. However, their low chlorophyll b content and lower light utilization efficiency suggest that C2-type Chlorella contains relatively low amounts of light-harvesting antennae, a disadvantage in severely light-limited ecosystems like wastewater reservoirs. We hypothesize that the two Chlorella types coexist by adopting different lifestyles: C1-type cells rely largely on their photosynthetic potential for energy conservation and growth, whereas C2-type cells may exploit their heterotrophic properties for this purpose.     

WT1 mutations in patients with Denys-Drash syndrome: a novel mutation in exon 8 and paternal allele origin

Denys-Drash syndrome (DDS) is characterized by early onset nephropathy, pseudohermaphroditism in males and a high risk for developing Wilms' tumour (WT). The exact cause of DDS is unknown but germline mutations in the Wilms' tumour suppressor gene (WT1) have recently been described in the majority of DDS patients studied. These mutations occur de novo and are clustered around the zinc finger (ZF) coding exons of the WT1 gene. Analysis of exons 2–10 of the WT1 gene in constitutional DNA from five patients with DDS was carried out using the polymerase chain reaction (PCR) and direct DNA sequencing. In four out of the five patients, heterozygous germline mutations were found: a novel point mutation in exon 8 (ZF₂) at codon 377 altering the wild-type histidine to arginine, and three previously described point mutations in exon 9 (ZF₃) in the codons corresponding to amino acids ³⁹⁴Arg and ³⁹⁶Asp. In one patient, no mutations could be demonstrated. In three patients where parental DNA was available, the mutations were shown to have occurred de novo. Furthermore, since tumour DNA in two of these cases had lost the wild-type allele, polymorphic markers from the short arm of chromosome 11 were used to determine the parental origin of the mutant chromosome. In both cases, the mutant chromosome was shown to be of paternal origin. Since the majority of published WT1 mutations in DDS patients alter a RsrII restriction site in exon 9, we were able to perform PCR-based diagnosis in a female patient with early renal insufficiency and normal external genitalia.     

Immortalization of antigen specific, helper T cell lines by transformation with the radiation leukemia virus (RadLV)

Antigen-specific immune T lymphocytes of male C57BL/6 mice were enriched in vitro on monolayers of antigen-pulsed syngeneic macrophages. The cells were treated in vitro with RadLV and inoculated intrathymically into irradiated female C56BL/6 animals. Thymomas arising in the inoculated recipients were characterized as donor- (male) type according to their karyotype. In vivo and in vitro cell lines were established from the primary lymphomas, two of which (designated ROT/6.1 and ROT/6.2) were capable of providing antigen- (carrier) specific help in normal or preimmunized mice. None of the lymphomas could induce antigen-specific DTH reaction. Five months after their establishment, ROT/6.2 alone retained its carrier specificity. ROT/6.2 consisted mainly of Lyt-1+ cells, whereas the ROT/6.1 population was more heterogeneous and contained Lyt-1+, Lyt-2+, and Lyt-3+ cells. The carrier specificity of the latter may have been lost due to selection against the specific helper cells during prolonged passages.     

Catechol oxidase from green olives: Properties and partial purification

Catechol oxidase was extracted from an acetone powder prepared from green olive. The enzyme was purified 240-fold by ammonium sulphate fractionation followed by ion exchange chromatography and gel filtration. The enzyme was characterized by substrate specificity and response to inhibitors. Between 7 and 9 bands having catechol oxidase activity could be detected by gel electrophoresis and electrofocusing. The purified enzyme had an estimated MW of 42 000. The enzyme was strongly inhibited by diethyldithiocarbamate. Inhibition by chloride was strongly dependent on pH. The enzyme did not oxidise monophenols.     

Unique properties of the camel erythrocyte membrane: II. Organization of membrane proteins

Camel erythrocyte membranes are distinguished by some unique properties of stability and composition. Notable is their abundance in proteins (protein: lipid ratio of 3 : 1). Membrane proteins of camel erythrocytes were compared with those of human erythrocytes, which have been intensively investigated. Proteins were extracted with various aqueous media (EDTA, alkaline or high ionic strength) and with ionic and non-ionic detergents and were analyzed by gel electrophoresis. In membranes of camel erythrocytes, the peripheral proteins constitute, proportionally, a much smaller fraction of total proteins than in the human erythrocyte, while their distribution is identical per unit of surface area. The camel erythrocyte membrane is particularly rich in integral proteins and in intramembranous particles. The proteins in this membrane are more closely organized than in the human system, as revealed by crosslinking and freeze-etching studies. It is proposed that protein-protein interaction of integral proteins, presumably constituting an “integral skeleton”, is a dominant structural feature stabilizing the camel erythrocyte membrane.     

Control of normal differentiation of myeloid leukemic cells. X. Glucose utilization,cellular ATP and associated membrane changes in D+ and D− cells

Glucose utilization, energy metabolism and associated membrane changes, have been studied in D⁺ myeloid leukemic cells that can be induced to undergo cell differentiation to mature granulocytes by incubation with the appropriate conditioned medium (CM) and in D^? myeloid leukemic cells that cannot be induced to differentiate to mature cells. Before incubation with CM, glycolysis and the glycolytic production of ATP were lower and the activity of the pentose cycle was higher in D⁺ than in D^? cells. ATP depletion induced a higher degree of agglutination by concanavalin A in D^? than in D⁺ cells, indicating a difference in their surface membrane. There were no detectable differences in the transport of glucose and the synthesis of sterols and fatty acids. After incubation with CM, the D⁺ cells, like normal granulocytes, showed a higher glycolysis, produced their ATP more through glycolysis than oxidative phosphorylation, became less dependent on the exogenous supply of glucose and oxygen and had a lower rate of sterol and fatty acid synthesis. The differentiating D⁺ cells also showed a change in their surface membrane resulting in an increased agglutinability without a change in ATP content and a stimulation of the pentose cycle by concanavalin A. These properties, which were not acquired by D^? cells, were found before most of the D⁺ cells had differentiated to mature granulocytes. The data indicate, that the block in the ability of the D^? cells to differentiate and the acquisition of the metabolic properties of normal granulocytes by differentiating D⁺ cells, were associated with differences in the organization of the cell surface membrane.     

COPII collar defines the boundary between ER and ER exit site and does not coat cargo containers

COPII and COPI mediate the formation of membrane vesicles translocating in opposite directions within the secretory pathway. Live-cell and electron microscopy revealed a novel mode of function for COPII during cargo export from the ER. COPII is recruited to membranes defining the boundary between the ER and ER exit sites, facilitating selective cargo concentration. Using direct observation of living cells, we monitored cargo selection processes, accumulation, and fission of COPII-free ERES membranes. CRISPR/Cas12a tagging, the RUSH system, and pharmaceutical and genetic perturbations of ER-Golgi transport demonstrated that the COPII coat remains bound to the ER–ERES boundary during protein export. Manipulation of the cargo-binding domain in COPII Sec24B prohibits cargo accumulation in ERES. These findings suggest a role for COPII in selecting and concentrating exported cargo rather than coating Golgi-bound carriers. These findings transform our understanding of coat proteins’ role in ER-to-Golgi transport.