cAMP-dependent protein phosphorylation in mitochondria of bovine heart

A study is presented of the cAMP-dependent phosphorylation in bovine heart mitochondria of three proteins of 42, 16 and 6.5 kDa associated to the inner membrane. These proteins are also phosphorylated by the cytosolic cAMP-dependent protein kinase and by the purified catalytic subunit of this enzyme. In the cytosol, proteins of 16 and 6.5 kDa are phosphorylated by the cAMP-dependent kinase. It is possible that cytosolic and mitochondrial cAMP-dependent kinases phosphorylate the same proteins in the two compartments.     

Role for autocrine TGF-β1 in regulating differentiation of a human leukemic cell line toward osteoclast-like cells

Increasing evidence suggests that transforming growth factor-β (TGF-β) is involved in bone formation during remodeling. Using a recently cloned human leukemic cell line (FLG 29.1 cells) we demonstrate that these cells synthesize and secrete TGF-β1 and that exogenous or autocrine TGF-β1 can induce the same features of osteoclastic-like cells, exerting its effects through the binding to TGF-β specific receptors. Scatchard analysis of ¹²⁵I-labeled TGF-β1 to FLG 29.1 cells revealed the presence of a single high affinity binding site with a Kd value of ～25 pM and a binding capacity of ～900 sites/cell. Affinity labeling experiments showed that FLG 29.1 cells express type I and type II TGF-β receptors. Stimulation of FLG 29.1 cells with low TGF-β1 doses reduced cell proliferation and increased cell adhesion and tartrate resistant acid phosphatase (TRAcP) activity. Pretreatment of FLG 29.1 cells with TGF-β1 caused a significant and dose-dependent response to calcitonin. Northern blot of total mRNA and analysis of the conditioned media (CM) showed that TGF-β1 was synthesized by FLG 29.1 cells. TPA treatment, which induces partial differentiation of these cells, markedly increased TGF-β1 mRNA expression and growth factor release. The majority of TGF-β1 secreted by TPA-treated cells was in its latent form. However, anti-TGF-β antibodies inhibited TGF-β1 and TPA-induced growth inhibition, calcitonin responsiveness, and TRAcP activity, suggesting that the TPA effect is mediated in part by autocrine TGF-β1 and indicating that the cells can activate and respond to the TGF-β that they secrete. These findings support a potential autocrine role for TGF-β1 in osteoclast differentiation. © 1994 Wiley-Liss, Inc.     

New species and a new combination in Myrtaceae from northeastern South America

Three new species of Myrtaceae (Calyptranthes bracteata, Eugenia gonglycocarpa, andMyrcia rupta) from northeastern South America are described and illustrated, and a new combination (Eugenia tetramera) is proposed. The closed-calyx and the completely or partially fused cotyledons ofMyrcia rupta, unusual features for the genus, are discussed and compared with related species inMyrcia andMarlierea.     

An Ultrastructural Investigation on Vitellophage Invasion of the Yolk Mass during and after Germ Band Formation in Embryos of the Stick Insect Carausius morosus Br.

Developing embryos of the stick insect Carausius morosus were examined ultrastructurally with a view to studying vitellophage invasion of the yolk mass during and after germ band formation. Newly laid eggs in C.morosus have a unique yolk fluid compartment surrounded by a narrow fringe of cytoplasm comprising several small yolk granules. Vitellophages originate mainly from a thin layer of stem cells, the so-called yolk cell membrane, interposed between the germ band and the yolk mass. Throughout development, a thin basal lamina separates the yolk cell membrane from the overlying embryo.
Vitellophages extend from the yolk cell membrane with long cytoplasmic processes or filopodia to invade the central yolk mass. Along their route of entrance, filopodia engulf portions of the yolk mass and sequester it into membrane-bounded granules. As this process continues, the yolk mass is gradually partitioned into a number of yolk granules inside the vitellophages.
Later in development, the yolk cell membrane is gradually replaced by the endodermal cells that emerge from the anterior and posterior embryonic rudiments. From this stage of development onwards, vitellophages remain attached to the basal lamina through long filopodia extending between the endodermal cells. Yolk confined in different vitellophagic cells appears heterogeneous both in density and texture, suggesting that yolk degradation may be spatially differentiated.     

Inhibition of Glutamate Dehydrogenase in Brain Mitochondria and Synaptosomes by Mg2+ and Polyamines: A Possible Cause for Its Low In Vivo Activity

Abstract: Magnesium and the polyamines putrescine, spermidine, and spermine inhibited the activity of glutamate dehydrogenase in permeabilized rat brain mitochondria in a concentration-dependent manner. The inhibitory effect was observed on both the reductive amination of 2-oxoglutarate and oxidative deamination of glutamate, as well as in the presence and absence of ADP and leucine, the allosteric activators of the enzyme. Kinetic studies at various concentrations of substrates showed that inhibition by magnesium and spermine was very pronounced at 2-oxoglutarate concentrations less than 0.5 m M and NADH levels less than 0.08 m M . The presence of the former compounds also accentuated the inhibitory effect of high concentrations of 2-oxoglutarate (>2.0 m M ) and NADH (>0.32 m M ). Addition of magnesium and spermine to suspensions of synaptosomes decreased the amount of ammonia produced from glutamate. It is suggested that polyamines and magnesium, normal constituents of mammalian brain, are responsible, at least in part, for the low glutamate dehydrogenase activity in vivo.     

Photosystem II in a mutant of Chlamydomonas reinhardtii lacking the 23 kDa psbP protein shows increased sensitivity to photoinhibition in the absence of chloride

The psbP gene product, the so called 23 kDa extrinsic protein, is involved in water oxidation carried out by Photosystem II. However, the protein is not absolutely required for water oxidation. Here we have studied Photosystem II mediated electron transfer in a mutant of Chlamydomonas reinhardtii, the FUD 39 mutant, that lacks the psbP protein. When grown in dim light the Photosystem II content in thylakoid membranes of FUD 39 is approximately similar to that in the wild-type. The oxygen evolution is dependent on the presence of chloride as a cofactor, which activates the water oxidation with a dissociation constant of about 4 mM. In the mutant, the oxygen evolution is very sensitive to photoinhibition when assayed at low chloride concentrations while chloride protects against photoinhibition with a dissociation constant of about 5 mM. The photoinhibition is irreversible as oxygen evolution cannot be restored by the addition of chloride to inhibited samples. In addition the inhibition seems to be targeted primarily to the Mn-cluster in Photosystem II as the electron transfer through the remaining part of Photosystem II is photoinhibited with slower kinetics. Thus, this mutant provides an experimental system in which effects of photoinhibition induced by lesions at the donor side of Photosystem II can be studied in vivo.Abbreviations Chl chlorophyll - DCIP 2,6-dichlorophenolindophenol - DPC 2,2-diphenylcarbonic dihydrazide - HEPES 4-(2-hydroxyethyl)-1-piperazinethanesulfonic acid - P₆₈₀ the primary electron donor to PS II - PpBQ phenyl-p-benzoquinone - PS II Photosystem II - Q_A the first quinone acceptor of PS II - Q_B the second quinone acceptor of PS II - SDS sodium dodecyl sulfate - Tris tris(hydroxymethyl)aminomethane - Tyr_D accessory electron donor on the D₂-protein - Tyr_Z tyrosine residue, acting as electron carrier between P₆₈₀ and the water oxidizing system     

Analysis by Polymerase Chain Reaction of α1 and α6 GABAA Receptor Subunit mRNAs in Individual Cerebellar Neurons After Whole-Cell Recordings

Abstract: With the use of the single-cell polymerase chain reaction (PCR), the GABA_A receptor subunit mRNA content was analyzed in granule and Purkinje neurons from rat cerebellar slices. We used an experimental protocol to assess simultaneously the presence of two subunits in each cell while electrophysiological recordings were performed with the whole-cell patch-clamp technique. Based on a computer alignment of the nucleotide sequence corresponding to α1 and α6 GABA_A receptor subunits, homologous regions were identified that allowed coamplification of both mRNAs using a single primer combination. The presence of selective restriction sites within the targeted templates allowed us to identify which receptor subunit mRNAs were coamplified by performing restriction enzyme-mediated cleavage of the amplification products. In all Purkinje neurons assayed, α1 subunit mRNA but not α6 mRNA was detected. In contrast, among individual granule neurons we found a heterogeneous distribution of the mRNA for the α1 and α6 GABA_A receptor subunits. A comparison of the results of the PCR amplification and the analysis of GABA-mediated inhibitory synaptic currents does not allow us to identify kinetic characteristics of synaptic currents that clearly correlate with the presence or the absence of α6 subunit mRNA.     

Speciation in the Artemia genus: Mitochondrial DNA analysis of bisexual and parthenogenetic brine shrimps

From the cloned mitochondrial DNAs (mtDNAs) isolated from two bisexual species, one Mediterranean, Artemia salina, and one American, Artemia franciscana, and two parthenogenetic (diploid and tetraploid) strains of Artemia parthenogenetica collected in Spain, physical maps have been constructed and compared. They are extremely different among themselves, much more than the differences between Drosophila melanogaster and D. yakuba and in the same range of different mammalian species such as mouse/rat or man/cow. The nucleotide sequences of two regions of mtDNA encoding parts of the cytochrome c oxidase subunit I (COI) and cytochrome b (Cytb) genes have been determined in the two bisexual species and the two parthenogenetic strains. Comparisons of these sequences have revealed a high degree of divergence at the nucleotide level, averaging more than 15%, in agreement with the differences found in the physical maps. The majority of the nucleotide changes are silent and there is a strong bias toward transitions, with the CT substitutions being highly predominant. The evolutionary distance between the two Artemia parthenogenetica is high and there is no clear relationship with any of the bisexual species, including the one present nowadays in Spain. Using a combination of molecular (mtDNA) and morphological markers it is possible to conclude that all of these Artemia isolates should be actually considered as belonging to different species, even the two Artemia parthenogenetica diploidica and tetraploidica.On sabbatical leave from Departamento de Bioquímica, Facultad de Veterinaria, Universidad Complutense de Madridearly Italian artemiologists to designate the Medi-Beatriz Batuecas died in an accident during the Christmas holy days of 1988 after she had initiated this workCorrespondence to: R. Garesse     

Involvement of protein phosphorylation in the sensitivity of photosystem II to strong illumination

Four types of differently phosphorylated hylakoids isolated from field grown spinach ( Spinacia oleracea L.) were tested for the sensitivity of photosystem II (PSII) to photoinactivation. Phosphorylation of light-harvesting II complexes (LHCII) protected PSII electron transfer from photoinhibitory damage, while the phosphorylation of the PSII core polypeptides slightly accelerated the decline of electron transfer during high irradiance treatment. Dephosphorylation of the CP43 apoprotein and PsbH protein by an alkaline phosphatase resulted in an extreme sensitivity of the thylakoids to strong illumination. The PSII photoinactivation of thylakoids with the impaired oxygen-evolving complex was found to be independent of phosphorylation.
The thylakoids of the thermophilic cyanobacterium Synechococcus elongates were used in order to compare the plants with an organism where LHCII complexes are missing and the PSII core proteins are not phosphorylated.     

Enhancement of extractable ethylene at light/dark transition in primary leaves of paclobutrazol-treated Phaseolus vulgaris seedlings

Paclobutrazol [(2RS,3RS)-1-(4-chlorophenyl)-4,4-dimethyl-2-(1,2,4-triazol-1-yl)pentan-3-ol], a triazole growth retardant, increased the 1-aminocyclopropane-1-carboxylic acid (ACC) level and resulted in reduced ethylene production, estimated as ethylene release in a closed system or by vacuum-extraction, in the primary leaves of Phaseolus vulgaris L. cv. Juliska seedlings exposed to light. At the light/dark transition, a definite enhancement of the endogenous ethylene level was observed by vacuum-extraction of primary leaves of treated plants and the ethylene deficiency of retardant-treated leaves ceased. The concentration of ACC after the light/dark transition followed the pattern for ethylene, and the increase in ACC content was paralleled by a decrease in malonyl-ACC.
It is concluded that the internal level of ethylene is not necessarily lower in the primary leaves of paclobutrazol-treated bean plants, but under special environmental conditions in vivo it may reach that of the control.