Pectin methylesterases from poplar cambium and inner bark: localization, properties and seasonal changes.

In the course of a study on the early events of cambial derivative differentiation in Populus x euramericana, seasonal changes in the pattern of pectin methylesterase (PME, EC 3.1.1.11) isoforms were followed. During the resting season, cell wall extracts contained mainly alkaline isoforms with an M(r) around 55 kDa and optimal pH between 5.6 and 6.0. Neutral isoforms with an M(r) around 35 kDa and optimal pH between 6.0 and 6.6 predominated in the extracts during the period of high meristematic activity. In the active cambial initials and in their immediate derivatives, the enzymes were immunolocalized exclusively in the dictyosomes. In older cells, they were present both in dictyosomes and in wall junctions. These results indicate that exportation of neutral PMEs towards the walls might be considered as an early marker of differentiation in cambial derivatives.

Neurospora crassa alpha-ketoglutarate dehydrogenase complex: description, resolution of components and catalytic properties.

A method is proposed for the purification of the Neurospora crassa alpha-ketoglutarate dehydrogenase complex, and the main points for preserving its activity, which seems to be particularly fragile in fungus, are discussed. Resolution of the constitutive enzymes was attempted and permitted the identification of the three protein bands resolved on SDS-polyacrylamide gel electrophoresis as E3, E1 and E2 with respective Mr values of 54,000, 53,000 and 49,000. Catalytic properties of the purified complex were established showing the importance of divalent cations in regulating the activity level. The role of Ca2+ in particular was investigated. It was shown that Ca2+ diminishes the Km value of the N. crassa alpha-ketoglutarate dehydrogenase complex for alpha-ketoglutarate in the physiological concentration range, as previously observed for the mammalian complexes.

Neurospora crassa pyruvate dehydrogenase complex: component characterization, catalytic properties and location of translation.

We propose a simplified procedure for the purification of the Neurospora crassa pyruvate dehydrogenase complex. The purified complex showed four protein bands with apparent Mr values of 53,400, 52,900, 49,000 and 36,900 upon SDS-polyacrylamide gel electrophoresis. Components, E2 and E3, of N. crassa pyruvate dehydrogenase complex were identified, respectively, as polypeptides 49,000 and 53,400. It can be deduced that component E1 is constituted of two subunits with Mr values of 52,900 and 36,900. The Km values towards different substrates and the optimal pH and temperature were determined. The protein kinase activity associated with the core enzyme was present in our most highly purified preparations. It was demonstrated that all the protein components of the complex are synthesized under the control of the nuclear genome.

[Measurement of maternal plasma volume during pregnancy]. / Mesure du volume plasmatique maternel au cours de la grossesse.

An increased maternal plasma volume (PV) is a characteristic phenomenon of normal pregnancy, which may be related to a physiological decrease of peripheral resistances. The authors have studied the plasma volume of 1,105 patients distributed as follows: normal (387), permanently hypertensive patients (84), hypertensive patients during pregnancy (390), patients with apparently isolated RCIU (154) or with a pathological past-history during previous pregnancies (90). It appears that the PV is a sign of a severe HBP, and presents a rather early and good predictive value regarding the weight of the fetus and some complications such as severe UCIU and fetal death in utero. In case of pathological past events or pre-existing hypertension, the PV enables to differentiate rather well patients who will be prone to a complicated pregnancy. In view of these results, utilization and interpretation criteria of this parameter during pregnancies with hypertension or pregnancies in which there is a suspicion or a risk of intra-uterine growth delay, are defined.

Purification of the 2-oxoglutarate dehydrogenase and pyruvate dehydrogenase complexes of Neurospora crassa mitochondria.

A simple purification procedure for the 2-oxoglutarate dehydrogenase and the pyruvate dehydrogenase complexes of Neurospora crassa mitochondria is described. After fractionated precipitations with polyethylene glycol, elimination of thiol proteins, and gel-filtration chromatography, the resulting preparations contained both activities. Covalent chromatography on thiol-activated Sepharose CL-4B allowed the specific binding of the 2-oxoglutarate dehydrogenase complex activity in the presence of 2-oxoglutarate, whereas the pyruvate dehydrogenase complex activity was retained in the presence of pyruvate. The purified 2-oxoglutarate dehydrogenase complex showed 4 protein bands by electrophoresis under dissociating conditions with apparent molecular weights of 160,000, 56,200, 55,600, 52,600 and a Km value of 3.8 X 10(-4) M for 2-oxoglutarate. The purified pyruvate dehydrogenase complex showed 5 protein bands with apparent molecular weights of 160,000, 57,600, 55,600, 52,500 and 37,100 and a Km value of 3.2 X 10(-4) M for pyruvate.

Evidence for a 2-oxoglutarate dehydrogenase complex activity in mitochondria of Neurospora crassa and compared localization with the pyruvate dehydrogenase complex.

A 2-oxoglutarate dehydrogenase complex activity is demonstrated in Neurospora crassa mitochondria. A submitochondrial fractionation by digitonin treatment followed by freeze-thawing enables measurement of a well preserved activity in the mitochondrial matrix. In contrast to other reports, the pyruvate dehydrogenase activity is also found to be localized in the matrix.

Heterogeneity of microtubule-associated proteins and brain development.

Developmental changes in the composition of brain microtubule-associated proteins have been studied in three species: the rat and the mouse, which are characterized by post-natal brain development, and the guinea-pig, whose brain is mature at birth. 1. At an adult stage, and whatever the species, two major microtubule-associated proteins, which have been referred to MAP2 and tau, have been identified by polyacrylamide gel electrophoresis. Rat tau is composed of four closely spaced bands; mouse tau contains only three components with one of them being present in higher proportion than the others; adult guinea-pig tau is essentially present as a single band. 2. Microtubule-associated proteins were also prepared at different stages of brain development. In the three species only two bands were seen in the tau region at immature stages of development (fast tau and slow tau). However adult tau factors progressively replace the young entities. In contrast, only small changes were seen in the proportion of MAP2. 3. Peptide mapping analysis of the purified tau entities confirmed that the four adult rat proteins are very similar. In contrast, peptide mapping of the two young rat tau proteins were very different from each other and from those of the adult ones. Peptide mappings of young and adult MAP2 were only slightly different. 4. The activities of young tau proteins and young MAP2 in promoting pure tubulin assembly were much lower than those of the adult ones. Young fast tau and young slow tau were purified and both show to be active in promoting pure tubulin polymerization. 5. These data demonstrate the existence of two types of heterogeneity of microtubule-associated proteins: plurality of protein species at every stage of brain development and changes in composition and activity dependent on development.

A biochemical model for neurite outgrowth during brain development.

Maximal amounts of tubulin in rat brain are observed during the 3 to 10-day postnatal period. The rates of in vitro tubulin polymerization are very low at these stages of development; they increase thereafter during the second postnatal week, reaching a maximum at adulthood. The increased rate of polymerization could depend either on modifications in the concentration and activity of microtubule-associated proteins (MAPs), which play a crucial role in microtubule assembly in vitro, or on changes in their composition. The results show that the composition and activity of TAU proteins (MW: 58-68000) change during development. Analysis of "young" and "adult" TAU protein peptide mapping suggests that their amino acid sequence is different. Our data indicate a good correlation between tubulin capacity to polymerize in vitro and changes in the composition and activity of TAU proteins which occur during the critical period when the neuronal network is constructed.

Changes in composition and activity of microtubule-associated proteins during brain development.

The onsert of neuronal differentiation is characterised by intensive neurite growth; because microtubule formation is strictly required during this process, in vitro assembly of the tubulin present in the rat brain has been studied at different stages of development: the rate of assembly is very slow in the early stages and increases progressively with age from birth until adulthood. Other data also suggested that the limiting factor in the young brain is the amount or activity of one or several of the minor components which co-polymerise into microtubules with tubulin. We show here that both the composition and the activity of the microtubule-associated proteins change during the time course of rat brain development.