Inorganic Phosphate (Pi) Enhancement of Dark Respiration in the Pi-Limited Green Alga Selenastrum minutum (Interactions between H+/Pi Cotransport,the Plasmalemma H+-ATPase,and Dark Respiratory Carbon Flow)

Inorganic phosphate (Pi) enrichment of the Pi-limited green alga Selenastrum minutum in the dark caused a 2.5-fold increase in the rate of O2 consumption. Alkalization of the media during Pi assimilation was consistent with a H+/Pi cotransport mechanism with a stoichiometry of at least 2 H+ cotransported per Pi. Dark O2 consumption remained enhanced beyond the period of Pi assimilation and did not recover until the medium was reacidified. This result, coupled with an immediate decrease in adenylate energy charge following Pi enrichment, suggested that respiration is regulated by the ATP requirements of a plasmalemma H+-ATPase that is activated to maintain intracellular pH and provide proton motive force to power Pi uptake. Concentrations of tricarboxylic acid cycle intermediates decreased following Pi enrichment and respiratory CO2 efflux increased, indicating that the tricarboxylic acid cycle was activated to supply reductant to the mitochondrial electron transport chain. These results are consistent with direct inhibition of electron transport by ADP limitation. Enhanced rates of starch breakdown and increases in glycolytic metabolites indicated that respiratory carbon flow was activated to supply reductant to the electron transport chain and to rapidly assimilate Pi into metabolic intermediates. The mechanism that initiates glycolytic carbon flow could not be clearly identified by product:substrate ratios due to the complex nature of Pi assimilation. High levels of triose-P and low levels of phosphoenolpyruvate were the primary regulators of pyruvate kinase and phosphofructokinase, respectively.     

Vibrational analysis and molecular force field of hypoxanthine as determined from ultraviolet resonance Raman spectra of native and deuterated species

Ultraviolet resonance Raman scattering spectra from aqueous solutions of hypoxanthine and its deuterated species (C8-deuterated, N-deuterated and C8-, N-deuterated derivatives) have been collected and reported in the spectral region between 400 and 1800 cm^–1. The laser excitation wavelengths at 281 nm and 257 nm correspond to preresonance and pure resonance conditions, respectively, with the purine strongly allowed ^* electronic transition: thus the observed experimental Raman features mainly correspond to inplane vibrational modes. The latter were then assigned according to the Wilson GF method by using an empirical harmonic valence force field. Normal mode calculations are based on a non-redundant set of internal coordinates. The calculated vibrational mode wavenumbers and their isotopic shifts upon selective deuterations are in good agreement with the experimental data. The present normal mode analysis rests on the transferability of the guanine and adenine force constants proposed in recent works based on resonance Raman spectroscopy and neutron inelastic scattering data from these major purine bases. Correspondence to: M. Ghomi     

Purification and Molecular and Immunological Characterization of a Unique Phosphoribulokinase from the Green Alga Selenastrum minutum

A unique phosphoribulokinase (ADP:D-ribulose 5-phosphate 1-phosphotransferase, EC 2.7.1.19) has been purified to homogeneity from the green alga Selenastrum minutum. The enzyme has a native molecular mass of about 83 kilodaltons and a native isoelectric point of 5.1. The enzyme consists of two different-sized subunits of 41 and 40 kilodaltons, implying that it is a heterodimer. This is the first report of a eukaryotic heterodimeric phosphoribulokinase. The in vivo existence of two nonidentical subunits of S. minutum phosphoribulokinase was confirmed by western blot analysis of crude protein extracts from trichloroacetic acid-killed cells. These two subunits were immunologically similar, as rabbit immunoglobulin G affinity purified against the 41 kilodalton subunit of S. minutum phosphoribulokinase (PRK) cross-reacts with the 40 kilodalton subunit and vice versa. Antibodies against S. minutum phosphoribulokinase also cross-react with the spinach enzyme. NH₂-terminal sequencing revealed that the two S. minutum PRK subunits shared a considerable degree of structure homology with each other and with the enzymes from spinach and Chlamydomonas reinhardtii, but not with PRK from Rhodobacter sphaeroides. There are, however, differences between the NH₂-terminal amino acid sequences of the two S. minutum PRK subunits, that imply that they are the products of separate genes or products of two different mRNAs spliced from a single gene.     

In vitro recognition of DNA base pairs by histones in histone-DNA complexes and reconstituted core particles: an ultraviolet resonance Raman study.

Resonance Raman spectra of complexes between DNA and the four core histones, alone or associated, have been investigated in vitro using excitations at 300 and 257 nm, which give complementary informations about the DNA bases. H2A and H2B fractions recognize the G-C base pairs, while H3 and H4 (arginine rich fractions) recognize the A-T base pairs. The associated fractions form complexes with DNA which yield about the same DNA spectral modifications as the DNA-H4 complexes. This reveals the important role of the arginine rich fractions in the core particle formation and confirms the preferential in vitro assembly of nucleosome cores on A-T rich regions of DNA (25).     

Two unrelated phosphoenolpyruvate carboxylase polypeptides physically interact in the high molecular mass isoforms of this enzyme in the unicellular green alga Selenastrum minutum

In the chlorophyte Selenastrum minutum, phosphoenolpyruvate carboxylase (PEPC) exists as two kinetically distinct classes of isoforms sharing the same 102-kDa catalytic subunit (p102). Class 1 PEPC is homotetrameric, whereas Class 2 PEPCs consist of three large protein complexes. The different Class 2 PEPCs contain p102 and 130-, 73-, and 65-kDa polypeptides in different stoichiometric combinations. Immunoblot, immunoprecipitation, and chemical cross-linking studies indicated that p102 physically interacts with the 130-kDa polypeptide (p130) in Class 2 PEPCs. Immunological data and mass spectrometric and sequence analyses revealed that p102 and p130 are not closely related even if a p130 tryptic peptide had significant similarity to a conserved PEPC C-terminal domain from several sources. Evidence supporting the hypothesis that p130 has PEPC activity includes the following. (i) Specific activity expressed relative to the amount of p102 was lower in Class 1 than in Class 2 PEPCs; (ii) reductive pyridoxylation of both p102 and p130 was inhibited by magnesium-phosphoenolpyruvate; and (iii) biphasic phosphoenolpyruvate binding kinetics were observed with Class 2 PEPCs. These data support the view that unicellular green algae uniquely express, regulate, and assemble divergent PEPC polypeptides. This probably serves an adaptive purpose by poising these organisms for survival in different environments varying in nutrient content.     

Radiothérapie interne vectorisée par 177Lu-PSMA-617 de l’adénocarcinome prostatique métastatique résistant à la castration : à propos d’un cas et revue de la littérature

The prostate-specific membrane antigen (PSMA) is a type II glycoprotein which is over-expressed in prostate cancer tissue, especially in high grade tumours, metastatic disease, as an effect of androgen-deprivation therapies and in castrate-resistant prostate cancer (CRPC). Recent studies about radioligand therapy using PSMA ligands labeled with lutetium-177 (¹⁷⁷Lu) in CRPC patients have suggested its interest as a third line of treatment after second generation anti-androgens and chemotherapy by taxane. We present the case of a CRPC patient who was treated by iterative radioligand therapy using PSMA-617 ligand labeled with ¹⁷⁷Lu. This case illustrates on the one hand the efficacy of the treatment, and on the other hand the fragility of the patients who are at an advanced stage of their disease. Then we present a short review of the literature on this topic, focusing on the published efficacy and tolerance of ¹⁷⁷Lu-PSMA radioligand therapy of CRPC patients.     

Arylsulfatases A and B in leukocytes: a comparative statistical study of late infantile and juvenile forms of metachromatic leukodystrophy and controls

We report a statistical study on the level of aryl-sulfatases A and B in leukocytes of 106 controls, 19 cases of metachromatic leukodystrophy (MLD) infantile and juvenile forms and 25 obligate heterozygotes for MLD. Arylsulfatase A has been found to be similarly deficient in patients of the two forms. Half of the mean of the controls have been found in both types for heterozygotes. Arylsulfatase B (ASB) is slightly higher than normal in late infantile MLD although it is not statistically significant. In the 5 cases of the juvenile forms that were examined, ASB was found to be significantly reduced. This enzyme may play a role in relation to the onset of the disease.     

Characterization of small and large human peripheral blood monocytes: effects of in vitro maturation on hydrogen peroxide release and on the response to macrophage activators

Human peripheral blood monocytes (HPBM) isolated from normal donors by centrifugal elutriation were divided into two populations according to volume. (Median volumes of small monocytes (SM) and large monocytes (LM) were 255 micron and 280 micron, respectively.) H2O2 production was determined during in vitro culture and in response to bacterial lipopolysaccharide (LPS), and to recombinant human interferon-gamma (rIFN-gamma). On day 1, H2O2 production by LM was significantly greater than that by SM. In vitro culture of SM resulted in an augmented ability to produce H2O2. By day 3, SM were the major H2O2 producers. Freshly isolated SM and LM, exposed for 24 hr to LPS and rIFN-gamma, showed different patterns of activation. Both SM and LM responded to LPS, with LM responding maximally at lower doses than SM. Only SM showed a significant augmentation of H2O2 production with rIFN-gamma treatment. We also assessed the effect of in vitro culture with activation. SM but not LM showed an increased H2O2 to LPS and rIFN-gamma after 7 days in culture. Continuous exposure of SM to rIFN-gamma resulted in maximal H2O2 production at day 3 of culture; this pattern was not seen for LPS. The production of H2O2 by HPBM is related to in vitro maturation. The enhanced H2O2 production by HPBM upon exposure to rIFN-gamma may be related to the induction of in vitro maturation.