Patterns of energy metabolism and growth kinetics of Kluyveromyces marxianus in whey chemostat culture

The influence of physiological parameters such as carbon substrate flux and O₂ uptake rates on energy metabolism are reported with reference to biomass productivity in whey chemostat culture. The combined results show that oxidoreductive energy metabolism may be attained independently of the yeast reaching its maximum respiratory capacity. A novel metabolic interpretation is presented proposing that a relative imbalance between glycolysis and subsequent oxidative steps alone is sufficient to account for the observed results. By means of a mathematical model the results could be reproduced under all experimental conditions. The new interpretation provides an insight into the manner in which energy mettbolism is regulated and influences growth-related process Kluyveromyces marxianus, as well as other yeasts with similar physiological characteristics. Correspondence to: J. I. Castrillo     

A Novel Isourazole Herbicide, Fluthiacet-Methyl, is a Potent Inhibitor of Protoporphyrinogen Oxidase after Isomerization by Glutathione S-Transferase

A novel isourazole herbicide, fluthiacet-methyl (methyl [[2-chloro-4-fluoro-5-[(5,6,7,8-tetrahydro-3-oxo-lH,3H-[l,3,4]thiadiazolo[3,4-a]pyridazin-l-ylidene)amino]phenyrjthio]acetate;experimental code name, KIH-9201) promoted the leakage of electrolytesfrom cotyledons of velvetleaf (Abtilon theophtasti Medic) andcotton (Gossypium hirsutum L.) plants that are sensitive tothis compound. It induced the accumulation of protoporphyrinIX in cotyledons of cotton and inhibited Chl biosynthesis incotyledons of velvetleaf and cotton at low concentrations (I₅₀values, 10–12 nM). Fluthiacet-methyl was converted toits urazole by glutathione S-transferase that had been partiallypurified from velvetleaf. The urazole inhibited protoporphyrinogenoxidase (Protox, EC 1.3.3.4 [EC] ) from some plants, including velvetleaf,at low concentrations (I₅₀ values, 5.1–11 nM), whereasfluthiacet-methyl was not as potent. The effects in vivo (electrolyteleakage and inhibition of Chi biosynthesis) of fluthiacet-methylwere correlated with the inhibition of Protox activity by theurazole and not with the action of fluthiacet-methyl itself.From these results, it is concluded that fluthiacet-methyl inhibitsProtox activity after conversion to the corresponding urazoleby glutathione S-transferase. It is in this way that fluthiacet-methylexerts its effect as a light-dependent peroxidizing herbicide. (Received November 1, 1994; Accepted March 6, 1995)     

Synthesis and Stereochemical Characterization of Hydroxy- and Epoxy-derivatives of Rotenone

One to four routes of synthesis are described for 8′-hydroxyrotenone, 5′-hydroxyrotenone, two epimers of 6′,7′-dihydro-6′,7′-dihydroxyrotenone, two epimers of 6′,7′-epoxyrotenone and the four rotenolones derived from each of these compounds. The stereochemical relationships are determined, in each case, by chemical interconversion, ORD and monochromatic rotation to assess the absolute configuration of the B/C ring juncture and by IR, MS and NMR for the cis- or trans-nature of this juncture. The new compounds described are useful standards for studies on the metabolites and photodecomposition products of rotenone insecticide chemical.     

A field portable method for the semi‐quantitative estimation of dehydration tolerance of photosynthetic tissues across distantly related land plants

Desiccation tolerant (DT) plants withstand complete cellular dehydration, reaching relative water contents (RWC) below 30% in their photosynthetic tissues. Desiccation sensitive (DS) plants exhibit different degrees of dehydration tolerance (DHT), never surviving water loss >70%. To date, no procedure for the quantitative evaluation of DHT extent exists that is able to discriminate DS species with differing degrees of DHT from truly DT plants. We developed a simple, feasible and portable protocol to differentiate between DT and different degrees of DHT in the photosynthetic tissues of seed plants and between fast desiccation (< 24 h) tolerant (FDT) and sensitive (FDS) bryophytes. The protocol is based on (1) controlled desiccation inside Falcon tubes equilibrated at three different relative humidities that, consequently, induce three different speeds and extents of dehydration and (2) an evaluation of the average percentage of maximal photochemical efficiency of PSII (F_v/fm) recovery after rehydration. Applying the method to 10 bryophytes and 28 tracheophytes from various locations, we found that (1) imbibition of absorbent material with concentrated salt‐solutions inside the tubes provides stable relative humidity and avoids direct contact with samples; (2) for 50 ml capacity tubes, the optimal plant amount is 50–200 mg fresh weight; (3) the method is useful in remote locations due to minimal instrumental requirements; and (4) a threshold of 30% recovery of the initial F_v/fm upon reaching RWC ≤ 30% correctly categorises DT species, with three exceptions: two poikilochlorophyllous species and one gymnosperm. The protocol provides a semi‐quantitative expression of DHT that facilitates comparisons of species with different morpho‐physiological traits and/or ecological attributes.     

Regulation of the xanthophyll cycle pool size in duckweed (Lemna minor) plants

Duckweed ( Lemna minor L.) plants grown under high light are characterized, when compared to low light acclimated plants, by a higher xanthophyll cycle (VAZ) pool content, but also by a higher proportion of photoconvertible violaxanthin and a superior ability to synthesize VAZ pigments. When duckweed plants were transferred to a high light environment a general response was the quick adjustment of the carotenoid composition, mainly xanthophyll cycle pigments. These changes resulted from a balance between a process of continuous light-independent carotenoid degradation and a light-induced accumulation. The use of norflurazon, an inhibitor of carotenogenesis, allowed us to demonstrate that the observed light induced increase of the VAZ pool was mainly caused by de novo synthesis through carotenogenesis. The extent of light-induced carotenogenesis was proportional to the light treatment and also to the operation of the VAZ cycle since it was partly abolished by treatments leading to a low activity of the VAZ cycle, such as low light, DTT or DCMU. These results suggest that not only the light itself, but also a mechanism triggered by a factor associated with the de-epoxidation state of the VAZ cycle controls carotenogenesis at some point before phytoene formation in the terpenoid biosynthesis pathway.     

Ivermectin-dependent release of IL-1beta in response to ATP by peritoneal macrophages from P2X<Subscript>7</Subscript>-KO mice

The response to ATP of peritoneal macrophages from wild-type (WT) and P2X₇-invalidated (KO) mice was tested. Low concentrations (1–100 μM) of ATP transiently increased the intracellular concentration of calcium ([Ca²⁺]_i) in cells from both mice. The inhibition of the polyphosphoinositide-specific phospholipase C with U73122 inhibited this response especially in WT mice suggesting that the responses coupled to P2Y receptors were potentiated by the expression of P2X₇ receptors. One millimolar ATP provoked a sustained increase in the [Ca²⁺]_i only in WT mice. The response to 10 μM ATP was potentiated and prolonged by ivermectin in both mice. One millimolar ATP increased the influx of extracellular calcium, decreased the intracellular concentration of potassium ([K⁺]_i) and stimulated the secretion of interleukin-1β (IL-1β) only in cells from WT mice. Ten micromolar ATP in combination with 3 μM ivermectin reproduced these responses both in WT and KO mice. The secretion of IL-1β was also increased by nigericin in WT mice and the secretory effect of a combination of ivermectin with ATP in KO mice was suppressed in a medium containing a high concentration of potassium. In WT mice, 150 μM BzATP stimulated the uptake of YOPRO-1. Incubation of macrophages from WT and KO mice with 10 μM ATP resulted in a small increase of YOPRO-1 uptake, which was potentiated by addition of 3 μM ivermectin. The uptake of this dye was unaffected by pannexin-1 blockers. In conclusion, prolonged stimulation of P2X₄ receptors by a combination of low concentrations of ATP plus ivermectin produced a sustained activation of the non-selective cation channel coupled to this receptor. The ensuing variations of the [K⁺]_i triggered the secretion of IL-1β. Pore formation was also triggered by activation of P2X₄ receptors. Higher concentrations of ATP elicited similar responses after binding to P2X₇ receptors. The expression of the P2X₇ receptors was also coupled to a better response to P2Y receptors.     

The Balearic Islands: a reservoir of cpDNA genetic variation for evergreen oaks

Aim To analyse the role of the Balearic Islands as a refuge area for evergreen Quercus (cork oak: Quercus suber L., holm oak: Q. ilex L., kermes oak: Q. coccifera L.), by using molecular, historical and palaeobotanical data. Location The Western Mediterranean Basin (Balearic Islands, eastern Iberia, Provence, Sardinia, Corsica, Sicily, Malta, Italy, Northern Africa). Methods We sampled 108 populations and used the PCR‐RFLP technique with five universal cpDNA primers to define haplotypes in the sampled populations. Diversity, differentiation parameters and spatial analysis of the populations, using a spatial version of amova , were linked to the geological history of the Western Mediterranean Basin in order to explain the present spatial pattern of the evergreen Quercus populations in the Balearics. Results Evergreen Quercus cpDNA shows a complex structure, with remnants of ancient diversity in the Balearics. Balearic populations of holm oak are related to Iberian populations, while for cork and kermes oaks, we found both Tyrrhenian and Iberian haplotypes. Main conclusions The complex spatial patterns of cpDNA in Balearic evergreen Quercus appears explicable in terms of a combination of physical (vicariance and long distance dispersal) and biological (introgressive hybridization) factors. The Balearics constitute a glacial refuge area and a reservoir of genetic variation with traces of ancient diversity from Messinian–Pliocene stages.     

The HIV-Brazil Cohort Study: Design,Methods and Participant Characteristics

Background

The HIV-Brazil Cohort Study was established to analyze the effectiveness of combination antiretroviral therapy (cART) and the impact of this treatment on morbidity, quality of life (QOL) and mortality. The study design, patients’ profiles and characteristics of cART initiation between 2003 and 2010 were described.

Methodology/Principal Findings

Since 2003, the HIV-Brazil Cohort has been following HIV-infected adults receiving cART at 26 public health care facilities, using routine clinical care data and self-reported QOL questionnaires. When not otherwise available, data are obtained from national information systems. The main outcomes of interest are diseases related or unrelated to HIV; suppression of viral replication; adverse events; virological, clinical and immunological failures; changes in the cART; and mortality. For the 5,061 patients who started cART between 2003 and 2010, the median follow-up time was 4.1 years (IQR 2.2–5.9 years) with an 83.4% retention rate. Patient profiles were characterized by a predominance of men (male/female ratio 1.7∶1), with a mean age of 36.9 years (SD 9.9 years); 55.2% had been infected with HIV via heterosexual contact. The majority of patients (53.4%) initiated cART with a CD4⁺ T-cell count ≤200 cells/mm³. The medications most often used in the various treatment regimens were efavirenz (59.7%) and lopinavir/ritonavir (18.2%). The proportion of individuals achieving viral suppression within the first 12 months of cART use was 77.4% (95% CI 76.1–78.6). Nearly half (45.4%) of the patients presented HIV-related clinical manifestations after starting cART, and the AIDS mortality rate was 13.9 per 1,000 person-years.

Conclusions/Significance

Results from cART use in the daily practice of health services remain relatively unknown in low- and middle-income countries, and studies with the characteristics of the HIV-Brazil Cohort contribute to minimizing these shortcomings, given its scope and patient profile, which is similar to that of the AIDS epidemic in the country.