Superstoichiometric Ca2+ uptake supported by hydrolysis of endogenous ATP in rat liver mitochondria.

The nature of the energy store causing rapid superstoichiometric leads to H+/2e minus ejection and leads to Ca2+/2e minus uptake ratios in rat liver mitochondria pulsed with Ca2+ has been investigated. The extent and the rate of the initial fast superstoichiometric phase of H plus ejection were greatly reduced by oligomycin and other ATPase inhibitors; the subsequent shoichiometric phase was unaffected. No such inhibition was seen with atractyloside. Similarly, the initial fast phase of Ca2+ uptake was reduced in extent by oligomycin, whereas the slower stoichiometric phase was unaffected. Moreover, the ATP content of mitochondria previously incubated with succinate decreased by about 80% within 5 s after pulsing with Ca2+. The energy store for superstoichiometric Ca2+ uptake and H plus injection is thus identified as endogenous ATP.     

Evaluation of the H+/site ratio of mitochondrial electron transport from rate measurements.

The mitochondrial H+/site ratio (i.e. the number of protons ejected per pair of electrons traversing each of the energy-conserving sites of the respiratory chain) has been evaluated employing a new experimental approach. In this method the rates of oxygen uptake and H+ ejection were measured simultaneously during the initial period of respiration evoked by addition of succinate to aerobic, rotenone-inhibited, de-energized mitochondria. Either K+, in the presence of valinomycin, or Ca2+, was used as mobile cation to dissipate the membrane potential and allow quantitative H+ ejection into the medium. The H+/site ratio observed with this method in the absence of precautions to inhibit the uptake of phosphate was close to 2.0, in agreement with values obtained using the oxygen pulse technique (Mitchell, P. and Moyle, J. (1967) Biochem. J. 105, 1147-1162). However, when phosphate movements were eliminated either by inhibition of the phosphate-hydroxide antiporter with N-ethylamaleimide or by depleting the mitochondria of their endogenous phosphate content, H+/site ratios close to 4.0 were consistently observed. This ratio was independent of the concentration of succinate, of mitochondrial protein, of pH between 6 and 8, and of ionic composition of the medium, provided that sufficient K+ (plus valinomycin) or Ca2+ were present. Specific inhibitors of the hydrolysis of endogenous ATP or transport of other ions (adenine nucleotides, tricarboxylates, HCO3-, etc.) were shown not to affect the observed H+/site ratio. Furthermore, the replacement of succinate by alpha-glycerol phosphate, a substrate which is oxidized on the outer surface of the inner membrane and thus does not need to enter the matrix, gave the same H+/site ratios as did succinate. It is concluded that the H+/site ratio of mitochondrial electron transport, when phosphate movements are eliminated, may be close to 4.0.     

Domesticity of Lutzomyia whitmani (Diptera: psychodidae) populations: field experiments indicate behavioural differences

The sandfly Lutzomyia whitmani (Antunes & Coutinho) is an important vector for cutaneous leishmaniasis throughout much of Brazil, and has recently been shown to consist of more than one mitochondrial lineage. It has frequently been asserted that the degree of adaptation of L. whitmani to human environments varies across its range. As a standardized test of indoor feeding for three geographically distant populations of L. whitmani, catches inside experimental chicken sheds of varying degrees of wall closure (0%, 33%, 67% and 98%) were compared. Each increment in shed closure reduced catches of females (relative to the most open shed) by a similar degree for each population: geometric mean catches dropped by 11-40% with 33% closure, by 41-62% with 67% closure, and by 69-100% with 98% closure. Geometric mean catches of males from the two more northerly populations also decreased with increasing shed closure, by 18% and 22% for 33% closure, 58% and 69% for 67% closure, 91% and 93% for 98% closure. Males from the most southerly population showed significantly different behaviour, with 33% closure causing a 54% increase in geometric mean catch, 67% closure causing a 6% increase, and 98% closure causing a 32% reduction. For this southerly population, sex ratios became more male biased with increasing density in more closed sheds, suggesting aggregation driven by intra-specific communication. Lutzomyia intermedia (Lutz & Neiva) was relatively more likely than L. whitmani to approach baits in the three more closed sheds, rather than the most open shed, offering a behavioural explanation for observed differences in indoor biting rates between the species.     

Hyphal heterogeneity in Aspergillus oryzae is the result of dynamic closure of septa by Woronin bodies

Hyphae of higher fungi are compartmentalized by septa. These septa contain a central pore that allows for inter‐compartmental and inter‐hyphal cytoplasmic streaming. The cytoplasm within the mycelium is therefore considered to be a continuous system. In this study, however, we demonstrate by laser dissection that 40% of the apical septa of exploring hyphae of Aspergillus oryzae are closed. Closure of septa correlated with the presence of a peroxisome‐derived organelle, known as Woronin body, near the septal pore. The location of Woronin bodies in the hyphae was dynamic and, as a result, plugging of the septal pore was reversible. Septal plugging was abolished in a ΔAohex1 strain that cannot form Woronin bodies. Notably, hyphal heterogeneity was also affected in the ΔAohex1 strain. Wild‐type strains of A. oryzae showed heterogeneous distribution of GFP between neighbouring hyphae at the outer part of the colony when the reporter was expressed from the promoter of the glucoamylase gene glaA or the α‐glucuronidase gene aguA. In contrast, GFP fluorescence showed a normal distribution in the case of the ΔAohex1 strain. Taken together, it is concluded that Woronin bodies maintain hyphal heterogeneity in a fungal mycelium by impeding cytoplasmic continuity.     

A Lipid Requirement for Photosystem I Activity in Heptane-extracted Spinach Chloroplasts

A lipid requirement for photosystem I activity in Spinacia oleracea chloroplasts has been characterized. The transfer of electrons from tetramethyl-p-phenylenediamine through the chloroplast photosystem to viologen dye was used as an assay of photosystem I activity. Activity is diminished by prolonged heptane extraction and is partially restored by readdition of the extracted lipid. Extracted chloroplasts require plastocyanin for maximal restoration of activity. The effect of lipid extract in restoration is partially replaced by triglycerides containing unsaturated, C₁₈ fatty acids. Various potential redox carriers which occur naturally in chloroplasts do not substitute for extracted lipid. Galacto-lipids, sulfolipids, and phospholipids are not involved in the restoration of activity.     

Use of AI technician scores for body condition, uterine tone and uterine discharge in a model with disease and milk production parameters to predict pregnancy risk at first AI in Holstein dairy cows

Technicians recorded body condition score (BCS) and several parameters related to estrus and/or metritis for 1694 first insemination cows on 23 farms. Additional variables for modeling the adjusted odds ratios (OR) for pregnancy were data on disease prior to or within 21 days of AI and test day milk yields. Significant predictors for pregnancy were farm, year and season, BCS, uterine tone, contaminated insemination gun after AI, fat-protein corrected kilograms milk (FPCM), days in milk (DIM), and diseases. Vaginal mucus, ease of cervical passage, and lameness were not significant predictors for pregnancy. Pregnancy risk at AI increased with increasing DIM, reaching a near optimum after 82 days. Lack of uterine tone was associated with a lowered pregnancy risk (OR = 0.69) as was contaminated insemination gun (OR = 0.67), first-parity lactation, FPCM >33 kg (OR = 0.71), BCS 2.5 at AI (OR = 0.65), clinical mastitis (OR = 0.53), cystic ovarian disease (OR = 0.53), and metritis (OR = 0.74). It was concluded that data on BCS and uterine findings, as collected by AI technicians, are significant predictors of AI outcome. Dairy producers and veterinarians should jointly examine the potential costs and value of such AI technician-based data to improve herd fertility.     

Cooperative strand invasion of supercoiled plasmid DNA by mixed linear PNA and PNA-peptide chimeras

Peptide nucleic acid (PNA) is a DNA analog with broad biotechnical applications, and possibly also treatment applications. Its suggested uses include that of a specific anchor sequence for biologically active peptides to plasmids in a sequence-specific manner. Such complexes, referred to as Bioplex, have already been used to enhance non-viral gene transfer in vitro. To investigate how hybridization of PNAs to supercoiled plasmids would be affected by the binding of multiple PNA-peptides to the same strand of DNA, we have developed a method of quantifying the specific binding of PNA using a PNA labeled with a derivative of the fluorophore thiazole orange (TO). Cooperative effects were found at a distance of up to three bases. With a peptide present at the end of one of the PNAs, steric hindrance occurred, reducing the increase in binding rate when the distance between the two sites was less than two bases. In addition, we found increased binding kinetics when two PNAs binding to overlapping sites on opposite DNA strands were used, without the use of chemically modified bases in the PNAs.     

Low genotyping error rates in non-invasively collected samples from roe deer of the Bavarian Forest National Park

Genetic wildlife monitoring is increasingly carried out on the basis of non-invasively collected samples, whereby the most commonly used DNA sources are skin appendages (hairs, feathers) and faeces. In order to guide decisions regarding future adequate ways to monitor the roe deer (Capreolus capreolus) population of the Bavarian Forest National Park in Germany, we tested these two different types of DNA source materials to compare their suitability for genetic monitoring. We determined the haplotypes (d-loop) of 19 roe deer and genotyped each individual (tissue, hairs, faeces) across 12 microsatellite loci. The amount of missing and erroneous microsatellite alleles obtained from hair and faeces samples, respectively, was estimated based on comparisons with the corresponding tissue sample control. We observed no missing alleles in hair samples, but in fecal samples PCR failed in 30 out of 228 instances (19 individuals x 12 loci), corresponding to a frequency of missing alleles of 13.2% across all loci and individuals. In genotypes generated from hairs erroneous alleles were detected in 2 out of 228 instances (0.9%), while genotypes retrieved from fecal samples displayed erroneous alleles in 6 out of 198 remaining instances (3%). We conclude that both hair and fecal samples are generally well suited for genetic roe deer monitoring, but that fecal sample based analyses require a larger sample size to account for higher PCR failure rates.