Gulonolactonase in the rat: influence of sex and sex hormones on development

Gulonolactonase (D(or L)-gulono-γ-lactone hydrolase, (EC 3.1.1.18) in the kidney and liver of the rat, which are known to be identical enzymes but exhibit different patterns of post-natal development. The hepatic enzyme is detectable one day after birth, increases sharply to the adult level after nine days, and shows no appreciable sex-related difference. The renal enzyme is not detectable until approximately four days after birth in the male and ten days after birth in the female. The level of renal enzyme increases slowly in both sexes until about day 27 at which time activity in the male begins to increase rapidly while it declines slowly in the female. At this time the adult male has about 20 times as much hepatic gulonolactonase as the female. Adult enzyme levels are reached at age 44 days.The normal development increase in male renal gulonolactonase is prevented by administration of 17β-estradiol but it can be restored by subsequent administration of testosterone. Testosterone alone, or in combination with glucose does not evoke precocious induction of gulonolactonase in the male, nor does it affect its level after development has begun.Of the androgens tested for their ability to induce gulonolactonase in the kidney of the adult female, the following potency was observed: 5-androstan-3α, 17β-diol > testosterone > androstandione = androstanolone. Androsterone was without effect.     

Cytochrome c oxidase: evolution of control via nuclear subunit addition

According to theory, present eukaryotic cells originated from a beneficial association between two free-living cells. Due to this endosymbiotic event the pre-eukaryotic cell gained access to oxidative phosphorylation (OXPHOS), which produces more than 15 times as much ATP as glycolysis. Because cellular ATP needs fluctuate and OXPHOS both requires and produces entities that can be toxic for eukaryotic cells such as ROS or NADH, we propose that the success of endosymbiosis has largely depended on the regulation of endosymbiont OXPHOS. Several studies have presented cytochrome c oxidase as a key regulator of OXPHOS; for example, COX is the only complex of mammalian OXPHOS with known tissue-specific isoforms of nuclear encoded subunits. We here discuss current knowledge about the origin of nuclear encoded subunits and the appearance of different isozymes promoted by tissue and cellular environments such as hypoxia. We also review evidence for recent selective pressure acting on COX among vertebrates, particularly in primate lineages, and discuss the unique pattern of co-evolution between the nuclear and mitochondrial genomes. Finally, even though the addition of nuclear encoded subunits was a major event in eukaryotic COX evolution, this does not lead to emergence of a more efficient COX, as might be expected from an anthropocentric point of view, for the "higher" organism possessing large brains and muscles. The main function of these subunits appears to be "only" to control the activity of the mitochondrial subunits. We propose that this control function is an as yet under appreciated key point of evolution. Moreover, the importance of regulating energy supply may have caused the addition of subunits encoded by the nucleus in a process comparable to a "domestication scenario" such that the host tends to control more and more tightly the ancestral activity of COX performed by the mtDNA encoded subunits.     

Simple butyrate esterase stain for monocytes.

The esterases used to identify monocytes are best demonstrated using alpha-naphthyl butyrate as substrate. However, the reagents commonly used for this stain are time-consuming to prepare and are unstable. This report describes a quick, easy, and reproducible staining method using stable reagents which are readily available commercially but which may also be prepared in the laboratory.     

Drivers of promiscuous soybean associated rhizobia diversity in un-inoculated soil in Malawi

Increasing legume cultivation and yields on smallholder farms is challenged by low soil rhizobia bacteria populations and limited access to rhizobia inoculants. However, by understanding the environmental drivers of rhizobia diversity in un-inoculated soils to improve nodulation success for smallholder farmers. Soils were collected from 39 smallholder farms in the Ekwendeni region of northern Malawi. Soils were categorized by cropping history and analyzed for Mehlich-3 phosphorus, calcium, magnesium, potassium, iron, particle size distribution, organic matter (OM) content and pH. Rhizobia bacteria were isolated using Tropical Glycine cross (TGx) soybean (Glycine max) variety 1740-2F as a trap crop. Genomic fingerprints of extracted rhizobia were created using rep-PCR with the BOX A1R primer and diversity indexes calculated from resulting fingerprints. Genomic fingerprinting of rhizobia resulted in 32 clusters with 70 % fingerprint similarity. Soil OM and carbon strongly influenced the presence of 6 clusters, Ca of 4 clusters, pH of 3 clusters, and Mg, K, Clay of three clusters each. Recent soybean production resulted in a greater number of nodules (16) than other histories (10), and uncultivated soils had a different rhizobia community structure than cultivated soils. Soil rhizobia are subject to a complex ecology in which plant communities as well as OM, clay, and nutrient (Mg, K, Fe and P) content select for community structure. Identifying the drivers and preferred environments of high performing rhizobia strains could improve nodulation in low-input agriculture environments.     

Restricted movement by mottled sculpin (pisces: cottidae) in a southern Appalachian stream

1. We used direct observation and mark‐recapture techniques to quantify movements by mottled sculpins (Cottus bairdi) in a 1 km segment of Shope Fork in western North Carolina. Our objectives were to: (i) quantify the overall rate of sculpin movement, (ii) assess variation in movement among years, individuals, and sculpin size classes, (iii) relate movement to variation in stream flow and population size structure, and (iv) quantify relationships between movement and individual growth rates. 2. Movements were very restricted: median and mean movement distances for all sculpin size classes over a 45 day period were 1.3 and 4.4 m respectively. Nevertheless, there was a high degree of intrapopulation and temporal variation in sculpin movement. Movement of juveniles increased with discharge and with the density of large adults. Movement by small and large adults was not influenced by stream flow, but large adults where more mobile when their own density was high. Finally, there were differences in the growth rates of mobile and sedentary sculpins. Mobile juveniles grew faster than sedentary individuals under conditions of low flow and high density of large adults, whereas adults exhibited the opposite pattern. 3. Our results support the hypothesis that juvenile movement and growth is influenced by both intraspecific interactions with adults and stream flow. In contrast, adult movement appears to be influenced by competitive interactions among residents for suitable space. The relationship between movement and growth may provide a negative feedback mechanism regulating mottled sculpin populations in this system.     

Cardiac-specific norepinephrine mass transport and its relationship to left ventricular size and systolic performance

Objectives of this study were to develop a technique for quantifying cardiac-specific norepinephrine (NE) mass transport and determine whether cardiac NE kinetic modeling parameters were related to physiological variables of left ventricular (LV) size and systolic performance in nine patients with chronic mitral regurgitation. Biplane contrast cineventriculograms were used to determine LV size and ejection fraction (EF), micromanometer LV pressures and radionuclide LV volumes from a range of loading conditions to calculate LV end-systolic elastance, and [(3)H]NE infusions with LV and coronary sinus sampling for [(3)H]NE and endogenous NE during and after termination of infusions to model NE mass transport. Total NE release rate into cardiac interstitial fluid (M(IF)(R)) averaged 859 +/- 214 and NE released de novo into cardiac interstitial fluid (M(IF)(u,r,en)) averaged 546 +/- 174 pmol/min. Both M(IF)(R) and M(IF)(u,r,en)correlated directly with LV end-systolic volume (r = 0.84, P = 0.005; r = 0.86, P = 0.003); inversely with LV EFs (r = -0.75, P = 0.02; r = -0.81, P = 0.008); and inversely with LV end-systolic elastance values, optimally fit by a nonlinear function (r = 0.89, P = 0.04; r = 0.96, P = 0.01). We conclude that total and newly released NE into interstitial fluid of the heart, determined by regional mass transport kinetic model, are specific measures of regional cardiac-specific sympathetic nervous system activity and are strongly related to measures of LV size and systolic performance. These data support the concept that this new model of organ-specific NE kinetics has physiological relevance.     

Polar Positioning of a Conjugation Protein from the Integrative and Conjugative Element ICEBs1 of Bacillus subtilis

ICEBs1 is an integrative and conjugative element found in the chromosome of Bacillus subtilis. ICEBs1 encodes functions needed for its excision and transfer to recipient cells. We found that the ICEBs1 gene conE (formerly yddE) is required for conjugation and that conjugative transfer of ICEBs1 requires a conserved ATPase motif of ConE. ConE belongs to the HerA/FtsK superfamily of ATPases, which includes the well-characterized proteins FtsK, SpoIIIE, VirB4, and VirD4. We found that a ConE-GFP (green fluorescent protein) fusion associated with the membrane predominantly at the cell poles in ICEBs1 donor cells. At least one ICEBs1 product likely interacts with ConE to target it to the membrane and cell poles, as ConE-GFP was dispersed throughout the cytoplasm in a strain lacking ICEBs1. We also visualized the subcellular location of ICEBs1. When integrated in the chromosome, ICEBs1 was located near midcell along the length of the cell, a position characteristic of that chromosomal region. Following excision, ICEBs1 was more frequently found near a cell pole. Excision of ICEBs1 also caused altered positioning of at least one component of the replisome. Taken together, our findings indicate that ConE is a critical component of the ICEBs1 conjugation machinery, that conjugative transfer of ICEBs1 from B. subtilis likely initiates at a donor cell pole, and that ICEBs1 affects the subcellular position of the replisome.Integrative and conjugative elements (also known as conjugative transposons) and conjugative plasmids are key elements in horizontal gene transfer and are capable of mediating their own transfer from donor to recipient cells. ICEBs1 is an integrative and conjugative element found in some Bacillus subtilis strains. Where found, ICEBs1 is integrated into the leucine tRNA gene trnS-leu2 (Fig. ​(Fig.1)1) (7, 14, 21).Open in a separate windowFIG. 1.Genetic map of ICEBs1. conE (formerly yddE), regulatory genes (gray arrows), and genes required for integration, excision, and nicking (hatched arrows) are indicated. The number of transmembrane (TM) segments for each protein predicted by cPSORTdb (46) is indicated below each gene. Other topology programs yield similar but not identical predictions.ICEBs1 gene expression, excision, and potential mating are induced by activation of RecA during the SOS response following DNA damage (7). In addition, ICEBs1 is induced by increased production or activation of the ICEBs1-encoded regulatory protein RapI. Production and activity of RapI are indicative of the presence of potential mating partners that do not contain a copy of ICEBs1 (7). Under inducing conditions, the ICEBs1 repressor ImmR (6) is inactivated by proteolytic cleavage mediated by the antirepressor and protease ImmA (12). Most ICEBs1 genes then become highly expressed (7). One of these genes (xis) encodes an excisionase, which in combination with the element''s integrase causes efficient excision and formation of a double-stranded circle (7, 38). The circular form is nicked at the origin of transfer, oriT, by a DNA relaxase, the product of nicK (39). Under appropriate conditions, ICEBs1 can then be transferred by mating into B. subtilis and other species, including the pathogens Listeria monocytogenes and Bacillus anthracis (7). Once transferred to a recipient, ICEBs1 can be stably integrated into the genome at its attachment site in trnS-leu2 by the ICEBs1-encoded integrase (38).In contrast to what is known about ICEBs1 genes and proteins involved in excision, integration, and gene regulation, less is known about the components that make up gram-positive organisms'' mating machinery, defined as the conjugation proteins involved in DNA transfer (18, 24). The well-characterized mating machinery of gram-negative organisms can serve as a preliminary model (15, 16, 37, 48). Gram-negative organisms'' mating machinery is a type IV secretion system composed of at least eight conserved proteins that span the cell envelope. For example, the conjugation apparatus of the Agrobacterium tumefaciens Ti plasmid (pTi) is composed of 11 proteins (VirB1 through VirB11), including the ATPase VirB4 (16). VirB4 family members interact with several components of their cognate secretion systems and may energize machine assembly and/or substrate transfer (16, 48). The secretion substrate is targeted to the conjugation machinery by a coupling protein. Coupling proteins, such as VirD4 of pTi, interact with a protein attached to the end of the DNA substrate and couple the substrate to other components of the conjugation machinery. Coupling proteins might also energize the translocation of DNA through the machinery. Both VirB4 and VirD4 belong to the large HerA/FtsK superfamily of ATPases (29). Two other characterized members of this superfamily are the chromosome-partitioning proteins FtsK and SpoIIIE (29), which are ATP-dependent DNA pumps (reviewed in reference 2).Some of the proteins encoded by the conjugative elements of gram-positive organisms are homologous to components of the conjugation machinery from gram-negative organisms (1, 9, 14, 29), indicating that some aspects of conjugative DNA transfer may be similar in gram-positive and gram-negative organisms. For example, ConE (formerly YddE) of ICEBs1 has sequence similarities to VirB4 (29). YdcQ may be the ICEBs1-encoded coupling protein, as it is phylogenetically related to other coupling proteins (29, 44). Despite some similarities, the cell envelopes and many of the genes encoding the conjugation machinery are different between gram-positive and gram-negative organisms, indicating that there are likely to be significant structural and mechanistic differences as well.To begin to define the conjugation machinery of ICEBs1 and to understand spatial aspects of conjugation, we examined the function and subcellular location of ConE of ICEBs1. Our results indicate that ConE is likely a crucial ATPase component of the ICEBs1 conjugation machinery. We found that ConE and excised ICEBs1 DNA were located at or near the cell poles. We propose that the conjugation machinery is likely located at the cell poles and that mating might occur from a donor cell pole.     

Cell cycle and sporulation in Bacillus subtilis

Recent work on cell division and chromosome orientation and partitioning in Bacillus subtilis has provided insights into cell cycle regulation during growth and development. The cell cycle is an integral part of development and entrance into sporulation is modulated by signals that transmit the status of DNA integrity, chromosome replication and segregation. In addition, B. subtilis modifies cell division and DNA segregation to establish cell-type-specific gene expression during sporulation.