Synthesis of ribosomal proteins during growth of Streptomyces coelicolor

Changes in expression of ribosomal protein genes during growth and stationary phase of Streptomyces coelicolor A3(2) in liquid medium were studied. Proteins being synthesized were pulse-labelled with [³⁵ S]-methionine, separated by two-dimensional poly-acrylamide gel electrophoresis, and quantified using the Bioimage computer software. Most of the ribosomal proteins were synthesized throughout the life cycle. Exceptions were two proteins whose synthesis drastically decreased at the approach of stationary phase. These two proteins were identified in purified ribosomes as homologues of Escherichia coli ribosomal proteins L10 and L7/L12, using antibodies raised against fusion proteins between these ribosomal proteins and Escherichia coliβ-galactosldase. The genes (rplJ and rplL) encoding the L10 and L7/L12 proteins were contained in a 1.2 kb BamHl fragment that was cloned and sequenced. The linkage and order of the genes coincide with other L10-L7/L12 operons. However, L11 and L1 genes were not present immediately upstream of the L10 gene, as is the case for E. coli and other bacteria. Instead, two open reading frames of unknown function were found immediately upstream of the L10 gene, in an adjacent 1.9 kb BamHl fragment.     

Human aldehyde dehydrogenase: chromosomal assignment of the gene for the isozyme that metabolizes γ-aminobutyraldehyde

A cDNA clone of the E3 isozyme of human liver aldehyde dehydrogenase consisting of a 1320-base pair (bp) coding region and a 180-bp non-coding region at the 3 end was used for chromosomal localization of the E3 gene. Using a panel of human/hamster somatic cell hybrids we have localized, the gene coding for the E3 isozyme to human chromosome 1.     

Evading pre-existing anti-hinge antibody binding by hinge engineering

Antigen-binding fragments (Fab) and F(ab′)₂ antibodies serve as alternative formats to full-length anti-bodies in therapeutic and immune assays. They provide the advantage of small size, short serum half-life, and lack of effector function. Several proteases associated with invasive diseases are known to cleave antibodies in the hinge-region, and this results in anti-hinge antibodies (AHA) toward the neoepitopes. The AHA can act as surrogate Fc and reintroduce the properties of the Fc that are otherwise lacking in antibody fragments. While this response is desired during the natural process of fighting disease, it is commonly unwanted for therapeutic antibody fragments. In our study, we identify a truncation in the lower hinge region of the antibody that maintains efficient proteolytic cleavage by IdeS protease. The resulting neoepitope at the F(ab′)₂ C-terminus does not have detectable binding of pre-existing AHA, providing a practical route to produce F(ab′)₂ in vitro by proteolytic digestion when the binding of pre-existing AHA is undesired. We extend our studies to the upper hinge region of the antibody and provide a detailed analysis of the contribution of C-terminal residues of the upper hinge of human IgG1, IgG2 and IgG4 to pre-existing AHA reactivity in human serum. While no pre-existing antibodies are observed toward the Fab of IgG2 and IgG4 isotype, a significant response is observed toward most residues of the upper hinge of human IgG1. We identify a T₂₂₅L variant and the natural C-terminal D₂₂₁ as solutions with minimal serum reactivity. Our work now enables the production of Fab and F(ab′)₂ for therapeutic and diagnostic immune assays that have minimal reactivity toward pre-existing AHA.     

Examining variation in the leaf mass per area of dominant species across two contrasting tropical gradients in light of community assembly

Understanding variation in key functional traits across gradients in high diversity systems and the ecology of community changes along gradients in these systems is crucial in light of conservation and climate change. We examined inter‐ and intraspecific variation in leaf mass per area (LMA) of sun and shade leaves along a 3330‐m elevation gradient in Peru, and in sun leaves across a forest–savanna vegetation gradient in Brazil. We also compared LMA variance ratios (T‐statistics metrics) to null models to explore internal (i.e., abiotic) and environmental filtering on community structure along the gradients. Community‐weighted LMA increased with decreasing forest cover in Brazil, likely due to increased light availability and water stress, and increased with elevation in Peru, consistent with the leaf economic spectrum strategy expected in colder, less productive environments. A very high species turnover was observed along both environmental gradients, and consequently, the first source of variation in LMA was species turnover. Variation in LMA at the genus or family levels was greater in Peru than in Brazil. Using dominant trees to examine possible filters on community assembly, we found that in Brazil, internal filtering was strongest in the forest, while environmental filtering was observed in the dry savanna. In Peru, internal filtering was observed along 80% of the gradient, perhaps due to variation in taxa or interspecific competition. Environmental filtering was observed at cloud zone edges and in lowlands, possibly due to water and nutrient availability, respectively. These results related to variation in LMA indicate that biodiversity in species rich tropical assemblages may be structured by differential niche‐based processes. In the future, specific mechanisms generating these patterns of variation in leaf functional traits across tropical environmental gradients should be explored.     

Climate and amphibian body size: a new perspective gained from the fossil record

In recent years several studies have been carried out to test the validity of Bergmann's rule for amphibians, and have generated varying results. Due to the lack of agreement on this topic, here we examine the relationship between climate and body size for one anuran species (Bufo calamita, commonly known as the natterjack toad) with a new methodological approach that uses the fossil record as the data source. We analysed bones from two archaeo‐paleontological sites located close to each other in the Sierra de Atapuerca (Burgos, Spain) that together encompass more than one million years from Early to Late Pleistocene. We used ordinary least squares (OLS) simple regression models to integrate body size and palaeoclimatic data (temperature and precipitation) and describe the relationship between the amphibian's body size and climate along the entire temporal gradient. We found the body size of B. calamita to be negatively related to the mean annual temperature and the mean temperature of the coldest month, and positively related to December‐to‐February precipitation. The climatic influence was stronger in females, which were smaller than males in most cases, and therefore an inverse sexual size dimorphism pattern was found. Juvenile individuals or the limited sexual size dimorphism of this species may be causes of this unusual pattern. Bufo calamita populations showed a clear Bergmann cline during the Pleistocene period, and winter stands out as the most influential season. Although this new methodology can only be used to describe patterns, we discuss several mechanisms that could explain our results. We propose that starvation resistance and delayed maturation are the main causes for this increase in size in periods with cooler winters, and a fecundity‐related hypothesis to explain why climate has a greater influence on female body size.     

Protectors,Aggressors, and Kinfolk: Dogs in a Tribal Community

Free-roaming dogs are a common phenomenon on many American Indian reservations as well as globally. Lack of canine restriction may be pathologized by outsiders, assumed to be a “problem” that reflects underlying individual or community dysfunction. Seldom investigated are the cultural logics underlying the lack of restriction, and the positive role that dogs may be playing in the community. This paper examines relationships between a northern plains reservation community and their dogs. We found these relationships to be complex and multifaceted, harkening back to a pre-contact past when human survival itself depended on the dog, and extending into a present shaped by a broad range of cultural notions about the human–dog relationship. We explore the concept of dog restriction, asking what it means for connections with dogs in a context where relationships with dogs run deep, but have been disrupted by settler colonialism. We found a community that very much desires dogs and views them positively, with their role as protector highly valued on nearly every level. While traditional notions guided many behaviors toward dogs, other conceptualizations were simultaneously in play, including rural ideas about animals as well as American popular culture. Our findings call into question the ethnocentric bias that construes all free-roaming dogs as strays, which is linked to cultural notions of “pet ownership” that equate love for dogs with restrictions on their movement.     

Inflammatory and Anti-inflammatory Responses Co-exist Inside Lung Granuloma of Fatal Cases of Coccidioidomycosis: A Pilot Report

Coccidioidomycosis is a fungal disease caused by Coccidioides immitis or Coccidioides posadasii. These fungi are endemic in the southern USA and northern Mexico. Immunocompromised patients are susceptible to develop severe forms of this fungal infection. Cytokines play an important role in controlling the fungal infection, but little is known about the predominant immunological environment in human lung tissue from fatal cases. Our aim was to analyze the pro-inflammatory and anti-inflammatory cytokines and monocyte/macrophages markers (CD14 and CD206) in the granulomas of six fatal cases of coccidioidomycosis. Cytokines and surface markers were higher in coccidioidomycosis cases when compared to control (P?<?0.05). CD14 positive cells were increased inside the coccidioidal granuloma when compared to the outside (P?<?0.05). No differences were found in the number of CD206+ cells inside the granuloma when compared to the outer population (P?>?0.05). Interestingly, an analysis of stain intensity signals showed an increased signaling of CD14, CD206, IL-10 and TNFα inside the granuloma when compared to the outside (P?<?0.05). iNOS and IL-12 gene expression were not detected in coccidioidomycosis cases, while IL-10, IL-6 and TGFβ gene expression were detected, but the differences when compared to healthy lungs were not significant (P?>?0.05). TNFα gene expression was lower in coccidioidomycosis cases when compared to healthy lung (P?=?0.05). In conclusion, pro- and anti-inflammatory responses co-exist inside of the granulomas of fatal cases of coccidioidomycosis and the absent of iNOS and IL-12 gene expression may be related with patient’s outcome.     

Orthogonikleithrus leichi n. gen., n. sp. (pisces: teleostei) from the late jurassic of Germany

Orthogonikleithrus leichi n. gen and n. sp. from the Late Jurassic of Zandt, W-Germany resemblesLeptolepides in the supra- and infraorbital sensory canals and in the length of the anterior process of the maxilla, andAscalabos in the massive aspect of the premaxilla. The caudal skeleton has some similarities with that ofLeptolepides (e. g. the broadening of the neural and haemal spines of the last caudal vertebrae, length of uroneurals 1 and 2), but also with that ofAnaethalion (in the neural arches above preural centrum 1 and ural centra). According to this combination of features, the fish remains as Elopocephala incertae sedis.     

Isolation and characterization of granules of the toad bladder

Summary The electron-dense granules that lie just below the apical plasma membrane of granular epithelial cells of toad urinary bladder contribute glycoproteins to that apical membrane. Also, exocytosis of granules (and tubules) elicited by antidiuretic hormone potentially doubles that apical surface, during the same period the transport changes characteristic of the hormonal response occur.Granules separated from other membrane systems of the cells provide the material to assess the importance of the granules as glycocalyx precursors and in hormone action. We used isosmotic media to effect preliminary separations by differential centrifugation. Then granules were isolated by centrifugation on self-forming gradients of Percoll of decreasing hypertonicity.We find qualitative and quantitative changes in protein composition and enzymic activities in the isolated fractions. The primary criterion for granule purification was electron microscopic morphology. In addition, polypeptide species found in the granule fraction are limited in number and quantity. The granules are enzymically and morphologically not lysosomal in nature. Granules may provide the glycoproteins of the apical glycocalyx but they differ from the isolated plasma membrane fraction enzymically, in protein composition and in proportion of esterified cholesterol.We conclude that the granules are not average plasma membrane precursors. Their role in the membrane properties of the toad urinary bladder may now be evaluated by characterizing permeability and other properties of the isolated organelles.     

Bovine Brain Coated Vesicles Contain Adenosine A1 Receptors. Presence of Adenylate Cyclase Coupled to the Receptor

Clathrin-coated vesicles purified from bovine brain express adenosine A1 receptor binding activity. N6-Cyclohexyl[3H]adenosine [( 3H]CHA), an agonist for the A1 receptor, binds specifically to coated vesicles. High and low agonist affinity states of the receptor for the radioligand [3H]CHA with KD values of 0.18 and 4.4 nM, respectively, were detected. The high purity of coated vesicles was established by assays for biochemical markers and by electron microscopy. Binding competition experiments using agonists (N6CHA, N-cyclopentyladenosine, 5'-(N-ethylcarboxamido)adenosine, and N6-[(R)- and N6-[(S)-phenylisopropyl]adenosine) and antagonists (theophylline, 3-isobutyl-1-methylxanthine, and caffeine) confirmed the typical adenosine A1 nature of the binding site. This binding site presents stereospecificity for N6-phenylisopropyladenosine, showing 33 times more affinity for N6-[(R)- than for N6-[(S)-phenylisopropyl]adenosine. The specific binding of [3H]CHA in coated vesicles is regulated by guanine nucleotides. [3H]CHA specific binding was decreased by 70% in the presence of the hydrolysis-resistant GTP analogue guanyl-5-yl-imidodiphosphate. Bovine brain coated vesicles present adenylate cyclase activity. This activity was modulated by forskolin and CHA. The results of this study support the evidence that adenosine A1 receptors present in coated vesicles are coupled to adenylate cyclase activity through a Gi protein.