Analysis of ligand-binding to the kringle 4 fragment from human plasminogen

The interaction of the isolated human plasminogen kringle 4 with the four -amino acid ligands -aminocaproic acid (ACA), N-acetyl-l-lysine (AcLys), trans-aminomethyl(cyclohexane)carboxylic acid (AMCHA) and p-benzylaminesulfonic acid (BASA) has been further characterized by ¹H-NMR spectroscopy at 300 and 600 MHz. Pronounced high-field shifts, reaching 3 ppm, are observed for AMCHA resonances upon binding to kringle 4, which underscores the relevance of ligand lipophilic interactions with aromatic side chains at the binding site. Ligand titration curves for the nine His and Trp singlets found in the kringle 4 aromatic spectrum reveal a striking uniformity in the kringle response to the various ligands. The average binding curves exhibit a clear Langmuir absorption isotherm saturation profile and the data were analyzed under the assumption of one (high affinity) binding site per kringle. Equilibrium association constants (K _a ) and first order dissociation rate constants (k _off) were derived from linearized expressions of the Langmuir isotherm and of the spectral line-shapes, respectively. The results for the four ligands, at 295 K, pH^* 7.2, indicate that: (a) AMCHA exhibits the strongest binding (K _a =159 mM ^-1) and ACA the weakest (K _a =21 mM ^–1) with AcLys and BASA falling in between; (b) ACA dissociates readily (k _off = 5.3 × 10³ s^–1) and AMCHA associates the fastest (k _off = 2.0 × 10⁸ M ^–1 s^–1) while the kinetics for BASA exchange is relatively slow (k _off = 0.8 × 10³ s^–1, k _on = 0.6 × 10⁸ M ^–1s^–1); (c) the ligand-binding kinetics is close to diffussion-controlled.Abbreviations ACA -aminocaproic acid - AcLys N-acetyl-l-lysine - AMCHA t-aminomethyl(cyclohexane)carboxylic acid - BASA p-benzylaminesulfonic acid - K4 kringle 4 - NOE nuclear Overhauser effect - ppm parts-per-million - pH^* glass electrode pH reading uncorrected for deuterium isotope effects - K _a ligand-kringle 4 equilibrium association constant - k _off ligand-kringle 4 dissociation rate constant - k _on ligand-kringle 4 association rate constant     

Correlation between Body Composition and Walking Capacity in Severe Obesity

Background

Obesity is associated with mobility reduction due to mechanical factors and excessive body fat. The six-minute walk test (6MWT) has been used to assess functional capacity in severe obesity.

Objective

To determine the association of BMI, total and segmental body composition with distance walked (6MWD) during the six-minute walk test (6MWT) according to gender and obesity grade.

Setting

University of São Paulo Medical School, Brazil; Public Practice.

Methods

Functional capacity was assessed by 6MWD and body composition (%) by bioelectrical impedance analysis in 90 patients.

Results

The mean 6MWD was 514.9 ± 50.3 m for both genders. The male group (M: 545.2 ± 46.9 m) showed a 6MWD higher (p = 0.002) than the female group (F: 505.6 ± 47.9 m). The morbid obese group (MO: 524.7 ± 44.0 m) also showed a 6MWD higher (p = 0.014) than the super obese group (SO: 494.2 ± 57.0 m). There was a positive relationship between 6MWD and fat free mass (FFM), FFM of upper limps (FFM_UL), trunk (FFM_TR) and lower limbs (FFM_LL). Female group presented a positive relationship between 6MWD and FFM, FFM_UL and FFM_LL and male group presented a positive relationship between 6MWD and FFM_TR. In morbid obese group there was a positive relationship between 6MWD with FFM, FFM_UL, FFM_TR and FFM_LL. The super obese group presented a positive relationship between 6MWD with FFM, FFM_TR and FFM_LL.

Conclusions

Total and segmental FFM is associated with a better walking capacity than BMI.     

Functional Characterization of a Newly Identified Group B Streptococcus Pullulanase Eliciting Antibodies Able to Prevent Alpha-Glucans Degradation

Streptococcal pullulanases have been recently proposed as key components of the metabolic machinery involved in bacterial adaptation to host niches. By sequence analysis of the Group B Streptococcus (GBS) genome we found a novel putative surface exposed protein with pullulanase activity. We named such a protein SAP. The sap gene is highly conserved among GBS strains and homologous genes, such as PulA and SpuA, have been described in other pathogenic streptococci. The SAP protein contains two N-terminal carbohydrate-binding motifs, followed by a catalytic domain and a C-terminal LPXTG cell wall-anchoring domain. In vitro analysis revealed that the recombinant form of SAP is able to degrade α-glucan polysaccharides, such as pullulan, glycogen and starch. Moreover, NMR analysis showed that SAP acts as a type I pullulanase. Studies performed on whole bacteria indicated that the presence of α-glucan polysaccharides in culture medium up-regulated the expression of SAP on bacterial surface as confirmed by FACS analysis and confocal imaging. Deletion of the sap gene resulted in a reduced capacity of bacteria to grow in medium containing pullulan or glycogen, but not glucose or maltose, confirming the pivotal role of SAP in GBS metabolism of α-glucans. As reported for other streptococcal pullulanases, we found specific anti-SAP antibodies in human sera from healthy volunteers. Investigation of the functional role of anti-SAP antibodies revealed that incubation of GBS in the presence of sera from animals immunized with SAP reduced the capacity of the bacterium to degrade pullulan. Of interest, anti-SAP sera, although to a lower extent, also inhibited Group A Streptococcus pullulanase activity. These data open new perspectives on the possibility to use SAP as a potential vaccine component inducing functional cross-reacting antibodies interfering with streptococcal infections.     

Excitation energy transfer in intact cells and in the phycobiliprotein antennae of the chlorophyll d containing cyanobacterium Acaryochloris marina

The cyanobacterium Acaryochloris marina is unique because it mainly contains Chlorophyll d (Chl d) in the core complexes of PS I and PS II instead of the usually dominant Chl a. Furthermore, its light harvesting system has a structure also different from other cyanobacteria. It has both, a membrane-internal chlorophyll containing antenna and a membrane-external phycobiliprotein (PBP) complex. The first one binds Chl d and is structurally analogous to CP43. The latter one has a rod-like structure consisting of three phycocyanin (PC) homohexamers and one heterohexamer containing PC and allophycocyanin (APC). In this paper, we give an overview on the investigations of excitation energy transfer (EET) in this PBP-light-harvesting system and of charge separation in the photosystem II (PS II) reaction center of A. marina performed at the Technische Universität Berlin. Due to the unique structure of the PBP antenna in A. marina, this EET occurs on a much shorter overall time scale than in other cyanobacteria. We also briefly discuss the question of the pigment composition in the reaction center (RC) of PS II and the nature of the primary donor of the PS II RC.     

HisE11 and HisF8 Provide Bis-histidyl Heme Hexa-coordination in the Globin Domain of Geobacter sulfurreducens Globin-coupled Sensor

Among heme-based sensors, recent phylogenomic and sequence analyses have identified 34 globin coupled sensors (GCS), to which an aerotactic or gene-regulating function has been tentatively ascribed. Here, the structural and biochemical characterization of the globin domain of the GCS from Geobacter sulfurreducens (GsGCS¹⁶²) is reported. A combination of X-ray crystallography (crystal structure at 1.5 Å resolution), UV-vis and resonance Raman spectroscopy reveals the ferric GsGCS¹⁶² as an example of bis-histidyl hexa-coordinated GCS. In contrast to the known hexa-coordinated globins, the distal heme-coordination in ferric GsGCS¹⁶² is provided by a His residue unexpectedly located at the E11 topological site. Furthermore, UV-vis and resonance Raman spectroscopy indicated that ferrous deoxygenated GsGCS¹⁶² is a penta-/hexa-coordinated mixture, and the heme hexa-to-penta-coordination transition does not represent a rate-limiting step for carbonylation kinetics. Lastly, electron paramagnetic resonance indicates that ferrous nitrosylated GsGCS¹⁶² is a penta-coordinated species, where the proximal HisF8-Fe bond is severed.     

Cycloartane triterpene glycosides from Astragalus trigonus

Three new cycloartane glycosides, trigonoside I, II and III, and the known astragalosides I and II were isolated from the roots of Astragalus trigonus. The structures of the new glycosides were totally elucidated by high field (600 MHz) NMR analyses as cycloastragenol-6-O-β-xylopyranoside, cycloastragenol-3-O-[-l-arabinopyranosyl(1 → 2)-β-d-xylopyranosyl]-6-O-β- d-xylopyranoside and cycloastragenol-3-O-[-l-arabinopyranosyl(1 → 2)-β-d-(3-O-acetyl)-xylopyranosyl]-6-O-β-d-xylopyranoside.     

Feline ocular chlamydiosis: clinical and microbiological effects of topical and systemic therapy

Conjunctival swabs taken from a two-month-old kitten showing ocular discharge were found to be positive for Chlamydophila felis by PCR and isolation. The cat was treated with topical 1% tetracycline ophthalmic ointment twice a day for 60 days. At the end of the treatment, the cat showed no ocular signs and conjunctival swabs resulted PCR and isolation negative for C. felis. Forty days later, the ocular discharge recurred and C. felis was isolated from conjunctival swabs taken from both the cat's eyes. Twenty days of doxycycline systemic treatment at 10 mg/kg once daily was started. The treatment resulted in a complete clinical recovery after a few days. C. felis was not isolated or amplified on the 10th day after beginning the treatment. The cat's conjunctival swabs were also PCR and isolation negative on the 10th, 30th, 60th, 90th, 120th and 240th days after the end of therapy.     

Genome physical mapping from large-insert clones by fingerprint analysis with capillary electrophoresis: a robust physical map of Penicillium chrysogenum

Physical mapping with large-insert clones is becoming an active area of genomics research, and capillary electrophoresis (CE) promises to revolutionize the physical mapping technology. Here, we demonstrate the utility of the CE technology for genome physical mapping with large-insert clones by constructing a robust, binary bacterial artificial chromosome (BIBAC)-based physical map of Penicillium chrysogenum. We fingerprinted 23.1× coverage BIBAC clones with five restriction enzymes and the SNaPshot kit containing four fluorescent-ddNTPs using the CE technology, and explored various strategies to construct quality physical maps. It was shown that the fingerprints labeled with one or two colors, resulting in 40–70 bands per clone, were assembled into much better quality maps than those labeled with three or four colors. The selection of fingerprinting enzymes was crucial to quality map construction. From the dataset labeled with ddTTP–dROX, we assembled a physical map for P.chrysogenum, with 2–3 contigs per chromosome and anchored the map to its chromosomes. This map represents the first physical map constructed using the CE technology, thus providing not only a platform for genomic studies of the penicillin-producing species, but also strategies for efficient use of the CE technology for genome physical mapping of plants, animals and microbes.     

Can amphibians take the heat? Vulnerability to climate warming in subtropical and temperate larval amphibian communities

Predicting the biodiversity impacts of global warming implies that we know where and with what magnitude these impacts will be encountered. Amphibians are currently the most threatened vertebrates, mainly due to habitat loss and to emerging infectious diseases. Global warming may further exacerbate their decline in the near future, although the impact might vary geographically. We predicted that subtropical amphibians should be relatively susceptible to warming‐induced extinctions because their upper critical thermal limits (CT_max) might be only slightly higher than maximum pond temperatures (T_max). We tested this prediction by measuring CT_max and T_max for 47 larval amphibian species from two thermally distinct subtropical communities (the warm community of the Gran Chaco and the cool community of Atlantic Forest, northern Argentina), as well as from one European temperate community. Upper thermal tolerances of tadpoles were positively correlated (controlling for phylogeny) with maximum pond temperatures, although the slope was steeper in subtropical than in temperate species. CT_max values were lowest in temperate species and highest in the subtropical warm community, which paradoxically, had very low warming tolerance (CT_max–T_max) and therefore may be prone to future local extinction from acute thermal stress if rising pond T_max soon exceeds their CT_max. Canopy‐protected subtropical cool species have larger warming tolerance and thus should be less impacted by peak temperatures. Temperate species are relatively secure to warming impacts, except for late breeders with low thermal tolerance, which may be exposed to physiological thermal stress in the coming years.