Small‐angle X‐ray scattering of BAMLET at pH 12: A complex of α‐lactalbumin and oleic acid

BAMLET (Bovine Alpha‐lactalbumin Made LEthal to Tumors) is a member of the family of the HAMLET‐like complexes, a novel class of protein‐based anti‐cancer complexes that incorporate oleic acid and deliver it to cancer cells. Small angle X‐ray scattering (SAXS) was performed on the complex at pH 12, examining the high pH structure as a function of oleic acid added. The SAXS data for BAMLET species prepared with a range of oleic acid concentrations indicate extended, irregular, partially unfolded protein conformations that vary with the oleic acid concentration. Increases in oleic acid concentration correlate with increasing radius of gyration without an increase in maximum particle dimension, indicating decreasing protein density. The models for the highest oleic acid content BAMLET indicate an unusual coiled elongated structure that contrasts with apo‐α‐lactalbumin at pH 12, which is an elongated globular molecule, suggesting that oleic acid inhibits the folding or collapse of the protein component of BAMLET to the globular form. Circular dichroism of BAMLET and apo‐α‐lactalbumin was performed and the results suggest that α‐lactalbumin and BAMLET unfold in a continuum of increasing degree of unfolded states. Taken together, these results support a model in which BAMLET retains oleic acid by non‐specific association in the core of partially unfolded protein, and represent a new type of lipoprotein structure. Proteins 2014; 82:1400–1408. © 2014 Wiley Periodicals, Inc.     

Catalytic electron transport in Chromatium vinosum [NiFe]-hydrogenase: application of voltammetry in detecting redox-active centers and establishing that hydrogen oxidation is very fast even at potentials close to the reversible H+/H2 value.

The nickel-iron hydrogenase from Chromatium vinosum adsorbs at a pyrolytic graphite edge-plane (PGE) electrode and catalyzes rapid interconversion of H(+)((aq)) and H(2) at potentials expected for the half-cell reaction 2H(+) right arrow over left arrow H(2), i.e., without the need for overpotentials. The voltammetry mirrors characteristics determined by conventional methods, while affording the capabilities for exquisite control and measurement of potential-dependent activities and substrate-product mass transport. Oxidation of H(2) is extremely rapid; at 10% partial pressure H(2), mass transport control persists even at the highest electrode rotation rates. The turnover number for H(2) oxidation lies in the range of 1500-9000 s(-)(1) at 30 degrees C (pH 5-8), which is significantly higher than that observed using methylene blue as the electron acceptor. By contrast, proton reduction is slower and controlled by processes occurring in the enzyme. Carbon monoxide, which binds reversibly to the NiFe site in the active form, inhibits electrocatalysis and allows improved definition of signals that can be attributed to the reversible (non-turnover) oxidation and reduction of redox centers. One signal, at -30 mV vs SHE (pH 7.0, 30 degrees C), is assigned to the [3Fe-4S](+/0) cluster on the basis of potentiometric measurements. The second, at -301 mV and having a 1. 5-2.5-fold greater amplitude, is tentatively assigned to the two [4Fe-4S](2+/+) clusters with similar reduction potentials. No other redox couples are observed, suggesting that these two sets of centers are the only ones in CO-inhibited hydrogenase capable of undergoing simple rapid cycling of their redox states. With the buried NiFe active site very unlikely to undergo direct electron exchange with the electrode, at least one and more likely each of the three iron-sulfur clusters must serve as relay sites. The fact that H(2) oxidation is rapid even at potentials nearly 300 mV more negative than the reduction potential of the [3Fe-4S](+/0) cluster shows that its singularly high equilibrium reduction potential does not compromise catalytic efficiency.     

Phosphate starvation-inducible synthesis of the alpha-subunit of the pyrophosphate-dependent phosphofructokinase in black mustard suspension cells.

PP(i)-dependent phosphofructokinase (PFP) activity, measured in the forward direction, increased approximately 19-fold when suspension cell cultures of black mustard (Brassica nigra) were subjected to 18 days of P(i) deprivation. Fructose 2,6-bisphosphate (2 microM) elicited a 10-fold activation of PFP from P(i)-deficient cells, compared to only a 2-fold activation of the enzyme from nutrient-sufficient cells. Also, PFP from P(i)-starved cells exhibited a greater affinity for the activator (Ka = 0.09 microM) than the enzyme from nutrient-sufficient cells (Ka = 0.32 microM). Western blots of extracts from P(i)-deficient cells were probed with rabbit anti-(potato tuber PFP) immune serum and revealed equal intensity staining immunoreactive polypeptides of M(r) 66,000 (alpha-subunit) and 60,000 (beta-subunit) that co-migrated with the alpha- and beta-subunits of homogeneous potato tuber PFP. By contrast, only the M(r) 60,000 beta-subunit was observed on immunoblots of extracts prepared from nutrient-sufficient cells. Quantification of immunoblots indicated that in black mustard cells experiencing transition from P(i) sufficiency to deficiency or vice versa, the relative amount of immunoreactive alpha-subunit correlated with the degree of activation of PFP by fructose 2,6-bisphosphate. These observations provide additional evidence that (i) plant PFP is an adaptive enzyme that may function in glycolysis during P(i) deprivation, and (ii) the alpha-subunit acts as a regulatory protein in controlling the catalytic activity of the beta-subunit and its regulation by fructose 2,6-bisphosphate.     

Chrysophyte cysts in 36 Canadian high arctic ponds

Chrysophyte stomatocysts were described from the surface sediments and other habitats (moss, rocks, and open water) of 36 ponds located on Cape Herschel, Ellesmere Island in the Canadian high Arctic (78°37'N). Thirty-five distinct stomatocyst types were described using scanning electron microscopy (SEM); 1 other cyst was common, but was not observed with SEM. Our study ponds were diverse limnologically and contained distinct cyst floras, but the cause(s) of the floristic differences are as yet unclear. Two ponds with extreme values of water chemistry (i.e. high salinity and low pH) supported unique cyst floras. Distinctive cyst floras were also observed in ponds with high nutrient input from nesting birds and/or with diverse microhabitats (e.g. moss banks), which may provide substrate for periphytic chrysophytes. Arctic pond cyst floras are typically less diverse than those from temperate regions. Highly ornamented cysts are also less common in arctic waters, but the reason for this is unknown. Stomatocysts could be used to augment paleolimnological research in arctic ponds, if the environmental factors controlling cyst distributions and possibly degree of ornamentation can be elucidated.     

Solution structure analysis of the periplasmic region of bacterial flagellar motor stators by small angle X-ray scattering

The bacterial flagellar motor drives the rotation of helical flagellar filaments to propel bacteria through viscous media. It consists of a dynamic population of mechanosensitive stators that are embedded in the inner membrane and activate in response to external load. This entails assembly around the rotor, anchoring to the peptidoglycan layer to counteract torque from the rotor and opening of a cation channel to facilitate an influx of cations, which is converted into mechanical rotation. Stator complexes are comprised of four copies of an integral membrane A subunit and two copies of a B subunit. Each B subunit includes a C-terminal OmpA-like peptidoglycan-binding (PGB) domain. This is thought to be linked to a single N-terminal transmembrane helix by a long unstructured peptide, which allows the PGB domain to bind to the peptidoglycan layer during stator anchoring. The high-resolution crystal structures of flagellar motor PGB domains from Salmonella enterica (MotB_C2) and Vibrio alginolyticus (PomB_C5) have previously been elucidated. Here, we use small-angle X-ray scattering (SAXS). We show that unlike MotB_C2, the dimeric conformation of the PomB_C5 in solution differs to its crystal structure, and explore the functional relevance by characterising gain-of-function mutants as well as wild-type constructs of various lengths. These provide new insight into the conformational diversity of flagellar motor PGB domains and experimental verification of their overall topology.     

In‐house monitoring of steroid hormone metabolites in urine informs breeding management of a giant anteater (Myrmechophaga tridactyla)

Although numbers of giant anteaters within North American facilities have been steadily increasing for the last 15 years, the population now exhibits an unstable age distribution with genetically valuable individuals nearing reproductive senescence. Contributing to this issue is the Association of Zoos and Aquariums (AZA) described lack of standardization of breeding introduction practices and high risk of female injury occurring during such pairings. This report describes the development of a successful breeding protocol at Cleveland Metroparks Zoo based on hormone monitoring and efficient communication between science and animal management teams that minimizes risk of female injury. By training a female giant anteater for urine sample and body weight data collection, staff members accurately predicted estrus, and timed breeding introductions to facilitate positive interactions between the male and female. Such training also allowed for careful monitoring of two pregnancies through parturition (169–184 days from breeding) and post‐partum return to estrus (114–129 days from parturition). Urinary hormone monitoring revealed a sharp progestogen increase averaging >five‐fold over basal levels (0.52 ± 0.05 ng/mg creatinine) which was sustained throughout the second half of pregnancy. Mean regular estrous cycle length (n = 14 cycles), was calculated as 46.17 ± 1.39 days, measured as days between estrogen peaks of mean concentration 2.27 ± 0.19 ng/mg creatinine. This report summarizes impressive collaborative efforts among multiple zoological departments to achieve extensive hormonal and body weight monitoring from a female giant anteater, adding valuable information on reproductive parameters, and specifics for novel hormone assay techniques.     

Nitrate reduction in sediments of lowland tropical streams draining swamp forest in Costa Rica: An ecosystem perspective

Nitrate reduction and denitrification were measured in swamp forest streams draining lowland rain forest on Costa Rica's Atlantic slope foothills using the C₂H₂-block assay and sediment-water nutrient fluxes. Denitrification assays using the C₂H₂-block technique indicated that the full suite of denitrifying enzymes were present in the sediment but that only a small fraction of the functional activity could be expressed without adding NO₃ ^–. Under optimal conditions, denitrification enzyme activity averaged 15 nmoles cm^–3 sediment h^–1. Areal NO₃ ^– reduction rates measured from NO₃ ^– loss in the overlying water of sediment-water flux chambers ranged from 65 to 470 umoles m^–2 h^–1. Oxygen loss rates accompanying NO₃ ^– depletion averaged 750 umoles m^–2 h^–1. Corrected for denitrification of NO₃ ^– oxidized from NH₄ ⁺ in the sediment, gross NO₃ ^– reduction rates increase by 130 umoles m^–2 h^–1, indicating nitrification may be the predominant source of NO₃ ^– for NO₃ ^– reduction in swamp forest stream sediments. Under field conditions approximately 80% of the increase in inorganic N mass along a 1250-m reach of the Salto River was in the form of NO₃ ^– with the balance NH₄ ⁺ . Scrutiny of potential inorganic N sources suggested that mineralized N released from the streambed was a major source of the inorganic N increase. Despite significant NO₃ ^– reduction potential, swamp forest stream sediments appear to be a source of inorganic N to downstream communities.     

Effect of surfactant and particle size reduction on hydrolysis of deinking sludge and nonrecyclable newsprint

Disposal of sludge from deinking mills represents a significant proportion of operating costs. Bioconversion of the cellulosic fraction of deinking sludge (DIS) to ethanol greatly reduces disposal costs while producing an environmentally friendly fuel. In this study, the cellulosic fraction of newsprint and deinking sludge was hydrolysed to produce fermentable sugars. For newsprint, a particle size of 1 to 1.5 mm provided optimal reaction rates in batch reactors over practical hydrolysis times, and reducing sugar concentrations as high as 35 g/L could be achieved using a fed-batch reactor configuration. For both newsprint and DIS, the hydrolysis rate increased nonlinearly with enzyme loading. Tween-80 only marginally improved sugar production but was able to release sugars from cellulosic substrates in the absence of lytic enzymes, in an amount proportional to the surfactant concentration and the substrate particle size. DIS was relatively recalcitrant to enzymatic hydrolysis, possibly due in part to inhibition by hydrophobic constituents. (c) 1995 John Wiley & Sons, Inc.     

Soybean root nodule acid phosphatase.

Acid phosphatases are ubiquitous enzymes that exhibit activity against a variety of substrates in vitro, although little is known about their intracellular function. In this study, we report the isolation, characterization, and partial sequence of the major acid phosphatase from soybean (Glycine max L.) root nodules. The phosphatase was purified predominantly as a heterodimer with subunits of 28 and 31 kD; homodimers of both subunits were also observed and exhibited phosphatase activity. In addition to the general phosphatase substrate, p-nitrophenyl phosphate, the heterodimeric form of the enzyme readily hydrolyzed 5'-nucleotides, flavin mononucleotide, and O-phospho-L-Tyr. Low or negligible activity was observed with ATP or polyphosphate. Purified nodule acid phosphatase was stimulated by magnesium, inhibited by calcium and EDTA, and competitively inhibited by cGMP and cAMP with apparent Ki values of 7 and 12 microM, respectively. Partial N-terminal and internal sequencing of the nodule acid phosphatase revealed homology to the soybean vegetative storage proteins. There was a 17-fold increase in enzyme activity and a noticeable increase in protein levels detected by immunoblotting methods during nodule development. Both of these parameters were low in young nodules and reached a peak in mature, functional nodules, suggesting that this enzyme is important for efficient nodule metabolism.