Connecting thiamine availability to the microbial community composition in Chinook salmon spawning habitats of the Sacramento River basin.

Thiamine deficiency complex (TDC) is a major emerging threat to global populations of culturally and economically important populations of salmonids. Salmonid eggs and embryos can assimilate exogenous thiamine, and evidence suggests that microbial communities in benthic environments can produce substantial amounts of thiamine. We therefore hypothesize that natural dissolved pools of thiamine exist in the surface water and hyporheic zones of riverine habitats where salmonids with TDC migrate, spawn, and begin their lives. To examine the relationship between dissolved thiamine-related compounds (dTRCs) and their microbial source, we determined the concentrations of these metabolites and the compositions of microbial communities in surface and hyporheic waters of the Sacramento River, California and its tributaries. Here we determine that all dTRCs are present in femto-picomolar concentrations in a range of critically important salmon spawning habitats. We observed that thiamine concentrations in the Sacramento River system are orders of magnitude lower than those of marine waters, indicating substantial differences in thiamine cycling between these two environments. Our data suggest that the hyporheic zone is likely the source of thiamine to the overlying surface water. Temporal variations in dTRC concentrations were observed where the highest concentrations existed when Chinook salmon were actively spawning. Significant correlations were seen between the richness of microbial taxa and dTRC concentrations, particularly in the hyporheic zone, which would influence the conditions where embryonic salmon incubate. Together, these results indicate a connection between microbial communities in freshwater habitats and the availability of thiamine to spawning TDC-impacted California Central Valley Chinook salmon.IMPORTANCEPacific salmon are keystone species with considerable economic importance and immeasurable cultural significance to Pacific Northwest indigenous peoples. Thiamine deficiency complex has recently been diagnosed as an emerging threat to the health and stability of multiple populations of salmonids ranging from California to Alaska. Microbial biosynthesis is the major source of thiamine in marine and aquatic environments. Despite this importance, the concentrations of thiamine and the identities of the microbial communities that cycle it are largely unknown. Here we investigate microbial communities and their relationship to thiamine in Chinook salmon spawning habitats in California's Sacramento River system to gain an understanding of how thiamine availability impacts salmonids suffering from thiamine deficiency complex.

Nonlocality in deuteron stripping reactions.

We propose a new method for the analysis of deuteron stripping reactions, A(d,p)B, in which the nonlocality of nucleon-nucleus interactions and three-body degrees of freedom are accounted for in a consistent way. The model deals with equivalent local nucleon potentials taken at an energy shifted by ∼40 MeV from the "E(d)/2" value frequently used in the analysis of experimental data, where E(d) is the incident deuteron energy. The "E(d)/2" rule lies at the heart of all three-body analyses of (d, p) reactions performed so far with the aim of obtaining nuclear structure properties such as spectroscopic factors and asymptotic normalization coefficients that are crucial for our understanding of nuclear shell evolution in neutron- and proton-rich regions of the nuclear periodic table and for predicting the cross sections of stellar reactions. The large predicted shift arises from the large relative kinetic energy of the neutron and proton in the incident deuteron in those components of the n+p+A wave function that dominate the (d, p) reaction amplitude. The large shift reduces the effective d-A potentials and leads to a change in predicted (d, p) cross sections, thus affecting the interpretation of these reactions in terms of nuclear structure.

[Distribution of Buruli ulcer in the Zè district of Benin]. / Distribution spatiale de l'ulcère de Buruli dans la commune de Zê (Bénin).

The goals of this cross-sectional study conducted in the Zè district of Benin were to determine the overall distribution and prevalence of Buruli ulcer (BU) and to identify environmental and behavioral risk factors. A total of 425 current or previous BU patients from the study district were included. Data was obtained by direct observation, semi-structured interviews, and document review. The main findings can be summarized as follows. The overall prevalence of BU in the Zè district in 2006 was 52 cases per 10000 inhabitants. The prevalence of current and previous cases was 28.1 and 23.9 per 10 000 inhabitants respectively. The distribution of BU within the district was highly variable from one subdistrict to another and from one village to another within the same subdistrict. The subdistricts showing the highest and lowest endemicity were Djigbé with 265 cases per 10 000 inhabitants and Koundokpoé with 3 cases per 10 000 inhabitants respectively. Proximity of the hamlets to water bodies was a risk factor for the disease.

Expression of individual forms of peptidylglycine alpha-amidating monooxygenase in AtT-20 cells: endoproteolytic processing and routing to secretory granules.

Peptidylglycine alpha-amidating monooxygenase (PAM: EC 1.14.17.3) is a bifunctional protein which catalyzes the COOH-terminal amidation of bioactive peptides; the NH2-terminal monooxygenase and mid-region lyase act in sequence to perform the peptide alpha-amidation reaction. Alternative splicing of the single PAM gene gives rise to mRNAs generating PAM proteins with and without a putative transmembrane domain, with and without a linker region between the two enzymes, and forms containing only the monooxygenase domain. The expression, endoproteolytic processing, storage, and secretion of this secretory granule-associated protein were examined after stable transfection of AtT-20 mouse pituitary cells with naturally occurring and truncated PAM proteins. The transfected proteins were examined using enzyme assays, subcellular fractionation, Western blotting, and immunocytochemistry. Western blots of crude membrane and soluble fractions of transfected cells demonstrated that all PAM proteins were endoproteolytically processed. When the linker region was present between the monooxygenase and lyase domains, monofunctional soluble enzymes were generated from bifunctional PAM proteins; without the linker region, bifunctional enzymes were generated. Soluble forms of PAM expressed in AtT-20 cells and soluble proteins generated through selective endoproteolysis of membrane-associated PAM were secreted in an active form into the medium; secretion of the transfected proteins and endogenous hormone were stimulated in parallel by secretagogues. PAM proteins were localized by immunocytochemistry in the perinuclear region near the Golgi apparatus and in secretory granules, with the greatest intensity of staining in the perinuclear region in cell lines expressing integral membrane forms of PAM. Monofunctional and bifunctional PAM proteins that were soluble or membrane-associated were all packaged into regulated secretory granules in AtT-20 cells.

Lung endothelial dipeptidyl peptidase IV is an adhesion molecule for lung-metastatic rat breast and prostate carcinoma cells.

Attachment of circulating tumor cells to endothelial cell adhesion molecules restricted to select vascular compartments is thought to be responsible for site-specific metastasis. Lung-metastatic rat R3230AC-MET breast and RPC-2 prostate carcinoma cells bound outside-out endothelial cell membrane vesicles, prepared by perfusion of the rat lung vasculature with a low-strength formaldehyde solution, in significantly higher numbers than their nonmetastatic counterparts R3230AC-LR and RPC-LR. In contrast, vesicles derived from the vasculature of a nonmetastasized organ (e.g., hind leg muscle) showed no binding preference for either of the four tumor cell lines. Lung-derived endothelial vesicles were used here to generate mAbs against lung endothelial cell adhesion molecules. The first group of mice were actively immunized against lung endothelial vesicles, whereas the second group was injected with syngeneic mouse antiserum against leg endothelial vesicles before active immunization with lung endothelial vesicles. 17 hybridoma supernatants obtained from the two fusions bound lung vesicles with at least a 10-fold higher affinity than leg vesicles. Seven (four obtained by a passive/active immunization protocol) stained rat capillary endothelia. One mAb, mAb 8.6A3, inhibited specific adhesion of lung-derived vesicles to lung-metastatic breast and prostate carcinoma cells. Purification of the antigen (endothelial cell adhesion molecule) from rat lung extracts revealed a protein with a 110-kD mol wt. NH2-terminal sequencing established identity with dipeptidyl peptidase IV which had been reported to serve as a fibronectin-binding protein. These results indicate that vesicles obtained from in situ perfused organs are a convenient immunogen for the production of antibodies to compartment-specific endothelial cell surface molecules, and reinforce the concept that endothelial cell surface components are selectively recognized by circulating cancer cells during metastasis formation.

The Hin invertasome: protein-mediated joining of distant recombination sites at the enhancer.

The Hin protein binds to two cis-acting recombination sites and catalyzes a site-specific DNA inversion reaction that regulates the expression of flagellin genes in Salmonella. In addition to the Hin protein and the two recombination sites that flank the invertible segment, a third cis-acting recombinational enhancer sequence and the Fis protein, which binds to two sites within the enhancer, are required for efficient recombination. Intermediates of this reaction were trapped during DNA strand cleavage and analyzed by gel electrophoresis and electron microscopy in order to determine their structure and composition. The analyses demonstrate that the recombination sites are assembled at the enhancer into a complex nucleo-protein structure (termed the invertasome) with the looping of the three segments of intervening DNA. Antibody studies indicated that Fis physically interacts with Hin and that both proteins are intimately associated with the invertasome. In order to achieve this protein-protein interaction and assemble the invertasome, the substrate DNA must be supercoiled.

Hin recombinase bound to DNA: the origin of specificity in major and minor groove interactions.

The structure of the 52-amino acid DNA-binding domain of the prokaryotic Hin recombinase, complexed with a DNA recombination half-site, has been solved by x-ray crystallography at 2.3 angstrom resolution. The Hin domain consists of a three-alpha-helix bundle, with the carboxyl-terminal helix inserted into the major groove of DNA, and two flanking extended polypeptide chains that contact bases in the minor groove. The overall structure displays features resembling both a prototypical bacterial helix-turn-helix and the eukaryotic homeodomain, and in many respects is an intermediate between these two DNA-binding motifs. In addition, a new structural motif is seen: the six-amino acid carboxyl-terminal peptide of the Hin domain runs along the minor groove at the edge of the recombination site, with the peptide backbone facing the floor of the groove and side chains extending away toward the exterior. The x-ray structure provides an almost complete explanation for DNA mutant binding studies in the Hin system and for DNA specificity observed in the Hin-related family of DNA invertases.

Near identity of cognitive structure in two ethnic groups.

As part of a large-scale family study in Hawaii, Americans of either Japanese or European ancestry were administered a battery of 15 cognitive tests. Principal component analyses (varimax rotations) yielded the same four major cognitive factors for each of the two ethnic groups, and these factors are defined by strikingly similar factor loadings.

Buruli ulcer: Evaluation of its medical and surgical management at the Allada (Benin) Screening and Treatment Center, 2010-2014.

The objective of our study was to evaluate the medico-surgical management of Buruli ulcer (BU) in the BU Screening and Treatment Center (CDTUB) of Allada in Benin. This retrospective and descriptive study retrospectively reviewed records of patients seen from 2010 to 2014 at the CDTUB of Allada. It included patients diagnosed with BU according to WHO epidemiological and clinical criteria as well as laboratory results and who were treated according to WHO medical and surgical recommendations. In all, 274 patients were diagnosed and treated, 57.7% of them children younger than 15 years. Ulcerative lesions (189, 69%) and WHO category II lesions (144, 52.5%) predominated. All patients received dual antibiotic therapy and 43.4% (119) underwent surgery as well. Category III lesions and multifocal lesions required more surgery, whereas most category I lesions healed under medical treatment. The overall rate of healing was 92%: 53.3% for patients who received only antibiotic therapy and 38.7% for those who also had surgery. The median healing time was 13 weeks and ranged from 4 to 56 weeks. In the CDTUB of Allada, between 2010 and 2014, most patients were treated with antibiotic therapy alone, but a significant number still received surgery.

The neuronal Rho-GEF Kalirin-7 interacts with PDZ domain-containing proteins and regulates dendritic morphogenesis.

Spine function requires precise control of the actin cytoskeleton. Kalirin-7, a GDP/GTP exchange factor for Rac1, interacts with PDZ proteins such as PSD-95, colocalizing with PSD-95 at synapses of cultured hippocampal neurons. PSD-95 and Kalirin-7 interact in vivo and in heterologous expression systems. In primary cortical neurons, transfected Kalirin-7 is targeted to spines and increases the number and size of spine-like structures. A Kalirin-7 mutant unable to interact with PDZ proteins remains in the cell soma, inducing local formation of aberrant filopodial neurites. Kalirin-7 with an inactivated GEF domain reduces the number of spines below control levels. These results provide evidence that PDZ proteins target Kalirin-7 to the PSD, where it regulates dendritic morphogenesis through Rac1 signaling to the actin cytoskeleton.

Mechanism of site-specific DNA inversion in bacteria.

A wealth of new information regarding the structure of the synaptic complex, the mechanism of DNA strand exchange, and the role of the recombinational enhancer in promoting DNA inversion has been obtained from a combination of approaches. These include: electron microscopy of reaction intermediates, topological analysis of recombination products, and X-ray crystallography coupled with genetic analysis.

Saxitoxin Exposure Confirmed by Human Urine and Food Analysis.

A case of an elderly female with suspected paralytic shellfish poisoning (PSP) is presented. The patient shared a meal of recreationally-harvested shellfish with her family and soon began to experience nausea and weakness. She was taken to the local emergency department and then transported to a larger hospital in Anchorage where she was admitted to the intensive care unit with respiratory depression and shock. Her condition improved, and she was discharged from the hospital 6 days later. No others who shared the meal reported symptoms of PSP. A clam remaining from the meal was collected and analyzed for paralytic shellfish toxins (PST) by the Alaska Department of Environmental Conservation Environmental Health Laboratory; the clam tested positive for saxitoxin (STX; 277 µg/100 g), neosaxitoxin (NEO; 309 µg/100 g), multiple gonyautoxins (GTX; 576-2490 µg/100 g), decarbamoyl congeners (7.52-11.3 µg/100 g) and C-toxins (10.8-221 µg/100 g) using high-pressure liquid chromatography with post-column oxidation (AOAC Method 2011.02). Urine from the patient was submitted to Centers for Disease Control for analysis of selected PSTs and creatinine. STX (64.0 µg/g-creatinine), NEO (60.0 µg/g-creatinine) and GTX1-4 (492-4780 µg/g-creatinine) were identified in the urine using online solid phase extraction with HPLC and tandem mass spectrometry. This was the first time GTX were identified in urine of a PSP case from Alaska, highlighting the need to include all STX congeners in testing to protect the public's health through a better understand of PST toxicity, monitoring and prevention of exposures.

Effects of heat stress and insulin sensitizers on pig adipose tissue.

Heat stress (HS) negatively impacts several swine production variables, including carcass fat quality and quantity. Pigs reared in HS have more adipose tissue than energetically predicted, explainable, in part, by HS-induced hyperinsulinemia. Study objectives were to evaluate insulin's role in altering fat characteristics during HS via feeding insulin-sensitizing compounds. Forty crossbred barrows (113 ± 9 kg BW) were randomly assigned to one of five environment by diet treatments: 1) thermoneutral (TN) fed ad libitum (TNAL), 2) TN and pair-fed (TNPF), 3) HS fed ad libitum (HSAL), 4) HS fed ad libitum with sterculic oil (SO) supplementation (HSSO; 13 g/d), and 5) HS fed ad libitum with dietary chromium (Cr) supplementation (HSCr; 0.5 mg/d; Kemin Industries, Des Moines, IA). The study consisted of three experimental periods (P). During P0 (2 d), all pigs were exposed to TN conditions (23 ± 3 °C, 68 ± 10% RH) and fed ad libitum. During P1 (7 d), all pigs received their respective dietary supplements, were maintained in TN conditions, and fed ad libitum. During P2 (21 d), HSAL, HSSO, and HSCr pigs were fed ad libitum and exposed to cyclical HS conditions (28 to 33 °C, 58 ± 10% RH). The TNAL and TNPF pigs remained in TN conditions and were fed ad libitum or pair-fed to their HSAL counterparts. Rectal temperature (TR), respiration rate (RR), and skin temperature (TS) were obtained daily at 0600 and 1800 h. At 1800 h, HS exposed pigs had increased TR, RR, and TS relative to TNAL controls (1.13 °C, 48 bpm, and 3.51 °C, respectively; P < 0.01). During wk 2 and 3 of P2, HSSO pigs had increased 1800 h TR relative to HSAL and HSCr (~0.40 and ~0.42 °C, respectively; P ≤ 0.05). Heat stress decreased ADFI and ADG compared to TNAL pigs (2.24 vs. 3.28 and 0.63 vs. 1.09 kg/d, respectively; P < 0.01) and neither variable was affected by SO or Cr supplementation. Heat stress increased or tended to increase moisture content of abdominal (7.7 vs. 5.9%; P = 0.07) and inner s.c. (11.4 vs. 9.8%; P < 0.05) adipose depots compared to TNAL controls. Interestingly, TNPF pigs also had increased adipose tissue moisture content and this was most pronounced in the outer s.c. depot (15.0 vs. 12.2%; P < 0.01) compared to TNAL pigs. Heat stress had little or no effect on fatty acid composition of abdominal, inner, and outer s.c. adipose tissue depots. In summary, the negative effects of HS on fat quality do not appear to be fatty acid composition related, but may be explained by increased adipose tissue moisture content.

Antimicrobial resistance in leprosy: results of the first prospective open survey conducted by a WHO surveillance network for the period 2009-15.

OBJECTIVES: Antimicrobial resistance (AMR) is a priority for surveillance in bacterial infections. For leprosy, AMR has not been assessed because Mycobacterium leprae does not grow in vitro. We aim to obtain AMR data using molecular detection of resistance genes and to conduct a prospective open survey of resistance to antileprosy drugs in countries where leprosy is endemic through a WHO surveillance network. METHODS: From 2009 to 2015, multi-bacillary leprosy cases at sentinel sites of 19 countries were studied for resistance to rifampicin, dapsone and ofloxacin by PCR sequencing of the drug-resistance-determining regions of the genes rpoB, folP1 and gyrA. RESULTS: Among 1932 (1143 relapse and 789 new) cases studied, 154 (8.0%) M. leprae strains were found with mutations conferring resistance showing 182 resistance traits (74 for rifampicin, 87 for dapsone and 21 for ofloxacin). Twenty cases showed rifampicin and dapsone resistance, four showed ofloxacin and dapsone resistance, but no cases were resistant to rifampicin and ofloxacin. Rifampicin resistance was observed among relapse (58/1143, 5.1%) and new (16/789, 2.0%) cases in 12 countries. India, Brazil and Colombia reported more than five rifampicin-resistant cases. CONCLUSIONS: This is the first study reporting global data on AMR in leprosy. Rifampicin resistance emerged, stressing the need for expansion of surveillance. This is also a call for vigilance on the global use of antimicrobial agents, because ofloxacin resistance probably developed in relation to the general intake of antibiotics for other infections as it is not part of the multidrug combination used to treat leprosy.

The Hin dimer interface is critical for Fis-mediated activation of the catalytic steps of site-specific DNA inversion.

BACKGROUND: Hin is a member of an extended family of site-specific recombinases--the DNA invertase/resolvase family--that catalyze inversion or deletion of DNA. DNA inversion by Hin occurs between two recombination sites and requires the regulatory protein Fis, which associates with a cis-acting recombinational enhancer sequence. Hin recombinase dimers bind to the two recombination sites and assemble onto the Fis-bound enhancer to generate an invertasome structure, at which time they become competent to catalyze DNA cleavage and strand exchange. In this report, we investigate the role of the Hin dimer interface in the activation of its catalytic functions. RESULTS: We show that the Hin dimer is formed at an interface that contains putative amphipathic alpha-helices in a manner that is very similar to gamma delta resolvase. Certain detergents weakened cooperative interactions between the subunits of the Hin dimer and dramatically increased the rate of the first chemical step of the reaction--double-strand cleavage events at the center of the recombination sites. Amino-acid substitutions within the dimer interface led to profound changes in the catalytic properties of the recombinase. Nearly all mutations strongly affected the ability of the dimer to cleave DNA and most abolished DNA strand exchange in vitro. Some amino-acid substitutions altered the concerted nature of the DNA cleavage events within both recombination sites, and two mutations resulted in cleavage activity that was independent of Fis activation in vitro. Disulfide-linked Hin dimers were catalytically inactive; however, subsequent to the addition of the Fis-bound enhancer sequence, catalytic activity was no longer affected by the presence of oxidizing agents. CONCLUSIONS: The combined results demonstrate that the Hin dimer interface is of critical importance for the activation of catalysis and imply that interactions with the Fis-bound enhancer may trigger a conformational adjustment within the region that is important for concerted DNA cleavage within both recombination sites, and possibly for the subsequent exchange of DNA strands.

Absence of P-selectin delays fatty streak formation in mice.

P-selectin is expressed on activated endothelium and platelets where it can bind monocytes, neutrophils, stimulated T cells, and platelets. Because recruitment of these cells is critical for atherosclerotic lesion development, we examined whether P-selectin might play a role in atherosclerosis. We intercrossed P-selectin-deficient mice with mice lacking the low density lipoprotein receptor (LDLR) because these mice readily develop atherosclerotic lesions on diets rich in saturated fat and cholesterol. The atherogenic diet stimulated leukocyte rolling in the mesenteric venules of LDLR-deficient mice, and the increase in adhesiveness of the vessels was P-selectin-dependent. Most likely due to the reduced leukocyte interaction with the vessel wall, P-selectin-deficient mice on diet for 8-20 wk formed significantly smaller fatty streaks in the cusp region of the aortae than did P-selectin-positive mice. This difference was more prominent in males. At 37 wk on diet, the lesions in the LDLR-deficient animals progressed to the fibrous plaque stage and were distributed throughout the entire aorta; their size or distribution was no longer dependent on P-selectin. Our results show that P-selectin-mediated adhesion is an important factor in the development of early atherosclerotic lesions, and that adhesion molecules such as P-selectin are involved in the complex process of atherosclerosis.

Hypoxia-induced exocytosis of endothelial cell Weibel-Palade bodies. A mechanism for rapid neutrophil recruitment after cardiac preservation.

The period of hypoxia is an important priming event for the vascular dysfunction that accompanies reperfusion, with endothelial cells (ECs) and neutrophils (PMNs) playing a central role. We hypothesized that EC Weibel-Palade (WP) body exocytosis during the hypoxic/ischemic period during organ preservation permits brisk PMN recruitment into postischemic tissue, a process further amplified in an oxidant-rich milieu. Exposure of human umbilical vein ECs to a hypoxic environment (pO2 approximately 20 torr) stimulated release of von Willebrand factor (vWF), stored in EC WP bodies, as well as increased expression of the WP body-derived PMN adhesion molecule P-selectin at the EC surface. Increased binding of 111In-labeled PMNs to hypoxic EC monolayers (compared with normoxic controls) was blocked with a blocking antibody to P-selectin, but was not affected by a nonblocking control antibody. Although increased P-selectin expression and vWF release were also noted during reoxygenation, hypoxia alone (even in the presence of antioxidants) was sufficient to increase WP body exocytosis. To determine the relevance of these observations to hypothermic cardiac preservation, during which the pO2 within the cardiac vasculature declines to similarly low levels, experiments were performed in a rodent (rat and mouse) cardiac preservation/transplantation model. Immunodepletion of recipient PMNs or administration of a blocking anti-P-selectin antibody before transplantation resulted in reduced graft neutrophil infiltration and improved graft survival, compared with identically preserved hearts transplanted into control recipients. To establish the important role of endothelial P-selectin expression on the donor vasculature, murine cardiac transplants were performed using homozygous P-selectin deficient and wild-type control donor hearts flushed free of blood/platelets before preservation/transplantation. P-selectin-null hearts transplanted into wild-type recipients demonstrated a marked (13-fold) reduction in graft neutrophil infiltration and increased graft survival compared with wild-type hearts transplanted into wild-type recipients. To determine whether coronary endothelial WP exocytosis may occur during cardiac preservation in humans, the release of vWF into the coronary sinus (CS) was measured in 32 patients during open heart surgery. CS samples obtained at the start and conclusion of the ischemic period demonstrated an increase in CS vWF antigen (by ELISA) consisting of predominantly high molecular weight multimers (by immunoelectrophoresis). These data suggest that EC WP exocytosis occurs during hypothermic cardiac preservation, priming the vasculature to recruit PMNs rapidly during reperfusion.

Mechanism for specificity by HMG-1 in enhanceosome assembly.

Assembly of enhanceosomes requires architectural proteins to facilitate the DNA conformational changes accompanying cooperative binding of activators to a regulatory sequence. The architectural protein HMG-1 has been proposed to bind DNA in a sequence-independent manner, yet, paradoxically, it facilitates specific DNA binding reactions in vitro. To investigate the mechanism of specificity we explored the effect of HMG-1 on binding of the Epstein-Barr virus activator ZEBRA to a natural responsive promoter in vitro. DNase I footprinting, mutagenesis, and electrophoretic mobility shift assay reveal that HMG-1 binds cooperatively with ZEBRA to a specific DNA sequence between two adjacent ZEBRA recognition sites. This binding requires a strict alignment between two adjacent ZEBRA sites and both HMG boxes of HMG-1. Our study provides the first demonstration of sequence-dependent binding by a nonspecific HMG-box protein. We hypothesize how a ubiquitous, nonspecific architectural protein can function in a specific context through the use of rudimentary sequence recognition coupled with cooperativity. The observation that an abundant architectural protein can bind DNA cooperatively and specifically has implications towards understanding HMG-1's role in mediating DNA transactions in a variety of enzymological systems.

Signaling mediated by the cytosolic domain of peptidylglycine alpha-amidating monooxygenase.

The luminal domains of membrane peptidylglycine alpha-amidating monooxygenase (PAM) are essential for peptide alpha-amidation, and the cytosolic domain (CD) is essential for trafficking. Overexpression of membrane PAM in corticotrope tumor cells reorganizes the actin cytoskeleton, shifts endogenous adrenocorticotropic hormone (ACTH) from mature granules localized at the tips of processes to the TGN region, and blocks regulated secretion. PAM-CD interactor proteins include a protein kinase that phosphorylates PAM (P-CIP2) and Kalirin, a Rho family GDP/GTP exchange factor. We engineered a PAM protein unable to interact with either P-CIP2 or Kalirin (PAM-1/K919R), along with PAM proteins able to interact with Kalirin but not with P-CIP2. AtT-20 cells expressing PAM-1/K919R produce fully active membrane enzyme but still exhibit regulated secretion, with ACTH-containing granules localized to process tips. Immunoelectron microscopy demonstrates accumulation of PAM and ACTH in tubular structures at the trans side of the Golgi in AtT-20 cells expressing PAM-1 but not in AtT-20 cells expressing PAM-1/K919R. The ability of PAM to interact with P-CIP2 is critical to its ability to block exit from the Golgi and affect regulated secretion. Consistent with this, mutation of its P-CIP2 phosphorylation site alters the ability of PAM to affect regulated secretion.

The effect of cold acclimation on the low molecular weight carbohydrate composition of safflower.

Understanding cold acclimation and identifying the low molecular weight carbohydrates that support the development of freezing tolerant safflower seedlings will aid in breeding winter-hardy cultivars for temperate cropping systems. Three field selected lines of winter safflower (WSRC01: PI 651878; WSRC02: PI 651879; WSRC03: PI 651880) were cold acclimated for four weeks at 4 °C and compared to seedlings grown for two weeks at 20 °C. The commercial spring-type cultivar, Olé, served as a non-hardy check. Leaf, stem, and root fructose, glucose, sucrose, raffinose, and stachyose concentrations all increased to variable extents across the PI accessions after cold acclimation. In comparison with Olé, winter safflower accessions tended to be more responsive to cold acclimation by increasing metabolite concentration. Verbascose was only recovered within leaf tissue and PI 651880 was the only entry to show a substantial alteration in verbascose concentration due to cold acclimation. Based on these data, no specific low molecular carbohydrate was responsive or responsible for the accumulation of freezing tolerance, but a concert of metabolites and their responsiveness may help explain the observed differences in development, freezing tolerance, and ultimately winterhardiness among safflower germplasm.