Using a Combination of Indirect Calorimetry, Infrared Thermography, and Blood Glucose Levels to Measure Brown Adipose Tissue Thermogenesis in Humans.

In mammals, brown adipose tissue (BAT) is activated rapidly in response to cold in order to maintain body temperature. Although BAT has been studied greatly in small animals, it is difficult to measure the activity of BAT in humans. Therefore, little is known about the heat-generating capacity and physiological significance of BAT in humans, including the degree to which components of the diet can activate BAT. This is due to the limitations in the currently most used method to assess the activation of BAT-radiolabeled glucose (fluorodeoxyglucose or 18FDG) measured by positron emission tomography-computerized tomography (PET-CT). This method is usually performed in fasted subjects, as feeding induces glucose uptake by the muscles, which can mask the glucose uptake into the BAT. This paper describes a detailed protocol for quantifying total-body human energy expenditure and substrate utilization from BAT thermogenesis by combining indirect calorimetry, infrared thermography, and blood glucose monitoring in carbohydrate-loaded adult males. To characterize the physiological significance of BAT, measures of the impact of BAT activity on human health are critical. We demonstrate a protocol to achieve this by combining carbohydrate loading and indirect calorimetry with measurements of supraclavicular changes in temperature. This novel approach will help to understand the physiology and pharmacology of BAT thermogenesis in humans.

5-HT3 Receptors on Mitochondria Influence Mitochondrial Function.

The 5-hydroxytryptamine 3 (5-HT3) receptor belongs to the pentameric ligand-gated cation channel superfamily. Humans have five different 5-HT3 receptor subunits: A to E. The 5-HT3 receptors are located on the cell membrane, but a previous study suggested that mitochondria could also contain A subunits. In this article, we explored the distribution of 5-HT3 receptor subunits in intracellular and cell-free mitochondria. Organelle prediction software supported the localization of the A and E subunits on the inner membrane of the mitochondria. We transiently transfected HEK293T cells that do not natively express the 5-HT3 receptor with an epitope and fluorescent protein-tagged 5HT3A and 5HT3E subunits. Fluorescence microscopy and cell fractionation indicated that both subunits, A and E, localized to the mitochondria, while transmission electron microscopy revealed the location of the subunits on the mitochondrial inner membrane, where they could form heteromeric complexes. Cell-free mitochondria isolated from cell culture media colocalized with the fluorescent signal for A subunits. The presence of A and E subunits influenced changes in the membrane potential and mitochondrial oxygen consumption rates upon exposure to serotonin; this was inhibited by pre-treatment with ondansetron. Therefore, it is likely that the 5-HT3 receptors present on mitochondria directly impact mitochondrial function and that this may have therapeutic implications.

Phylogenetic analyses of 5-hydroxytryptamine 3 (5-HT3) receptors in Metazoa.

The 5-hydroxytrptamine 3 (5-HT3) receptor is a member of the 'Cys-loop' family and the only pentameric ligand gated ion channel among the serotonin receptors. 5-HT3 receptors play an important role in controlling growth, development, and behaviour in animals. Several 5-HT3 receptor antagonists are used to treat diseases (e.g., irritable bowel syndrome, nausea and emesis). Humans express five different subunits (A-E) enabling a variety of heteromeric receptors to form but all contain 5HT3A subunits. However, the information available about the 5-HT3 receptor subunit occurrence among the metazoan lineages is minimal. In the present article we searched for 5-HT3 receptor subunit homologs from different phyla in Metazoa. We identified more than 1000 5-HT3 receptor subunits in Metazoa in different phyla and undertook simultaneous phylogenetic analysis of 526 5HT3A, 358 5HT3B, 239 5HT3C, 70 5HT3D, and 173 5HT3E sequences. 5-HT3 receptor subunits were present in species belonging to 11 phyla: Annelida, Arthropoda, Chordata, Cnidaria, Echinodermata, Mollusca, Nematoda, Orthonectida, Platyhelminthes, Rotifera and Tardigrada. All subunits were most often identified in Chordata phylum which was strongly represented in searches. Using multiple sequence alignment, we investigated variations in the ligand binding region of the 5HT3A subunit protein sequences in the metazoan lineage. Several critical amino acid residues important for ligand binding (common structural features) are commonly present in species from Nematoda and Platyhelminth gut parasites through to Chordata. Collectively, this better understanding of the 5-HT3 receptor evolutionary patterns raises possibilities of future pharmacological challenges facing Metazoa including effects on parasitic and other species in ecosystems that contain 5-HT3 receptor ligands.

Both caffeine and Capsicum annuum fruit powder lower blood glucose levels and increase brown adipose tissue temperature in healthy adult males.

Using a combination of respiratory gas exchange, infrared thermography, and blood glucose (BGL) analysis, we have investigated the impact of Capsicum annuum (C. annuum) fruit powder (475 mg) or caffeine (100 mg) on metabolic activity in a placebo controlled (lactose, 100 mg) double-blinded three-way cross-over-design experiment. Metabolic measurements were made on day 1 and day 7 of supplementation in eight adult male participants (22.2 ± 2 years of age, BMI 23 ± 2 kg/m2, x̅ ± SD). Participants arrived fasted overnight and were fed a high carbohydrate meal (90 g glucose), raising BGL from fasting baseline (4.4 ± 0.3 mmol/L) to peak BGL (8.5 ± 0.3 mmol/L) 45 min after the meal. Participants consumed the supplement 45 min after the meal, and both caffeine and C. annuum fruit powder restored BGL (F (8,178) = 2.2, p = 0.02) to near fasting levels within 15 min of supplementation compared to placebo (120 min). In parallel both supplements increased energy expenditure (F (2, 21) = 175.6, p < 0.001) over the 120-min test period (caffeine = 50.74 ± 2 kcal/kg/min, C. annuum fruit = 50.95 ± 1 kcal/kg/min, placebo = 29.34 ± 1 kcal/kg/min). Both caffeine and C. annuum fruit powder increased supraclavicular fossa temperature (F (2,42) = 32, p < 0.001) on both day 1 and day 7 of testing over the 120-min test period. No statistical difference in core temperature or reference point temperature, mean arterial pressure or heart rate was observed due to supplementation nor was any statistical difference seen between day 1 and day 7 of intervention. This is important for implementing dietary ingredients as potential metabolism increasing supplements. Together the results imply that through dietary supplements such as caffeine and C. annuum, mechanisms for increasing metabolism can be potentially targeted to improve metabolic homeostasis in people.

Modulation of Inflammatory Cytokine Production in Human Monocytes by cGMP and IRAK3.

Interleukin-1 receptor-associated kinase-3 (IRAK3) is a critical checkpoint molecule of inflammatory responses in the innate immune system. The pseudokinase domain of IRAK3 contains a guanylate cyclase (GC) centre that generates small amounts of cyclic guanosine monophosphate (cGMP) associated with IRAK3 functions in inflammation. However, the mechanisms of IRAK3 actions are poorly understood. The effects of low cGMP levels on inflammation are unknown, therefore a dose-response effect of cGMP on inflammatory markers was assessed in THP-1 monocytes challenged with lipopolysaccharide (LPS). Sub-nanomolar concentrations of membrane permeable 8-Br-cGMP reduced LPS-induced NFκB activity, IL-6 and TNF-α cytokine levels. Pharmacologically upregulating cellular cGMP levels using a nitric oxide donor reduced cytokine secretion. Downregulating cellular cGMP using a soluble GC inhibitor increased cytokine levels. Knocking down IRAK3 in THP-1 cells revealed that unlike the wild type cells, 8-Br-cGMP did not suppress inflammatory responses. Complementation of IRAK3 knockdown cells with wild type IRAK3 suppressed cytokine production while complementation with an IRAK3 mutant at GC centre only partially restored this function. Together these findings indicate low levels of cGMP form a critical component in suppressing cytokine production and in mediating IRAK3 action, and this may be via a cGMP enriched nanodomain formed by IRAK3 itself.

A systematic review and meta-analyses of interleukin-1 receptor associated kinase 3 (IRAK3) action on inflammation in in vivo models for the study of sepsis.

BACKGROUND: Interleukin-1 receptor associated kinase 3 (IRAK3) is a critical modulator of inflammation and is associated with endotoxin tolerance and sepsis. Although IRAK3 is known as a negative regulator of inflammation, several studies have reported opposing functions, and the temporal actions of IRAK3 on inflammation remain unclear. A systematic review and meta-analyses were performed to investigate IRAK3 expression and its effects on inflammatory markers (TNF-α and IL-6) after one- or two-challenge interventions, which mimic the hyperinflammatory and immunosuppression phases of sepsis, respectively, using human or animal in vivo models. METHODS: This systematic review and meta-analyses has been registered in the Open Science Framework (OSF) (Registration DOI: 10.17605/OSF.IO/V39UR). A systematic search was performed to identify in vivo studies reporting outcome measures of expression of IRAK3 and inflammatory markers. Meta-analyses were performed where sufficient data was available. RESULTS: The search identified 7778 studies for screening. After screening titles, abstracts and full texts, a total of 49 studies were included in the systematic review. The review identified significant increase of IRAK3 mRNA and protein expression at different times in humans compared to rodents following one-challenge, whereas the increases of IL-6 and TNF-α protein expression in humans were similar to rodent in vivo models. Meta-analyses confirmed the inhibitory effect of IRAK3 on TNF-α mRNA and protein expression after two challenges. CONCLUSIONS: A negative correlation between IRAK3 and TNF-α expression in rodents following two challenges demonstrates the association of IRAK3 in the immunosuppression phase of sepsis. Species differences in underlying biology affect the translatability of immune responses of animal models to human, as shown by the dissimilarity in patterns of IRAK3 mRNA and protein expression between humans and rodents following one challenge that are further influenced by variations in experimental procedures.

Corrigendum: Both caffeine and Capsicum annuum fruit powder lower blood glucose levels and increase brown adipose tissue temperature in healthy adult males.

[This corrects the article DOI: 10.3389/fphys.2022.870154.].

Intrinsic and extrinsic apoptosis responses in leukaemia cells following daunorubicin treatment.

BACKGROUND: Daunorubicin is used clinically in the treatment of myeloma, acute lymphatic and myelocytic leukaemia. The toxic lesions caused by daunorubicin induce various modes of cell death, including apoptosis. Apoptosis is highly regulated programmed cell death that can be initiated mainly via two pathways, through death receptors (extrinsic) or involvement of the mitochondria (intrinsic). Induction of apoptosis via these pathways has been alluded following treatment with daunorubicin, but never compared in acute lymphoblastic leukaemia over a time course. METHODS: This study investigated the mechanisms of daunorubicin induced apoptosis in the treatment of CCRF-CEM, MOLT-4 (acute T-lymphoblastic leukaemia) and SUP-B15 (acute B-lymphoblastic leukaemia) cells. Cells were treated with daunorubicin for 4 h, and then placed in recovery medium (without daunorubicin) for 4 h, 12 h and 24 h. Apoptotic response was analysing using annexin-V expression, caspase activity, mitochondrial membrane potential change and an array to detect 43 apoptotic proteins. RESULTS: Daunorubicin induced apoptosis in all leukemic cell lines, but with different levels and duration of response. Both apoptosis levels and caspase activity increased after four hours recovery then declined in CCRF-CEM and MOLT-4 cells. However, SUP-B15 cells displayed initially comparable levels but remained elevated over the 24 h assessment period. Changes in mitochondrial membrane potential occurred in both MOLT-4 and CCRF-CEM cells but not in SUP-B15 cells. Expression of apoptotic proteins, including Bcl-2, Bax, caspase 3 and FADD, indicated that daunorubicin potentially induced both extrinsic and intrinsic apoptosis in both CCRF-CEM and MOLT-4 cells, but only extrinsic apoptosis in SUP-B15 cells. CONCLUSIONS: This study describes variations in sensitivities and timing of apoptotic responses in different leukaemia cell lines. These differences could be attributed to the lack of functional p53 in coordinating the cells response following cytotoxic treatment with daunorubicin, which appears to delay apoptosis and utilises alternative signalling mechanisms that need to be further explored.

Effects of Caffeine on Brown Adipose Tissue Thermogenesis and Metabolic Homeostasis: A Review.

The impact of brown adipose tissue (BAT) metabolism on understanding energy balance in humans is a relatively new and exciting field of research. The pathogenesis of obesity can be largely explained by an imbalance between caloric intake and energy expenditure, but the underlying mechanisms are far more complex. Traditional non-selective sympathetic activators have been used to artificially elevate energy utilization, or suppress appetite, however undesirable side effects are apparent with the use of these pharmacological interventions. Understanding the role of BAT, in relation to human energy homeostasis has the potential to dramatically offset the energy imbalance associated with obesity. This review discusses paradoxical effects of caffeine on peripheral adenosine receptors and the possible role of adenosine in increasing metabolism is highlighted, with consideration to the potential of central rather than peripheral mechanisms for caffeine mediated BAT thermogenesis and energy expenditure. Research on the complex physiology of adipose tissue, the embryonic lineage and function of the different types of adipocytes is summarized. In addition, the effect of BAT on overall human metabolism and the extent of the associated increase in energy expenditure are discussed. The controversy surrounding the primary ß-adrenoceptor involved in human BAT activation is examined, and suggestions as to the lack of translational findings from animal to human physiology and human in vitro to in vivo models are provided. This review compares and distinguishes human and rodent BAT effects, thus developing an understanding of human BAT thermogenesis to aid lifestyle interventions targeting obesity and metabolic syndrome. The focus of this review is on the effect of BAT thermogenesis on overall metabolism, and the potential therapeutic effects of caffeine in increasing metabolism via its effects on BAT.

Visualising functional 5-HT3 receptors containing A and C subunits at or near the cell surface.

Five different subunits of the human serotonin 3 (5-hydroxytrptamine 3; 5-HT3) receptor exist and these are present in both central and peripheral systems. Different subunits alter the efficacy of 5-HT3 receptor antagonists used to treat diarrhoea predominant-irritable bowel syndrome, chemotherapy induced nausea and vomiting and depression. Cell surface arrangement of 5-HT3 receptor complexes and the contribution of C, D and E subunits to receptor function is poorly understood. Here, we examine interactions of A and C subunits using 5-HT3 receptor subunits containing fluorescent protein inserts between the 3rd and 4th transmembrane spanning region. HEK293T cells that do not normally express 5-HT3 receptor subunits, were transiently transfected with A or C or both subunits. Patch clamp experiments show that cells transfected with either fluorescent protein tagged A or A and C subunits generate whole cell currents in response to 5-HT. These findings correlate with the apparent distribution of fluorescent protein tagged A and C subunits at or near cell surfaces detected using TIRF microscopy. In co-transfected cells, the A and C subunits are associated forming AC heteromer complexes at or near the cell surface and a proportion can also form A or C homomers. In conclusion, it is likely that both A homomers and AC heteromers contribute to whole cell currents in response to 5-HT with minimal contribution from C homomers.

Innervation of supraclavicular adipose tissue: A human cadaveric study.

Functional brown adipose tissue (BAT) was identified in adult humans only in 2007 with the use of fluorodeoxyglucose positron emission tomography imaging. Previous studies have demonstrated a negative correlation between obesity and BAT presence in humans. It is proposed that BAT possesses the capacity to increase metabolism and aid weight loss. In rodents it is well established that BAT is stimulated by the sympathetic nervous system with the interscapular BAT being innervated via branches of intercostal nerves. Whilst there is evidence to suggest that BAT possesses beta-3 adrenoceptors, no studies have identified the specific nerve branch that carries sympathetic innervation to BAT in humans. The aim of this study was to identify and trace the peripheral nerve or nerves that innervate human BAT in the supraclavicular region. The posterior triangle region of the neck of cadaveric specimens were dissected in order to identify any peripheral nerve branches piercing and/or terminating in supraclavicular BAT. A previously undescribed branch of the cervical plexus terminating in a supraclavicular adipose depot was identified in all specimens. This was typically an independent branch of the plexus, from the third cervical spinal nerve, but in one specimen was a branch of the supraclavicular nerve. Histological analysis revealed the supraclavicular adipose depot contained tyrosine hydroxylase immunoreactive structures, which likely represent sympathetic axons. This is the first study that identifies a nerve branch to supraclavicular BAT-like tissue. This finding opens new avenues for the investigation of neural regulation of fat metabolism in humans.

Determination of the DNA repair pathways utilised by acute lymphoblastic leukaemia cells following daunorubicin treatment.

OBJECTIVE: DNA double strand breaks (DNA-DSBs) are among the most lethal DNA lesions leading to genomic instability and repaired by either homologous recombination (HR) or the non-homologous end joining (NHEJ) mechanisms. The purpose of this study was to assess the importance and the level of activation of non-homologous end joining (NHEJ) and homologous recombination (HR) DNA repair pathways in three cell lines, CCRF-CEM and MOLT-4 derived from T lymphocytes and SUP-B15 derived from B lymphocytes following treatment with chemotherapy agent daunorubicin. RESULTS: The Gamma histone H2AX (γH2AX) assay was used assess the effects of DNA-PK inhibitor NU7026 and RAD51 inhibitor RI-2 on repair of DNA-DSB following treatment with daunorubicin. In all cell lines, the NHEJ DNA repair pathway appeared more rapid and efficient. MOLT-4 and CCFR-CEM cells utilised both NHEJ and HR pathways for DNA-DSB repair. Whereas, SUP-B15 cells utilised only NHEJ for DSB repair, suggestive of a deficiency in HR repair pathways.

Time dependent response of daunorubicin on cytotoxicity, cell cycle and DNA repair in acute lymphoblastic leukaemia.

BACKGROUND: Daunorubicin is commonly used in the treatment of acute lymphoblastic leukaemia (ALL). The aim of this study was to explore the kinetics of double strand break (DSB) formation of three ALL cell lines following exposure to daunorubicin and to investigate the effects of daunorubicin on the cell cycle and the protein kinases involved in specific checkpoints following DNA damage and recovery periods. METHODS: Three ALL cell lines CCRF-CEM and MOLT-4 derived from T lymphocytes and SUP-B15 derived from B lymphocytes were examined following 4 h treatment with daunorubicin chemotherapy and 4, 12 and 24 h recovery periods. Cell viability was measured via MTT (3-(4,5-dimethylthiazol-2-yl)-2-5 diphenyltetrazolium bromide) assay, reactive oxygen species (ROS) production by flow cytometry, double stranded DNA breaks by detecting γH2AX levels while stages of the cell cycle were detected following propidium iodide staining and flow cytometry. Western blotting was used to detect specific proteins while RNA was extracted from all cell lines and converted to cDNA to sequence Ataxia-telangiectasia mutated (ATM). RESULTS: Daunorubicin induced different degrees of toxicity in all cell lines and consistently generated reactive oxygen species. Daunorubicin was more potent at inducing DSB in MOLT-4 and CCRF-CEM cell lines while SUP-B15 cells showed delays in DSB repair and significantly more resistance to daunorubicin compared to the other cell lines as measured by γH2AX assay. Daunorubicin also causes cell cycle arrest in all three cell lines at different checkpoints at different times. These effects were not due to mutations in ATM as sequencing revealed none in any of the three cell lines. However, p53 was phosphorylated at serine 15 only in CCRF-CEM and MOLT-4 but not in SUP-B15 cells. The lack of active p53 may be correlated to the increase of SOD2 in SUP-B15 cells. CONCLUSIONS: The delay in DSB repair and lower sensitivity to daunorubicin seen in the B lymphocyte derived SUP-B15 cells could be due to loss of function of p53 that may be correlated to increased expression of SOD2 and lower ROS production.

The capacity for oestrogen to influence obesity through brown adipose tissue thermogenesis in animal models: A systematic review and meta-analysis.

Pharmacological interventions to aid weight loss have historically targeted either appetite suppression or increased metabolic rate. Brown adipose tissue (BAT) possesses the capacity to expend energy in a futile cycle, thus increasing basal metabolic rate. In animal models, oestrogen has been implicated in the regulation of body weight, and it is hypothesized that oestrogen is acting by modulating BAT metabolism. A systematic search was performed, to identify research articles implementing in vivo oestrogen-related interventions and reporting outcome measures that provide direct or indirect measures of BAT metabolism. Meta-analyses were conducted where sufficient data were available. The final library of 67 articles were predominantly in rodent models and provided mostly indirect measures of BAT metabolism. Results of this review found that oestrogen's effects on body weight, in rats and possibly mice, are likely facilitated by both metabolic and appetitive mechanisms but are largely only found in ovariectomized models. There is a need for further studies to clarify the potential effects of oestrogen on BAT metabolism in gonad-intact and castrated male animal models.

Effect of the 5-HT4 receptor agonist tegaserod on the expression of GRK2 and GRK6 in the rat gastrointestinal tract.

OBJECTIVE: Tegaserod is a 5-hydroxytryptamine type 4 (5-HT4) receptor agonist, formerly used in treating constipation predominant irritable bowel syndrome, which desensitizes 5-HT4 receptors in rat oesophagus and colon in vitro. Desensitization of 5-HT4 receptors is regulated by G-protein coupled receptor kinases. This study was designed to assess the effect of 5-HT4 receptor activation on the expression of GRK2 and GRK6 in the rat oesophagus and distal colon by acute administration of tegaserod. RESULTS: Rats were treated with a single dose of tegaserod (5 mg/kg) and tissue samples of the oesophagus and distal colon were prepared and level of GRK2 and GRK6 protein expression was determined using western blotting. The immunodensity of GRK2 and GRK6 was normalized against the loading control ß-actin and compared with control animals. Acute administration of tegaserod for 1, 2, 3, 4, 6, and 8 h did not change significantly the immunodensity of GRK2 or GRK6 in the oesophagus or GRK2 in the distal colon when compared with control animals. This may indicate that the basal level of GRK2 and GRK6 expression is sufficient to regulate the desensitization of 5-HT4 receptors in acute drug treatment.

Moonlighting Proteins and Their Role in the Control of Signaling Microenvironments, as Exemplified by cGMP and Phytosulfokine Receptor 1 (PSKR1).

Signal generating and processing complexes and changes in concentrations of messenger molecules such as calcium ions and cyclic nucleotides develop gradients that have critical roles in relaying messages within cells. Cytoplasmic contents are densely packed, and in plant cells this is compounded by the restricted cytoplasmic space. To function in such crowded spaces, scaffold proteins have evolved to keep key enzymes in the correct place to ensure ordered spatial and temporal and stimulus-specific message generation. Hence, throughout the cytoplasm there are gradients of messenger molecules that influence signaling processes. However, it is only recently becoming apparent that specific complexes involving receptor molecules can generate multiple signal gradients and enriched microenvironments around the cytoplasmic domains of the receptor that regulate downstream signaling. Such gradients or signal circuits can involve moonlighting proteins, so called because they can enable fine-tune signal cascades via cryptic additional functions that are just being defined. This perspective focuses on how enigmatic activity of moonlighting proteins potentially contributes to regional intracellular microenvironments. For instance, the proteins associated with moonlighting proteins that generate cyclic nucleotides may be regulated by cyclic nucleotide binding directly or indirectly. In this perspective, we discuss how generation of cyclic nucleotide-enriched microenvironments can promote and regulate signaling events. As an example, we use the phytosulfokine receptor (PSKR1), discuss the function of its domains and their mutual interactions and argue that this complex architecture and function enhances tuning of signals in microenvironments.

Intramolecular crosstalk between catalytic activities of receptor kinases.

Signal modulation is important for the growth and development of plants and this process is mediated by a number of factors including physiological growth regulators and their associated signal transduction pathways. Protein kinases play a central role in signaling, including those involving pathogen response mechanisms. We previously demonstrated an active guanylate cyclase (GC) catalytic center in the brassinosteroid insensitive receptor (AtBRI1) within an active intracellular kinase domain resulting in dual enzymatic activity. Here we propose a novel type of receptor architecture that is characterized by a functional GC catalytic center nested in the cytosolic kinase domain enabling intramolecular crosstalk. This may be through a cGMP-AtBRI1 complex forming that may induce a negative feedback mechanism leading to desensitisation of the receptor, regulated through the cGMP production pathway. We further argue that the comparatively low but highly localized cGMP generated by the GC in response to a ligand is sufficient to modulate the kinase activity. This type of receptor therefore provides a molecular switch that directly and/or indirectly affects ligand dependent phosphorylation of downstream signaling cascades and suggests that subsequent signal transduction and modulation works in conjunction with the kinase in downstream signaling.

The brassinosteroid receptor BRI1 can generate cGMP enabling cGMP-dependent downstream signaling.

The brassinosteroid receptor brassinosteroid insensitive 1 (BRI1) is a member of the leucine-rich repeat receptor-like kinase family. The intracellular kinase domain of BRI1 is an active kinase and also encapsulates a guanylate cyclase catalytic centre. Using liquid chromatography tandem mass spectrometry, we confirmed that the recombinant cytoplasmic domain of BRI1 generates pmol amounts of cGMP per µg protein with a preference for magnesium over manganese as a co-factor. Importantly, a functional BRI1 kinase is essential for optimal cGMP generation. Therefore, the guanylate cyclase activity of BRI1 is modulated by the kinase while cGMP, the product of the guanylate cyclase, in turn inhibits BRI1 kinase activity. Furthermore, we show using Arabidopsis root cell cultures that cGMP rapidly potentiates phosphorylation of the downstream substrate brassinosteroid signaling kinase 1 (BSK1). Taken together, our results suggest that cGMP acts as a modulator that enhances downstream signaling while dampening signal generation from the receptor.

High Molecular Weight DNA Enrichment with Peptide Nucleic Acid Probes.

Here, we describe how peptide nucleic acid (PNA) probes can be used to enrich genomic DNA fractions to facilitate downstream analysis, such as the haplotype phasing of the isolated genomic pieces. This method enriches for polymorphic regions of fragmented chromosomes by physically separating the desired sequence and flanking regions. The PNA probes used for enrichment are novel synthetic nucleic acids with highly specific targeting and hybridization properties. Using a enrichment technique, we capture high molecular weight genomic DNA using nothing more than a simple modification to standard genomic DNA extraction from blood.

Phosphorylation of the dimeric cytoplasmic domain of the phytosulfokine receptor, PSKR1.

Phytosulfokines (PSKs) are plant peptide hormones that co-regulate plant growth, differentiation and defense responses. PSKs signal through a plasma membrane localized leucine-rich repeat receptor-like kinase (phytosulfokine receptor 1, PSKR1) that also contains a functional cytosolic guanylate cyclase with its cyclase catalytic center embedded within the kinase domain. To functionally characterize this novel type of overlapping dual catalytic function, we investigated the phosphorylation of PSKR1 in vitro Tandem mass spectrometry of the cytoplasmic domain of PSKR1 (PSKR1cd) revealed at least 11 phosphorylation sites (8 serines, 2 threonines and 1 tyrosine) within the PSKR1cd. Phosphomimetic mutations of three serine residues (Ser686, Ser696 and Ser698) in tandem at the juxta-membrane position resulted in enhanced kinase activity in the on-mutant that was suppressed in the off-mutant, but both mutations reduced guanylate cyclase activity. Both the on and off phosphomimetic mutations of the phosphotyrosine (Tyr888) residue in the activation loop suppressed kinase activity, while neither mutation affected guanylate cyclase activity. Size exclusion and analytical ultracentrifugation analysis of the PSKR1cd suggest that it is reversibly dimeric in solution, which was further confirmed by biflourescence complementation. Taken together, these data suggest that in this novel type of receptor domain architecture, specific phosphorylation and dimerization are possibly essential mechanisms for ligand-mediated catalysis and signaling.