RGS4 controls airway hyperresponsiveness through GAP-independent mechanisms.

Regulators of G protein signaling (RGS) proteins constrain G protein-coupled receptor (GPCR)-mediated and other responses throughout the body primarily, but not exclusively, through their GTPase-activating protein activity. Asthma is a highly prevalent condition characterized by airway hyper-responsiveness (AHR) to environmental stimuli resulting in part from amplified GPCR-mediated airway smooth muscle contraction. Rgs2 or Rgs5 gene deletion in mice enhances AHR and airway smooth muscle contraction, whereas RGS4 KO mice unexpectedly have decreased AHR because of increased production of the bronchodilator prostaglandin E2 (PGE2) by lung epithelial cells. Here, we found that knockin mice harboring Rgs4 alleles encoding a point mutation (N128A) that sharply curtails RGS4 GTPase-activating protein activity had increased AHR, reduced airway PGE2 levels, and augmented GPCR-induced bronchoconstriction compared with either RGS4 KO mice or WT controls. RGS4 interacted with the p85α subunit of PI3K and inhibited PI3K-dependent PGE2 secretion elicited by transforming growth factor beta in airway epithelial cells. Together, these findings suggest that RGS4 affects asthma severity in part by regulating the airway inflammatory milieu in a G protein-independent manner.

Markers of endothelial glycocalyx dysfunction in Clarkson disease.

BACKGROUND: Clarkson disease (monoclonal gammopathy-associated idiopathic systemic capillary leak syndrome, ISCLS) is a rare idiopathic condition marked by transient, relapsing-remitting episodes of systemic microvascular hyper-permeability, which liberates plasma fluid and macromolecules into the peripheral tissues. This pathology manifests clinically as the abrupt onset of hypotensive shock, hemoconcentration, and hypoalbuminemia. METHODS: We analysed endothelial glycocalyx (eGCX)-related markers in plasma from patients with ISCLS during acute disease flares and convalescence by ELISA and comprehensive proteomic profiling. We evaluated eGCX-related components and gene expression in cultured endothelial cells using RNA-sequencing, real-time PCR, and fluorescence staining. RESULTS: Serum levels of eGCX-related core components including hyaluronic acid (HA) and the core proteoglycan soluble syndecan-1 (sCD138) were elevated at baseline and during acute ISCLS flares. Serial measurements demonstrated that sCD138 levels peaked during the recovery (post-leak) phase of the illness. Proteomic analysis of matched acute and convalescent ISCLS plasma revealed increased abundance of eGCX-related proteins, including glypicans, thrombospondin-1 (TSP-1), and eGCX-degrading enzymes in acute compared to remission plasma. Abundance of endothelial cell damage markers did not differ in acute and baseline plasma. Expression of several eGCX-related genes and surface carbohydrate content in endothelial cells from patients with ISCLS did not differ significantly from that observed in healthy control cells. CONCLUSIONS: eGCX dysfunction, but not endothelial injury, may contribute to clinical symptoms of acute ISCLS. Serum levels of of eGCX components including sCD138 may be measured during acute episodes of ISCLS to monitor clinical status and therapeutic responses.

Short stature and combined immunodeficiency associated with mutations in RGS10.

We report the clinical and molecular phenotype of three siblings from one family, who presented with short stature and immunodeficiency and carried uncharacterized variants in RGS10 (c.489_491del:p.E163del and c.G511T:p.A171S). This gene encodes regulator of G protein signaling 10 (RGS10), a member of a large family of GTPase-activating proteins (GAPs) that targets heterotrimeric G proteins to constrain the activity of G protein-coupled receptors, including receptors for chemoattractants. The affected individuals exhibited systemic abnormalities directly related to the RGS10 mutations, including recurrent infections, hypergammaglobulinemia, profoundly reduced lymphocyte chemotaxis, abnormal lymph node architecture, and short stature due to growth hormone deficiency. Although the GAP activity of each RGS10 variant was intact, each protein exhibited aberrant patterns of PKA-mediated phosphorylation and increased cytosolic and cell membrane localization and activity compared to the wild-type protein. We propose that the RGS10 p.E163del and p.A171S mutations lead to mislocalization of the RGS10 protein in the cytosol, thereby resulting in attenuated chemokine signaling. This study suggests that RGS10 is critical for both immune competence and normal hormonal metabolism in humans and that rare RGS10 variants may contribute to distinct systemic genetic disorders.

RGS4 promotes allergen- and aspirin-associated airway hyperresponsiveness by inhibiting PGE2 biosynthesis.

BACKGROUND: Allergens elicit host production of mediators acting on G-protein-coupled receptors to regulate airway tone. Among these is prostaglandin E2 (PGE2), which, in addition to its role as a bronchodilator, has anti-inflammatory actions. Some patients with asthma develop bronchospasm after the ingestion of aspirin and other nonsteroidal anti-inflammatory drugs, a disorder termed aspirin-exacerbated respiratory disease. This condition may result in part from abnormal dependence on the bronchoprotective actions of PGE2. OBJECTIVE: We sought to understand the functions of regulator of G protein signaling 4 (RGS4), a cytoplasmic protein expressed in airway smooth muscle and bronchial epithelium that regulates the activity of G-protein-coupled receptors, in asthma. METHODS: We examined RGS4 expression in human lung biopsies by immunohistochemistry. We assessed airways hyperresponsiveness (AHR) and lung inflammation in germline and airway smooth muscle-specific Rgs4-/- mice and in mice treated with an RGS4 antagonist after challenge with Aspergillus fumigatus. We examined the role of RGS4 in nonsteroidal anti-inflammatory drug-associated bronchoconstriction by challenging aspirin-exacerbated respiratory disease-like (ptges1-/-) mice with aspirin. RESULTS: RGS4 expression in respiratory epithelium is increased in subjects with severe asthma. Allergen-induced AHR was unexpectedly diminished in Rgs4-/- mice, a finding associated with increased airway PGE2 levels. RGS4 modulated allergen-induced PGE2 secretion in human bronchial epithelial cells and prostanoid-dependent bronchodilation. The RGS4 antagonist CCG203769 attenuated AHR induced by allergen or aspirin challenge of wild-type or ptges1-/- mice, respectively, in association with increased airway PGE2 levels. CONCLUSIONS: RGS4 may contribute to the development of AHR by reducing airway PGE2 biosynthesis in allergen- and aspirin-induced asthma.

Erratum: Author Correction: A natural mouse model reveals genetic determinants of systemic capillary leak syndrome (Clarkson disease).

[This corrects the article DOI: 10.1038/s42003-019-0647-4.].

A natural mouse model reveals genetic determinants of systemic capillary leak syndrome (Clarkson disease).

The systemic capillary leak syndrome (SCLS, Clarkson disease) is a disorder of unknown etiology characterized by recurrent episodes of vascular leakage of proteins and fluids into peripheral tissues, resulting in whole-body edema and hypotensive shock. The pathologic mechanisms and genetic basis for SCLS remain elusive. Here we identify an inbred mouse strain, SJL, which recapitulates cardinal features of SCLS, including susceptibility to histamine- and infection-triggered vascular leak. We named this trait "Histamine hypersensitivity" (Hhs/Hhs) and mapped it to Chromosome 6. Hhs is syntenic to the genomic locus most strongly associated with SCLS in humans (3p25.3), revealing that the predisposition to develop vascular hyperpermeability has a strong genetic component conserved between humans and mice and providing a naturally occurring animal model for SCLS. Genetic analysis of Hhs may reveal orthologous candidate genes that contribute not only to SCLS, but also to normal and dysregulated mechanisms underlying vascular barrier function more generally.

Neuroprotective Effect of ß-secretase Inhibitory Peptide from Pacific Hake (Merluccius productus) Fish Protein Hydrolysate.

BACKGROUND: Various methodologies have been employed for the therapeutic interpolation of the progressive brain disorder Alzheimer's disease. Thus, ß-secretase inhibition is significant to prevent disease progression in the early stages. OBJECTIVE: This study seeks to purify and characterize a novel ß-secretase inhibitory peptide from Pacific hake enzymatic hydrolysate. METHODS: A potent ß-secretase inhibitory peptide was isolated by sequential purifications using Sephadex G-25 column chromatography and octadecylsilane (ODS) C18 reversed-phase HPLC. A total of seven peptides were synthesized using the isolated peptide sequences. SH-SY5Y cells stably transfected with the human ''Swedish'' amyloid precursor protein (APP) mutation APP695 (SH-SY5YAPP695swe) were used as an in-vitro model system to investigate the effect of Leu-Asn peptide on APP processing. RESULTS: The ß-secretase inhibitory activity (IC50) of the purified peptide (Ser-Leu-Ala-Phe-Val-Asp- Asp-Val-Leu-Asn) from fish protein hydrolysate was 18.65 µM and dipeptide Leu-Asn was the most potent ß-secretase inhibitor (IC50 value = 8.82 µM). When comparing all the seven peptides, the inhibition pattern of Leu-Asn dipeptide was found to be competitive by Lineweaver-Burk plot and Dixon plot (Ki value = 4.24 µM). The 24 h treatment with Leu-Asn peptide in SH-SY5Y cells resulted in reducing the ß-amyloid (Aß) production in a dose-dependent manner. CONCLUSION: Therefore, the results of this study suggest that ß-secretase inhibitory peptides derived from marine organisms could be potential candidates to develop nutraceuticals or pharmaceuticals as antidementia agents.

Aspergillus fumigatus-Secreted Alkaline Protease 1 Mediates Airways Hyperresponsiveness in Severe Asthma.

The hallmark features of allergic asthma are type 2 (eosinophilic) inflammation and airways hyperresponsiveness (AHR). Although these features often comanifest in mouse lungs in vivo, we demonstrate in this study that the serine protease Alp1 from the ubiquitous mold and allergen, Aspergillus fumigatus, can induce AHR in mice unable to generate eosinophilic inflammation. Strikingly, Alp1 induced AHR in mice devoid of protease-activated receptor 2/F2 trypsin-like receptor 1 (PAR2/F2RL1), a receptor expressed in lung epithelium that is critical for allergic responses to protease-containing allergens. Instead, using precision-cut lung slices and human airway smooth muscle cells, we demonstrate that Alp1 directly increased contractile force. Taken together, these findings suggest that Alp1 induces bronchoconstriction through mechanisms that are largely independent of allergic inflammation and point to a new target for direct intervention of fungal-associated asthma.

Whole-Exome Sequencing of Adult and Pediatric Cohorts of the Rare Vascular Disorder Systemic Capillary Leak Syndrome.

OBJECTIVE: Systemic capillary leak syndrome (SCLS) is a rare disorder that presents with episodes of hypovolemic shock. The extent to which genetic abnormalities contribute to SCLS is unknown. We identified pediatric and adult cohorts with characteristic clinical courses. We sought to describe the clinical characteristics of both cohorts, identify a possible genetic contribution to SCLS, and demonstrate that whole-exome sequencing (WES) may be conducted by critical care providers. DESIGN: Prospective observational study of WES of nine adult and eight pediatric SCLS patients and available unaffected first-degree relatives. SETTING: Tertiary children's hospitals and referral research laboratory. PATIENTS: Children and adults with SCLS. INTERVENTIONS: None. MEASUREMENTS: Patients and available first-degree relatives underwent WES. Data were analyzed for rare homozygous, biallelic, de novo, and heterozygous variants with allelic enrichment and metabolic pathway analyses. MAIN RESULTS: Children with SCLS presented at a younger age with episodes similar to those experienced by adults. All patients and available relatives underwent satisfactory WES. No overlapping gene variants or metabolic pathways were identified across all SCLS patients. Multiple candidate genes with homozygous or biallelic mutations were identified in individual subjects with SCLS. There was no significant enrichment of genes with rare heterozygous variants. CONCLUSIONS: The clinical characteristics of children and adults with SCLS are similar. We did not identify a uniform germline exomic genetic etiology for SCLS. WES identified several candidate genes in individual patients for future research. WES is a viable way for critical care providers to investigate the etiology of diseases with presumed genetic contributions.

Regulator of G protein signaling 5 restricts neutrophil chemotaxis and trafficking.

Neutrophils are white blood cells that are mobilized to damaged tissues and to sites of pathogen invasion, providing the first line of host defense. Chemokines displayed on the surface of blood vessels promote migration of neutrophils to these sites, and tissue- and pathogen-derived chemoattractant signals, including N-formylmethionylleucylphenylalanine (fMLP), elicit further migration to sites of infection. Although nearly all chemoattractant receptors use heterotrimeric G proteins to transmit signals, many of the mechanisms lying downstream of chemoattractant receptors that either promote or limit neutrophil motility are incompletely defined. Here, we show that regulator of G protein signaling 5 (RGS5), a protein that modulates G protein activity, is expressed in both human and murine neutrophils. We detected significantly more neutrophils in the airways of Rgs5-/- mice than WT counterparts following acute respiratory virus infection and in the peritoneum in response to injection of thioglycollate, a biochemical proinflammatory stimulus. RGS5-deficient neutrophils responded with increased chemotaxis elicited by the chemokines CXC motif chemokine ligand 1 (CXCL1), CXCL2, and CXCL12 but not fMLP. Moreover, adhesion of these cells was increased in the presence of both CXCL2 and fMLP. In summary, our results indicate that RGS5 deficiency increases chemotaxis and adhesion, leading to more efficient neutrophil mobilization to inflamed tissues in mice. These findings suggest that RGS5 expression and activity in neutrophils determine their migrational patterns in the complex microenvironments characteristic of inflamed tissues.

Transepithelial transport across Caco-2 cell monolayers of angiotensin converting enzyme (ACE) inhibitory peptides derived from simulated in vitro gastrointestinal digestion of cooked chicken muscles.

Korat-chicken breast and thigh were subjected to heating at 70, 100 or 121 °C for 30 min and simulated in vitro gastrointestinal digestion. At 70 or 100 °C heating, digests of breast possessed higher ACE inhibitory activity than those of thigh. The highest ACE inhibitory activity was found in the digest of breast cooked at 70 °C (B/H-70), whereas breast heated at 121 °C (B/H-121) exhibited the lowest. The 1-kDa permeate of the B/H-70 digest revealed higher permeability through colorectal adenocarcinoma monolayers and ACE inhibitory activity than did B/H-121. Among nine transported peptides, APP derived from myosin showed the highest ACE inhibition, with a non-competitive characteristic (Ki 0.93 µM). Molecular docking showed that APP interacts with ACE via hydrogen bonds, electrostatic and van der Waals interactions. In conclusion, mild thermal treatment of chicken breast resulted in a higher amount of transported peptides, exerting higher ACE inhibitory activity, which could lead to potential health benefits.

Adrenomedullin surges are linked to acute episodes of the systemic capillary leak syndrome (Clarkson disease).

BACKGROUND: Systemic Capillary Leak Syndrome (SCLS) is an extremely rare and life-threatening vascular disorder of unknown etiology. SCLS is characterized by abrupt and transient episodes of hypotensive shock and edema due to plasma leakage into peripheral tissues. The disorder has garnered attention recently because its initial presentation resembles more common vascular disorders including systemic anaphylaxis, sepsis, and acute infections with the Ebola/Marburg family of filoviruses. Although approximately 70-85% of patients with SCLS have a concurrent monoclonal gammopathy of unknown significance (MGUS), any contribution of the paraprotein to acute flares is unknown. PROCEDURE: To identify circulating factors that might trigger acute SCLS crises, we profiled transcriptomes of paired peripheral blood mononuclear cell fractions obtained from patients during acute attacks and convalescent intervals by microarray. RESULTS: This study uncovered 61 genes that were significantly up- or downregulated more than 2.5-fold in acute samples relative to respective baselines. One of the most upregulated genes was ADM, which encodes the vasoactive peptide adrenomedullin. A stable ADM protein surrogate (pro-ADM) was markedly elevated in SCLS acute sera compared to remission samples or sera from healthy controls. Monocytes and endothelial cells (ECs) from SCLS subjects expressed significantly more ADM in response to proinflammatory stimuli compared to healthy control cells. Application of ADM to ECs elicited protective effects on vascular barrier function, suggesting a feedback protective mechanism in SCLS. CONCLUSIONS: Since ADM has established hypotensive effects, differentiating between these dual actions of ADM is crucial for therapeutic applications aimed at more common diseases associated with increased ADM levels.

Production and assessment of Pacific hake (Merluccius productus) hydrolysates as cryoprotectants for frozen fish mince.

The aim of this study was to investigate application of fish protein hydrolysates (FPHs) as cryoprotectants for cod fish mince subjected to freeze-thaw abuse. Response surface methodology revealed little difference in cryoprotectant ability between FPHs produced from Pacific hake muscle within the range of conditions studied, namely Flavourzyme® enzyme/substrate ratio (E/S 1-4%), time (1-6h) and pH (5-7). When added at 4% or higher concentrations, FPH minimized expressible moisture and cook loss, while maximizing salt extractable protein from freeze-thaw abused fish mince, providing similar or better cryoprotection compared to an 8% sucrose-sorbitol blend, and a stabilizing effect of FPH on myosin was observed by differential scanning calorimetry. Sensory evaluation showed that addition of 8% FPH in fish ball products increased the perception of fishiness, saltiness, bitterness and firmness while decreasing moistness. FPH could be a viable alternative to the sugar-based cryoprotectants currently used for frozen fish products.

Optimization of vitamins A and D3 loading in re-assembled casein micelles and effect of loading on stability of vitamin D3 during storage.

The objectives of this study were to apply response surface methodology to optimize fat-soluble vitamin loading in re-assembled casein micelles, and to evaluate vitamin D stability of dry formulations during ambient or accelerated storage and in fortified fluid skim milk stored under refrigeration. Optimal loading of vitamin A (1.46-1.48mg/100mgcasein) was found at 9.7mM phosphate, 5.5mM citrate and 30.0mM calcium, while optimal loading of vitamin D (1.38-1.46mg/100mg casein) was found at 4.9mM phosphate, 4.0mM citrate and 26.1mM calcium. In general, more vitamin D was retained in vitamin D-re-assembled casein micelles than control powders during storage, while vitamin D loss was not different for vitamin D-re-assembled casein micelles and control fortified milks after 21days of refrigerated storage with light exposure. In conclusion, re-assembled casein micelles with high loading efficiency show promise for improving vitamin D stability during dry storage.

Investigation into the bioavailability of milk protein-derived peptides with dipeptidyl-peptidase IV inhibitory activity using Caco-2 cell monolayers.

In recent years, peptides derived from a variety of dietary proteins have been reported to exhibit inhibitory activity against the dipeptidyl-peptidase IV (DPP-IV) enzyme, a target in the management of type 2 diabetes. While much attention has been given to the production and identification of peptides with DPP-IV inhibitory activity from food proteins, particularly dairy proteins, little is known on the bioavailability of these molecules. In this study, the stability and transport of five previously identified milk-derived peptides (LKPTPEGDL, LPYPY, IPIQY, IPI and WR) and a whey protein isolate (WPI) digest with DPP-IV-inhibitory activity were investigated using Caco-2 cell monolayers as a model system for human intestinal absorption. Even though a small percentage (ranging from 0.05% for LPYPY to 0.47% for WR) of the bioactive peptides added to the apical side was able to cross the monolayer intact, all five peptides investigated were susceptible to peptidase action during the transport study. Conversely, only minor changes to the WPI digest composition were observed. Determination of the DPP-IV inhibitory activity of the peptides and amino acids identified in the apical and basolateral solutions showed that most degradation products were less effective at inhibiting DPP-IV than the peptide they originated from. Findings from this research suggest that the susceptibility of food-derived DPP-IV inhibitory peptides to degradation by intestinal brush border membrane enzymes may alter their biological activity in vivo. Further research should be conducted to enhance the bioavailability of DPP-IV inhibitory peptides.

Household Consumption of Thiamin-Fortified Fish Sauce Increases Erythrocyte Thiamin Concentrations among Rural Cambodian Women and Their Children Younger Than 5 Years of Age: A Randomized Controlled Efficacy Trial.

OBJECTIVES: To assess whether ad libitum consumption of thiamin-fortified fish sauce over 6 months yields higher erythrocyte thiamin diphosphate concentrations (eTDP) among women of childbearing age and their children aged 12-59 months compared with control sauce containing no thiamin. STUDY DESIGN: In this double-blind, randomized controlled efficacy trial, 276 nonpregnant, nonlactating women (18-45 years of age) and their families in Prey Veng, Cambodia, were randomized to receive 1 of 3 fish sauce formulations: low thiamin concentration (low, 2 g/L), high thiamin concentration (high, 8 g/L), or a control (no thiamin) fish sauce. Baseline (t = 0) and endline (t = 6 months) eTDP were measured with the use of high-performance liquid chromatography with a fluorescence detector. RESULTS: Fish sauce consumption did not differ between treatment groups (P = .19). In intent-to-treat analysis, women's baseline-adjusted endline eTDP (mean; 95% CI) was higher among women in the low (259; 245-274 nmol/L) and high (257; 237-276 nmol/L) groups compared with control (184; 169-198 nmol/L; P < .001); low and high groups did not differ (P = .83). Similarly, children's baseline-adjusted eTDP was higher in the low (259; 246-271 nmol/L) and high (257; 243-270 nmol/L) groups compared with control (213; 202-224 nmol/L; P < .001). CONCLUSION: Fortified fish sauce appears to be an efficacious means of improving biochemical thiamin status in nonpregnant, nonlactating women and their children (1-5 years of age) living in rural Cambodia. TRIAL REGISTRATION: ClinicalTrials.gov: NCT02221063.

Perinatal Consumption of Thiamine-Fortified Fish Sauce in Rural Cambodia: A Randomized Clinical Trial.

IMPORTANCE: Infantile beriberi, a potentially fatal disease caused by thiamine deficiency, remains a public health concern in Cambodia and regions where thiamine-poor white rice is a staple food. Low maternal thiamine intake reduces breast milk thiamine concentrations, placing breastfed infants at risk of beriberi. OBJECTIVE: To determine if consumption of thiamine-fortified fish sauce yields higher erythrocyte thiamine diphosphate concentrations (eTDP) among lactating women and newborn infants and higher breast milk thiamine concentrations compared with a control sauce. DESIGN, SETTING, AND PARTICIPANTS: In this double-blind randomized clinical trial, 90 pregnant women were recruited in the Prey Veng province, Cambodia. The study took place between October 2014 and April 2015. INTERVENTIONS: Women were randomized to 1 of 3 groups (n = 30) for ad libitum fish sauce consumption for 6 months: control (no thiamine), low-concentration (2 g/L), or high-concentration (8 g/L) fish sauce. MAIN OUTCOMES AND MEASURES: Maternal eTDP was assessed at baseline (October 2014) and endline (April 2015). Secondary outcomes, breast milk thiamine concentration and infant eTDP, were measured at endline. RESULTS: Women's mean (SD) age and gestational stage were 26 (5) years and 23 (7) weeks, respectively. April 2015 eTDP was measured among 28 women (93%), 29 women (97%), and 23 women (77%) in the control, low-concentration, and high-concentration groups, respectively. In modified intent-to-treat analysis, mean baseline-adjusted endline eTDP was higher among women in the low-concentration (282nM; 95% CI, 235nM to 310nM) and high-concentration (254nM; 95% CI, 225nM to 284nM) groups compared with the control group (193nM; 95% CI, 164nM to 222M; P < .05); low-concentration and high-concentration groups did not differ (P = .19). Breast milk total thiamine concentrations were 14.4 µg/dL for the control group (95% CI, 12.3 µg/dL to 16.5 µg/dL) (to convert to nanomoles per liter, multiply by 29.6); 20.7 µg/dL for the low-concentration group (95% CI, 18.6 µg/dL to 22.7 µg/dL ); and 17.7 µg/dL for the high-concentration group (95% CI, 15.6 µg/dL to 19.9 µg/dL). Mean (SD) infant age at endline was 16 (8) weeks for the control group, 17 (7) weeks for the low-concentration group, and 14 (8) for the high-concentration group. Infant eTDP was higher among those in the high-concentration group (257nM; 95% CI, 222nM to 291nM; P < .05) compared with the low-concentration (212nM; 95% CI, 181nM to 244nM) and control (187nM; 95% CI, 155nM to 218nM) groups. CONCLUSIONS AND RELEVANCE: Compared with women in the control group, women who consumed thiamine-fortified fish sauce through pregnancy and early lactation had higher eTDP and breast milk thiamine concentrations and their infants had higher eTDP, which was more pronounced in the high group. Thiamine-fortified fish sauce has the potential to prevent infantile beriberi in this population. TRIAL REGISTRATION: Clinicaltrials.gov Identifier: NCT02221063.

Effect of high intensity ultrasound on transglutaminase-catalyzed soy protein isolate cold set gel.

The effects of high intensity ultrasound (HIU, 105-110 W/cm(2) for 5 or 40 min) pre-treatment of soy protein isolate (SPI) on the physicochemical properties of ensuing transglutaminase-catalyzed soy protein isolate cold set gel (TSCG) were investigated in this study. The gel strength of TSCG increased remarkably from 34.5 to 207.1 g for TSCG produced from SPI with 40 min HIU pre-treatment. Moreover, gel yield and water holding capacity also increased after HIU pre-treatments. Scanning electron microscopy showed that HIU of SPI resulted in a more uniform and denser microstructure of TSCG. The content of free sulfhydryl (SH) groups was higher in HIU TSCG than non-HIU TSG, even though greater decrease of the SH groups present in HIU treated SPI was observed when the TSCG was formed, suggesting the involvement of disulfide bonds in gel formation. Protein solubility of TSCG in both denaturing and non-denaturing solvents was higher after HIU pretreatment, and changes in hydrophobic amino acid residues as well as in polypeptide backbone conformation and secondary structure of TSCG were demonstrated by Raman spectroscopy. These results suggest that increased inter-molecular ε-(γ-glutamyl) lysine isopeptide bonds, disulfide bonds and hydrophobic interactions might have contributed to the HIU TSCG gel network. In conclusion, HIU changed physicochemical and structural properties of SPI, producing better substrates for TGase. The resulting TSCG network structure was formed with greater involvement of covalent and non-covalent interactions between SPI molecules and aggregates than in the TSCG from non-HIU SPI.

R4 Regulator of G Protein Signaling (RGS) Proteins in Inflammation and Immunity.

G protein-coupled receptors (GPCRs) have important functions in both innate and adaptive immunity, with the capacity to bridge interactions between the two arms of the host responses to pathogens through direct recognition of secreted microbial products or the by-products of host cells damaged by pathogen exposure. In the mid-1990s, a large group of intracellular proteins was discovered, the regulator of G protein signaling (RGS) family, whose main, but not exclusive, function appears to be to constrain the intensity and duration of GPCR signaling. The R4/B subfamily--the focus of this review--includes RGS1-5, 8, 13, 16, 18, and 21, which are the smallest RGS proteins in size, with the exception of RGS3. Prominent roles in the trafficking of B and T lymphocytes and macrophages have been described for RGS1, RGS13, and RGS16, while RGS18 appears to control platelet and osteoclast functions. Additional G protein independent functions of RGS13 have been uncovered in gene expression in B lymphocytes and mast cell-mediated allergic reactions. In this review, we discuss potential physiological roles of this RGS protein subfamily, primarily in leukocytes having central roles in immune and inflammatory responses. We also discuss approaches to target RGS proteins therapeutically, which represents a virtually untapped strategy to combat exaggerated immune responses leading to inflammation.