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Comparative transcriptomics uncovers poplar and fungal genetic determinants of ectomycorrhizal compatibility.

Ectomycorrhizal symbiosis supports tree growth and is crucial for nutrient cycling and temperate and boreal ecosystems functioning. The establishment of functional ectomycorrhiza (ECM) first requires the association of compatible partners. However, host and fungal genetic determinants governing mycorrhizal compatibility are unknown. To identify such factors in poplar and its fungal associates, we mined existing and de novo tree and fungal transcriptional datasets. We identified co-expressed genes enabling ECM symbiosis at early and mature stages of the interaction. These sets of genes can be divided into general fungal-sensing and ECM-specific components. We highlight the importance of fungal modulation of plant JA-related defenses and the regulation of secretory pathways for ECM compatibility, including upregulation of key fungal small secreted proteins, the downregulation of plant secreted peroxidases, and the downregulation of plant cell wall remodeling proteins concomitantly with the upregulation of fungal glycosyl hydrolases acting on pectin. Not only gene regulation, but also its temporal scale and dynamics seem to play a crucial role for mycorrhizal compatibility. The expression profile of the host Common Symbiosis Pathway and nutrient transporters was also studied, revealing constitutive levels of expression and moderate upregulation in compatible ECM interactions. Overall, these results underscore the importance of novel biological functions during the establishment of ECM symbiosis, help us gain insights into the molecular events determining mycorrhiza compatibility, and serve as a data-rich transcriptomic resource to open new research questions in the field.

Mycorrhizae

Annotation of RxLR Effectors in Oomycete Genomes.

Pathogens have evolved effector proteins to suppress host immunity and facilitate plant infections. RxLR effectors are small, secreted effector proteins with conserved RxLR and dEER amino acid motifs at the N terminus and highly variable C termini and are commonly found in oomycete species. We provide computational approaches to annotate RxLR candidate effector genes in a genome assembly in FASTA format with an available GFF file. Hidden Markov Modeling (HHM) is used in combination with regular expressions to search for RxLR and EER amino acid patterns.

Oomycetes

Expansion of secreted cystine knot proteins reveals virulence factors in the human fungal pathogen Histoplasma.

Identifying fungal secreted factors that influence host infection remains a key challenge in microbial pathogenesis. While secreted effectors, particularly small cysteine-rich proteins, are well characterized in plant fungal pathogens, their counterparts in mammalian pathogens are understudied. We apply criteria from plant fungal effectors to the mammalian fungal pathogen Histoplasma, yielding a set of putative effectors highly enriched for knottins, proteins that adopt a distinctive cystine knot fold. Using an algorithm, we further identify 25 putative knottins in the Histoplasma genome, revealing a significant expansion of knottin genes. Knottin domains are found in diverse molecules but play an unknown role in virulence. Functional studies of individual Histoplasma knottins demonstrate their critical roles in intracellular survival and host cell lysis during macrophage infection as well as virulence in vivo. These findings highlight the importance of knottins in fungal pathogenesis and suggest their broader relevance for discovering conserved mechanisms of host manipulation.

Histoplasma

Spontaneous secretion from the dog small intestine in vivo.

Forty loops of small intestine in the dog were perfused under control conditions with a balanced electrolyte solution. Most of the loops absorbed sodium and water, but 10 loops were in a state of spontaneous intestinal secretion. Compared to absorbing loops, spontaneously secreting loops exhibited reduced values for lumen-to-plasma fluxes of sodium and chloride and increased values for the plasma-to-lumen fluxes of these ions. Analysis of flux ratios suggested that sodium and chloride were actively scecreted during spontaneous intestinal secretion in the dog. Spontaneous secretion was similar to the secretion induced by CT or VIP, except that the latter were associated with a change in PD whereas the PD in the spontaneously secreting loops was the same as in the spontaneously absorbing loops.

Animals

[Effect of parenterally administered protein hydrolysates on the secretory function of the small intestine].

The effect of intravenous infusion of proteinic hydrolysates on the secretory function of the intestines was studied in dogs with an intestinal loop isolated starting from the initial portion of the jejunum. The preparation of proteinic hydrolysates, such as the caseine hydrolysate TSOLIPK, aminopeptide and hydrolysine L-103 with their drop-by-drop infusion into the posterior vena cava to dogs on an empty stomach were found to have in most cases an effect on the periodicity of the small intestine secretory activity. The infusion of proteinic hydrolyastes failed to exert any essential influence on the secretion of the dense proportion of the juice, whereas the secretion of the liquid component of the intestinal juice increased in all experiments involving introduction of all the proteinic hydrolysates. The concentration of the total protein in the liquid part of the intestinal juice with intravenous infusion of the preparations remained unchanged. At the same time, the amount of protein secreted together with the intestinal juice at the rate of single secretory action was in the instance of the caseine hydrolysate TSOLIPK and aminopeptide infusion proved 1 1/2--3 times as great as in the tests without infusion.

Animals

Use of hydrophobic interaction methods in the isolation of proteins from endocrine and paraendocrine tissues and cells by high-performance liquid chromatography.

We have recently described the separation of a large number of polypeptide hormones, related peptides and some protein standards by hydrophobic interaction high-performance liquid chromatography (HPLC). This paper reports the practical application of these methods to the reproducible isolation and separation of components of a mixture of immunoreactive calcitonin-like proteins (less than 25 kD) synthesised and secreted by human tumour cells in vitro. Using hydrophobic interaction HPLC on ODS-silica for both preliminary bulk fractionation and subsequent analytical separation greater than 80% recoveries of small (ng) quantities of immunoreactive proteins were obtained from samples containing less than 100 mg total protein, and characteristic profiles of synthesised and secreted materials were established. Using a partially purified hypothalamic extract, containing a number of small proteins (12--25 kD), we have also examined the effects of varying chromatographic conditions in an attempt to modify the separations obtained with ODS-silica using an acid-saline-acetonitrile gradient elution system at ambient temperature, and achieve further resolution of its components. No useful selective effects were observed when temperature, organic modifier, gradient profile or hydrophobic stationary phase were altered. These techniques may not therefore be inherently capable of completely resolving all components of natural protein mixtures. They do, however, offer an adjunct to and in certain cases a substitute for conventional methods of protein separation.

Animals

Correlation between morphology and function in isolated islets of the Zucker rat.

Obesity in the Zucker rat is accompanied by hyperlipemia, hyperinsulinism, insulin resistance, pancreatic hyperplasia, and islet hypertrophy. This study correlates the morphologic heterogeneity of isolated pancreatic islets with secretion of insulin and glucagon in the perifusion system. Islet size was arbitrarily defined as large (greater than 0.45 mm) or small (smaller than 0.12 mm). Protein content and volume (V = 4/3pir3) were calculated for groups and individual islets, respectively. Islets from obese rats secreted more insulin in response to glucose and aminophylline than islets from lean rats (peak 7.8 +/- 2.4 vs. 1.5 +/- 0.37 microU/islet/min, P less than 0.005). Insulin release was related directly to islet size and protein content. Small islets from lean and obese animals produced less insulin per islet than large islets (P less than 0.005). In terms of islet volume, however, large islets were inefficient insulin releasers as compared to small islets (P less than 0.005). Stimulation with Br-cAMP released glucagon from islets of lean but not from large islets of obese animals (peak 11 +/- 3.3 vs. 4.1 +/- 0.3 pg/microgram protein per minute, P less than 0.05). Arginine produced the same effect on glucagon release (P less than 0.05) as stimulation with Br-cAMP. The observed increased insulin release rates and the blunted glucagon response are related to islet size in the pancreas of the Zucker rat.

Aminophylline

[Nitrogen and amino acid absorption in swine. 3. Endogenous secretion and absorption of nitrogen].

The endogenous nitrogen in the duodenum and the distal segment of the small intestine of 3 fistulated pigs fed a 15-N-labelled feed protein was determined using the isotope dilution technique. Over 24 hours, the endogenous N secretion was found at least 12.5 g at the end of the duodenum. For the upper segment of the small intestine, a statistically secured correlation between endogenous nitrogen and chyme amount was found to exist. It was possible to determine the true and apparent absorption of the nitrogen for both individual segments of and the whole digestive tract.

Amino Acids

Secretion of nitrogenous compounds in the small intestine of pigs.

Secretion of nitrogenous compounds in the small intestine of pigs. Acta Physiol. Pol., 1979, 30 (2): 319--326. The study was performed on pigs weighting 50 to 75 kg with temporarily isolated loops of one of the three parts of the small intestine: proximal, middle and terminal. For measurement of intestinal secretion the isolated loops were perfused with N-free solutions and the effluent was collected and analyzed. On a protein diet, under conditions of an isotonic osmotic pressure of the solution, secretion of nitrogen compounds in the upper part was twice that in the lower part of the small intestine, 0.97 against 0.48 g N/m/24 h. A hypertonic pressure increased secretion of N into the intestinal lumen. On a protein-free diet secretion of nitrogen compounds was markedly decreased. Soluble N amounted to about 90% of total N and alpha-amino-N to 50 to 79% of soluble N. Free amino acids made up about 65% and protein 33% of the total alpha-amino-N. There was a large proportion of threonine, 6.0--6.3%, and lysine, 8.0--8.7% in the total amino acids secreted into the intestine.

Animals

Cellular basis of regulation of expression of idiotype. I. T-suppressor cells specific for MOPC 460 idiotype regulate the expression of cells secreting anti-TNP antibodies bearing 460 idiotype.

An idiotype of the dinitrophenyl-binding myeloma protein MOPC 460 was expressed on a small but significant proportion of anti-TNP antibodies which appeared after in vivo or in vitro immunization of BALB/c mice with three T-independent TNP antigens. In vitro experiments show that the depletion of T cells before culture increased significantly the number of plaques secreting anti-TNP antibodies bearing MOPC 460 idiotype (460Id). T cells from BALB/c mice, but not from C.B20 mice, exhibit this suppressor activity. Plate-binding experiments indicate that the suppressive action of the T-lymphocyte population depends on a cell which can bind to MOPC 460 myeloma protein. The possible role of these normally occurring, idiotype-specific T cells on expression of 460Id in the anti-TNP antibody response of BALB/c mice is discussed.

Animals

Proteins in the uterine secretions of the cow.

Cows of normal reproductive history were treated with progesterone for periods of 2--3 months. The uterine secretions yielded 5 major fractions of macromolecular components. Two of the fractions comprised serum proteins. In the other 3 fractions at least 9 non-serum proteins were observed, 7 in one fraction, but in only small amounts. Of the remaining 2 non-serum proteins one is an acid phosphatase recoverable in small amounts, and the other is lactoferrin and is the major non-serum protein present in the uterine secretions of progesterone-treated cows.

Acid Phosphatase

Synthesis and secretion of corticotropins, melanotropins, and endorphins by rat intermediate pituitary cells.

The synthesis and secretion of various intermediate pituitary proteins was studied by using dispersed intermediate pituitary cell suspensions. Control studies indicated that the isolated cells were obtained in good yield and that after more than 24 h in culture the isolated cells continued to synthesize a collection of proteins similar to those found in freshly extracted intermediate pituitary tissue. Rat intermediate pituitary cells synthesized a molecule (Mr = 30,000; called 30K) that contained antigenic determinants for beta-endorphin, gamma-lipotropin, corticotropin (ACTH), and 16K fragment (the NH2-terminal region of mouse tumor cell pro-ACTH/endorphin). This 30K molecule, two high molecular weight forms of ACTH(13K and 20K), and 16K fragment were all shown to be glycoproteins. Continuous labeling and pulse-chase incubations were used to define the intracellular biosynthetic processing of the 30K molecule. After a 15-min pulse incubation the 30K molecule was the only labeled protein containing antigenic determinants for beta-endorphin, gamma-lipotropin, ACTH, or 16K fragment. A beta-lipotropin-like molecule served as a biosynthetic intermediate in the production of proteins similar to beta-endorphin and gamma-lipotropin. Methionine-enkephalin and alpha-endorphin were not major products in the intermediate lobe cells. Molecules similar to alpha-melanocyte-stimulating hormone and corticotropin-like intermediate lobe peptide (ACTH(18-39)) were also derived from the same 30K molecule; 20K ACTH served as a biosynthetic intermediate in this conversion. In rat intermediate pituitary cells ACTH(1-39) was not a major final product of the intracellular biosynthetic processing of the 30K molecule. The 30K molecule also served as a precursor to a protein similar to mouse tumor cell 16K fragment and related smaller proteins. With rat intermediate pituitary cells, pulse-chase experiments utilizing [35S]methionine demonstrated almost quantitative conversion of the 30K precursor into labeled proteins similar to beta-endorphin and alpha-melanocyte-stimulating hormone. In the absence of added secretagogues, small amounts of all of the smaller proteins derived from the 30K precursor were secreted coordinately into the culture medium.

Adrenocorticotropic Hormone

The purified protein derivative of turberculin, a B-cell mitogen that distinguishes in its action resting, small B cells from activated B-cell blasts.

The purified protein derivative of tuberculin (PPD tuberculin) stimulates approximately one of two lipopolysaccharide (LPS)-activated B-cell blasts of C57BL/6J nu/nu spleen cells to continued clonal growth and maturation to IgM and IgG secretion. It alwo stimulates background, in vivo-activated large cells of normal C57BL/6J nu/nu spleen to growth and Ig secretion, at a frequency of approximately 1 of 100 large spleen cells. PPD tuberculin, therefore, is a polyclonal B-cell activator for B-cell blasts. Many single murine splenic B cells (approximately 50%) appear to have reactivities, and therefore probably receptors, for LPS and PPD tuberculin. PPD tuberculin does not stimulate small, resting B cells to growth as measured by the number of cells in culture and by thymidine uptake. However, it stimulates approximately one-fourth of all spleen cells to blast transformation. The large-size blast cells secrete IgM and, therefore, form plaques in the protein A plaque assay. IgG-secreting, plaque-forming cells develop at later stages of stimulation, indicating that the switch from IgM to IgG may occur without division in single, stimulated B cells. Stimulation of resting B cells to maturation by PPD tuberculin is polyclonal. Thus, approximately 1 in 10(2) IgM-secreting plaque-forming cells form plaques with trinitrophenyl-substituted sheep erythrocytes, 1 in 450 do so with horse erythrocytes, and 1 in 10(3) with sheep erythrocytes. Furthermore, the number of Ig-secreting cells developing from small, resting cells without growth in cultures with or without filler thymus cells suggests polyclonal activation by PPD tuberculin to maturation only of at least one out of four small, splenic B cells.

Animals

Human submandibular gland tissue in culture 2. Nickel affinity to secretory proteins.

In view of the exposure to nickel through the oral cavity the relation between this metal (as 63NiCl2) and salivary macromolecules was studied by tissue culture, isoelectric focusing and equilibrium dialysis. During cultivation of human submandibular gland tissue, small amounts of nickel were incorporated into macromolecules with an isoelectric point of about 4.4 and approximately 7.5. No affinity independent of the protein synthesis was revealed between nickel and submandibular proteins, including the acidic, viscous, sulphate-incorporating glycoproteins. It is therefore possible that a nickel-containing metalloprotein is present in the submandibular secretion, but a complex formation between nickel and macromolecules similar to the nickel-albumin transport system in plasma was not found. The affinity of nickel to small molecular substances in the secretion was not studied.

Culture Techniques

Ultrastructural aspects of crystal-like inclusions in a case of IgA plasma cell proliferation.

A case of apparently benign plasma cell proliferation is described. Needle-like paracrystalline inclusions were observed in numerous plasma cells outside the cisternae of the rough endoplasmic reticulum. They had a dense protein core surrounded by a layer of carbohydrate. They were found only in plasma cells secreting IgA-kappa immunoglobulin, but did not react with labelled specific antisera. A small amount of paraprotein IgA-kappa was found in the serum but no Bence-Jones protein could be detected in the urine. These observations suggest that these plasma cells synthesize normal IgA immunoglobulin and accumulate abnormal protein in the form of paracrystalline inclusions.

Aged

The effect of thyroid hormone on bile salt-independent bile flow and Na+, K+ -ATPase activity in liver plasma membranes enriched in bile canaliculi.

The relationship between bile salt-independent canalicular flow and ATPase activity in liver plasma membranes (LPM) enriched in bile canaliculi, was studied in control, hyperthyroid, and hypothyroid rats. Canalicular bile production was significantly increased in hyperthyroid rats (3.19 +/- 0.23 mul/min per g liver) compared to controls (2.27 +/- 0.24 mul/min per g liver), while it diminished in hypothyroid animals (1.58 +/- 0.17 mul/min per g liver). Although bile salt excretion was also increased in hyperthyroid animals (62.4 +/- 13.3 vs. 41.2 +/- 8.4 nmol/min per g liver), the stimulation in canalicular secretion was primarily related to enhancement of the bile salt-independent fraction of flow (2.47 mul/min per g liver in hyperthyroid rats vs. 1.67 mul/min per g liver in controls). LPM Na+, K+-ATPase activity doubled in hyperthyroid animals (21.5 +/- 5.8 vs. 10.7 +/- 3.1 mumol Pi/mg protein per h) while Mg++-ATPase activity remained unchanged and 5'-nucleotidase activity increased to a small but significant extent. In hypothyroid rats, bile salt excretion remained unchanged from control values so that the reduced secretion was entirely secondary to an inhibition of bile salt-independent secretion (1.19 mul/min per g liver). Na+, K+-ATPase activity in the LPMs from hypothyroid animals decreased by nearly 50% (5.4 +/- 1.6 mumol Pi/mg protein per h), although comparable reductions in the specific activity of Mg++-ATPase and 5'-nucleotidase were also observed. Administration of L-thyroxine to hypothyroid animals restored both bile salt-independent canalicular secretion and membrane enzymes to control values within 2 and 4 days, respectively. Sodium dodecyl sulfate gel electrophoresis demonstrated no significant changes in LPM protein fractions from any of the treatment groups. These studies indicate that thyroid hormone has a parallel effect on bile salt-independent canalicular secretion and LPM Na+, K+-ATPase activity, supporting the hypothesis that Na+ transport and Na+, K+-ATPase may be determinants of bile salt-independent canalicular flow.

Adenosine Triphosphatases

Effect of thyroid hormone, actinomycin D, cycloheximide and puromycin on TRH-induced secretion of TSH, as studied by pituitary concentration of cyclic AMP and intrathyroidal colloid droplet formation.

In an attempt to study the functional relation between pituitary cyclic AMP and TSH secretion in response to thyrotropin releasing hormone (TRH) or thyroid hormone administration, pituitary concentration of cyclic AMP was measured by protein binding assay after in vivo and in vitro administration of test materials (TRH, thyroxine, triiodothyronine, actinomycin D, puromycin and cycloheximide singly or in combination). Small dose of TRH apparently augmented TSH secretion as evidenced by a marked increase of intrathyroidal colloid droplet, but failed to elevate the pituitary concentration of cyclic AMP. Triiodothyronine (T3) and thyroxine (T4) blocked an increase of TSH secretion produced by TRH, but they elevated pituitary concentration of cyclic AMP in vivo and in vitro. Actinomycin D (Act D), puromycin and cycloheximide elevated pituitary cyclic AMP concentration without stimulating TSH secretion. From the data accumulated, it appears that the measurement of total anterior pituitary concentration of cyclic AMP is not useful to evaluate the activity of TSH cells in response to thyroid hormone, TRH and TRH plus thyroid hormone.

Animals

The effect of partially replacing urea nitrogen with protein N on N capture in the rumen of sheep fed a purified diet.

1. The influence of replacing 10% of the urea nitrogen in a purified diet with casein, maize gluten or white fish meal on the efficiency of conversion of dietary-N into microbial N was examined using sheep equipped with rumen fistulas and duodenal re-entrant cannulas. 2. Total nitrogen (TN), non-ammonia nitrogen (NAN) and amino acid nitrogen (AAN) flowing to the proximal duodenum were significantly higher (P smaller than 0.05) when maize gluten was added to the diet, and this appeared to be due to an increased efficiency of microbial protein production. 3. Pepsin secretion was not significantly different between treatments and the daily amount of pepsin N flowing to the proximal duodenum was very small (40-53 mg). The peak of pepsin activity in duodenal digesta was reached 6-8 h after feeding. 4. The possible practical implications of the results are discussed.

Amino Acids