Activity and components of the granulocyte colony-stimulating factor pathway in hidradenitis suppurativa.

BACKGROUND: Hidradenitis suppurativa (HS) is a chronic inflammatory disease, characterized by painful, purulent and destructive skin alterations in intertriginous areas. OBJECTIVES: We investigated the expression and role in HS of granulocyte colony-stimulating factor (G-CSF), the regulator of neutrophil biology, as clinical signs of a neutrophilic granulocyte-driven inflammation are distinctive in the disease. METHODS: Skin and blood samples obtained from different cohorts of patients with HS and control individuals were assessed by RNA sequencing, quantitative polymerase chain reaction on reverse transcribed mRNA, and/or enzyme-linked immunosorbent assay. Mechanistic studies using keratinocytes, dermal fibroblasts, immune cell populations and skin biopsies were performed. RESULTS: G-CSF was abundant in HS skin, particularly in inflamed nodules and abscesses. Its levels even exceeded those found in other inflammatory skin diseases. Interleukin (IL)-1 and IL-17, respectively, induced G-CSF production by fibroblasts and keratinocytes. These effects were enhanced by tumour necrosis factor (TNF)-α and IL-36. Accordingly, fibroblasts separated from HS lesions expressed G-CSF, and IL-1 receptor antagonist reduced G-CSF levels in explanted HS skin. G-CSF blood levels positively correlated with severity of HS. Elevated lesional G-CSF receptor levels were linked to upregulation of molecules that contribute to prolonged activation of neutrophils by components of bacteria and damaged host cells [formyl peptide receptor 1 (FPR1), FPR2 and free fatty acid receptor 2 (FFAR2)], neutrophil survival [TNF receptor superfamily member 10C (TNFRSF10C/TRAIL-R3) and TNF receptor superfamily member 6B], kinases (tyrosine-protein kinase HCK and hexokinase 3), and skin destruction [MMP25 (matrix metalloproteinase 25) and ADAM8 (disintegrin and metalloproteinase domain-containing protein 8)]. G-CSF elevated the expression of FPR1, FFAR2, and TNFRSF10C/TRAIL-R3 in neutrophils and synergized with bacterial components to induce skin-destructive enzymes. CONCLUSIONS: The G-CSF pathway engages both tissue and immune cells, is strongly activated in HS lesions, and offers the opportunity to target the neutrophil-driven inflammation.

Cell-free soluble expression of the membrane protein PsbS.

Photosystem II subunit S (PsbS) is a membrane protein that plays an exclusive role in non-photochemical quenching for photoprotection of plants under high-light conditions. The activation mechanism of PsbS and its pH-induced conformational changes are currently unknown. For structural investigation of PsbS, effective synthesis of PsbS with selective isotope or electron-spin labels or non-natural amino acids incorporated would be a great asset. Here we present cell-free (CF) expression as a successful method for in vitro production of PsbS that would allow such incorporation. The addition of several detergents, liposomes and lipid nanodiscs was tested for achieving soluble CF expression of PsbS. We have optimized the CF method to yield soluble PsbS of ∼500 ng/µl using a continuous-exchange method at 30 °C, along with a successful purification and refolding of PsbS in n-Dodecyl ß-D-maltoside (ß-DM) detergent. We expect that the presented protocols are transferrable for in vitro expression of other membrane proteins of the Light-Harvesting Complex family.

Red Blood Cell Distribution Width and the Platelet Count in Iron-deficient Children Aged 0.5-3 Years.

Early detection of iron deficiency (ID) and iron deficiency anemia (IDA) in young children is important to prevent impaired neurodevelopment. Unfortunately, many biomarkers of ID are influenced by infection, thus limiting their usefulness. The aim of this study was to investigate the value of red blood cell distribution width (RDW) and the platelet count for detecting ID(A) among otherwise healthy children. A multicenter prospective observational study was conducted in the Netherlands to investigate the prevalence of ID(A) in 400 healthy children aged 0.5-3 years. ID was defined as serum ferritin (SF) <12 µg/L in the absence of infection (C-reactive protein [CRP] <5 mg/L) and IDA as hemoglobin <110 g/L combined with ID. RDW (%) and the platelet count were determined in the complete blood cell count. RDW was inversely correlated with SF and not associated with CRP. Calculated cutoff values for RDW to detect ID and IDA gave a relatively low sensitivity (53.1% and 57.1%, respectively) and specificity (64.7% and 69.9%, respectively). Anemic children with a RDW >14.3% had a 2.7 higher odds (95% confidence interval [CI]: 1.2-6.3) to be iron deficient, compared with anemic children with a RDW <14.3%. The platelet count showed a large range in both ID and non-ID children. In conclusion, RDW can be helpful for identifying ID as the cause of anemia in 0.5- to 3-year-old children, but not as primary biomarker of ID(A). RDW values are not influenced by the presence of infection. There appears to be no role for the platelet count in diagnosing ID(A) in this group of children.

Anaesthesia and postoperative analgesia in surgical neonates with or without Down's syndrome: is it really different?

BACKGROUND: Reports conflict on optimal postoperative analgesic treatment in children with intellectual disability. We retrospectively compared postoperative analgesics consumption between neonates with and without Down's syndrome in relation to anaesthesia requirements and pain scores. METHODS: We analysed hypnotic and analgesic drug administration, pain scores [COMFORT-Behaviour (COMFORT-B) scale], and duration of mechanical ventilation during the first 48 h after surgical repair of congenital duodenal obstruction in neonates, between 1999 and 2011. Data of 15 children with Down's syndrome were compared with data of 30 children without Down's syndrome. RESULTS: General anaesthesia requirements did not differ. The median (inter-quartile range) maintenance dose of morphine during the first 24 h after operation was 9.5 (7.8-10.1) µg kg(-1) h(-1) in the Down's syndrome group vs 7.7 (5.0-10.0) µg kg(-1) h(-1) in the control group (P=0.46). Morphine doses at postoperative day 2 and COMFORT-B scores at day 1 did not significantly differ between the two groups. COMFORT-B scores at day two were lower in children with Down's syndrome (P=0.04). The duration of postoperative mechanical ventilation did not statistically differ between the two groups (P=0.89). CONCLUSIONS: In this study, neonates with and without Down's syndrome received adequate postoperative analgesia, as judged from comparable analgesic consumption and pain scores. We recommend prospective studies in children of different age groups with Down's syndrome and in other groups of intellectually disabled children to provide further investigation of the hypothesis that intellectual disability predisposes to different analgesic requirements.

Ultrasound as guidance for a combined bilateral supraclavicular and caudal block, in order to reduce the total anaesthetic dose in a two year old child after a pneumococcal sepsis.

We present a case of the combination of a bilateral supraclavicular block and a caudal block in a two year old boy who needed amputations of four extremities after a pneumococcal sepsis. With the use of ultrasound guidance, reduction of local anaesthetic dose could be obtained in order not to reach the toxic dose of the local anaesthetic. Amputations of four extremities is not common practice. A good postoperative pain management is more than a challenge.

A role for myosin-I in actin assembly through interactions with Vrp1p, Bee1p, and the Arp2/3 complex.

Type I myosins are highly conserved actin-based molecular motors that localize to the actin-rich cortex and participate in motility functions such as endocytosis, polarized morphogenesis, and cell migration. The COOH-terminal tail of yeast myosin-I proteins, Myo3p and Myo5p, contains an Src homology domain 3 (SH3) followed by an acidic domain. The myosin-I SH3 domain interacted with both Bee1p and Vrp1p, yeast homologues of human WASP and WIP, adapter proteins that link actin assembly and signaling molecules. The myosin-I acidic domain interacted with Arp2/3 complex subunits, Arc40p and Arc19p, and showed both sequence similarity and genetic redundancy with the COOH-terminal acidic domain of Bee1p (Las17p), which controls Arp2/3-mediated actin nucleation. These findings suggest that myosin-I proteins may participate in a diverse set of motility functions through a role in actin assembly.

Association of the yeast pheromone response G protein beta gamma subunits with the MAP kinase scaffold Ste5p.

The mating response pathway of the yeast Saccharomyces cerevisiae includes a heterotrimeric guanine nucleotide-binding protein (G protein) that activates a mitogen-activated protein MAP kinase cascade by an unknown mechanism. An amino-terminal fragment of the MAP kinase scaffold protein Ste5p that interfered with pheromone-induced cell cycle arrest was identified. A haploid-specific interaction between the amino terminus of Ste5p and the G protein beta subunit Ste4p was also detected in a two-hybrid assay, and the product of a signaling-defective allele of STE4 was defective in this interaction. In cells with a constitutively activated pheromone response pathway, epitope-tagged Ste4p was coimmunoprecipitated with Ste5p. Thus, association of the G protein and the MAP kinase cassette via the scaffolding protein Ste5p may transmit the G protein signal.

Pheromone response in yeast: association of Bem1p with proteins of the MAP kinase cascade and actin.

Haploid cells of the yeast Saccharomyces cerevisiae respond to mating pheromones with polarized growth toward the mating partner. This morphological response requires the function of the cell polarity establishment protein Bem1p. Immunochemical and two-hybrid protein interaction assays revealed that Bem1p interacts with two components of the pheromone-responsive mitogen-activated protein (MAP) kinase cascade, Ste20p and Ste5p, as well as with actin. Mutants of Bem1p that are associated with defective pheromone-induced polarized morphogenesis interacted with Ste5p and actin but not with Ste20p. Thus, the association of Bem1p with Ste20p and Ste5p may contribute to the conveyance of spatial information that regulates polarized rearrangement of the actin cytoskeleton during yeast mating.

The research gap in chronic paediatric pain: A systematic review of randomised controlled trials.

BACKGROUND AND OBJECTIVE: Chronic pain is associated with significant functional and social impairment. The objective of this review was to assess the characteristics and quality of randomized controlled trials (RCTs) evaluating pain management interventions in children and adolescents with chronic pain. METHODS: We performed a systematic search of PubMed, Embase and the Cochrane Library up to July 2017. We included RCTs that involved children and adolescents (3 months-18 years) and evaluated the use of pharmacological or non-pharmacological intervention(s) in the context of pain persisting or re-occurring for more than 3 months. Methodological quality was evaluated using the Cochrane Risk of Bias (ROB) Tool. RESULTS: A total of 58 RCTs were identified and numbers steadily increased over time. The majority were conducted in single hospital institutions, with no information on study funding. Median sample size was 47.5 participants (Q1,Q3: 32, 70). Forty-five percent of RCTs included both adults and children and the median of the mean ages at inclusion was 12.9 years (Q1,Q3: 11, 15). Testing of non-pharmacological interventions was predominant and only 5 RCTs evaluated analgesics or co-analgesics. Abdominal pain, headache/migraine and musculoskeletal pain were the most common types of chronic pain among participants. Methodological quality was poor with 90% of RCTs presenting a high or unclear ROB. CONCLUSIONS: Evaluation of analgesics targeting chronic pain relief in children and adolescents through RCTs is marginal. Infants and children with long-lasting painful conditions are insufficiently represented in RCTs. We discuss possible research constraints and challenges as well as methodologies to circumvent them. SIGNIFICANCE: There is a substantial research gap regarding analgesic interventions for children and adolescents with chronic pain. Most clinical trials in the field focus on the evaluation of non-pharmacological interventions and are of low methodological quality. There is also a specific lack of trials involving infants and children and adolescents with long-lasting diseases.

Localization and signaling of G(beta) subunit Ste4p are controlled by a-factor receptor and the a-specific protein Asg7p.

Haploid yeast cells initiate pheromone signaling upon the binding of pheromone to its receptor and activation of the coupled G protein. A regulatory process termed receptor inhibition blocks pheromone signaling when the a-factor receptor is inappropriately expressed in MATa cells. Receptor inhibition blocks signaling by inhibiting the activity of the G protein beta subunit, Ste4p. To investigate how Ste4p activity is inhibited, its subcellular location was examined. In wild-type cells, alpha-factor treatment resulted in localization of Ste4p to the plasma membrane of mating projections. In cells expressing the a-factor receptor, alpha-factor treatment resulted in localization of Ste4p away from the plasma membrane to an internal compartment. An altered version of Ste4p that is largely insensitive to receptor inhibition retained its association with the membrane in cells expressing the a-factor receptor. The inhibitory function of the a-factor receptor required ASG7, an a-specific gene of previously unknown function. ASG7 RNA was induced by pheromone, consistent with increased inhibition as the pheromone response progresses. The a-factor receptor inhibited signaling in its liganded state, demonstrating that the receptor can block the signal that it initiates. ASG7 was required for the altered localization of Ste4p that occurs during receptor inhibition, and the subcellular location of Asg7p was consistent with its having a direct effect on Ste4p localization. These results demonstrate that Asg7p mediates a regulatory process that blocks signaling from a G protein beta subunit and causes its relocalization within the cell.

Cloning and expression of a cDNA encoding bovine retinal pigment epithelial 11-cis retinol dehydrogenase.

PURPOSE: Identification of a 32-kd protein in the bovine retinal pigment epithelium. METHODS: A bovine retinal pigment epithelium cDNA library was constructed in the bacteriophage lambda ZAP Express. A monoclonal antibody, designated 21-C3/AV, was used to isolate the cDNA encoding the 21-C3/AV antigen. A positive full-length clone, designated 21-C3RDH/CD, was sequenced. Northern blot analysis was used to determine the length of the mRNA and the tissue expression pattern. The entire open reading frame of clone 21-C3RDH/CD was used to isolate a recombinant baculovirus clone and expressed in Spodoptera frugiperda insect cells. Enzymatic activity toward 11-cis retinaldehyde was investigated. RESULTS: The complete nucleotide sequence of 21-C3RDH/CD was obtained. The deduced amino acid sequence reveals homology with short-chain alcohol dehydrogenases. Northern blot analysis detected a 1.2-kb transcript. Although the monoclonal antibody used to isolate 21-C3RDH/CD also reacts with other ocular and nonocular tissues, the authors were unable to demonstrate any reactivity with RNA samples isolated from different (non)ocular tissues. Recombinant baculovirus-infected insect cells synthesized the 21-C3/AV antigen. This protein showed 11-cis retinol dehydrogenase activity. CONCLUSIONS: Homology to the human D-beta-hydroxybutyrate dehydrogenase precursor and other alcohol dehydrogenases shows that 21-C3RDH/CD encodes a short-chain alcohol dehydrogenase. Furthermore, tissue specificity and molecular weight of the antigen suggest that 21-C3RDH/CD encodes the bovine retinal pigment epithelial 11-cis retinol dehydrogenase. Direct proof came from experiments in which we used the baculovirus-based expression system for in vitro synthesis of the protein encoded by 21-C3RDH/CD. Protein extracts obtained from recombinant baculovirus-infected insect cells were found capable of reducing 11-cis retinaldehyde.

Rhinomanometric detection rate of rhinoscopically-assessed septal deviations.

Normal values for the flow at a transnasal pressure of 150 Pa were established with active anterior rhinomanometry (with decongestion) in a group of 33 normal subjects. These values were used to detect abnormalities in a group of 193 patients whose septum anatomy had been evaluated with rhinoscopy. About 25% of the rhinoscopically normal patients were found to have significantly low ("abnormal") flow values on one side. The same was true for patients with a small septal deviation restricted to one anatomical area. An abnormal flow was measured in about 35% of the patients with a moderate (restricted) septal deviation. In the patients whose septal deviation was not restricted to one anatomical area, about 45% had an abnormal flow. The highest detection rate was about 80% in patients with major deviations in the region of the vestibule and the valve. Such deviations were found only in a minority of the patients with complaints of nasal obstruction, which limits the importance of rhinomanometric evaluation in clinical practice.

The value of Ret-Hb and sTfR in the diagnosis of iron depletion in healthy, young children.

OBJECTIVES: Reticulocyte hemoglobin (Ret-Hb) content and soluble transferrin receptor (sTfR) are described as promising biomarkers in the analysis of iron status. However, the value of Ret-Hb and sTfR in the early detection of iron depletion, as frequently observed in children in high-income countries, is unclear. We hypothesized that young children to iron depletion, using the WHO cutoff of ferritin <12 µg/l, would have lower Ret-Hb and higher sTfR concentrations compared to children with a ferritin ⩾level 12 µg/l. SUBJECTS/METHODS: In this cross-sectional study, we analyzed mean concentrations of Ret-Hb and sTfR in 351 healthy children aged 0.5-3 years in a high-income country. The Student's t-test was used to compare Ret-Hb and sTfR concentrations between groups. RESULTS: We showed that concentrations of Ret-Hb and sTfR are similar in children with and without iron depletion. A decrease in Ret-Hb concentration was present only when ferritin concentrations were <8 µg/l. sTfR concentrations were similar in children with ferritin concentrations <6 µg/l and ⩾12 µg/l. CONCLUSIONS: Our results showed that the discriminative value of Ret-Hb and sTfR for the detection of iron depletion is limited. Our findings suggest that ferritin is the most useful biomarker in the screening of iron depletion in healthy children in high-income countries. However, ideally, reference ranges of iron status biomarkers should be based on studies showing that children with concentrations outside reference ranges have poor neurodevelopmental outcomes.

Coordinate changes in the expression of troponin subunit and myosin heavy-chain isoforms during fast-to-slow transition of low-frequency-stimulated rabbit muscle.

The purpose of this study was to follow the time course of changes in the expression of myosin heavy chain (HC) and troponin (Tn) subunit isoforms during fast-to-slow transition as induced in rabbit fast-twitch muscle by low-frequency stimulation. The evaluation of changes in the relative concentrations of myosin and troponin subunit isoforms were supplemented by measurements of relative protein synthesis rates using an in situ labeling technique. Changes in the amounts of mRNA encoding fast troponin C (TnC) were followed by Northern blot analysis, those for fast and slow troponin I (TnI) by in vitro translation of total RNA. The various fast myosin heavy chain (HC) and fast troponin T (TnT) isoforms were exchanged sequentially. Myosin HCIId which is the predominant fast isoform in rabbit tibialis anterior, was exchanged with HCIIa and, finally, the latter was replaced by the slow myosin HCI. The replacement of HCIId by HCIIa was accompanied by an exchange of TnT1f and TnT2f with TnT3f. The expression of HCI was accompanied by an exchange of TnT3f with the slow TnT isoforms, TnT1s and TnT2s. The changes in the relative concentrations of the TnT isoforms were preceded by similar changes of their relative synthesis rates. Pronounced decreases in the fast TnI and TnC isoforms occurred only with prolonged stimulation and were preceded by changes of the specific mRNAs and decreases in relative synthesis rates. The parallel time courses of the sequential transitions in both the myosin heavy chain and troponin T isoforms suggest the existence of coordinate programs of expression serving specific functional requirements.

Kinetic microphotometric evaluation of in situ hybridization for mRNA of slow myosin heavy chain in type I and C fibres of rabbit muscle.

The present study was undertaken in order to test the possibility of microphotometric evaluation of in situ hybridizations. The histochemical detection of mRNA specific to the slow myosin heavy chain (HCI), in fibre cross sections of normal and transforming rabbit muscles with a digoxigenin-labelled complementary RNA (cRNA) probe was used as a model. Scanning densitometry of Northern blot hybridizations showed that the detection of cRNA/mRNA hybrids by a staining reaction catalysed by alkaline phosphatase coupled to an anti-digoxigenin antibody occurs in a concentration-dependent manner and follows a linear time course. These findings were the basis for elaborating a comparative microphotometric evaluation of in situ hybridization in tissue sections by measuring the reaction rate of the alkaline phosphatase-catalysed formazan production. Relative amounts of HCI mRNA were thus determined by comparing reaction rates instead of by single point microphotometry. This method was applied to studies on the distribution of HCI mRNA in different fibre types of normal rabbit muscles and and muscles undergoing fast-to-slow fibre transformation in response to low-frequency stimulation. The different fibre types were identified by histochemical staining for myofibrillar actomyosin ATPase (mATPase) in cross sections adjacent to the sections processed for in situ hybridization. On the average, type I fibres displayed 2.3-fold higher reaction rates than the mean value recorded for C fibres. According to the pronounced scattering of the values measured in single C fibres, these fibres represented a heterogeneous population in the transforming muscle.(ABSTRACT TRUNCATED AT 250 WORDS)

Coordinate changes of myosin light and heavy chain isoforms during forced fiber type transitions in rabbit muscle.

Skeletal muscle fibers are versatile entities, capable of changing their phenotype in response to altered functional demands. In the present study, fast-to-slow fiber type transitions were induced in rabbit tibialis anterior (fA) muscles by chronic low-frequency stimulation (CLFS). The time course of changes in relative protein concentrations of fast and slow myosin light chain (MLC) isoforms and changes in their relative synthesis rates by in vivo labeling with [35S]methionine were followed during stimulation periods of up to 60 days. Generally, relative synthesis rates and protein concentrations changed in parallel; i.e., fast isoforms decreased and slow isoforms increased. MLC3f, however, which turns over at a higher rate than the other light chains, exhibited a conspicuous discrepancy between a markedly reduced relative synthesis but only a moderate decrease in protein amount during the initial 2 weeks of CLFS. Apparently, MLC3f is regulated independent of MLC1f, with protein degradation playing an important role in its regulation. The exchange of fast MLC isoforms with their slow counterparts seemed to correspond to the ultimate fast-to-slow (MHCIIa-->MHCI) transition at the MHC level. However, due to an earlier onset of the fast-to-slow transition of the regulatory light chain and the delayed fast-to-slow exchange of the alkali light chains, a spectrum of hybrid isomyosins composed of fast and slow light and heavy chains must have existed transiently in transforming fibers. Such hybrid isomyosins appeared to be restricted to MHCIIa- and MHCI-based combinations. In conclusion, fiber type specific programs that normally coordinate the expression of myofibrillar protein isoforms seem to be maintained during fiber type transitions. Possible differences in post-transcriptional regulation may result in the transient accumulation of atypical combinations of fast and slow MLC and MHC isoforms, giving rise to the appearance of hybrid fibers under the conditions of forced fiber type conversion.

Role of innervation for development and maintenance of troponin subunit isoform patterns in fast- and slow-twitch muscles of the rabbit.

This study investigates the neural influence on the establishment and maintenance of muscle type-specific expression patterns of the three troponin (Tn) subunits, troponin T (TnT), troponin C (TnC), and troponin I (TnI) during postnatal development and in the adult rabbit. For this purpose, we followed changes in the expression of fast and slow TnT, TnC, and TnI isoforms at the protein and mRNA level in slow- and fast-twitch muscles. During postnatal development all fast Tn isoforms increased in fast-twitch muscle. Sequential transitions (TnTs-->TnT3f-->TnT1f) occurred in the TnT isoform pattern. These changes occurred in parallel with sequential transitions in the pattern of myosin heavy chain (HC) isoforms. Neonatal slow-twitch muscle displayed more mature (slow) isoform patterns for both TnT subunits and myosin HCs than fast-twitch muscle. Although the expression of slow TnC in slow-twitch muscle required innervation, denervation had little effect on slow TnT and TnI which seemed to be controlled by an intrinsic program. In fast-twitch muscle, denervation enhanced the expression of all slow Tn subunit isoforms. In addition, it led to a pronounced increase of the slow TnT2s isoform such that the amount of TnT2s exceeded that of TnT1s. The effects of denervation together with previous data on low-frequency stimulated muscle indicate that the expression of fast Tn isoforms in fast-twitch muscle is neurally controlled. The pattern of slow Tn isoforms in slow-twitch muscle seems to be regulated by an intrinsic program and, in addition, by neural influences.

A conserved Gbeta binding (GBB) sequence motif in Ste20p/PAK family protein kinases.

Serine/threonine protein kinases of the Ste20p/PAK family are highly conserved from yeast to man. These protein kinases have been implicated in the signaling from heterotrimeric G proteins to mitogen-activated protein (MAP) kinase cascades and to cytoskeletal components such as myosin-I. In the yeast Saccharomyces cerevisiae, Ste20p is involved in transmitting the mating-pheromone signal from the betagamma-subunits of a heterotrimeric G protein to a downstream MAP kinase cascade. We have previously shown that binding of the G-protein beta-subunit (Gbeta) to a short binding site in the non-catalytic carboxy-terminal region of Ste20p is essential fortransmitting the pheromone signal. In this study, we searched protein sequence databases for sequences that are similar to the Gbeta binding site in Ste20p. We identified a sequence motif with the consensus sequence S S L phi P L I/V x phi phi beta (x: any residue; phi: A, I, L, S, or T; beta: basic residues) that is solely present in members of Ste20p/PAK family protein kinases. We propose that this sequence motif, which we have designated GBB (Gbeta binding) motif, is specifically responsible for binding of Gbeta to Ste20p/PAK protein kinases in response to activation of heterotrimeric G protein coupled receptors. Thus, the GBB motif is a novel type of signaling domain that serves to link protein kinases of the Ste20p/PAK family to G protein coupled receptors.

Genetic interactions indicate a role for Mdg1p and the SH3 domain protein Bem1p in linking the G-protein mediated yeast pheromone signalling pathway to regulators of cell polarity.

The pheromone signal in the yeast Saccharomyces cerevisiae is transmitted by the beta and gamma subunits of the mating response G-protein. The STE20 gene, encoding a protein kinase required for pheromone signal transduction, has recently been identified in a genetic screen for high-gene-dosage suppressors of a partly defective G beta mutation. The same genetic screen identified BEM1, which encodes an SH3 domain protein required for polarized morphogenesis in response to pheromone, and a novel gene, designated MDG1 (multicopy suppressor of defective G-protein). The MDG1 gene was independently isolated in a search for multicopy suppressors of a bem1 mutation. The MDG1 gene encodes a predicted hydrophilic protein of 364 amino acids with a molecular weight of 41 kDa that has no homology with known proteins. A fusion of Mdg1p with the green fluorescent protein from Aequorea victoria localizes to the plasma membrane, suggesting that Mdg1p is an extrinsically bound membrane protein. Deletion of MDG1 causes sterility in cells in which the wild-type G beta has been replaced by partly defective G beta derivatives but does not cause any other obvious phenotypes. The mating defect of cells deleted for STE20 is partially suppressed by multiple copies of BEM1 and CDC42, which encodes a small GTP-binding protein that binds to Ste20p and is necessary for the development of cell polarity. Elevated levels of STE20 and BEM1 are capable of suppressing a temperature-sensitive mutation in CDC42. This complex network of genetic interactions points to a role for Bem1p and Mdg1p in G-protein mediated signal transduction and indicates a functional linkage between components of the pheromone signalling pathway and regulators of cell polarity during yeast mating.

Cell cycle- and Cln2p-Cdc28p-dependent phosphorylation of the yeast Ste20p protein kinase.

Ste20p from Saccharomyces cerevisiae is a member of the Ste20/p21-activated protein kinase family of protein kinases. The Ste20p kinase is post-translationally modified by phosphorylation in a cell cycle-dependent manner, as judged by the appearance of phosphatase-sensitive species with reduced mobility on SDS-polyacrylamide gel electrophoresis. This modification is maximal during S phase, and correlates with the accumulation of Ste20p fused to green fluorescent protein at the site of bud emergence. Overexpression of Cln2p, but not Clb2p or Clb5p, causes a quantitative shift of Ste20p to the reduced mobility form, and this shift is dependent on Cdc28p activity. The post-translational mobility shift can be generated in vitro by incubation of Ste20p with immunoprecipitated Cln2p kinase complexes, but not by immunoprecipitated Clb2p or Clb5p kinase complexes. Ste20p is therefore a substrate for the Cdc28p kinase, and undergoes a Cln2p-Cdc28p mediated mobility shift as cells initiate budding and DNA replication. In cells that express only the Cln2p G1 cyclin, minor overexpression of Ste20p causes aberrant morphology, suggesting a proper coordination of Ste20p and Cln-Cdc28p activity may be required for the control of cell shape.