Haemophilic arthropathy: the usefulness of intra-articular oxytetracycline (synoviorthesis) in the treatment of chronic synovitis in children.

Synoviorthesis is already widely used in the treatment of chronic haemophilic synovitis. The aim of this study was evaluate the effectiveness of oxytetracicline synoviorthesis on the frequency of haemarthrosis in haemophilic children with chronic synovitis and its impact on joint function. Between January 2001 and October 2006, we performed 34 synoviorthesis in 28 paediatric patients (6-16 years old) with diagnosis of haemophilic arthropathy stage I-II. At each joint were administered five doses of oxytetracycline for five consecutive weeks at doses of 100 mg in elbow and ankle and 250 mg in the knee. The frequency of haemarthrosis and range of joint mobility were evaluated before and after of treatment. The results were analysed with Student t-test and descriptive statistics. Thirty-four joints were treated, including 20 knees (58.8%), eight elbows (23.5%) and six ankles (17.6%). Median follow-up was 46.3 months (range 12-71 months). The frequency of haemarthrosis was recorded before treatment 47.3 year(-1) (range 12-96, P < 0.0001) and decreased to 3.5 year(-1) (range 0-15, P = 0.0119) after treatment. The range of joint motion in flexion-extension before treatment was 84.9°, while after this was 97.5° (P = 0.0119). The synoviorthesis with oxytetracycline has shown a favourable effect in the treatment of chronic haemophilic synovitis in reducing the frequency of haemarthrosis and improvement was observed consistently in the range of motion.

Internal trehalose protects endocytosis from inhibition by ethanol in Saccharomyces cerevisiae.

Endocytosis in Saccharomyces cerevisiae is inhibited by concentrations of ethanol of 2 to 6% (vol/vol), which are lower than concentrations commonly present in its natural habitats. In spite of this inhibition, endocytosis takes place under enological conditions when high concentrations of ethanol are present. Therefore, it seems that yeast has developed some means to circumvent the inhibition. In this work we have investigated this possibility. We identified two stress conditions under which endocytosis was resistant to inhibition by ethanol: fermentation during nitrogen starvation and growth on nonfermentable substrates. Under these conditions, yeast accumulates stress protectors, primarily trehalose and Hsp104, a protein required for yeast to survive ethanol stress. We found the following. (i) The appearance of ethanol resistance was accompanied by trehalose accumulation. (ii) Mutant cells unable to synthesize trehalose also were unable to develop resistance. (iii) Mutant cells that accumulated trehalose during growth on sugars were resistant to ethanol even under this nonstressing condition. (iv) Mutant cells unable to synthesize Hsp104 were able to develop resistance. We conclude that trehalose is the major factor in the protection of endocytosis from ethanol. Our results suggest another important physiological role for trehalose in yeast.

Monoubiquitination is sufficient to signal internalization of the maltose transporter in Saccharomyces cerevisiae.

Monoubiquitination of the 12-transmembrane segment (12-TMS) Saccharomyces cerevisiae maltose transporter promoted the maximal internalization rate of this protein. This modification is similar to that of the 7-TMS alpha-factor receptor but different from that of the 12-TMS uracil and general amino acid permeases. This result shows that binding of ubiquitin-Lys63 chains is not required for maximal internalization of all 12-TMS-containing proteins.

Clathrin and two components of the COPII complex, Sec23p and Sec24p, could be involved in endocytosis of the Saccharomyces cerevisiae maltose transporter.

The Saccharomyces cerevisiae maltose transporter is a 12-transmembrane segment protein that under certain physiological conditions is degraded in the vacuole after internalization by endocytosis. Previous studies showed that endocytosis of this protein is dependent on the actin network, is independent of microtubules, and requires the binding of ubiquitin. In this work, we attempted to determine which coat proteins are involved in this endocytosis. Using mutants defective in the heavy chain of clathrin and in several subunits of the COPI and the COPII complexes, we found that clathrin, as well as two cytosolic subunits of COPII, Sec23p and Sec24p, could be involved in internalization of the yeast maltose transporter. The results also indicate that endocytosis of the maltose transporter and of the alpha-factor receptor could have different requirements.

Catabolite inactivation of the maltose transporter in nitrogen-starved yeast could be due to the stimulation of general protein turnover.

Addition of glucose to Saccharomyces cerevisiae inactivates the maltose transporter. The general consensus is that this inactivation, called catabolite inactivation, is one of the control mechanisms developed by this organism to use glucose preferentially whenever it is available. Using nitrogen-starved cells (resting cells), it has been shown that glucose triggers endocytosis and degradation of the transporter in the vacuole. We now show that maltose itself triggers inactivation and degradation of its own transporter as efficiently as glucose. This fact, and the observation that glucose inactivates a variety of plasma membrane proteins including glucose transporters themselves, suggests that catabolite inactivation of the maltose transporter in nitrogen-starved cells is not a control mechanism specifically directed to ensure a preferential use of glucose. It is proposed that, in this metabolic condition, inactivation of the maltose transporter might be due to the stimulation of the general protein turnover that follows nitrogen starvation.

Moderate concentrations of ethanol inhibit endocytosis of the yeast maltose transporter.

The maltose transporter in Saccharomyces cerevisiae is degraded in the vacuole after internalization by endocytosis upon nitrogen starvation in the presence of a fermentable substrate. This degradation, known as catabolite inactivation, is inhibited by the presence of moderate concentrations (2 to 6%, vol/vol) of ethanol. We have investigated the mechanism of this inactivation and have found that it is due to the inhibition of the internalization of the transporter by endocytosis. The results also indicate that this inhibition is due to alterations produced by ethanol in the organization of the plasma membrane which also affects to endocytosis of other plasma membrane proteins. Apparently, endocytosis is particularly sensitive to these alterations compared with other processes occurring at the plasma membrane.

Role of the cytoskeleton in endocytosis of the yeast maltose transporter.

Certain components of the cytoskeleton play a role in yeast fluid-phase endocytosis as well as in endocytosis of the alpha-factor when this pheromone is bound to its 7-transmembrane segment receptor. The yeast maltose transporter is a 12-transmembrane segment protein that, under certain physiological conditions, is degraded in the vacuole after internalization by endocytosis. In this work, the possible role of the cytoskeleton in endocytosis of this transporter has been investigated. Using mutants defective in beta-tubulin, actin and the actin-binding proteins Sac6 and Abp85. as well as nocodazole, which inhibits formation of microtubules, we have shown that actin microfilaments are involved in endocytosis of the maltose transporter whereas microtubules are not.

Catabolite inactivation of the yeast maltose transporter requires ubiquitin-ligase npi1/rsp5 and ubiquitin-hydrolase npi2/doa4.

The maltose transporter in Saccharomyces cerevisiae is degraded in the vacuole after internalization by endocytosis when protein synthesis is impaired and a fermentable substrate is present. The possible implication of the ubiquitin pathway in this inactivation, known as catabolite inactivation, has been investigated. Using mutants deficient in npi1/rsp5 ubiquitin-protein ligase and npi2/doa4 ubiquitin-protein hydrolase, we have shown that these two enzymes are required for normal endocytosis and degradation of the transporter. These facts indicate that the ubiquitin pathway is involved in catabolite inactivation of the maltose transporter. The results also revealed that both enzymes act in the internalization step of endocytosis.

The low-affinity component of the glucose transport system in Saccharomyces cerevisiae is not due to passive diffusion.

It has been claimed that the low-affinity component of glucose transport in Saccharomyces cerevisiae is due to passive diffusion of the sugar across the plasma membrane. We have investigated this possibility. For this purpose we have measured the permeability coefficient of hexoses in this organism. We have found that this coefficient is at least two to three orders of magnitude lower than required to account for the low-affinity component of glucose transport, and have concluded that this component is not due to passive diffusion.

Catabolite inactivation of the yeast maltose transporter occurs in the vacuole after internalization by endocytosis.

The maltose transporter of Saccharomyces cerevisiae is rapidly degraded during fermentation in the absence of a nitrogen source. The location and mechanism of degradation of the transporter have been investigated. Using mutants defective in endocytosis, we have shown that degradation of this transporter requires internalization by endocytosis. In addition, studies of mutants defective in proteasome or vacuolar proteolysis revealed that degradation occurs in the vacuole and is independent of proteasome function. The results also revealed that degradation of the maltose transporter requires Sec18p and raised the question of whether in the absence of Sec18p activity the internalized maltose transporter is recycled back to the plasma membrane.

Involvement of endocytosis in catabolite inactivation of the K+ and glucose transport systems in Saccharomyces cerevisiae.

The possible relationship between endocytosis and catabolite inactivation of plasma membrane proteins in Saccharomyces cerevisiae has been investigated. Using mutants with an increased rate of endocytosis we have shown that there is a positive correlation between the rate of endocytosis and the rate of inactivation of the K+ and glucose transport systems. It is concluded that endocytosis is involved in catabolite inactivation of these two transport systems.

cAMP-dependent protein kinase is not involved in catabolite inactivation of the transport of sugars in Saccharomyces cerevisiae.

It has been reported that catabolite inactivation of sugar transport systems in Saccharomyces cerevisiae requires cAMP-dependent protein kinase activity (cAPK) and that the levels of these transport systems are decreased in the absence of a functional cAPK regulatory subunit. We have re-examined these possibilities and have found that catabolite inactivation does not require cAPK activity and that normal levels of the transports occur independently from the presence of the regulatory subunit. With the available information, it is difficult to ascertain the reasons for the discrepancy between our results and the ones previously reported. The inadequacy of the method used to measure the sugar transport activities might contribute to this discrepancy.

Separation and analysis of 4'-epimeric UDP-sugars by ion-pair reversed-phase HPLC.

A simple and sensitive method for determination of 4'-epimeric UDP-sugars using ion-pair reversed-phase HPLC has been developed. The method presents advantages over existing ion-exchange HPLC procedures mainly concerning sensitivity and rapidity of analysis as well as efficiency and stability of the column. It is based on the ability of borate ions to react with cis-diols resulting in the formation of UDP-sugar-borate complexes with different charges. Good resolution and rapid separation (5-25 min) of all 4'-epimeric UDP-sugars tested was achieved with this method, was suitable for concentrations over 20 pmol. The applicability to biochemical analysis was demonstrated by the quantitative determination of the UDP-2-deoxyglucose and UDP-2-deoxygalactose formed in yeast cells upon incubation in the presence of 2-deoxygalactose.

Catabolite inactivation of the yeast maltose transporter is due to proteolysis.

The maltose transport capacity of fermenting Saccharomyces cerevisiae rapidly decreases when protein synthesis is impaired. Using polyclonal antibodies against a recombinant maltose transporter-protein we measured the cellular content of the transporter along this inactivation process. Loss of transport capacity was paralleled by a decrease of cross-reacting material which suggests degradation of the transporter. We also show that in ammonium-starved cells the half-life of the maltose transporter is 1.3 h during catabolism of glucose and > 15 h during catabolism of ethanol.

In vivo inactivation of the yeast plasma membrane ATPase in the absence of exogenous catabolism.

Yeast plasma membrane ATPase is inactivated up to 80% in the absence of catabolism of exogenous nutrients (exogenous catabolism). This inactivation, that is not accompanied by a decrease in the cellular content of ATPase, is due to an irreversible decrease of the Vmax and does not require protein synthesis. The inactivated enzyme maintains the ability to be regulated by fermentable sugars but shows important alterations in the characteristics of this regulation. Upon addition of glucose, the Vmax of the inactivated enzyme increases as well as its Ki for vanadate but, in contrast to the normal enzyme, its affinity for ATP or its pH optimum do not increase. It is concluded that in the absence of exogenous catabolism an irreversible modification of the yeast plasma membrane ATPase takes place that affects several of its kinetic properties.

Trehalose-6-phosphate, a new regulator of yeast glycolysis that inhibits hexokinases.

Trehalose-6-phosphate (P) competitively inhibited the hexokinases from Saccharomyces cerevisiae. The strongest inhibition was observed upon hexokinase II, with a Ki of 40 microM, while in the case of hexokinase I the Ki was 200 microM. Glucokinase was not inhibited by trehalose-6-P up to 5 mM. This inhibition appears to have physiological significance, since the intracellular levels of trehalose-6-P were about 0.2 mM. Hexokinases from other organisms were also inhibited, while glucokinases were unaffected. The hexokinase from the yeast, Yarrowia lipolytica, was particularly sensitive to the inhibition by trehalose-6-P: when assayed with 2 mM fructose an apparent Ki of 5 microM was calculated. Two S. cerevisiae mutants with abnormal levels of trehalose-6-P exhibited defects in glucose metabolism. It is concluded that trehalose-6-P plays an important role in the regulation of the first steps of yeast glycolysis, mainly through the inhibition of hexokinase II.

Sugar transport in Saccharomyces cerevisiae.

The yeast Saccharomyces cerevisiae consumes mono- and disaccharides preferentially to any other carbon source. Since sugars do not freely permeate biological membranes, cellular uptake of these compounds requires the action of 'transporters'. The purpose of this review is to summarize the present knowledge on sugar transport in this organism. Yeast cells show two transporters for monosaccharides, the so-called glucose and galactose transporters that act by a facilitated diffusion mechanism. In the case of glucose transport, which also acts upon D-fructose and D-mannose, two components with high- and low-affinity constants have been identified kinetically. Activity of the high-affinity component is dependent on the presence of active kinases whereas activity of the low-affinity component is independent of the presence of these enzymes. Three genes, SNF3, HXT1 and HXT2, encode three different glucose transporters with a high affinity for the substrates and are repressed by high concentrations of glucose in the medium. Kinetic studies suggest that at least one additional gene exists that encodes a transporter with a low affinity and is expressed constitutively. The present view is that there are several additional transporters for glucose that have not yet been identified. Galactose transport has only one natural substrate, D-galactose, and is encoded by the gene GAL2. Expression of this gene is induced by galactose and repressed by glucose. Two transporters for disaccharides have been identified in S. cerevisiae: maltose and alpha-methylglucoside transporters. These transporters are H(+)-symports that depend on the electrochemical proton gradient and are independent of the ATP level. The gene that encodes the maltose transporter is clustered with the other two genes required for maltose utilization in a locus that is found repeated at different chromosomal locations. Its expression is induced by maltose and repressed by glucose. The rate of sugar uptake in yeast cells is controlled by changes in affinity of the corresponding transporters as well as by an irreversible inactivation that affects their Vmax. The mechanisms involved in these regulatory processes are unknown at present.

The low-affinity component of Saccharomyces cerevisiae maltose transport is an artifact.

It has been reported by several laboratories that maltose transport in Saccharomyces cerevisiae consists of two components with high- and low-affinity constants for maltose. We have investigated the characteristics of the low-affinity component and have found that it shows an abnormal behavior without similarity to any transport mechanism described in this organism. The results strongly indicate that this apparent transport activity is due not to a genuine transport process but to nonspecific binding of maltose to the cell wall and plasma membrane.