Search

The objectives of this study were to (1) compare a test for serum measurement of total Ca (tCa), Mg, and P (VetTest Chemistry Analyzer, IDEXX Laboratories Inc., Westbrook, ME) to reference methods (spectrophotometric assays on a Beckman Coulter 640e automated clinical chemistry analyzer; Beckman Coulter, Brea, CA), (2) determine the relationship between ionized Ca (iCa) and reference method tCa in the immediate postpartum period, and (3) assess the relative value of these blood Ca indices as predictors of neutrophil oxidative burst activity. Samples were collected from multiparous Holstein cows (n = 33) over the first 5 d in milk. A total of 183 samples for objective 1 and 181 samples for objective 2 were available. Neutrophil oxidative burst activity was assessed once between 2 and 5 d in milk (n = 29). Linear regression demonstrated strong relationships between serum tCa, Mg, and P concentrations measured by the VetTest compared with the reference method. Bland Altman analysis indicated that the VetTest values were higher than the reference method by 0.22 mmol/L for tCa, 0.12 mmol/L for Mg, and 0.16 mmol/L for P. Compared with hypocalcemia categorized at ≤2.0 or ≤2.125 mmol/L with the reference method tCa, thresholds for the VetTest measured tCa of ≤2.23 mmol/L (sensitivity = 87%, specificity = 89%) or ≤2.30 mmol/L (sensitivity = 86%, specificity = 96%) could be used. The relationship between whole-blood iCa and reference method serum tCa differed by sampling time point after calving. Compared with identification of hypocalcemia with serum tCa measurements from the reference method (thresholds of ≤2.0 and 2.125 mmol/L), a whole-blood iCa threshold of ≤1.17 mmol/L resulted in the highest combined sensitivities (94 and 82%) and specificities (80 and 94%) at either threshold. Ionized Ca measurements were more consistently related to outcomes of neutrophil oxidative burst activity measured in vitro. The VetTest measurements of serum tCa reliably identified hypocalcemia when thresholds were adjusted to account for the bias of the test. The variation in the relationship between iCa and reference method tCa in the days following parturition suggest that these measures cannot be used interchangeably as indicators of Ca status. The more consistent associations between iCa and in vitro measures of neutrophil function, compared with tCa, indicated that this may be a more sensitive predictor of functional outcomes associated with postpartum Ca status. [ABSTRACT FROM AUTHOR]

The objectives of these experiments were as follows: (1) to determine the association between circulating concentrations of pregnancy-associated glycoproteins (PAG) and late embryonic mortality (EM) in lactating dairy cattle following fixed-time artificial insemination (TAI) on d 0 or timed embryo transfer (TET) on d 7, (2) to identify a circulating concentration of PAG on d 31 below which late EM would be likely to occur, and (3) to identify when during gestation (d 31-59) late EM is occurring. Cows were diagnosed pregnant on d 31 of gestation based on presence of a fetal heartbeat and reconfirmed to be pregnant on d 59 of gestation. Late EM occurred when a cow had a viable embryo on d 31 of gestation but not on d 59 following TAI or TET. Only pregnant cows on d 31 were included in the analysis (TAI-maintained, n = 413; TAI-EM, n = 77; TET-maintained, n = 238; TET-EM, n = 47). Cows that were pregnant at d 31 of gestation and maintained the pregnancy until d 59 had significantly higher circulating concentrations of PAG at d 31 of gestation compared with cows that experienced late EM between d 31 and 59 of gestation in both TAI and TET. To conduct a more stringent test of the effectiveness of a single circulating PAG concentration (d 31) to predict EM, a receiver-operating characteristic curve was generated to identify a PAG concentration on d 31 that would predict EM with ≥95% accuracy in cows that received TAI or TET. Based on positive and negative predicative value analysis, a circulating concentration of PAG below 1.4 ng/mL (TAI; minimal detectable level 0.28 ng/mL) and 1.85 ng/mL (TET) was 95% accurate in predicting EM (between d 31 and 59) at d 31 of gestation, respectively. Following TET, embryonic loss was tracked by Doppler ultrasound, progesterone, and PAG from d 24 to 59 of gestation, with more than 50% of the loss occurring between d 31 and 38 of gestation. In summary, circulating concentrations of PAG on d 31 of gestation may provide a good marker for predicting EM between d 31 and 59 of gestation, and the data suggest that this model could help predict which cows will undergo late EM. [ABSTRACT FROM AUTHOR]

Muscle mass is influenced by many factors including genetically programmed changes, hormonal state, level of activity, and disease processes. Ultimately, whether or not a muscle hypertrophies or atrophies is determined by a simple relationship between the rates of protein synthesis and degradation. When synthesis exceeds degradation, the muscle hypertrophies, and vice versa. In contrast to this simple relationship, the processes that control muscle protein synthesis and degradation are complex. Recently, significant progress has been made in understanding the biochemical mechanisms that control the rate of translation initiation, which is generally the limiting phase in protein synthesis.

Skeletal muscles adapt to changes in their workload by regulating fibre size by unknown mechanisms. The roles of two signalling pathways implicated in muscle hypertrophy on the basis of findings in vitro, Akt/mTOR (mammalian target of rapamycin) and calcineurin/NFAT (nuclear factor of activated T cells), were investigated in several models of skeletal muscle hypertrophy and atrophy in vivo. The Akt/mTOR pathway was upregulated during hypertrophy and downregulated during muscle atrophy. Furthermore, rapamycin, a selective blocker of mTOR, blocked hypertrophy in all models tested, without causing atrophy in control muscles. In contrast, the calcineurin pathway was not activated during hypertrophy in vivo, and inhibitors of calcineurin, cyclosporin A and FK506 did not blunt hypertrophy. Finally, genetic activation of the Akt/mTOR pathway was sufficient to cause hypertrophy and prevent atrophy in vivo, whereas genetic blockade of this pathway blocked hypertrophy in vivo. We conclude that the activation of the Akt/mTOR pathway and its downstream targets, p70S6K and PHAS-1/4E-BP1, is requisitely involved in regulating skeletal muscle fibre size, and that activation of the Akt/mTOR pathway can oppose muscle atrophy induced by disuse.

The regulatory-targeting subunit (RGL), also called GM) of the muscle-specific glycogen-associated protein phosphatase PP1G targets the enzyme to glycogen where it modulates the activity of glycogen-metabolizing enzymes. PP1G/RGL has been postulated to play a central role in epinephrine and insulin control of glycogen metabolism via phosphorylation of RGL. To investigate the function of the phosphatase, RGL knockout mice were generated. Animals lacking RGL show no obvious defects. The RGL protein is absent from the skeletal and cardiac muscle of null mutants and present at approximately 50% of the wild-type level in heterozygotes. Both the level and activity of C1 protein are also decreased by approximately 50% in the RGL-deficient mice. In skeletal muscle, the glycogen synthase (GS) activity ratio in the absence and presence of glucose-6-phosphate is reduced from 0.3 in the wild type to 0.1 in the null mutant RGL mice, whereas the phosphorylase activity ratio in the absence and presence of AMP is increased from 0.4 to 0.7. Glycogen accumulation is decreased by approximately 90%. Despite impaired glycogen accumulation in muscle, the animals remain normoglycemic. Glucose tolerance and insulin responsiveness are identical in wild-type and knockout mice, as are basal and insulin-stimulated glucose uptakes in skeletal muscle. Most importantly, insulin activated GS in both wild-type and RGL null mutant mice and stimulated a GS-specific protein phosphatase in both groups. These results demonstrate that RGL is genetically linked to glycogen metabolism, since its loss decreases PP1 and basal GS activities and glycogen accumulation. However, PP1G/RGL is not required for insulin activation of GS in skeletal muscle, and rather another GS-specific phosphatase appears to be involved.

Lima beans (Phaseolus lunatus) have been shown to contain cysteine proteinase inhibitor (CPI) activity, but the CPI has not been isolated or characterized. Accordingly, our objective was to isolate and partially characterize a CPI from lima bean. The isolation scheme included water extraction of lima bean flour followed by a chromatography series using DEAE Sepharose, Phenyl Sepharose, hydroxyapatite, and reversed-phase high performance liquid chromatography. This scheme resulted in the partial purification of a approximately 20 000-dalton protein with high inhibitory activity against papain. This isolated lima bean CPI had an N-terminal sequence homologous with other members of the cystatin class of CPIs. The protein was relatively heat labile; suggesting it could be inactivated with normal cooking, which is favorable for its use in transforming plants to create insect resistance.

The role and control of the four rapamycin-sensitive phosphorylation sites that govern the association of PHAS-I with the mRNA cap-binding protein, eukaryotic initiation factor 4E (eIF4E), were investigated by using newly developed phospho-specific antibodies. Thr(P)-36/45 antibodies reacted with all three forms of PHAS-I that were resolved when cell extracts were subjected to SDS-polyacrylamide gel electrophoresis. Thr(P)-69 antibodies bound the forms of intermediate and lowest mobility, and Ser(P)-64 antibodies reacted only with the lowest mobility form. A portion of PHAS-I that copurified with eIF4E reacted with Thr(P)-36/45 and Thr(P)-69 antibodies but not with Ser(P)-64 antibodies. Insulin and/or amino acids increased, and rapamycin decreased, the reactivity of all three antibodies with PHAS-I in both HEK293 cells and 3T3-L1 adipocytes. Immunoprecipitated epitope-tagged mammalian target of rapamycin (mTOR) phosphorylated Thr-36/45. mTOR also phosphorylated Thr-69 and Ser-64 but only when purified immune complexes were incubated with the activating antibody, mTAb1. Interestingly, the phosphorylation of Thr-69 and Ser-64 was much more sensitive to inhibition by rapamycin-FKBP12 than the phosphorylation of Thr-36/45, and the phosphorylation of Ser-64 by mTOR was facilitated by phosphorylation of Thr-36, Thr-45, and Thr-69. In these respects the phosphorylation of PHAS-I by mTOR in vitro resembles the ordered phosphorylation of PHAS-I in cells.

The ability of four wound dressings (CombiDERM, Allevyn Hydrocellular, Tegaderm and Tielle) to resist penetration of methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-resistant Enterococcus faecalis and Pseudomonas aeruginosa was investigated in vitro using a dedicated test apparatus. With the exception of Tielle, each dressing prevented bacterial transmission over an 11-day challenge period. When both the wound contact surface and the external surface of Tielle were directly challenged with a bacterial suspension, penetration of the dressing was observed within three to five days. The breakdown of its outer membrane could explain the inability of Tielle to maintain a barrier to the passage of the test bacteria used in this wound model. On the basis of these data, CombiDERM, Allevyn Hydrocellular and Tegaderm dressings may facilitate infection control by acting as a physical barrier to the transmission of potentially pathogenic and antibiotic-resistant wound bacteria. However, further research is urgently required to determine whether or not the same results are observed in clinical practice.

Control of the translational repressor, PHAS-I, was investigated by expressing proteins with Ser/Thr --> Ala mutations in the five (S/T)P phosphorylation sites. Results of experiments with HEK293 cells reveal at least three levels of control. At one extreme is nonregulated phosphorylation, exemplified by constitutive phosphorylation of Ser82. At an intermediate level, amino acids and insulin stimulate the phosphorylation of Thr36, Thr45, and Thr69 via mTOR-dependent processes that function independently of other sites in PHAS-I. At the third level, the extent of phosphorylation of one site modulates the phosphorylation of another. This control is represented by Ser64 phosphorylation, which depends on the phosphorylation of all three TP sites. The five sites have different influences on the electrophoretic properties of PHAS-I and on the affinity of PHAS-I for eukaryotic initiation factor 4E (eIF4E). Phosphorylation of Thr45 or Ser64 results in the most dramatic decreases in eIF4E binding in vitro. However, each of the sites influences mRNA translation, either directly by modulating the binding affinity of PHAS-I and eIF4E or indirectly by affecting the phosphorylation of other sites.

A recent study by Frantz et al. investigated the relationship between length of stay (LOS) and several factors in a small, rural hospice and found significant differences in LOS by primary physician specialty, referral source, and diagnosis (American Journal of Hospice & Palliative Care, March/April 1999). The purpose of the present study was to replicate and extend the Frantz et al. study in a midsized, urban hospice setting and to examine the relationship of LOS with additional variables, such as living status, discharge status, race, and religion. Significant differences in LOS by gender, diagnosis, physician specialty, referral source, type of insurance, living status, and discharge status were found. No significant differences in LOS were found by race, religion, and place of death. Results are interpreted in the light of previous research findings regarding LOS and in the context of the sample size. Strategies are suggested for increasing patients' LOS.

The effects of transgenic overexpression of glycogen synthase in different types of fast-twitch muscle fibers were investigated in individual fibers from the anterior tibialis muscle. Glycogen synthase was severalfold higher in all transgenic fibers, although the extent of overexpression was twofold greater in type IIB fibers. Effects of the transgene on increasing glycogen and phosphorylase and on decreasing UDP-glucose were also more pronounced in type IIB fibers. However, in any grouping of fibers having equivalent malate dehydrogenase activity (an index of oxidative potential), glycogen was higher in the transgenic fibers. Thus increasing synthase is sufficient to enhance glycogen accumulation in all types of fast-twitch fibers. Effects on glucose transport and glycogen synthesis were investigated in experiments in which diaphragm, extensor digitorum longus (EDL), and soleus muscles were incubated in vitro. Transport was not increased by the transgene in any of the muscles. The transgene increased basal [(14)C]glucose into glycogen by 2.5-fold in the EDL, which is composed primarily of IIB fibers. The transgene also enhanced insulin-stimulated glycogen synthesis in the diaphragm and soleus muscles, which are composed of oxidative fiber types. We conclude that increasing glycogen synthase activity increases the rate of glycogen synthesis in both oxidative and glycolytic fibers, implying that the control of glycogen accumulation by insulin in skeletal muscle is distributed between the glucose transport and glycogen synthase steps.

Glycogen synthase is an excellent in vitro substrate for protein phosphatase-1 (PP1), which is potently inhibited by the phosphorylated forms of DARPP-32 (dopamine- and cAMP-regulated phosphoprotein, M(r) = 32,000) and Inhibitor-1. To test the hypothesis that the activation of glycogen synthase by insulin is due to a decrease in the inhibition of PP1 by the phosphatase inhibitors, we have investigated the effects of insulin on glycogen synthesis in skeletal muscles from wild-type mice and mice lacking Inhibitor-1 and DARPP-32 as a result of targeted disruption of the genes encoding the two proteins. Insulin increased glycogen synthase activity and the synthesis of glycogen to the same extent in wild-type and knockout mice, indicating that neither Inhibitor-1 nor DARPP-32 is required for the full stimulatory effects of insulin on glycogen synthase and glycogen synthesis in skeletal muscle.

Results obtained with PHAS-I proteins having Ser to Ala mutations in the five known phosphorylation sites indicate that mTOR preferentially phosphorylates Thr36 and Thr45. The effects of phosphorylating these sites on eIF4E binding were assessed in a far-Western analysis with a labeled eIF4E probe. Phosphorylation of Thr36 only slightly attenuated binding of PHAS-I to eIF4E, while phosphorylation of Thr45 markedly inhibited binding. Phosphorylation of neither site affected the electrophoretic mobility of the protein, indicating that results of studies that rely solely on a gel-shift assay to assess changes in PHAS-I phosphorylation must be interpreted with caution.

Many hospice patients are referred comparatively late in the course of their disease progression, therefore minimizing the time of services to the patient, caregivers, and families. Untimely referrals can create organizational, clinical, and emotional problems for all involved; a better understanding of the factors related to length of stay (LOS) in hospice is necessary. This study investigated the relationship between LOS and selected variables. There were significant differences in LOS by diagnosis, physician type, and referral source. No significant differences were found in LOS by gender or insurance type. Factors related to LOS can assist hospices in identifying those particular patients more likely to have longer stays. Additionally, administrators may tailor their programs to meet the needs of the individual hospice.

PRESSURE ULCER RISK ASSESSMENT IN INTENSIVE CARE SETTINGS MRSA IN BURN PATIENTS LONG-STRETCH AND SHORT- STRETCH BANDAGING HEALING RATES AND TREATMENT COSTS IN COMPRESSION BANDAGING.

Rapamycin is a potent cytostatic agent that arrests cells in the G1 phase of the cell cycle. The relationships between cellular sensitivity to rapamycin, drug accumulation, expression of mammalian target of rapamycin (mTOR), and inhibition of growth factor activation of ribosomal p70S6 kinase (p70(S6k)) and dephosphorylation of pH acid stable protein I (eukaryotic initiation factor 4E binding protein) were examined. We show that some cell lines derived from childhood tumors are highly sensitive to growth inhibition by rapamycin, whereas others have high intrinsic resistance (>1000-fold). Accumulation and retention of [14C]rapamycin were similar in sensitive and resistant cells, with all cells examined demonstrating a stable tight binding component. Western analysis showed levels of mTOR were similar in each cell line (<2-fold variation). The activity of p70(S6k), activated downstream of mTOR, was similar in four cell lines (range, 11.75-41. 8 pmol/2 x 10(6) cells/30 min), but activity was equally inhibited in cells that were highly resistant to rapamycin-induced growth arrest. Rapamycin equally inhibited serum-induced phosphorylation of pH acid stable protein I in Rh1 (intrinsically resistant) and sensitive Rh30 cells. In serum-fasted Rh30 and Rh1 cells, the addition of serum rapidly induced c-MYC (protein) levels. Rapamycin blocked induction in Rh30 cells but not in Rh1 cells. Serum-fasted Rh30/rapa10K cells, selected for high level acquired resistance to rapamycin, showed >/=10-fold increased c-MYC compared with Rh30. These results suggest that the ability of rapamycin to inhibit c-MYC induction correlates with intrinsic sensitivity, whereas failure of rapamycin to inhibit induction or overexpression of c-MYC correlates with intrinsic and acquired resistance, respectively.

HONEY AS A BURN TREATMENT SURGEONS' GLOVES THE ROLE OF ZINC IN WOUND HEALING.

The primary site in PHAS-I for phosphorylation by protein kinase CK2 in vitro was identified as Ser111. A relatively small amount of phosphorylation of Ser99 was also detected, and mutating Ser99 to Ala in PHAS-I slightly decreased phosphorylation by CK2 in vitro. In contrast, mutating Ser111 to Ala almost abolished phosphorylation, confirming Ser111 as the preferred site for CK2. Phosphorylation of Ser111 did not decrease binding of PHAS-I to eIF4E, and results of peptide mapping experiments with PHAS-I immunoprecipitated from 32P-labeled adipocytes indicated that Ser111 was not phosphorylated in cells. These results support the conclusion that CK2 is not involved in the control of PHAS-I.

THE DOCTOR-PATIENT RELATIONSHIP PREVENTING LEG ULCER RECURRENCE EVIDENCE-BASED PRACTICE IN BURN MANAGEMENT.

The iodine content of a tulle gras-type dressing medicated with povidone-iodine (Poviderm) has been measured and its potential efficacy in wound care explored by means of laboratory models. Simple tests demonstrated the ready diffusibility and antibacterial activity of povidone-iodine. Wound models clearly showed that the limiting factor for useful dressing life is extent of exudation. It seems likely that this dressing would provide good topical antibacterial prophylaxis and may reduce the bacterial burden of colonised wounds. The dressing should help contain wound bacteria and thus assist infection control.

The effects of insulin on the mammalian target of rapamycin, mTOR, were investigated in 3T3-L1 adipocytes. mTOR protein kinase activity was measured in immune complex assays with recombinant PHAS-I as substrate. Insulin-stimulated kinase activity was clearly observed when immunoprecipitations were conducted with the mTOR antibody, mTAb2. Insulin also increased by severalfold the 32P content of mTOR that was determined after purifying the protein from 32P-labeled adipocytes with rapamycin.FKBP12 agarose beads. Insulin affected neither the amount of mTOR immunoprecipitated nor the amount of mTOR detected by immunoblotting with mTAb2. However, the hormone markedly decreased the reactivity of mTOR with mTAb1, an antibody that activates the mTOR protein kinase. The effects of insulin on increasing mTOR protein kinase activity and on decreasing mTAb1 reactivity were abolished by incubating mTOR with protein phosphatase 1. Interestingly, the epitope for mTAb1 is located near the COOH terminus of mTOR in a 20-amino acid region that includes consensus sites for phosphorylation by protein kinase B (PKB). Experiments were performed in MER-Akt cells to investigate the role of PKB in controlling mTOR. These cells express a PKB-mutant estrogen receptor fusion protein that is activated when the cells are exposed to 4-hydroxytamoxifen. Activating PKB with 4-hydroxytamoxifen mimicked insulin by decreasing mTOR reactivity with mTAb1 and by increasing the PHAS-I kinase activity of mTOR. Our findings support the conclusion that insulin activates mTOR by promoting phosphorylation of the protein via a signaling pathway that contains PKB.

In the Adult Intensive Care Unit of The Prince of Wales Hospital, Sydney, Australia, inspiratory gas is humidified to saturation at 37 degrees C. This stops the buildup of dried sputum within the endotracheal tubes and thus prevents blocked tubes and the increased resistance caused by partial obstruction. Inspiratory and expiratory hose heater wires are used to produce a completely dry circuit, allowing the elimination of water traps and circuit support arms without the resistance of a heat and moisture exchanger.

This study explores the absorptive capacity of standard cotton wool-tipped swabs in vitro and in vivo. An alternative wound exudate sampling technique using small filter paper discs is also briefly examined. Results showed a poor uniformity of fluid absorption by swabs and a limited correlation of wound exudate quantities in relation to visual estimates and size of wound. It was found, however, that swabs reliably removed material from a wound surface. The filter paper technique appeared to offer no advantages.

Chemical control of micro-organisms relies on strategies that either inhibit growth or cause death of the organisms. Chemicals that cause death of micro-organisms are known as cidal agents. Chemicals that prevent microbial growth without causing death are known as static agents.

THE USE OF TAP WATER FOR CLEANSING TRAUMATIC WOUNDS TISSUE ADHESIVES IN THE MANAGEMENT OF LACERATIONS EVALUATING COMPRESSION THERAPY FOR LEG ULCERS THE EFFECTS OF DRESSINGS ON THE HEALING OF VENOUS LEG ULCERS.

Insulin and growth factors elicit rapid increases in protein synthesis by stimulating mRNA translation. PHAS/4E-BPs, a recently discovered family of elF4E-binding, proteins, appear to play a key role in this process, as well as in the control of cell proliferation.

The immunosuppressant rapamycin interferes with G1-phase progression in lymphoid and other cell types by inhibiting the function of the mammalian target of rapamycin (mTOR). mTOR was determined to be a terminal kinase in a signaling pathway that couples mitogenic stimulation to the phosphorylation of the eukaryotic initiation factor (eIF)-4E-binding protein, PHAS-I. The rapamycin-sensitive protein kinase activity of mTOR was required for phosphorylation of PHAS-I in insulin-stimulated human embryonic kidney cells. mTOR phosphorylated PHAS-I on serine and threonine residues in vitro, and these modifications inhibited the binding of PHAS-I to eIF-4E. These studies define a role for mTOR in translational control and offer further insights into the mechanism whereby rapamycin inhibits G1-phase progression in mammalian cells.

PHAS-I is a recently discovered regulator of translation initiation. Non-phosphorylated PHAS-I binds and inhibits eukaryotic initiation factor-4E, the mRNA cap-binding protein that mediates a rate-limiting step in translation initiation. When PHAS-I is phosphorylated in response to insulin, the PHAS-I/eukaryotic initiation factor-4E complex dissociates. The present study was conducted to investigate mechanisms involved in the control of PHAS-I. Phosphorylation of PHAS-I was monitored by immunoblotting after subjecting extracts to polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. This was possible because phosphorylation markedly decreases the electrophoretic mobility of PHAS-I. Incubating 3T3-L1 adipocytes with rapamycin and wortmannin inhibited insulin-stimulated phosphorylation of PHAS-I at concentrations similar to those that inhibited activation of p70S6K. Both agents increased the amount of PHAS-I that co-purified with eukaryotic initiation factor-4E when extracts were fractionated using a cap affinity resin, indicating that PHAS-I binding to the initiation factor was increased. Incubating adipocytes with the protein phosphatase inhibitors, calyculin A and okadaic acid, increased PHAS-I phosphorylation and opposed the effects of rapamycin on decreasing PHAS-I phosphorylation. However, neither okadaic acid nor calyculin A abolished the effects of rapamycin on PHAS-I. These results suggest that the phosphorylation of PHAS-I in response to insulin occurs via the p70S6K signalling pathway. By regulating eukaryotic initiation factor-4E, PHAS-I may have important roles in the control of both protein synthesis and mitogenesis.

Incubating either Chinese hamster ovary (CHO) cells or 3T3-L1 adipocytes with insulin increased the phosphorylation of the eIF-4E-binding protein, PHAS-I. Insulin also activated p70S6K and the Erk-1 and Erk-2 isoforms of mitogen-activated protein kinase (MAP kinase). However, the concentrations of the hormone needed to activate MAP kinase were 10-100 times higher than those needed to increase PHAS-I phosphorylation and p70S6K activity. Incubating cells with the inhibitor of MAP kinase kinase (MEK) activation, PD 098059, blocked the effects of low concentrations of insulin on PHAS-I and p70S6K. The effects of the inhibitor were overcome by increasing concentrations of insulin. The results indicate that insulin activates a PD 098059-sensitive kinase that is involved in the regulation of both p70S6K and PHAS-I.

The metabolism of the storage polysaccharide glycogen is intimately linked with insulin action and blood glucose homeostasis. Insulin activates both glucose transport and glycogen synthase in skeletal muscle. The central issue of a long-standing debate is which of these two effects determines the rate of glycogen synthesis in response to insulin. Recent studies with transgenic animals indicate that, under appropriate conditions, each process can contribute to determining the extent of glycogen accumulation. Insulin causes stable activation of glycogen synthase by promoting dephosphorylation of multiple sites in the enzyme. A model linking this action to the mitogen-activated protein kinase signaling pathway via the phosphorylation of the regulatory subunit of glycogen synthase phosphatase gained widespread acceptance. However, the most recent evidence argues strongly against this mechanism. A newer model, in which insulin inactivates the enzyme glycogen synthase kinase-3 via the protein kinase B pathway, has emerged. Though promising, this model still does not completely explain the molecular basis for the insulin-mediated activation of glycogen synthase, which remains one of the many unknowns of insulin action.

Two critiques review the classic paper cited below and discuss its influence on the development of surgical dressings.

Insulin stimulated protein synthesis in gastrocnemius muscle of perfused rat hindlimb preparations by approximately twofold. The stimulation of protein synthesis was associated with a 12-fold increase in the amount of eukaryotic initiation factor eIF-4G bound to the mRNA cap-binding protein eIF-4E. In part, the increased binding of eIF-4G to eIF-4E was a result of release of eIF-4E bound to the translational regulator, PHAS-I, through a mechanism involving enhanced phosphorylation of PHAS-I. However, the insulin-induced association of eIF-4E and eIF-4G was not due to increased net phosphorylation of eIF-4E because insulin decreased the amount present in the phosphorylated form from 86 to 59% of total eIF-4E. Overall, the results suggest that insulin stimulates protein synthesis in gastrocnemius muscle through a mechanism involving increased binding of eIF-4G to eIF-4E, which is in part due to phosphorylation of PHAS-I, resulting in a release of eIF-4E from the inactive PHAS-I x eIF-4E complex.

Two critiques review the classic paper cited below and discuss its influence on the development of surgical dressings.

The immunosuppressant, rapamycin, inhibits cell growth by interfering with the function of a novel kinase, termed mammalian target of rapamycin (mTOR). The putative catalytic domain of mTOR is similar to those of mammalian and yeast phosphatidylinositol (PI) 3-kinases. This study demonstrates that mTOR is a component of a cytokine-triggered protein kinase cascade leading to the phosphorylation of the eukaryotic initiation factor-4E (eIF-4E) binding protein, PHAS-1, in activated T lymphocytes. This event promotes G1 phase progression by stimulating eIF-4E-dependent translation initiation. A mutant YAC-1 T lymphoma cell line, which was selected for resistance to the growth-inhibitory action of rapamycin, was correspondingly resistant to the suppressive effect of this drug on PHAS-1 phosphorylation. In contrast, the PI 3-kinase inhibitor, wortmannin, reduced the phosphorylation of PHAS-1 in both rapamycin-sensitive and -resistant T cells. At similar drug concentrations (0.1-1 microM), wortmannin irreversibly inhibited the serine-specific autokinase activity of mTOR. The autokinase activity of mTOR was also sensitive to the structurally distinct PI 3-kinase inhibitor, LY294002, at concentrations (1-30 microM) nearly identical to those required for inhibition of the lipid kinase activity of the mammalian p85-p110 heterodimer. These studies indicate that the signaling functions of mTOR, and potentially those of other high molecular weight PI 3-kinase homologs, are directly affected by cellular treatment with wortmannin or LY294002.

To investigate the role of glycogen synthase in controlling glycogen accumulation, we generated three lines of transgenic mice in which the enzyme was overexpressed in skeletal muscle by using promoter-enhancer elements derived from the mouse muscle creatine kinase gene. In all three lines, expression was highest in muscles composed primarily of fast-twitch fibers, such as the gastrocnemius and anterior tibialis. In these muscles, glycogen synthase activity was increased by as much as 10-fold, with concomitant increases (up to 5-fold) in the glycogen content. The uridine diphosphoglucose concentrations were markedly decreased, consistent with the increase in glycogen synthase activity. Levels of glycogen phosphorylase in these muscles increased (up to 3-fold), whereas the amount of the insulin-sensitive glucose transporter 4 either remained unchanged or decreased. The observation that increasing glycogen synthase enhances glycogen accumulation supports the conclusion that the activation of glycogen synthase, as well as glucose transport, contributes to the accumulation of glycogen in response to insulin in skeletal muscle.