Akt activation by growth factors is a multiple-step process: the role of the PH domain

The protein kinase encoded by the Akt proto-oncogene is activated by phospholipid binding, membrane translocation and phosphorylation. To address the relative roles of these mechanisms of Akt activation, we have employed a combination of genetic and pharmacological approaches. Transient transfection of NIH3T3 cells with wild-type Akt, pleckstrin homology (PH) domain mutants, generated on the basis of a PH domain structural model, and phosphorylation site Akt mutants provided evidence for a model of Akt activation consisting of three sequential steps: (1) a PH domain-dependent, growth factor-independent step, marked by constitutive phosphorylation of threonine 450 (T450) and perhaps serine 124 (S124), that renders the protein responsive to subsequent activation events; (2) a growth factor-induced, PI3-K-dependent membrane-translocation step; and (3) a PI3-K-dependent step, characterized by phosphorylation at T308 and S473, that occurs in the cell membrane and is required for activation. When forced to translocate to the membrane, wild-type Akt and PH domain Akt mutants that are defective in the first step become constitutively active, suggesting that the purpose of this step is to prepare the protein for membrane translocation. Both growth factor stimulation and forced membrane translocation, however, failed to activate a T308A mutant. This, combined with the finding that T308D/S473D double mutant is constitutively active, suggests that the purpose of the three-step process of Akt activation is the phosphorylation of the protein at T308 and S473. The proposed model provides a framework for a comprehensive understanding of the temporal and spatial requirements for Akt activation by growth factors.     

Ocular manifestations of toxic epidermal necrolysis associated with allopurinol use

A 54-year-old man was receiving allopurinol therapy to treat hyperuricemia that followed an inferior wall, myocardial infarction. After three weeks of allopurinol therapy, the patient developed signs and symptoms of toxic epidermal necrolysis that included pseudomembranous conjunctivitis with ulcerative lesions on the lids and conjunctiva, and punctate corneal staining with subsequent corneal abrasions. Treatment with topical antibiotics and artificial tears relieved the symptoms somewhat, but punctate staining and dry eyes persisted after 14 months of follow-up. Bilateral corneal ulcers developed and necessitated conjunctival flaps in each eye. Visual acuity in each eye was 20/40.     

Studies on mushroom flavours 2. Flavour compounds in coprinus comatus

Within 3 hours, 10 healthy male volunteers were infused 500 ml of a 5 percent emulsion in which 25 percent of the fat proportion had been replaced by MCT-fats. As expected, the ketone body concentration in the blood rose, while pyruvate remained constant and lactate dropped. The results show that, basically, a MCT-containing fat infusion is suited for parenteral nutrition and, because of their specific properties, medium chain triglycerides may be used as rapid energy donators.     

In vivo biocompatibility and biostability of modified polyurethanes

Modified segmented polyurethanes were examined for biostability and biocompatibility using an in vivo cage implant system for time intervals of 1, 2, 3, 5, and 10 weeks. Two types of materials were used: polyether polyurethanes and polycarbonate polyurethanes. Two unmodified polyether polyurethanes (PEUU A' and SPU-PRM), one PDMS endcapped polyether polyurethane (SPU-S), and two polycarbonate polyurethanes (SPU-PCU and SPU-C) were investigated in this study. Techniques used to characterize untreated materials were dynamic water contact angle, stress-strain analysis, and gel permeation chromatography. Cellular response was measured by exudate analysis and by macrophage and foreign body giant cell (FBGC) densities. Material characterization, postimplantation, was done by attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR) in order to quantify biodegradation and scanning electron microscopy (SEM) to qualitatively describe the cellular response and biodegradation. The exudate analysis showed that the acute and chronic inflammatory responses for all materials were similar. Lower FBGC densities and cell coverage on SPU-S were attributed to the hydrophobic surface provided by the PDMS endgroups. The polycarbonate polyurethanes did not show any significant differences in cell coverage or FBGC densities even though the macrophage densities were slightly lower compared to polyether polyurethanes. By 10 weeks, biodegradation in the case of PEUU A' and SPU-PRM was extensive as compared to SPU-S because the PDMS endcaps of SPU-S provided a shield against the oxygen radicals secreted by macrophages and FBGCs and lowered the rate of biodegradation. In the case of polycarbonate polyurethanes, the oxidative stability of the carbonate linkage lowered the rate of biodegradation tremendously as compared to the polyether polyurethanes (including SPU-S). The minor amount of biodegradation seen in polycarbonate polyurethanes at 10 weeks was attributed to hydrolysis of the carbonate linkage.     

Isolation and characterization of rat liver amphisomes. Evidence for fusion of autophagosomes with both early and late endosomes

Amphisomes, the autophagic vacuoles (AVs) formed upon fusion between autophagosomes and endosomes, have so far only been characterized in indirect, functional terms. To enable a physical distinction between autophagosomes and amphisomes, the latter were selectively density-shifted in sucrose gradients following fusion with AOM-gold-loaded endosomes (endosomes made dense by asialoorosomucoid-conjugated gold particles, endocytosed by isolated rat hepatocytes prior to subcellular fractionation). Whereas amphisomes, by this criterion, accounted for only a minor fraction of the AVs in control hepatocytes, treatment of the cells with leupeptin (an inhibitor of lysosomal protein degradation) caused an accumulation of amphisomes to about one-half of the AV population. A quantitative electron microscopic study confirmed that leupeptin induced a severalfold increase in the number of hepatocytic amphisomes (recognized by their gold particle contents; otherwise, their ultrastructure was quite similar to autophagosomes). Leupeptin caused, furthermore, a selective retention of endocytosed AOM-gold in the amphisomes at the expense of the lysosomes, consistent with an inhibition of amphisome-lysosome fusion. The electron micrographs suggested that autophagosomes could undergo multiple independent fusions, with multivesicular (late) endosomes to form amphisomes and with small lysosomes to form large autolysosomes. A biochemical comparison between autophagosomes and amphisomes, purified by a novel procedure, showed that the amphisomes were enriched in early endosome markers (the asialoglycoprotein receptor and the early endosome-associated protein 1) as well as in a late endosome marker (the cation-independent mannose 6-phosphate receptor). Amphisomes would thus seem to be capable of receiving inputs both from early and late endosomes.     

Preservation of the stapedius tendon in laser stapes surgery

OBJECTIVES/HYPOTHESIS: The stapedius tendon is routinely transected during stapes surgery. The objective of this study was to evaluate the technique of stapedial tendon preservation during stapes surgery and to compare results of these cases with cases where the stapedial tendon was not preserved. STUDY DESIGN: Retrospective study. METHODS: Four groups of patients were evaluated. Two groups had undergone stapes surgery with preservation of the stapedial tendon. One of these groups underwent a laser stapedotomy minus prosthesis (laser STAMP) procedure, while the other group had a prosthesis inserted. The other two groups had undergone laser stapedotomy with one of two different prostheses being used. Audiometric data were obtained and reviewed both preoperatively and at approximately 6 weeks postoperatively. RESULTS: All groups had overall successful results demonstrating that stapedial tendon preservation is technically possible and does not compromise outcomes. CONCLUSIONS: Based on the results, it is recommended that the stapedius tendon be preserved whenever possible during laser stapes surgery. Reasons justifying its preservation are discussed.     

Is the use of dummy or carry-cot of importance for sudden infant death?

The study was undertaken to test the significance of dummy use and carry-cots for counts of sudden infant death (SID). Based on the impression that very few SID victims have a dummy (comforter, pacifier) in their mouth at the time of death, and that a high proportion of SID babies were found dead in a carry-cot (portable crib, pram), a case-control study was performed. Questionnaires were sent to 167 parents of SID victims and to 352 parents of live-infants matched for sex and time of birth. Questionnaires were returned by 121 parents of SID victims (73%) and 307 parents of controls (87%). Only 4 SID victims (3%) were found dead with a dummy in their mouth. A quarter of the controls always used a dummy, 24% during night-time and 23% during daytime between 0 and 2 months of age, whereas this was only true for 10% in the SID group, the odds ratios being 0.27 for night-time and 0.36 for daytime. This trend was also seen until 1 year of life. Of the SID victims, 48% were found dead in a carry-cot, 79% during the cold time of the year and two thirds outdoors. Most deaths occurred during the afternoon (12 pm-5 pm). In both SID and control groups daytime use of carry-cots was equally frequent. Approximately 40% in both groups slept in such a cot between 5-7 days a week during the daytime. CONCLUSION: The use of dummy could be a favourable factor in the prevention of SID. Because of the frequent use of carry-cots during the daytime both in the SID group and the control group, the high percentage of SID in such cots does not seem to be of significance. However the high frequency of deaths in outdoor placed carry-cots during the cold period of the year may give clues to understanding the death mechanism in SID.