Asymmetric pulses delivered by a cochlear implant allow a reduction in evoked firing rate and in spatial activation in the guinea pig auditory cortex.

Despite that fact that the cochlear implant (CI) is one of the most successful neuro-prosthetic devices which allows hearing restoration, several aspects still need to be improved. Interactions between stimulating electrodes through current spread occurring within the cochlea drastically limit the number of discriminable frequency channels and thus can ultimately result in poor speech perception. One potential solution relies on the use of new pulse shapes, such as asymmetric pulses, which can potentially reduce the current spread within the cochlea. The present study characterized the impact of changing electrical pulse shapes from the standard biphasic symmetric to the asymmetrical shape by quantifying the evoked firing rate and the spatial activation in the guinea pig primary auditory cortex (A1). At a fixed charge, the firing rate and the spatial activation in A1 decreased by 15 to 25 % when asymmetric pulses were used to activate the auditory nerve fibers, suggesting a potential reduction of the spread of excitation inside the cochlea. A strong "polarity-order" effect was found as the reduction was more pronounced when the first phase of the pulse was cathodic with high amplitude. These results suggest that the use of asymmetrical pulse shapes in clinical settings can potentially reduce the channel interactions in CI users.

Long term behavior of dexamethasone-loaded cochlear implants: In vitro & in vivo.

The aim of this study was to better understand the long term behavior of silicone-based cochlear implants loaded with dexamethasone: in vitro as well as in vivo (gerbils). This type of local controlled drug delivery systems offers an interesting potential for the treatment of hearing loss. Because very long release periods are targeted (several years/decades), product optimization is highly challenging. Up to now, only little is known on the long term behavior of these systems, including their drug release patterns as well as potential swelling or shrinking upon exposure to aqueous media or living tissue. Different types of cylindrical, cochlear implants were prepared by injection molding, varying their dimensions (being suitable for use in humans or gerbils) and initial drug loading (0, 1 or 10%). Dexamethasone release was monitored in vitro upon exposure to artificial perilymph at 37 °C for >3 years. Optical microscopy, X-ray diffraction and Raman imaging were used to characterize the implants before and after exposure to the release medium in vitro, as well as after 2 years implantation in gerbils. Importantly, in all cases dexamethasone release was reliably controlled during the observation periods. Diffusional mass transport and limited drug solubility effects within the silicone matrices seem to play a major role. Initially, the dexamethasone is homogeneously distributed throughout the polymeric matrices in the form of tiny crystals. Upon exposure to aqueous media or living tissue, limited amounts of water penetrate into the implant, dissolve the drug, which subsequently diffuses out. Surface-near regions are depleted first, resulting in an increase in the apparent drug diffusivity with time. No evidence for noteworthy implant swelling or shrinkage was observed in vitro, nor in vivo. A simplified mathematical model can be used to facilitate drug product optimization, allowing the prediction of the resulting drug release rates during decades as a function of the implant's design.

Dexamethasone-loaded cochlear implants: How to provide a desired "burst release".

Cochlear implants containing iridium platinum electrodes are used to transmit electrical signals into the inner ear of patients suffering from severe or profound deafness without valuable benefit from conventional hearing aids. However, their placement is invasive and can cause trauma as well as local inflammation, harming remaining hair cells or other inner ear cells. As foreign bodies, the implants also induce fibrosis, resulting in a less efficient conduction of the electrical signals and, thus, potentially decreased system performance. To overcome these obstacles, dexamethasone has recently been embedded in this type of implants: into the silicone matrices separating the metal electrodes (to avoid short circuits). It has been shown that the resulting drug release can be controlled over several years. Importantly, the dexamethasone does not only act against the immediate consequences of trauma, inflammation and fibrosis, it can also be expected to be beneficial for remaining hair cells in the long term. However, the reported amounts of drug released at "early" time points (during the first days/weeks) are relatively low and the in vivo efficacy in animal models was reported to be non-optimal. The aim of this study was to increase the initial "burst release" from the implants, adding a freely water-soluble salt of a phosphate ester of dexamethasone. The idea was to facilitate water penetration into the highly hydrophobic system and, thus, to promote drug dissolution and diffusion. This approach was efficient: Adding up to 10% dexamethasone sodium phosphate to the silicone matrices substantially increased the resulting drug release rate at early time points. This can be expected to improve drug action and implant functionality. But at elevated dexamethasone sodium phosphate loadings device swelling became important. Since the cochlea is a tiny and sensitive organ, a potential increase in implant dimensions over time must be limited. Hence, a balance has to be found between drug release and implant swelling.

MALDI imaging mass spectrometry reveals multiple clinically relevant masses in colorectal cancer using large-scale tissue microarrays.

For identification of clinically relevant masses to predict status, grade, relapse and prognosis of colorectal cancer, we applied Matrix-assisted laser desorption ionization (MALDI) imaging mass spectrometry (IMS) to a tissue micro array containing formalin-fixed and paraffin-embedded tissue samples from 349 patients. Analysis of our MALDI-IMS data revealed 27 different m/z signals associated with epithelial structures. Comparison of these signals showed significant association with status, grade and Ki-67 labeling index. Fifteen out of 27 IMS signals revealed a significant association with survival. For seven signals (m/z 654, 776, 788, 904, 944, 975 and 1013) the absence and for eight signals (m/z 643, 678, 836, 886, 898, 1095, 1459 and 1477) the presence were associated with decreased life expectancy, including five masses (m/z 788, 836, 904, 944 and 1013) that provided prognostic information independently from the established prognosticators pT and pN. Combination of these five masses resulted in a three-step classifier that provided prognostic information superior to univariate analysis. In addition, a total of 19 masses were associated with tumor stage, grade, metastasis and cell proliferation. Our data demonstrate the suitability of combining IMS and large-scale tissue micro arrays to simultaneously identify and validate clinically useful molecular marker. Copyright © 2017 John Wiley & Sons, Ltd.

[On-scene times for helicopter services. Influence of central dispatch center strategy]. / Präklinische Versorgungszeiten bei Einsätzen der Luftrettung. Einfluss der Dispositionsstrategie der Rettungsleitstelle.

BACKGROUND: Previous studies have suggested that when using several emergency systems and air rescue prehospital and on-scene times are extended, depending on the dispatch strategy. Emergency medical services (EMS) in Germany are delivered by ambulances (AMB) staffed by paramedics alone or with physicians (EMD) and by helicopter emergency medical services (HEMS) always staffed by both. The advantages of HEMS in countries with short transport distances and high hospital density are controversial. The best dispatching strategy for HEMS has not been determined OBJECTIVE: The BoLuS study in the German state of Hessen was designed to evaluate the influence of dispatch strategy on prehospital times for responses involving both HEMS and EMS. METHODS: Rescue responses involving HEMS were prospectively evaluated in 12 regions of Hessen from July 2010 to September 2011. Although all regions had access to HEMS, only one had its own service. Data from both central dispatch centers and helicopter services were collected and combined to calculate the on-scene time (OST) and correlate it with dispatch strategy. RESULTS: A total of 2111 emergency interventions were evaluated. Internal medicine emergencies accounted for 42.9 % of cases and trauma for 36.7 %. Just one patient was involved in 87.9 % of rescues. Two services were involved in 65.3 % of rescues and three or more in 31.5 %. The most common dispatch categories were initial dispatch of EMS and HEMS (50.6 %), initial dispatch of EMS with later request for HEMS (19.7 %) and initial dispatch of both EMS and EMD with later request for HEMS (17.4 %). The OST for these categories were 31.0 ± 13.7 min, 43.7 ± 16.2 min and 54.6 ± 21.3 min (p < 0.01), respectively. CONCLUSION: OST varies significantly depending on the number of EMS involved and the dispatch strategy. Sequential dispatching of ground and later HEMS wastes time. Getting an emergency physician to the scene as quickly as possible, reducing transport time to an appropriate hospital and caring for more complex emergencies are the main indications for HEMS. If HEMS appears likely to be needed, it should be dispatched immediately.

Altered PTEN function caused by deletion or gene disruption is associated with poor prognosis in rectal but not in colon cancer.

Colorectal cancer is the third most common malignancy worldwide. Anti-epidermal growth factor receptor (EGFR)-targeted therapy shows clinical evidence in this malignancy and improves outcome. The tumor suppressor gene phosphatase and tensin homologue (PTEN) is considered a potential predictor of nonresponse to anti-EGFR agents. The purpose of this study was to assess whether associations between PTEN alterations (PTEN gene deletion or PTEN gene disruption) and clinical outcome could be caused by a prognostic (and not predictive) effect of PTEN inactivation. Therefore, we analyzed 404 colorectal cancers not previously treated with anti-EGFR drugs in a tissue microarray format. PTEN deletion and PTEN gene rearrangements were analyzed by fluorescence in situ hybridization. Heterogeneity analysis of all available large tissue sections was performed in 6 cases with genomic PTEN alteration. Twenty-seven (8.8%) of 307 analyzable colorectal cancer spots showed genomic PTEN alterations including 24 hemizygous and 1 homozygous deletion as well as 2 PTEN gene disruptions. Genomic PTEN alterations were associated with reduced patient survival in rectal cancer in univariate and multivariate analyses (P = .012; hazard ratio, 2.675; 95% confidence interval, 1.242-5.759) but not in colon cancer. Large-section evaluation revealed a homogeneous distribution pattern in all 4 analyzed cases with PTEN deletion and in both cases with a PTEN gene disruption. In conclusion, genomic PTEN gene alterations caused by deletion or gene disruption characterize a fraction of rectal cancers with particularly poor outcome.

Genetic variants in the human glucocorticoid-induced leucine zipper (GILZ) gene in fertile and infertile men.

Sertoli cell only (SCO) syndrome is the predominant histology for men with non-obstructive azoospermia (NOA) and is usually of unexplained aetiology. Studies in mouse models indicated that the X-linked gene glucocorticoid-induced leucine zipper (GILZ) is essential for survival and differentiation of spermatogonia, and meiosis. GILZ deficiency results in a rapid and progressive loss of germ cells with SCO tubules and sterility in adults. The role of GILZ in human fertility has not been examined. Here we show that GILZ is localized to spermatogonia and spermatocytes in the human testis in a pattern analogous to that seen in mice. To assess the potential for an association between GILZ variants and human infertility, we sequenced the entire protein-coding regions of the GILZ gene in 65 SCO and 87 fertile Australian men. We identified six genetic variants, three of which had not been reported previously. Three variants, 107018665 G>A, 107018485 C>G and 106959283 C>T, were found at a low frequency only in SCO men. Although none of the identified variants changed the protein code, sequence analysis indicated that two variants, 107018665 G>A and 107018485 C>G, would completely abolish the exonic splicing enhancer (ESE)-binding motifs for the splicing factors SF2/ASF and SC35 respectively. This result prompted an assessment of whether these two variants were associated with male infertility in a separate population of men. We used a PCR-based SNP detection approach to screen an additional 52 NOA and 153 fertile Australian men, and 86 SCO and 54 fertile American men. None of these men carried either of these two variants. The cumulative allelic frequency of these variants is less than 1% in SCO men and no association with fertility status was observed. Our study suggests that GILZ variants are not common causes of SCO and NOA in Australian or American men.

[Time in care of trauma patients in the air rescue service: implications for disposition?]. / Versorgungszeiten bei Traumapatienten im Luftrettungsdienst: Implikationen für die Disposition?

BACKGROUND: Time plays a crucial role in treating multiple traumatized patients and delays in management worsen the prognosis. Furthermore, current studies show that trauma patients profit from primary delivery to a trauma center. Therefore, the goal of physician-staffed ground and air rescue services in Germany is to treat these patients as quickly as possible and deliver them to a suitable trauma center. The aim of the present study was to investigate prehospital treatment times for the air rescue team in terms of disposition and efficiency when a ground rescue team was already present at the scene. METHODS: In a nationwide, multicenter analysis emergency missions carried out for traumatological emergencies in 2006 by 28 air rescue centers (ARC) of the TeamDRF and 6 ARC of the federal police were evaluated using the medical database MEDAT of the German Air Rescue Service. A distinction was made between combined missions with (MEDAT 1 group) and without (MEDAT 2 group) physician-staffed ground emergency medical services already being present at the emergency site and in particular the rescue helicopter treatment times for both groups were investigated. Furthermore, combined missions (MAN 1 group) and solo missions (MAN 2 group) for traumatological emergencies in the period 01.05.2006 to 31.01.2007 were investigated in a complementary prospective regional study at the ARC Heidelberg/Mannheim "Christoph 53". In both groups the total treatment times for all physician-staffed emergency systems involved in treatment at the scene were investigated. RESULTS: Nationwide, 26,010 primary missions could be evaluated and of these, 11,464 missions were traumatological emergencies (44.1%) with 2,229 (19.4%) carried out by the MEDAT 1 group and 9,235 (80.6%) by the MEDAT 2 group. For both groups the helicopter treatment times depended on the severity of the injuries (NACA classification) and were between 17+/-12 min (NACA I) and 34+/-19 min (NACA VII) in MEDAT group 1 versus 21+/-10 and 36+/-19 min in MEDAT group 2 (p<0.05, p<0.001), respectively. In the MEDAT 1 group, the average treatment times were between 2.8 min (NACA VII) and 8.1 min (NACA VI) shorter compared with the MEDAT 2 group. Moreover, when taking the severity of the injury into consideration, a regular and significantly higher treatment effort (e.g. intubation, repositioning and chest tube insertion) and a greater proportion of patients who were transported to the clinic via rescue helicopter were observed for the MEDAT 1 group than for the MEDAT 2 group. In the regional study 670 primary missions were evaluated including 382 traumatological emergencies (57%). From these, 90 multiple trauma patients (NACA V) were not resuscitated or died at the scene, 58 from the MAN 1 group and 32 from the MAN 2 group, and were investigated more closely. The helicopter treatment times were comparable to those observed in the nationwide study and were found to be 26+/-12 min and 35+/-20 min (p<0.05), respectively. In the MAN 1 group the treatment times for the ground rescue services up to the time when the helicopter arrived was 22+/-11 min on average; the total treatment time was 48+/-15 min and 12+/-8 min longer than the time for the MAN 2 group, which was statistically significant. In the MAN 1 group the helicopter was alerted on average 17+/-15 min after the physician-staffed ground rescue services arrived at the emergency site. Treatment by the rescue helicopter teams was significantly more extensive in the MAN 1 group. CONCLUSIONS: The treatment times for the helicopter were several minutes shorter when a physician-staffed ground rescue team had already arrived at the emergency site. However, it must be assumed that the total prehospital time is significantly longer for such missions. These results directly affect the disposition at the emergency dispatch center and indicate that when air rescue is required to transport a patient to hospital, the helicopter should be alerted at an early stage. In such settings, it is likely that initiating the operation in this way would improve the prognosis of severely injured patients and save costs.

[Maillard reaction products in food as pro-inflammatory and pro-arteriosclerotic factors of degenerative diseases]. / Maillardprodukte aus Lebensmitteln als pro-entzündliche und pro-arteriosklerotische Faktoren bei degenerativen Erkrankungen.

Heating of food induces the formation of Maillard reaction products (MRPs) caused by the reaction of reducing sugars with proteins or amino acids. Analogous reactions occur in the human body, eventually forming "Advanced Glycation Endproducts" (AGEs). AGEs accumulate in aging tissues accelerating degenerative-inflammatory and proliferative processes. MRPs present in food can also directly cause inflammatory processes in the intestines and, once absorbed, would support and reinforce any inflammatory and degenerative process occurring in the body. The contribution of AGEs (and additional MRPs) in the development of diabetic complications as well as nephropathy, neuropathy, micro- and macroangiopathies is now well established. Which of the MRPs or AGEs in particular induce these cellular processes is currently unknown. Thus the exact knowledge of the chemical structures of the MRPs could help to minimize the formation of "harmful MRPs" that occur due to heating in food processing. Because MRPs play a decisive role in the successful marketing of edibles due to their characteristics as flavor components, it is important to increase the amount of innocuous and palatable MRPs, and minimize signal active pro-inflammatory MRPs by the use of defined preparation methods. It is practicable to use low-priced immunological methods for the quantitative determination of specific MRPs or AGEs. In the medical area, the knowledge of the signal active MRP/AGE structures provides the opportunity to measure their concentrations in body fluids and tissues and thus determine their influence on inflammatory and age-related degenerative processes (e. g., late diabetic complications, arteriosclerosis, degeneration of neurons). From a clinical perspective, the application of RAGE antagonists after an appropriate chemical diagnosis could be effective in supporting the treatment of affected patient groups, especially older diabetic and dialysis patients.

Evaluation of the maillard reaction in infant formulas by means of front-face fluorescence.

Foods are complex mixtures of macro- and micronutrients, which interact, leading to oxidation, glycation, and hydrolysis upon heating (e.g., sterilization, cooking) and storage. Their nutritional quality and safety are consequently affected, justifying the need for accurate monitoring of the evolution of the food composition during processing and shelf life. Classical chromatographic analysis as well as newly proposed rapid methods based on fluorescence spectrometry analyses were applied on whey powder-based models and commercial samples (in powdered form and ultrahigh temperature [UHT] sterilized), some of which had been previously submitted to protein hydrolysis. These samples were incubated for 48 h at 60 degrees C to mimic accelerated storage. Fluorescence fingerprints addressing modifications in the product composition during processing were recorded and analyzed by chemometric methods. Carboxymethyllysine (Nepsilon-[carboxymethyl]lysine; CML) was measured using an ELISA method. Fluorescence, recorded in a front-face mode on intact samples, is very sensitive to pertinent physicochemical changes induced by heat treatment, formulation (the moisture level in powders, presence of vitamin C and iron), and storage. Similar trends were observed between powders' fluorescence and CML-for example, a strong effect of protein hydrolysis and increasing water content. Addition of vitamin C was associated with an antioxidant effect despite the presence of iron. Good calibration models were obtained for predicting CML from fluorescence spectra both in food models and in commercial samples, although more work is needed to obtain accurate and robust calibration models. Results show the potential of nondestructively applied fluorescence spectrometry for measuring CML in formulas, a rapid, simple, and cost-effective method to monitor formula quality.

Elevated serum levels of N(epsilon)-carboxymethyl-lysine, an advanced glycation end product, are associated with proliferative diabetic retinopathy and macular oedema.

AIMS/HYPOTHESIS: Diabetic retinopathy is a frequent microvascular complication. In search of novel risk markers, we analysed the association between serum levels of the major advanced glycation end product N(epsilon)-carboxymethyl-lysine (CML) and prevalence of advanced stages of retinopathy in Type 2 diabetic patients without nephropathy. METHODS: We carried out a case-control study of Type 2 diabetic patients with and without advanced stages of diabetic retinopathy. Retinopathy and macular oedema were defined according to standard criteria. Serum levels of CML were estimated by means of a novel competition-based ELISA assay. RESULTS: Serum levels of CML were significantly different between age-matched controls (n=792; mean value +/- SD: 521+/-134 ng/ml), Type 2 diabetic patients without severe retinopathy (821+/-141 ng/ml; p<0.0001) and Type 2 diabetic patients with proliferative retinopathy (1182+/-346 ng/ml; p<0.0001). Levels of CML greater than 1000 ng/ml represented a 25-fold increase in risk of proliferative retinopathy. Receiver operating characteristics analysis revealed a CML threshold of 1087 ng/ml (100% sensitivity, 93% specificity) for clinically significant macular oedema. CONCLUSIONS/INTERPRETATION: High serum levels of CML were associated with advanced stages of retinopathy. Serum levels were shown to be a progressive risk marker, whereby a level of more than 1000 ng/ml induced a 25-fold increase in risk of proliferative retinopathy and clinically significant macular oedema. Our data suggest that serum levels of CML provide a novel risk marker for advanced stages of diabetic retinopathy in Type 2 diabetic patients.

Analysis of soil whole- and inner-microaggregate bacterial communities.

Although soil structure largely determines energy flows and the distribution and composition of soil microhabitats, little is known about how microbial community composition is influenced by soil structural characteristics and organic matter compartmentalization dynamics. A UV irradiation-based procedure was developed to specifically isolate inner-microaggregate microbial communities, thus providing the means to analyze these communities in relation to their environment. Whole- and inner-microaggregate fractions of undisturbed soil and soils reclaimed after disturbance by surface coal mining were analyzed using 16S rDNA terminal restriction fragment polymorphism (T-RFLP) and sequence analyses to determine salient bacterial community structural characteristics. We hypothesized that inner-microaggregate environments select for definable microbial communities and that, due to their sequestered environment, inner-microaggregate communities would not be significantly impacted by disturbance. However, T-RFLP analysis indicated distinct differences between bacterial populations of inner-microaggregates of undisturbed and reclaimed soils. While both undisturbed and reclaimed inner-microaggregate bacterial communities were found dominated by Actinobacteria, undisturbed soils contained only Actinobacteridae, while in inner-microaggregates of reclaimed soils Rubrobacteridae predominate. Spatial stratification of division-level lineages within microaggregates was also evidenced, with Proteobacteria clones being prevalent in libraries derived from whole microaggregates. The fractionation methods employed in this study therefore represent a valuable tool for defining relationships between biodiversity and soil structure.

Receptor tyrosine kinase signaling and trafficking--paradigms revisited.

The recognition of growth factors and other cell signaling agents by their cognate cell surface receptors triggers a cascade of signal transducing events. Ligand binding and subsequent activation of many signal transducing receptors increases their rate of internalization. Endocytosis of the receptor has always been viewed as primarily a mechanism for signal attenuation and receptor degradation, but recent evidence suggests that internalization may result in the formation of specialized signaling platforms on intracellular vesicles. Thus, understanding how interactions between receptors and intracellular signaling molecules, such as adaptors, GTPases, and kinases, are regulated will undoubtedly provide insight into the ways that cells sense and adapt to the extracellular milieu.

Lipid kinases play crucial and multiple roles in membrane trafficking and signaling.

Phosphotidylinositols (PIs) are known to play an essential role in membrane trafficking and signaling transduction. PIs serve multiple functions, such as recruitment of cytosolic proteins with PI phosphate (PIP) binding domains and modification of the physical properties of the membranes in which they reside. As substrates for phosphoinositide-specific lipases they function as a switch point in phosphoinositide metabolism. Recent work with epidermal growth factor receptor (EGFR) and colony stimulating factor-1 receptor (CSFR) has identified a possible connection between endocytosis of activated receptors and type-1 phosphatidylinositol-4-phosphate-5-kinase. Furthermore, serine/tyrosine phosphorylation of phosphatidylinositol-4-phosphate-5-kinase seems to be essential for its activities. Indeed, one of the products of the phosphatidylinositol-4-phosphate-5-kinases, PIP2, has been shown to be involved in multiple steps of endocytosis, including the assembly of the clathrin coat, regulation of adaptor proteins, and production of endocytic vesicles via the regulation of dynamin. The discussion in this review focuses primarily on receptors with intrinsic enzymatic activity, specifically on receptor tyrosine kinases (RTKs). We will discuss their structure; mechanism of action and potential role in membrane trafficking and/or signaling through the regulation of phosphatidylinositol phosphate kinases.

Regulation of Trypanosoma cruzi invasion of nonphagocytic cells by the endocytically active GTPases dynamin, Rab5, and Rab7.

During invasion of nonphagocytic cells by Trypanosoma cruzi (T. cruzi), host cell lysosomes are recruited to the plasma membrane attachment site followed by lysosomal enzyme secretion. The membrane trafficking events involved in invasion have not been delineated. We demonstrate here that T. cruzi invasion of nonphagocytic cells was completely abolished by overexpression of a dominant negative mutant of dynamin. Likewise, overexpression of a dominant negative mutant of Rab5, the rate-limiting GTPase for endocytosis, resulted in reduced infection rates compared with cells expressing Rab5 wild-type. Moreover, cells expressing the activated mutant of Rab5 experienced higher infection rates. A similar pattern was also observed when Rab7-transfected cells were examined. Confocal microscopy experiments showed that parasites colocalized with green fluorescent protein-Rab5-positive early endosomes after 5 min of invasion. These data clearly indicate that newly forming T. cruzi phagosomes first interact with an early endosomal compartment and subsequently with other late component markers before lysosomal interaction occurs.

Rab22a affects the morphology and function of the endocytic pathway.

Soon after endocytosis, internalized material is sorted along different pathways in a process that requires the coordinated activity of several Rab proteins. Although abundant information is available about the subcellular distribution and function of some of the endocytosis-specific Rabs (e.g. Rab5 and Rab4), very little is known about some other members of this family of proteins. To unveil some of the properties of Rab22a, one of the less studied endosome-associated small GTPases, we have expressed the protein tagged with the green fluorescent protein in CHO cells. The results indicate that Rab22a associates with early and late endosomes (labeled by a 5 minute rhodamine-transferrin uptake and the cation-independent mannose 6-phosphate receptor, respectively) but not with lysosomes (labeled by 1 hour rhodamine horseradish peroxidase uptake followed by 1 hour chase). Overexpression of the protein causes a prominent morphological enlargement of the early and late endosomes. Two mutants were generated by site-directed mutagenesis, a negative mutant (Rab22aS19N, with reduced affinity for GTP) and a constitutively active mutant (Rab22aQ64L, with reduced endogenous GTPase activity). The distribution of the negative mutant was mostly cytosolic, whereas the positive mutant associated with early and late endosomes and, interestingly also with lysosomes and autophagosomes (labeled with monodansylcadaverine). Cells expressing Rab22a wild type and Rab22aS19N displayed decreased endocytosis of a fluid phase marker. Conversely, overexpression of Rab22aQ64L, which strongly affects the morphology of endosomes, did not inhibit bulk endocytosis. Our results show that Rab22a has a unique distribution along the endocytic pathway that is not shared by any other Rab protein, and that it strongly affects the morphology and function of endosomes.

Ras-activated endocytosis is mediated by the Rab5 guanine nucleotide exchange activity of RIN1.

RIN1 was originally identified by its ability to inhibit activated Ras and likely participates in multiple signaling pathways because it binds c-ABL and 14-3-3 proteins, in addition to Ras. RIN1 also contains a region homologous to the catalytic domain of Vps9p-like Rab guanine nucleotide exchange factors (GEFs). Here, we show that this region is necessary and sufficient for RIN1 interaction with the GDP-bound Rabs, Vps21p, and Rab5A. RIN1 is also shown to stimulate Rab5 guanine nucleotide exchange, Rab5A-dependent endosome fusion, and EGF receptor-mediated endocytosis. The stimulatory effect of RIN1 on all three of these processes is potentiated by activated Ras. We conclude that Ras-activated endocytosis is facilitated, in part, by the ability of Ras to directly regulate the Rab5 nucleotide exchange activity of RIN1.

Total synthesis and biological evaluation of the nakijiquinones.

The Her-2/Neu receptor tyrosine kinase is vastly overexpressed in about 30% of primary breast, ovary, and gastric carcinomas. The nakijiquinones are the only naturally occurring inhibitors of this important oncogene, and structural analogues of the nakijiquinones may display inhibitory properties toward other receptor tyrosine kinases involved in cell signaling and proliferation. Here, we describe the first enantioselective synthesis of the nakijiquinones. Key elements of the synthesis are (i) the reductive alkylation of a Wieland-Miescher-type enone with a tetramethoxyaryl bromide, (ii) the oxidative conversion of the aryl ring into a p-quinoid system, (iii) the regioselective saponification of one of the two vinylogous esters incorporated therein, and (iv) the selective introduction of different amino acids via nucleophilic conversion of the remaining vinylogous ester into the corresponding vinylogous amide. The correct stereochemistry and substitution patterns are completed by conversion of two keto groups into a methyl group and an endocyclic olefin via olefination/reduction and olefination/isomerization sequences, respectively. This synthesis route also gave access to analogues of nakijiquinone C with inverted configuration at C-2 or with an exocyclic instead of an endocyclic double bond. Investigation of the kinase-inhibiting properties of the synthesized derivatives revealed that the C-2 epimer 30 of nakijiquinone C is a potent and selective inhibitor of the KDR receptor, a receptor tyrosine kinase involved in tumor angiogenesis. Molecular modeling studies based on the crystal structure of KDR and a model of the ATP binding site built from a crystal structure of FGF-R revealed an insight into the structural basis for the difference in activity between the natural product nakijiquinone C and the C-2 epimer 30.

Phosphatidylinositol-4-phosphate 5-kinase-1beta is essential for epidermal growth factor receptor-mediated endocytosis.

Phosphatidylinositol-4,5-bisphosphate (PIP(2)) is known to play an important role in signal transduction and membrane trafficking. We show that one enzyme responsible for PIP(2) production, phosphatidylinositol-4-phosphate 5-kinase type 1beta (PIPKbeta), is essential for epidermal growth factor receptor (EGFR)-mediated endocytosis. Expression of murine PIPKbeta in NR6 cells expressing EGFR strikingly increased receptor internalization. Moreover, the kinase was shown to form an immunoprecipitable complex with EGFR. Expression of either a truncated kinase or a kinase dead mutant inhibited EGFR endocytosis and also blocked the membrane recruitment of PIPKbeta and both clathrin light chain and dynamin. Our results delineate a novel mechanism by which PIPKbeta regulates receptor-mediated endocytosis and receptor tyrosine kinase membrane traffic.