Uptake Of Horseradish Peroxidase Research Articles

Thermosensitive Polymer Conjugated Prodrug-Activating Enzyme with Enhanced Tumor Retention and Antitumor Efficacy.

Enzyme-activated prodrug therapy has emerged as an effective strategy for cancer therapy. However, the inefficient delivery of prodrug-activating enzymes into tumor tissues leads to unsatisfactory antitumor efficacy and undesirable toxicity to normal tissues. Herein, we report in situ growth of a thermosensitive polymer of poly(diethylene glycol) methyl ether methacrylate (PDEGMA) from horseradish peroxidase (HRP) to yield a HRP-PDEGMA conjugate with well-retained activity as compared to HRP. The conjugate shows a sharp phase transition behavior with a lower critical solution temperature of 23 °C. The conjugate catalyzes the conversion of non-cytotoxic indole-3-acetic acid (IAA) into cytotoxic species for killing tumor cells. Notably, the PDEGMA conjugation not only increases the stability and cellular uptake of HRP but also prolongs the tumor retention time of HRP upon intratumoral injection. As a result, in mice bearing melanoma, the conjugate inhibits the growth of melanoma much more efficiently than HRP. These results demonstrate that the thermosensitive polymer conjugation of an enzyme is an effective strategy that can enhance the antitumor efficacy of an enzyme-activated prodrug.

Time-Dependent Alterations of Gut Wall Integrity in Small Bowel Obstruction in Mice

BackgroundSmall bowel obstruction (SBO) is one of the most common disorders in surgical emergency departments. Without resolution of the obstructed bowel segments, patients may develop multiorgan failure. The aim of this study was to investigate whether morphological damage of the intestinal wall during SBO may lead to molecular translocation and how this may impair intestinal motility. MethodsC57Bl6 mice were laparotomized, and the small intestine was ligated 5 cm oral to the coecum for SBO. Controls received minilaparotomy only. Animals were sacrificed 3 h, 9 h, and 24 h after SBO. Morphological changes were evaluated on hematoxylin and eosin histology by a standardized score. Intestinal motility was determined by recording intraluminal pressure of the small intestine in vitro. Permeability was measured by fluorospectroscopy and ELISA of blood samples after oral gavage with fluorescein isothiocyanate (FITC)-dextrane and horse radish peroxidase. Data are mean ± SD. ResultsThree hours after SBO, FITC-dextrane uptake was increased to 187.6 ± 15.2 ng/mL compared to controls (P = 0.011). At 9 h, uptake of horse radish peroxidase (23.0 ± 8.6 ng/mL, 9.0 ± 6.3 ng/mL, P = 0.039) and FITC-dextrane (86.8 ± 17.8 ng/mL, 62.0 ± 1.6 ng/mL, P = 0.029) was higher compared to controls. Motility was increased to 162.2 ± 20.2 area under the curve (AUC) compared to 121.3 ± 20.3 AUC in controls, P = 0.009 and an increased histology score was observed at 9 h (3.2 ± 1.8 versus 0.6 ± 0.7, P = 0.003). Twenty-four hours after SBO, histology score was 3.8 ± 1.7, which was higher than 0.9 ± 0.7 in controls (P = 0.001). Intestinal motility was decreased 24 h after SBO compared to sham controls (146.0 ± 21.4 AUC versus 198.9 ± 21.2 AUC, P = 0.003). ConclusionsSBO entails a time dependent epithelial damage to the mucosa. In parallel, molecular changes in the gut mucosal barrier occur as early as 3 h after the onset of SBO with a subsequent increase in permeability. Initial intestinal hypermotility is followed by a decrease in motility.

Measuring Synaptic Vesicle Endocytosis in Cultured Hippocampal Neurons.

During endocytosis, fused synaptic vesicles are retrieved at nerve terminals, allowing for vesicle recycling and thus the maintenance of synaptic transmission during repetitive nerve firing. Impaired endocytosis in pathological conditions leads to decreases in synaptic strength and brain functions. Here, we describe methods used to measure synaptic vesicle endocytosis at the mammalian hippocampal synapse in neuronal culture. We monitored synaptic vesicle protein endocytosis by fusing a synaptic vesicular membrane protein, including synaptophysin and VAMP2/synaptobrevin, at the vesicular lumenal side, with pHluorin, a pH-sensitive green fluorescent protein that increases its fluorescence intensity as the pH increases. During exocytosis, vesicular lumen pH increases, whereas during endocytosis vesicular lumen pH is re-acidified. Thus, an increase of pHluorin fluorescence intensity indicates fusion, whereas a decrease indicates endocytosis of the labelled synaptic vesicle protein. In addition to using the pHluorin imaging method to record endocytosis, we monitored vesicular membrane endocytosis by electron microscopy (EM) measurements of Horseradish peroxidase (HRP) uptake by vesicles. Finally, we monitored the formation of nerve terminal membrane pits at various times after high potassium-induced depolarization. The time course of HRP uptake and membrane pit formation indicates the time course of endocytosis.

Measuring Synaptic Vesicle Endocytosis in Cultured Hippocampal Neurons

During endocytosis, fused synaptic vesicles are retrieved at nerve terminals, allowing for vesicle recycling and thus the maintenance of synaptic transmission during repetitive nerve firing. Impaired endocytosis in pathological conditions leads to decreases in synaptic strength and brain functions. Here, we describe methods used to measure synaptic vesicle endocytosis at the mammalian hippocampal synapse in neuronal culture. We monitored synaptic vesicle protein endocytosis by fusing a synaptic vesicular membrane protein, including synaptophysin and VAMP2/synaptobrevin, at the vesicular lumenal side, with pHluorin, a pH-sensitive green fluorescent protein that increases its fluorescence intensity as the pH increases. During exocytosis, vesicular lumen pH increases, whereas during endocytosis vesicular lumen pH is re-acidified. Thus, an increase of pHluorin fluorescence intensity indicates fusion, whereas a decrease indicates endocytosis of the labelled synaptic vesicle protein. In addition to using the pHluorin imaging method to record endocytosis, we monitored vesicular membrane endocytosis by electron microscopy (EM) measurements of Horseradish peroxidase (HRP) uptake by vesicles. Finally, we monitored the formation of nerve terminal membrane pits at various times after high potassium-induced depolarization. The time course of HRP uptake and membrane pit formation indicates the time course of endocytosis.

Synaptic vesicle pool-specific modification of neurotransmitter release by intravesicular free radical generation.

Synaptic transmission is mediated by the release of neurotransmitters from synaptic vesicles in response to stimulation or through the spontaneous fusion of a synaptic vesicle with the presynaptic plasma membrane. There is growing evidence that synaptic vesicles undergoing spontaneous fusion versus those fusing in response to stimuli are functionally distinct. In this study, we acutely probe the effects of intravesicular free radical generation on synaptic vesicles that fuse spontaneously or in response to stimuli. By targeting vesicles that preferentially release spontaneously, we can dissociate the effects of intravesicular free radical generation on spontaneous neurotransmission from evoked neurotransmission and vice versa. Taken together, these results further advance our knowledge of the synapse and the nature of the different synaptic vesicle pools mediating neurotransmission. Earlier studies suggest that spontaneous and evoked neurotransmitter release processes are maintained by synaptic vesicles which are segregated into functionally distinct pools. However, direct interrogation of the link between this putative synaptic vesicle pool heterogeneity and neurotransmission has been difficult. To examine this link, we tagged vesicles with horseradish peroxidase (HRP) - a haem-containing plant enzyme - or antibodies against synaptotagmin-1 (syt1). Filling recycling vesicles in hippocampal neurons with HRP and subsequent treatment with hydrogen peroxide (H2 O2 ) modified the properties of neurotransmitter release depending on the route of HRP uptake. While strong depolarization-induced uptake of HRP suppressed evoked release and augmented spontaneous release, HRP uptake during mild activity selectively impaired evoked release, whereas HRP uptake at rest solely potentiated spontaneous release. Expression of a luminal HRP-tagged syt1 construct and subsequent H2 O2 application resulted in a similar increase in spontaneous release and suppression as well as desynchronization of evoked release, recapitulating the canonical syt1 loss-of-function phenotype. An antibody targeting the luminal domain of syt1, on the other hand, showed that augmentation of spontaneous release and suppression of evoked release phenotypes are dissociable depending on whether the antibody uptake occurred at rest or during depolarization. Taken together, these findings indicate that vesicles that maintain spontaneous and evoked neurotransmitter release preserve their identity during recycling and syt1 function in suppression of spontaneous neurotransmission can be acutely dissociated from syt1 function to synchronize synaptic vesicle exocytosis upon stimulation.

Correlations between Blood–Brain Barrier Disruption and Neuroinflammation in an Experimental Model of Penetrating Ballistic-Like Brain Injury

Abstract Blood-brain barrier (BBB) disruption is a pathological hallmark of severe traumatic brain injury (TBI) and is associated with neuroinflammatory events contributing to brain edema and cell death. The goal of this study was to elucidate the profile of BBB disruption after penetrating ballistic-like brain injury (PBBI) in conjunction with changes in neuroinflammatory markers. Brain uptake of biotin-dextran amine (BDA; 3 kDa) and horseradish peroxidase (HRP; 44 kDa) was evaluated in rats at 4 h, 24 h, 48 h, 72 h, and 7 days post-PBBI and compared with the histopathologic and molecular profiles for inflammatory markers. BDA and HRP both displayed a uniphasic profile of extravasation, greatest at 24 h post-injury and which remained evident out to 48 h for HRP and 7 days for BDA. This profile was most closely associated with markers for adhesion (mRNA for intercellular adhesion molecule-1) and infiltration of peripheral granulocytes (mRNA for matrix metalloproteinase-9 [MMP-9] and myeloperoxidase staining). Improvement of BBB dysfunction coincided with increased expression of markers implicated in tissue remodeling and repair. The results of this study reveal a uniphasic and gradient opening of the BBB after PBBI and suggest MMP-9 and resident inflammatory cell activation as candidates for future neurotherapeutic intervention after PBBI.

Methamphetamine-induced nitric oxide promotes vesicular transport in blood–brain barrier endothelial cells

Methamphetamine's (METH) neurotoxicity is thought to be in part due to its ability to induce blood–brain barrier (BBB) dysfunction. Here, we investigated the effect of METH on barrier properties of cultured rat primary brain microvascular endothelial cells (BMVECs). Transendothelial flux doubled in response to METH, irrespective of the size of tracer used. At the same time, transendothelial electrical resistance was unchanged as was the ultrastructural appearance of inter-endothelial junctions and the distribution of key junction proteins, suggesting that METH promoted vesicular but not junctional transport. Indeed, METH significantly increased uptake of horseradish peroxidase into vesicular structures. METH also enhanced transendothelial migration of lymphocytes indicating that the endothelial barrier against both molecules and cells was compromised. Barrier breakdown was only observed in response to METH at low micromolar concentrations, with enhanced vesicular uptake peaking at 1 μM METH. The BMVEC response to METH also involved rapid activation of endothelial nitric oxide synthase and its inhibition abrogated METH-induced permeability and lymphocyte migration, indicating that nitric oxide was a key mediator of BBB disruption in response to METH. This study underlines the key role of nitric oxide in BBB function and describes a novel mechanism of drug-induced fluid-phase transcytosis at the BBB.

Activity-Dependent Bulk Endocytosis and Clathrin-Dependent Endocytosis Replenish Specific Synaptic Vesicle Pools in Central Nerve Terminals

Multiple synaptic vesicle (SV) retrieval modes exist in central nerve terminals to maintain a continual supply of SVs for neurotransmission. Two such modes are clathrin-mediated endocytosis (CME), which is dominant during mild neuronal activity, and activity-dependent bulk endocytosis (ADBE), which is dominant during intense neuronal activity. However, little is known about how activation of these SV retrieval modes impact the replenishment of the total SV recycling pool and the pools that reside within it, the readily releasable pool (RRP) and reserve pool. To address this question, we examined the replenishment of all three SV pools by triggering these SV retrieval modes during both high- and low-intensity stimulation of primary rat neuronal cultures. SVs generated by CME and ADBE were differentially labeled using the dyes FM1-43 and FM2-10, and their replenishment of specific SV pools was quantified using stimulation protocols that selectively depleted each pool. Our studies indicate that while the RRP was replenished by CME-generated SVs, ADBE provided additional SVs to increase the capacity of the reserve pool. Morphological analysis of the uptake of the fluid phase marker horseradish peroxidase corroborated these findings. The differential replenishment of specific SV pools by independent SV retrieval modes illustrates how previously experienced neuronal activity impacts the capability of central nerve terminals to respond to future stimuli.

Altered renal proximal tubular endocytosis and histology in mice lacking myosin‐VI

Myosin VI (Myo6) is an actin-based molecular motor involved in clathrin-mediated endocytosis that is highly expressed in the renal proximal tubule brush border. We investigated the renal physiological consequences of loss of Myo6 function by performing renal clearance and physiological measurements on Myo6 functional null Snell's waltzer (sv/sv) and control heterozygous (+/sv) mice. Sv/sv mice showed reduced body weight and elevated blood pressure compared with controls; no differences were observed for glomerular flow rate, urine volume, blood acid-base parameters, and plasma concentrations and urinary excretions of Na(+) and K(+). To assess the integrity of endocytosis-mediated protein absorption by the kidney, urinary albumin excretion was measured, and the proximal tubular uptake of intravenously injected endocytic marker horseradish peroxidase (HRP) was examined. Albumin excretion was increased nearly 4-fold in sv/sv mice relative to controls. Conversely, HRP uptake was reduced and delayed in proximal tubule cells of the sv/sv kidney observed by electron microscopy at 5 and 30 min after injection. Consistent with impaired endocytosis, we also observed defects indicating alterations along the endocytic pathway in sv/sv proximal tubule cells: (1) decreased membrane association of the clathrin adaptor subunit, adaptin beta, and Disabled-2 (Dab2) after sedimentation of renal homogenates and (2) reduced apical vacuole number. In addition, proximal tubular dilation and fibrosis, likely secondary effects of the loss of Myo6, were observed in sv/sv kidneys. These results indicate that Myo6 plays a key role in endocytosis-mediated protein absorption in the mouse kidney proximal tubule.

The Cdc42-interacting Protein-4 (CIP4) Gene Knock-out Mouse Reveals Delayed and Decreased Endocytosis

The newly described F-BAR (Fer/CIP4 and Bin, amphiphysin, Rvs) family of proteins includes Cdc42-interacting protein-4 (CIP4), formin-binding protein-17 (FBP-17) and transactivator of cytoskeletal assembly-1 (Toca-1), and drives membrane deformation and invagination. Membrane remodeling affects endocytosis, vesicle budding, and cargo selection. The F-BAR family presents a novel family of proteins, which little is known about their in vivo function. We investigated the physiological role of CIP4, by creating Cip4-null mice through homologous recombination. Compared with their wild-type littermates, the Cip4-null mice displayed lower early post-prandial glucose levels. Adipocytes isolated from Cip4-null mice exhibited increased [(14)C]2-deoxyglucose uptake compared with cells from wild-type mice. The enhanced insulin sensitivity was not due to higher levels of insulin or phospho-Akt, a critical player in insulin signaling. However, higher glucose transporter 4 (GLUT4) levels were detected in muscle membrane fractions in Cip4-null mice under insulin stimulation. Mouse embryonic fibroblasts from Cip4-null mice demonstrated decreased transferrin uptake, fluorescein isothiocyanate-dextran, and horseradish peroxidase uptake, indicating that CIP4 affects multiple modes of endocytosis. These studies demonstrate a physiological role for CIP4 in endocytosis leading to a whole animal phenotype.

Fractal analysis and ionic dependence of endocytotic membrane activity of human breast cancer cells

The endocytic membrane activities of two human breast cancer cell lines (MDA-MB-231 and MCF-7) of strong and weak metastatic potential, respectively, were studied in a comparative approach. Uptake of horseradish peroxidase was used to follow endocytosis. Dependence on ionic conditions and voltage-gated sodium channel (VGSC) activity were characterized. Fractal methods were used to analyze quantitative differences in vesicular patterning. Digital quantification showed that MDA-MB-231 cells took up more tracer (i.e., were more endocytic) than MCF-7 cells. For the former, uptake was totally dependent on extracellular Na(+) and partially dependent on extracellular and intracellular Ca(2+) and protein kinase activity. Analyzing the generalized fractal dimension (D(q )) and its Legendre transform f(alpha) revealed that under control conditions, all multifractal parameters determined had values greater for MDA-MB-231 compared with MCF-7 cells, consistent with endocytic/vesicular activity being more developed in the strongly metastatic cells. All fractal parameters studied were sensitive to the VGSC blocker tetrodotoxin (TTX). Some of the parameters had a "simple" dependence on VGSC activity, if present, whereby pretreatment with TTX reduced the values for the MDA-MB-231 cells and eliminated the differences between the two cell lines. For other parameters, however, there was a "complex" dependence on VGSC activity. The possible physical/physiological meaning of the mathematical parameters studied and the nature of involvement of VGSC activity in control of endocytosis/secretion are discussed.

Critical roles of decompression in functional recovery of ex vivo spinal cord white matter

The correlations between functional deficits, the magnitude of compression, and the role of sustained compression during traumatic spinal cord injury remain largely unknown. Thus, the functional outcome of this type of injury with or without surgical intervention is rather unpredictable. To elucidate how severity and duration of compression affect cord function, the authors have developed a method to study electrophysiological characteristics and axonal membrane damage in white matter from guinea pig spinal cord. Ventral white matter strips isolated from adult guinea pigs were compressed by a compression rod at a level of either 60 or 80% and held briefly, for 30 minutes, or for 60 minutes. In half the experimental groups, a decompression phase consisting of probe withdrawal and 30 minutes of recovery was also applied. For all cord samples, functional response was continuously monitored through compound action potential (CAP) recording. In addition, axonal membrane damage was assessed by a horseradish peroxidase (HRP) exclusion assay. After 30 minutes of sustained compression at levels of 60 or 80%, a spinal cord decompression procedure caused a significant CAP recovery, with specimens reaching 97.5 +/- 6.84% (p < 0.05) and 56.2 +/- 6.14% (p < 0.05) of preinjury amplitude, respectively. After 60 minutes of compression, the amount of CAP recovery following the decompression stage was only 65.5 +/- 9.33% for 60% compression (p < 0.05) and 29.8 +/- 6.31% for 80% compression (p < 0.05). Unlike the CAP response, HRP uptake did not increase during sustained compression, and the data showed that HRP staining was primarily time dependent. The degree of axonal membrane damage is not exacerbated during sustained compression. However, the electrical conductivity of the cord white matter weakens throughout the duration of compression. Therefore, decompression is a viable procedure for preservation of neurological function following compressive injury.

Beneficial Effects of Cocoa in Perivascular Mato Cells of Cerebral Arterioles in SHR-SP (Izm) Rats

As previously reported, the cerebral arterioles are surrounded by unique perivascular Mato cells. They contain many inclusion bodies rich in hydrolytic enzymes, and have strong uptake capacity. They are thus considered scavenger cells of vascular and neural tissues in steady-state. In this study, employing hypertensive SHR-SP (Izm) rats, the viability of Mato cells was investigated. In hypertensive rats, the capacity for uptake of horse radish peroxidase (HRP) and the activity of acid phosphatase (ACPase) of Mato cells were markedly reduced, and on electron-microscopic examination Mato cells were found to include heterogeneous contents and appeared electron-dense and degenerated. Vascular cells exhibited some signs of pathology. However, in hypertensive rats fed chow containing 0.25% cocoa, the uptake capacity and ACPase activity of Mato cells for HRP were enhanced, and on electron-microscopic examination Mato cells appeared healthy, with mitochondria with nearly normal profiles. Signs of pathology in vascular cells were also decreased. Superoxides may impair Mato cells and vascular cells.

Caspase 8 Promotes Peripheral Localization and Activation of Rab5

Caspase 8 is a cysteine protease that initiates apoptotic signaling via the extrinsic pathway in a manner dependent upon association with early endosomes. Previously, we identified caspase 8 as an effector of migration, promoting motility in a manner dependent upon phosphorylation on Tyr-380 by Src family kinases and its subsequent association with Src homology 2 domain-containing proteins. Here we demonstrate the regulation of the small GTPase Rab5, which mediates early endosome formation, homotypic fusion, and maturation by caspase 8. Regulation requires the Tyr-380 phosphorylation site but not caspase proteolytic activity. Tyr-380 is essential for interaction with the Src homology 2 domains of p85α, a multifunctional adaptor for phosphatidylinositol 3-kinase, that possesses Rab-GAP activity. Interaction between caspase 8 and p85α promotes Rab5 GTP loading, alters endosomal trafficking, and results in the accumulation of Rab5-positive endosomes at the edge of the cell. Conversely, caspase 8-dependent GTP loading of Rab5 is overcome by increased expression of p85α in a Rab-GAP-dependent manner. Thus, we demonstrate a novel function for caspase 8 as a modulator of p85α Rab-GAP activity and endosomal trafficking.

Function and role of voltage-gated sodium channel NaV1.7 expressed in aortic smooth muscle cells

Voltage-gated Na(+) channel currents (I(Na)) are expressed in several types of smooth muscle cells. The purpose of this study was to evaluate the expression of I(Na), its functional role, pathophysiology in cultured human (hASMCs) and rabbit aortic smooth muscle cells (rASMCs), and its association with vascular intimal hyperplasia. In whole cell voltage clamp, I(Na) was observed at potential positive to -40 mV, was blocked by tetrodotoxin (TTX), and replacing extracellular Na(+) with N-methyl-d-glucamine in cultured hASMCs. In contrast to native aorta, cultured hASMCs strongly expressed SCN9A encoding Na(V)1.7, as determined by quantitative RT-PCR. I(Na) was abolished by the treatment with SCN9A small-interfering (si)RNA (P < 0.01). TTX and SCN9A siRNA significantly inhibited cell migration (P < 0.01, respectively) and horseradish peroxidase uptake (P < 0.01, respectively). TTX also significantly reduced the secretion of matrix metalloproteinase-2 6 and 12 h after the treatment (P < 0.01 and P < 0.05, respectively). However, neither TTX nor siRNA had any effect on cell proliferation. L-type Ca(2+) channel current was recorded, and I(Na) was not observed in freshly isolated rASMCs, whereas TTX-sensitive I(Na) was recorded in cultured rASMCs. Quantitative RT-PCR and immunostaining for Na(V)1.7 revealed the prominent expression of SCN9A in cultured rASMCs and aorta 48 h after balloon injury but not in native aorta. In conclusion, these studies show that I(Na) is expressed in cultured and diseased conditions but not in normal aorta. The Na(V)1.7 plays an important role in cell migration, endocytosis, and secretion. Na(V)1.7 is also expressed in aorta after balloon injury, suggesting a potential role for Na(V)1.7 in the progression of intimal hyperplasia.

Bulk Synaptic Vesicle Endocytosis Is Rapidly Triggered during Strong Stimulation

Bulk endocytosis in central nerve terminals is activated by strong stimulation; however, the speed at which it is initiated and for how long it persists is still a matter of debate. To resolve this issue, we performed a characterization of bulk endocytic retrieval using action potential trains of increasing intensity. Bulk endocytosis was monitored by the loading of the fluorescent dyes FM2-10 and FM1-43, uptake of tetramethylrhodamine-dextran (40 kDa), or morphological analysis of uptake of the fluid-phase marker horseradish peroxidase. When neuronal cultures were subjected to mild stimulation (200 action potentials at 10 Hz), bulk endocytosis was not observed using any of our assay systems. However, when more intense trains of action potentials (400 or 800 action potentials at 40 and 80 Hz, respectively) were applied to neurons, bulk endocytosis was activated immediately, with the majority of bulk endocytosis complete by the end of stimulation. This contrasts with single synaptic vesicle endocytosis, the majority of which occurred after stimulation was terminated. Thus, bulk endocytosis is a fast event that is triggered during strong stimulation and provides the nerve terminal with an appropriate mechanism to meet the demands of synaptic vesicle retrieval during periods of intense synaptic vesicle exocytosis.

TAT Opens the Door

TAT Opens the Door

ALS2/alsin deficiency in neurons leads to mild defects in macropinocytosis and axonal growth

Loss of function mutations in the ALS2 gene account for a number of juvenile/infantile recessive motor neuron diseases, indicating that its gene product, ALS2/alsin, plays a crucial role in maintenance and survival for a subset of neurons. ALS2 acts as a guanine nucleotide exchange factor (GEF) for the small GTPase Rab5 and is implicated in endosome dynamics in cells. However, the role of ALS2 in neurons remains unclear. To elucidate the neuronal ALS2 functions, we investigate cellular phenotypes of ALS2-deficient primary cultured neurons derived from Als2-knockout (KO) mice. Here, we show that ALS2 deficiency results not only in the delay of axon outgrowth in hippocampal neurons, but also in a decreased level of the fluid phase horseradish peroxidase (HRP) uptake, which represents the activity for macropinocytic endocytosis, in cortical neurons. Thus, ALS2 may act as a modulator in neuronal differentiation and/or development through regulation of membrane dynamics.

Mechanisms of the ifosfamide-induced inhibition of endocytosis in the rat proximal kidney tubule

The Fanconi syndrome is a common side effect of the chemotherapeutic agent ifosfamide. Current evidences suggest that chloroacetaldehyde (CAA), one of the main metabolites of ifosfamide activation, contributes to its nephrotoxicity. However, the pathophysiology of CAA-induced Fanconi syndrome is not fully understood. The present work examined the adverse effects of CAA on precision-cut rat renal cortical slices, which allowed studying the toxic effect of CAA on proximal endocytosis. We demonstrated that clinically relevant concentrations of CAA (< or =200 microM) are able to inhibit the uptake of horseradish peroxidase, a marker of proximal tubular cell endocytosis in renal tubular proximal cells. CAA > or =75 microM has adverse effects, both on viability parameters and on energy metabolism, as shown by the great decrease in total glutathione and ATP levels. In addition, the V-ATPase, which plays a crucial role in intracellular vesicle trafficking, was inhibited by 100 microM of CAA. By contrast, the slight decrease in Na-K-ATPase activity observed for CAA> or = 125 microM (maximum inhibition: 33%) could not totally explain the inhibition of the reabsorption processes. In conclusion, the addition of the two main adverse effects of CAA (decrease in ATP levels and inhibition of the V-ATPase) could explain the inhibition of endocytosis and the Fanconi syndrome observed during ifosfamide treatments.

Biomechanics of Spinal Cord Injury: A Multimodal Investigation Using Ex Vivo Guinea Pig Spinal Cord White Matter

The primary injury phase of traumatic spinal cord injury (SCI) was investigated using a novel compression injury model. Ventral white matter from adult guinea pigs was crushed to 25%, 50%, 70%, and 90% ex vivo. During compression, the physical deformation, applied force and the compound action potentials (CAP) were simultaneously recorded. In addition, axonal membrane continuity was analyzed with a horseradish peroxidase (HRP) exclusion assay. Experimental results showed both a CAP decrease and increased HRP uptake as a function of increased compression. The percent CAP reduction was also consistent to the percent HRP uptake, which implies that either metric could be used to assess acute axon damage. Analysis of the HRP stained axon distribution demonstrated a gradient of damage, with the highest levels of staining near the gray matter. The patterns of axon damage revealed by histology also coincided with higher levels of von Mises stress, which were predicted with a recently developed finite element model of ventral white matter. Numerical values obtained from the finite element model suggest stress magnitudes near 2 kPa are required to initiate anatomical tissue injury. We believe that data from this study could further elucidate the deformation-function relationship in acute spinal cord injury.