Reduced cytotoxicity in doxorubicin-exposed HepG2 cells pretreated with menthol due to upregulation of P-glycoprotein.

The aim of the present study was to examine changes in the expression and activity of P-glycoprotein (P-gp) in human hepatocellular carcinoma HepG2 cells after exposure to menthol, and their relationship to the cytotoxicity of and apoptotic responses to doxorubicin (DOX), a substrate of P-gp, in the cells. The expression of P-gp in HepG2 cells was significantly increased by menthol treatment. Intracellular accumulation of DOX in HepG2 cells was significantly lower in the menthol-treated group than in the control group, but this phenomenon was abolished in the presence of verapamil. Decreased cell viability by DOX was significantly attenuated by 24-h menthol treatment prior to DOX exposure, which coincided with the changes in mRNA expression of Bcl-xl and caspase-3. These results demonstrate that menthol causes hepatocellular carcinoma cells to acquire resistance to DOX by increasing its efflux through the upregulation of P-gp.

Visual perceptual strengths and weaknesses in adults with intellectual disabilities compared with a birth year-matched norm.

BACKGROUND: The ventral and dorsal streams are considered to be the brain substrates of vision for perception and action, respectively. Using the Developmental Test of Visual Perception (DTVP), the current study examined whether visual perceptual strengths and weaknesses in adults with intellectual disabilities (ID) were attributable to the dichotomy of the visual streams. METHOD: In study 1, DTVP performance was compared among mild, moderate and severe adult ID groups; study 2 contrasted adult ID groups with and without Down syndrome (DS). To prevent possible contamination by the Flynn effect, participants were matched by birth year with the norm of the DTVP original edition. RESULTS: Independent of the extent of ID among the three groups in study 1 and the aetiological group difference in study 2, relative strength was found for two DTVP tasks: eye-hand coordination and distinguishing target figures from interference background. Relative weakness was obtained in identifying a figural category. Participants with DS demonstrated exceptional weakness in discerning a target from either mirror-imaged or rotated alternatives, in addition to figural-category detection. CONCLUSIONS: Visual perceptual strengths and weaknesses in persons with ID were difficult to explain on the basis of two visual streams. An interpretation originating in a different research context (e.g. frontal-lobe dysfunction) appears to be required for explaining visual perceptual weaknesses in persons with ID. For persons with DS, strong frontal-lobe dysfunction with atypical lateralisation might be the pathological determinant of visual perceptual weaknesses.

Microkeratome assisted deep lamellar keratoprosthesis.

AIMS: To establish a keratoprosthesis (Kpro) surgical technique that maintains an intact superficial corneal layer. METHODS: A manual microkeratome (Moria LSK-1) was used to create a 130 mum flap of approximately 10 mm diameter in the right eye of Japanese white rabbits. The stoma beneath the flap area was dissected before the removal of a 5.0 mm stromal disc. A 5.0 mm collagen I immobilised poly(vinyl alcohol) (COL-PVA) disc was placed on the exposed posterior stroma close to Descemet's membrane. The flap was repositioned and fixed using 10-0 nylon sutures, which were removed 2 days following surgery. The corneas were followed clinically by slit lamp microscopy and photographs. Rabbits were sacrificed after 6 months, and the transplanted corneas were examined histologically by haematoxylin and eosin staining and immunohistochemistry against vimentin and alpha-smooth muscle actin (alpha-SMA). RESULTS: The transplanted COL-PVA discs remained transparent throughout the study, with no complications related to the flap or overlying epithelium. The interface between COL-PVA and Descemet's membrane remained clear without signs of opacification caused by scarring or cellular deposition. Pathology revealed the intact COL-PVA polymer in the posterior stroma, with minimal cellular infiltration along the anterior and posterior interfaces. Immunohistology shows vimentin and alpha-SMA staining at levels comparable to lamellar keratoplasty control. CONCLUSIONS: Microkeratome assisted deep lamellar keratoprosthesis may be a safe technique for the transplantation of artificial hydrogels for therapeutic purposes.

Positron Emission Tomography and Autoradiography of (18)F-Fluorodeoxyglucose Labeled Islets With or Without Warm Ischemic Stress in Portal Transplanted Rats.

OBJECTIVE: The use of positron-emission tomography (PET) with (18)F-fluorodeoxyglucose (FDG) -labeled islets has been considered to be a potential modality to visualize and quantify early engraftment of islet transplantation. The objective of this study was to evaluate the early islets' survival of the FDG-labeled islets with or without warm ischemic stress in portal transplanted rats using PET and autoradiography. METHODS: Islets were isolated from Lewis rat pancreata with or without 30-minute warm ischemia times (WITs). For islets' labeling, 300 islets were incubated with 3 MBq FDG for 60 minutes. FDG-labeled islets were transplanted into the liver via portal vein. In in vivo study, a PET study was scanned for 90 minutes and the FDG uptake was expressed as percentage of liver injection dose (ID). In ex vivo study, the liver was exposed for 30 minutes with single fluorescence autoradiography. RESULTS: In the PET study, the percentage of liver ID of the islets without WIT was 27.8 and that of the WIT islets was 20.1 at the end of islet transplantation. At 90 minutes after transplantation, the percentage of liver ID was decreased to 14.7 in the islets without WIT and 10.1 in the WIT islets. In the autoradiogram, the number of hot spots was more obviously visualized in the liver transplanted without WIT islets than in the liver transplanted with WIT islets. CONCLUSION: Almost 50% of the islets were immediately lost in both the islets without WIT and those with WIT transplantation in the early period. However, islet survival was 1.4 times higher in the islets without WIT than that in those with WIT in the early engraftment phase.

Distinct functions of the two protein tyrosine phosphatase domains of LAR (leukocyte common antigen-related) on tyrosine dephosphorylation of insulin receptor.

Most receptor-like, transmembrane protein tyrosine phosphatases (PTPases), such as CD45 and the leukocyte common antigen-related (LAR) molecule, have two tandemly repeated PTPase domains in the cytoplasmic segment. The role of each PTPase domain in mediating PTPase activity remains unclear; however, it has been proposed that PTPase activity is associated with only the first of the two domains, PTPase domain 1, and the membrane-distal PTPase domain 2, which has no catalytic activity, would regulate substrate specificity. In this paper, we examine the function of each PTPase domain of LAR in vivo using a potential physiological substrate, namely insulin receptor, and LAR mutant proteins in which the conserved cysteine residue was changed to a serine residue in the active site of either or both PTPase domains. LAR associated with and preferentially dephosphorylated the insulin receptor that was tyrosine phosphorylated by insulin stimulation. Its association was mediated by PTPase domain 2, because the mutation of Cys-1813 to Ser in domain 2 resulted in weakening of the association. The Cys-1522 to Ser mutant protein, which is defective in the LAR PTPase domain 1 catalytic site, was tightly associated with tyrosine-phosphorylated insulin receptor, but failed to dephosphorylate it, indicating that LAR PTPase domain 1 is critical for dephosphorylation of tyrosine-phosphorylated insulin receptor. This hypothesis was further confirmed by using LAR mutants in which either PTPase domain 1 or domain 2 was deleted. Moreover, the association of the extracellular domains of both LAR and insulin receptor was supported by using the LAR mutant protein without the two PTPase domains. LAR was phosphorylated by insulin receptor tyrosine kinase and autodephosphorylated by the catalytic activity of the PTPase domain 1. These results indicate that each domain of LAR plays distinct functional roles through phosphorylation and dephosphorylation in vivo.

Detection of CD45iota mRNA in murine Th1 but not Th2 clones.

CD45, a prototype of the receptor-like protein tyrosine phosphatase (PTPase) family, is one of the essential molecules in signal transduction through T cell receptors. Because at least 8 types of CD45 isoforms can potentially be produced by alternative mRNA splicing of exons 4, 5, and 6, the analyses at the transcription and protein levels of CD45 during the development and differentiation of T cells have been performed using RT-PCR and isoform-specific monoclonal antibodies, respectively. We report here that the ninth and smallest isoform of CD45, designated as CD45iota (CD45t), which is alternatively spiced from exons 4, 5, and 6 as well as exon 7, is present in the fetal thymus and splenic T cells of mice, and in murine Th1 clones, but not in Th2 clones. The expression of full-length CD45t mRNA as the functional CD45 PTPase was confirmed by RT-PCR analysis. Furthermore, the expression vector of CD45t was constructed, and its expression was detected in combination with anti-pan CD45 mAb and our newly established anti-LAR/CD45 PTPase domain mAb. These results suggested that CD45t might be an important isoform of CD45 for differentiation signaling of Th cells, and might be used as a marker to distinguish between Th1 and Th2 cells.

Cross-striolar and commissural inhibition in the otolith system.

Neural connections from the saccular and utricular nerves to the ipsilateral vestibular neurons and the commissural effects were studied by using intracellular recordings of excitatory (E) and inhibitory (I) postsynaptic potentials (PSPs) in vestibular neurons of cats after focal stimulation of the saccular and the utricular maculae. Neural circuits from the maculae to vestibular neurons, termed cross-striolar inhibition, may provide a mechanism for increasing the sensitivity to linear acceleration and tilt of the head. It was examined whether secondary vestibular neurons activated by an ipsilateral otolith organ received a commissural inhibition from a contralateral otolith organ that occupied the same geometric plane. Results suggest that utricular-activated vestibular neurons receiving commissural inhibition may provide a mechanism for increasing the sensitivity to horizontal linear acceleration and tilt of the head. The commissural inhibition of the saccular system was much weaker than that of the utricular system.

Superior vestibular nucleus neurones related to the excitatory vestibulo-ocular reflex of anterior canal origin and their ascending course in the cat.

Stimulation of the superior vestibular nucleus and the anterior canal nerve evoked mono- and disynaptic excitatory postsynaptic potentials, respectively, in contralateral inferior oblique motoneurones of the cat. Combined stimulation revealed that the superior vestibular nucleus relayed excitatory anterior canal signals to the motoneurones. Thirty-six superior vestibular neurones receiving anterior canal inputs were activated antidromically by microstimulation of the contralateral inferior oblique motoneurone pool. Their axons ascended neither in the brachium conjunctivum nor in the medial longitudinal fasciculus, but proceeded rostrally in the ventral part of the brain stem.

Axon collaterals of anterior semicircular canal-activated vestibular neurons and their coactivation of extraocular and neck motoneurons in the cat.

We studied the ascending and descending axonal trajectories of excitatory vestibular neurons related to the anterior semicircular canal, by means of local stimulation and spike-triggered signal averaging techniques in anesthetized cats. More than 200 vestibular neurons related to the ampullary nerve of the anterior semicircular canal (ACN) were identified as vestibulo-ocular neurons by antidromic stimulation of the contralateral inferior oblique (IO) muscle motoneuron pool. In the descending, medial and ventral lateral nuclei, about 60% of these vestibulo-ocular neurons were also activated antidromically by upper cervical spinal cord stimulation (vestibulo-ocular-collic (cervical) = VOC). These VOC neurons produced unitary EPSPs in the majority of neck extensor motoneurons located at the C1 segment. None of the VOC neurons had axons descending as far as the thoracic level. Most of these VOC neurons were activated monosynaptically following stimulation of the ACN. The conduction velocity of the descending axons of VOC neurons was approximately 63 m/s, which was significantly faster than that of the ascending axons. The remaining 40% of the vestibulo-ocular neurons were not activated antidromically following spinal cord stimulation at intensities of 1 mA or more (vestibulo-ocular = VO). Most of the VO neurons were activated polysynaptically by ACN stimulation. The superior vestibular nucleus contained VO neurons that were activated mono- and polysynaptically following ACN stimulation.

Floccular influence on excitatory relay neurones of vestibular reflexes of anterior semicircular canal origin in the cat.

Floccular influence on excitatory vestibular reflex arcs of anterior semicircular canal origin was examined in the anaesthetized cat. Stimulation of the anterior semicircular canal nerve (ACN) evoked disynaptic excitatory postsynaptic potentials (EPSPs) in all sampled inferior oblique (IO), superior rectus (SR), and biventor cervicis (BIV) muscle motoneurones of the contralateral side. Conditioning stimulus to the flocculus depressed the amplitude of the EPSPs in both IO and SR motoneurones by 50% on the average but not in any BIV motoneurones. The excitatory vestibulo-ocular neurones identified by orthodromic and antidromic responses to stimulation of the ACN and the contralateral IO motoneurone pool, respectively, were classified as VOC (vestibulo-ocular neurones with axons descending to the cervical segment) or VO (vestibulo-ocular proper) neurones on the basis of whether or not they responded antidromically to stimulation of the spinal cord in the C1 segment. All of the VO neurones in the superior vestibular nucleus (n = 19) were inhibited from the flocculus while the activities of three-fourths of the VO neurones (36/48) in the other vestibular nuclei were not suppressed by floccular stimulation. In contrast, none of VOC neurones (n = 49) received floccular inhibition. Besides inhibition, floccular stimulation induced the antidromic or orthodromic responses in some VO and VOC neurones.

Axon branching of medullary expiratory neurons in the lumbar and the sacral spinal cord of the cat.

Intraspinal axon collaterals of expiratory (E) neurons in the caudal nucleus retroambigualis extending their desending spinal axons to the lower lumbar (L6-L7) and the sacral (S1-S3) segments were investigated in anesthetized cats. To search for axon collaterals of single E neurons in the lumbar segments, the spinal gray matter was microstimulated from the dorsal to the ventral sites at 100 microns intervals with an intensity of 150-250 microA at 1 mm intervals rostrocaudally along the spinal cord, and effective stimulating sites of antidromic activation in axon collaterals were systematically mapped. In addition, the detailed trajectory of collaterals in the upper lumbar (L1-L3), the middle lumbar (L4-L5), and the sacral (S1-S3) spinal cord was examined by microstimulation at a matrix of points 100-200 microns apart with a maximum stimulus intensity of 50 microA. The trajectory of axon collaterals was reconstructed on the basis of the location of low-threshold foci and the latency of antidromic spikes. Virtually all E neurons examined had 1-7 collaterals at widely separated segments of the lumbar cord. Many axon collaterals were found in the upper lumbar spinal cord as compared to the middle and the lower lumbar spinal cord. The locations of axon collaterals in the upper lumbar spinal cord overlapped with those of abdominal motoneurons. Axon collaterals in the sacral gray matter were found in 3 of 9 E neurons. Axon collaterals were found within the nucleus of Onuf, in the region dorsal to the nucleus of Onuf, and in the intermediate region. The functional significance of the divergent distribution of multiple axon collaterals of single E neurons in different spinal levels of the lumbar and the sacral spinal cord is discussed in relation to the respiratory function of E neurons and other spinal motor activities.

Horizontal canal input to cat extraocular motoneurons.

Synaptic potentials were recorded in identified extraocular motoneurons in the anesthetized cat, following stimulation of the horizontal canal nerve (HCN). Weak stimulation of the HCN evoked disynaptic EPSPs in ipsilateral medial rectus (i-MR), contralateral lateral rectus (c-LR) and disynaptic IPSPs in i-LR motoneurons. Weak stimulation of the HCN produced longer latency IPSPs (probably trisynaptic) in c-MR motoneurons. It is suggested that the HCN projects to an excitatory interneuron in the vestibular nucleus whose axon in turn projects to a third order inhibitory interneuron in the IIIrd nucleus which finally projects to c-MR motoneurons. Essentially there is no influence of the HCN stimulation on bilateral superior rectus (SR), inferior rectus (IR), superior oblique (SO) and inferior oblique (IO) motoneurons.

Properties of secondary vestibular neurons fired by stimulation of ampullary nerve of the vertical, anterior or posterior, semicircular canals in the cat.

Experiments on cats were performed to study the pathway and location of the secondary vestibulo-ocular neurons in response to stimulation of the ampullary nerves of the vertical, anterior or posterior, semicircular canals. Experiments on the medial longitudinal fasciculus transection disclosed that vertical canal-evoked, disynaptic excitation and inhibition were transmitted to the extraocular motoneurons through the contra- and ipsilateral medial longitudinal fasciculus respectively. Secondary vestibular neurons, which receive input from the ampullary nerve of the vertical semicircular canals and send their axons to contralateral medial longitudinal fasciculus, were intermingled in the rostral half of the descending and lateral part of the medial vestibular nuclei. A direct excitatory connection of some of these neurons to the target extraocular motoneurons was confirmed by means of a spike-triggered signal averaging technique. It was also found that neurons activated by antidromic stimulation of ipsilateral medial longitudinal fasciculus were located in the superior vestibular nucleus, some of which made direct inhibitory connections to the target extraocular motoneurons. Both excitatory and inhibitory vestibuloocular neurons made synaptic contact in about half of the impaled target motoneurons.

Lower lumbar branching of caudal medullary expiratory neurons of the cat.

Extracellular spike activities of medullary expiratory (E) neurons in the caudal ventral respiratory group were recorded in cats anesthetized with sodium pentobarbital. The majority of E neurons extended their axons in the lower lumbar or the sacral segments and distributed collaterals in L5-L7. These results suggest that E neurons are involved not only in respiratory activities but also in the respiratory modulated motor activities of the lower lumbar segments.

Properties of central vestibular neurons fired by stimulation of the saccular nerve.

In 4 cats all vestibular afferents in one labyrinth except those innervating the saccular macula were transected and allowed to degenerate. 23--53 days after the initial surgery the central connections of the remaining saccular nerve were studied under chloralose anesthesia. Stimulation of the saccular nerve evoked N1 field potentials in the ipsilateral lateral and descending vestibular nuclei; little or no field potential activity was seen in the superior nucleus. The distribution of field potentials overlapped with that of neurons of origin of the vestibulospinal tracts. Forty-two neurons in the ipsilateral vestibular nuclei, many in the lateral nucleus, responded, often monosynaptically, to stimulation of the saccular nerve with single or double shocks; some of the neurons projected to the spinal cord. All saccular-fired neurons were tested for commissural actions by stimulation of the contralateral vestibular nerve. Many were facilitated, almost none were inhibited. In agreement with earlier work, we conclude that commissural inhibition may be a property of the canal system only.

Localization of proprioceptive reflexes in the splenius muscle of the cat.

Activity of the cat splenius muscle was modulated by sinusoidal rotation of the head around the C1-C2 joint in decerebrate cats with labyrinth intact or with all semicircular canals plugged, or, in one intact and alert cat, by rotation of the body with the head fixed in space. EMG modulation, recorded from the areas of splenius innervated by the C1-C4 nerves, was due to the cervicocollic reflex. Modulation was not uniform, but decreased with progressively more caudal recording locations; with stimuli of small amplitude it was often possible to obtain modulation of the rostral part of the muscle only. The results demonstrate localization of proprioceptive reflexes, including the stretch reflex, within the splenius muscle.

Axon collaterals to the extraocular motoneuron pools of inhibitory vestibuloocular neurons activated from the anterior, posterior and horizontal semicircular canals in the cat.

The branching pattern of inhibitory vestibuloocular neurons and their synaptic contacts with extraocular motoneurons were studied by means of spike-triggered averaging and local stimulation techniques. Individual vestibuloocular neurons activated by stimulation of the ampullary nerve of the anterior semicircular canal (ACN) inhibited motoneurons in both the ipsilateral (i-) trochlear nucleus and i-inferior rectus motoneuron pools. Individual vestibuloocular neurons receiving input from the ampullary nerve of the posterior semicircular canal (PCN) inhibited motoneurons in both the i-inferior oblique and i-superior rectus motoneuron pools. Probably, these axonal trajectories underlie conjugate eye movement during vertical head rotation. No conclusive evidence was found to indicate that single inhibitory vestibular neurons receiving input from the horizontal semicircular canal (HCN) give off axon collaterals to the i-abducens and the contralateral medial rectus motoneurons. A separate projection of HCN-related neurons to motoneurons supplying the lateral and medial rectus muscles might be useful for convergence during horizontal head movement.

Axonal branching in the trochlear and oculomotor nuclei of single vestibular neurons activated from the posterior semicircular canal nerve in the cat.

Axonal branches of single vestibular neurons activated by stimulation of the ampullary nerve of the posterior semicircular canal in the cat were studied by means of local antidromic stimulation in the trochlear and the oculomotor nucleus. These vestibulo-ocular neurons were located in the rostral half of the descending vestibular nucleus and the lateral part of the medial vestibular nucleus. The majority of vestibulo-ocular neurons projecting to the inferior rectus motoneuron pool in the contralateral oculomotor nucleus was activated antidromically from the contralateral trochlear nucleus as well. This suggests that axonal branches of a single vestibular neuron project to both nuclei.

The commissural inhibition on secondary vestibulo-ocular neurons in the vertical semicircular canal systems in the cat.

The commissural inhibition on secondary vestibulo-ocular neurons (VOns) from the contralateral (c-) vertical canal system in the same geometric plane was studied in the anesthetized cat. The secondary VOns were identified by their orthodromic responses to stimulation of the ampullary nerves of the anterior (ACN) or posterior (PCN) semicircular canals and also by their antidromic responses to stimulation of the IIIrd and IVth nuclei. The majority of ACN-activated excitatory VOns in the descending and medial nuclei (32/36, 89%) and in the superior nucleus (20/23, 87%), received commissural inhibition from the c-PCN, while only few ACN-activated inhibitory VOns (3/35, 9%) in the superior nucleus received commissural inhibition from the c-PCN. On the other hand, all of the PCN-activated excitatory (50/50) and inhibitory (30/30) VOns in the vestibular nuclei received commissural inhibition following c-ACN stimulation.

Neuronal organization of the utricular macula concerned with innervation of single vestibular neurons in the cat.

We investigated whether cross-striolar inhibition, which may increase sensitivity to linear acceleration, contributed to utricular (UT) afferent innervation of single vestibular neurons (VNs). Excitatory and inhibitory postsynaptic potentials (EPSPs, IPSPs, respectively) were recorded from VNs after focal stimulation of the UT macula (M). From a total of 83 VNs, 25 (30%) neurons received inputs from both sides of the UTM, and the response patterns were opposite, i.e. cross-striolar inhibition was observed. In roughly 2/3 of these neurons, stimulation of the medial side of the UTM evoked EPSPs, while stimulation of the lateral side evoked IPSPs. In the remaining 1/3 neurons, the response patterns were opposite. Thirty-two (39%) of the 83 neurons received the identical pattern of inputs from both sides of the UTM: EPSPs in 26 neurons and IPSPs in six neurons. Twenty-six (31%) of the 83 neurons received inputs from either the medial or the lateral side of the UTM. These findings suggest that cross-striolar inhibition existed in the UT system, although it was not a dominant circuit that increased the sensitivity as in the saccular system [15].