Protein transduction by pseudotyped lentivirus-like nanoparticles.

A simple, efficient and reproducible method to transduce proteins into mammalian cells has not been established. Here we describe a novel protein transduction method based on a lentiviral vector. We have developed a method to package several thousand foreign protein molecules into a lentivirus-like nanoparticle (LENA) and deliver them into mammalian cells. In this proof-of-concept study, we used ß-lactamase (BlaM) as a reporter molecule. The amino-terminus of BlaM was fused to the myristoylation signal of lyn, which was placed upstream of the amino-terminus of Gag (BlaM-gag-pol). By co-transfection of plasmids encoding BlaM-gag-pol and vesicular stomatitis virus-G (VSV-G) into 293T cells, LENA were produced containing BlaM enzyme molecules as many as Gag per capsid, which has been reported to be ∼5000 molecules, but lacking the viral genome. Infection of 293T and MT-4 cells by VSV-G-pseudotyped BlaM-containing LENA led to successful transduction of BlaM molecules into the cell cytoplasm, as detected by cleavage of the fluorescent BlaM substrate CCF2-AM. LENA-mediated transient protein transduction does not damage cellular DNA, and the preparation of highly purified protein is not necessary. This technology is potentially useful in various basic and clinical applications.

Selective intracellular vaporisation of antibody-conjugated phase-change nano-droplets in vitro.

While chemotherapy is a major mode of cancer therapeutics, its efficacy is limited by systemic toxicities and drug resistance. Recent advances in nanomedicine provide the opportunity to reduce systemic toxicities. However, drug resistance remains a major challenge in cancer treatment research. Here we developed a nanomedicine composed of a phase-change nano-droplet (PCND) and an anti-cancer antibody (9E5), proposing the concept of ultrasound cancer therapy with intracellular vaporisation. PCND is a liquid perfluorocarbon nanoparticle with a liquid-gas phase that is transformable upon exposure to ultrasound. 9E5 is a monoclonal antibody targeting epiregulin (EREG). We found that 9E5-conjugated PCNDs are selectively internalised into targeted cancer cells and kill the cells dynamically by ultrasound-induced intracellular vaporisation. In vitro experiments show that 9E5-conjugated PCND targets 97.8% of high-EREG-expressing cancer cells and kills 57% of those targeted upon exposure to ultrasound. Furthermore, direct observation of the intracellular vaporisation process revealed the significant morphological alterations of cells and the release of intracellular contents.

Roles of glutamate receptor delta 2 subunit (GluRdelta 2) and metabotropic glutamate receptor subtype 1 (mGluR1) in climbing fiber synapse elimination during postnatal cerebellar development.

Climbing fiber (CF) synapse formation onto cerebellar Purkinje cells (PCs) is critically dependent on the synaptogenesis from parallel fibers (PFs), the other input to PCs. Previous studies revealed that deletion of the glutamate receptor delta2 subunit (GluRdelta2) gene results in persistent multiple CF innervation of PCs with impaired PF synaptogenesis, whereas mutation of the metabotropic glutamate receptor subtype 1 (mGluR1) gene causes multiple CF innervation with normal PF synaptogenesis. We demonstrate that atypical CF-mediated EPSCs (CF-EPSCs) with slow rise times and small amplitudes coexisted with typical CF-EPSCs with fast rise times and large amplitudes in PCs from GluRdelta2 mutant cerebellar slices. CF-EPSCs in mGluR1 mutant and wild-type PCs had fast rise times. Atypical slow CF responses of GluRdelta2 mutant PCs were associated with voltage-dependent Ca(2+) signals that were confined to PC distal dendrites. In the wild-type and mGluR1 mutant PCs, CF-induced Ca(2+) signals involved both proximal and distal dendrites. Morphologically, CFs of GluRdelta2 mutant mice extended to the superficial regions of the molecular layer, whereas those of wild-type and mGluR1 mutant mice did not innervate the superficial one-fifth of the molecular layer. It is therefore likely that surplus CFs of GluRdelta2 mutant mice form ectopic synapses onto distal dendrites, whereas those of wild-type and mGluR1 mutant mice innervate proximal dendrites. These findings suggest that GluRdelta2 is required for consolidating PF synapses and restricting CF synapses to the proximal dendrites, whereas the mGluR1-signaling pathway does not affect PF synaptogenesis but is involved in eliminating surplus CF synapses at the proximal dendrites.

Preferential impairment of nitric oxide-mediated endothelium-dependent relaxation in human cervical arteries after irradiation.

BACKGROUND: Vascular abnormalities are a major cause of postoperative complications in irradiated tissues. Endothelial cell dysfunction characterized by diminished endothelium-dependent relaxation may be involved. We examined the endothelium-dependent relaxation and morphology of the endothelium in irradiated human cervical arteries. METHODS AND RESULTS: Irradiated arteries were taken from the neck region of patients who had radiation therapy. Arteries from patients who did not receive radiation therapy were used as controls. Endothelium-dependent relaxation to acetylcholine and A23187 was impaired in irradiated arteries. Norepinephrine-induced contraction and sodium nitroprusside-induced relaxation were unchanged. In control arteries, N(omega)-nitro-L-arginine and indomethacin each caused a partial inhibition of endothelium-dependent relaxation. In irradiated arteries, the impaired endothelium-dependent relaxation was unaffected by these agents, but it was abolished by high K(+). Acetylcholine produced similar degrees of hyperpolarization in control and irradiated arteries. Immunohistochemical examination for endothelial nitric oxide synthase indicated no expression in the endothelium of irradiated arteries. Electron scanning microscopy showed morphologically intact endothelial cells in irradiated arteries. CONCLUSIONS: In irradiated human cervical arteries, the nitric oxide- and prostacyclin-mediated endothelium-dependent relaxation, but not endothelium-derived hyperpolarizing factor-mediated relaxation, are specifically impaired, without significant morphological damage of the endothelium. The impaired nitric oxide-mediated relaxation was associated with a lack of endothelial nitric oxide synthase expression. Our results suggest the importance of impaired endothelial function in irradiated human blood vessels, which may partly explain the development of vascular stenosis and poor surgical wound healing in irradiated tissues.

Different expressions of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid and N-methyl-D-aspartate receptor subunit mRNAs between visceromotor and somatomotor neurons of the rat lumbosacral spinal cord.

The glutamatergic transmission system plays a key role in afferent and efferent pathways involved in micturition. By in situ hybridization combined with retrograde Fast Blue labeling, expression of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor (GluR-A to -D) and N-methyl-D-aspartate (NMDA) receptor (NR1 and NR2A-D) subunit mRNAs were examined in visceromotor and somatomotor neurons of the rat lumbosacral spinal cord. Parasympathetic preganglionic neurons (PGNs) in the intermediolateral nucleus highly expressed GluR-A and GluR-B subunit mRNAs, with very low levels for GluR-C and GluR-D subunits. As for the NMDA receptor, PGNs were associated with abundant signals for NR1 subunit mRNA, but without any NR2 subunit mRNAs. On the other hand, somatomotor neurons in the ventral horn (dorsolateral nucleus) express all four AMPA receptor subunit mRNAs, showing relatively abundant expressions of GluR-C and GluR-D subunit mRNA compared with PGNs. In addition to high levels of NR1 subunit mRNA, dorsolateral nucleus neurons moderately expressed NR2A and NR2B subunit mRNAs. These results suggest that molecular organization of both AMPA and NMDA receptor channels are distinct between PGNs and dorsolateral nucleus neurons. Considering that native NMDA receptors are heteromeric channels composed of NR1 and NR2 subunits, it seems likely that dorsolateral nucleus neurons, not PGNs, are provided with functional NMDA receptors, which could induce activity-dependent changes in synaptic transmission in the efferent pathway for the lower urinary tract.

Immunoelectron microscopic evidence for organ differences in the composition of peroxisome-specific membrane polypeptides among three rat organs: liver, kidney, and small intestine.

We examined the distribution of peroxisome-specific membrane polypeptides (PMPs) among peroxisomes of the liver, renal cortex, and jejunal mucosa, using antibodies for 70 KD, 26 KD and 22 KD PMPs. Immunoblot analysis showed signals for 70 KD polypeptide in all three kinds of tissue, but for the other two only in the liver and renal cortex, with neither being detected in jejunal mucosa. The total amounts of PMPs increased in all three organs with DEHP (di-(2-ethylhexyl)phthalate) administration. By immunoelectron microscopic analysis using protein A-gold, the three PMPs were localized along the peroxisomal membrane. Quantitation of the gold particles associated with the peroxisomal membrane showed an increase in the density of 70 KD and 26 KD PMPs but a decrease in 22 KD PMP with the administration of DEHP. The presence of tissue-specific localizations of PMPs suggest the 70 KD PMP is a common constituent of peroxisomes of these three tissues, whereas 26 KD and 22 KD PMPs are absent in microperoxisomes of jejunal mucosal epithelium.

Immunoelectron microscopic study of a new D-amino acid oxidase-immunoreactive subcompartment in rat liver peroxisomes.

We report the presence of a new subcompartment in rat liver peroxisomal matrix in which only D-amino acid oxidase is localized and other matrix enzymes are absent. By electron microscopic observation, the rat liver peroxisome has generally been considered to consist of a single limiting membrane, an electron-dense crystalline core, and a homogeneous matrix. Immunohistochemical staining for D-amino acid oxidase by the protein A-gold technique revealed the presence of a small area in the matrix that was immunoreactive for the enzyme and was less electron-dense than the surrounding matrix. The localization of D-amino acid oxidase in this small area of the peroxisomal matrix was confirmed by immunoelectron microscopy on freeze-substituted tissues processed without chemical fixation. To analyze the characteristics of the electron-lucent area, immunoreactivity for various peroxisomal enzymes, including catalase, acyl-CoA oxidase, enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase bifunctional protein, 3-ketoacyl-CoA thiolase, L-alpha-hydroxy acid oxidase (isozyme B), and glycolate oxidase (isozyme A), was assayed. The electron-lucent area was negative for all of these. By double staining for D-amino acid oxidase and catalase, using colloidal gold particles of different sizes, these enzymes were shown to be located in separate areas in the matrix.

Extra-junctional localization of glutamate transporter EAAT4 at excitatory Purkinje cell synapses.

We used silver-enhanced immunogold electron microscopy to reveal synaptic localization of the glutamate transporter EAAT4 in mouse cerebellar Purkinje cells (PCs). Gold-silver particles representing the EAAT4 were densely localized on extra-junctional membrane, but not on junctional membrane of PC spines in contact with parallel fiber or climbing fiber terminals. No particle accumulations were observed at inhibitory synapses formed on cell body and dendritic shafts of PCs. Therefore, the EAAT4 is selectively targeted to the extra-junctional site of excitatory PC synapses. The finding suggests that the EAAT4 transports glutamate or its related amino acids from outside the synaptic cleft, which would facilitate glutamate diffusion from the synaptic cleft to the extrasynaptic space and restrict glutamate spillover to adjacent synapses.

Abnormal synaptic architecture in the cerebellar cortex of a new dystonic mutant mouse, Wriggle Mouse Sagami.

The 'Wriggle Mouse Sagami (WMS)' is a new neurological mutant with severe dystonic movements of the trunk and extremities whose pathological characters are transmitted by an autosomal recessive gene (wri). Manifestations first appear at 10 days to 2 weeks after birth and progress until 12 weeks of age. In spite of the severe dystonic movements, no marked abnormalities had been found in the cyto- or myeloarchitecture of the central nervous system or that of the peripheral nerves, except for the impaired development of the dendritic trees of the Purkinje cells. In this study we quantitatively demonstrated decreased synaptic connections of parallel fibers on the dendritic spines of the Purkinje cells as early as 2 weeks after birth. On the other hand, synaptic boutons on the dendritic shafts and somata of the Purkinje cells and synaptic bouton-like structures which contained synaptic vesicles but without synaptic membrane specialization, were significantly increased in the molecular layer at 9 weeks of age. Glutamic acid decarboxylase immunohistochemistry suggested that some of these increased synaptic boutons and other bouton-like structures may have originated in GABA interneurons, such as stellate cells, basket cells and Golgi cells, and in the cerebellar nuclei. Because of the severity of the manifestations, it appears that synaptic alteration in interneurons also occurs in the other parts of the CNS.

Cerebellum of the adult reeler mutant mouse contains two Purkinje cell populations with respect to gene expression for the N-methyl-D-aspartate receptor channel.

Recent studies have identified five NMDA receptor subunits, which exhibit distinct cellular expressions in the normal rodent brain. The purpose of this investigation is to clarify the molecular-anatomical organization in the cerebellum of the reeler mutant mouse, in which various categories of the Purkinje cells are present as to the cell position and synaptic connectivity. In comparison with the distribution of the inositol 1,4,5-trisphosphate receptor mRNA, a molecular marker specific to the Purkinje cells, the epsilon 1 subunit mRNA of the NMDA receptor channel was found in the adjacent sections to be expressed in a subset of the Purkinje cells. In the rostrocaudal extent, the Purkinje cells expressing the epsilon 1 subunit mRNA were distributed preferentially in the rostral cerebellum, irrespective of the normal and heterotopic positions. In the mediolateral extent, they formed segregated cell clusters, interposed by epsilon 1 subunit mRNA-negative clusters. Hybridizing signals for the zeta 1 subunit mRNA were found in all the Purkinje cell population, whereas those for the epsilon 2, epsilon 3, and epsilon 4 subunit mRNAs were not detected in the cells. These findings suggest that the reeler cerebellum is topographically compartmentalized by two subpopulations of the Purkinje cells, one expressing the epsilon 1 and zeta 1 subunit mRNAs, and the other expressing the zeta 1 subunit mRNA alone.

Early establishment of lesion-insensitive mature barrelettes corresponding to upper lip vibrissae in developing mice.

Vibrissae are tactile sense organs on the face of non-human mammals, and build up topographical representations in the brainstem trigeminal sensory nucleus called barrelettes. In the present study, we examined postnatal development of barrelettes corresponding to upper lip vibrissae by cytochrome oxidase (CO) histochemistry. At nuclear regions corresponding to upper lip vibrissae, a few segregated barrelettes first appeared at postnatal day 2 (P2), and segregation became clear for most upper lip barrelettes at P4. Compared with major barrelettes corresponding to mystacial vibrissae on the snout, the development of segregated pattern formation for upper lip barrelettes was retarded by 1-2 days. When vibrissa-related patterns were examined 5 days after infraorbital nerve transection, upper lip barrelettes became obscure in all mice lesioned at P1 and P2. Lesion-insensitive upper lip barrelettes first emerged in a few mice lesioned at P3 (33%), and the percentage attained 100% at P6. This temporal transition from lesion-sensitive to lesion-insensitive barrelettes was 3 days ahead of mystacial barrelettes. Therefore, upper lip barrelettes achieve rapid development within a narrow time frame during the first postnatal week. The early and rapid establishment of lesion-insensitive, mature barrelettes can be interpreted as suggesting the importance of oral sensory function in neonatal life.

Distribution of guanine nucleotide-binding protein in the brain of the reeler mutant mouse.

The localization of a GTP-binding protein (G(o)) in the cerebellar and cerebral cortex and hippocampus of the normal and reeler mutant mouse was immunohistochemically examined using affinity-purified antibody raised against the alpha subunit of G(o). Although the general distribution pattern of G(o)-immunoreactive products in the brain of the normal mouse, i.e., abundant in the neuropil but absent from neuronal cell bodies, is also seen in the reeler brain, some differences are present, as described below. Strong G(o)-immunoreactive products are found in the molecular layer of the cerebellar cortex of the normal mouse. In the reeler cerebellum, in addition to the strong G(o)-immunoreactivity of the thin molecular layer, moderate G(o)-immunoreactivities are also found in the granular cell layer and the central cerebellar mass. G(o)-immunoreactive products are distributed throughout all layers of the cerebral cortex of the normal and reeler mouse. However, layer I of the normal cerebral cortex is more strongly stained with this antibody than the underlying layers, whereas the upper third of the reeler cerebral cortex is more strongly stained than the lower two-thirds. In the hippocampus of the normal mouse, G(o)-immunoreactive products are localized in the neuropil of the stratum oriens, stratum radiatum and stratum lacunosum-moleculare, but absent from the cell bodies of the pyramidal cells and their apical dendritic shafts. Such a distribution pattern of G(o)-immunoreactive products is also seen in the hippocampus of the reeler mouse, except that G(o)-immunonegative pyramidal cells split into 2 or 3 laminae.(ABSTRACT TRUNCATED AT 250 WORDS)

N-ethylmaleimide inhibits xanthine oxidase activity with no detectable change in xanthine dehydrogenase activity in rabbit liver.

The effect of an alkylating agent, N-ethylmaleimide (NEM), on the activities of xanthine oxidase (XO) and xanthine dehydrogenase (XD) in the presence and absence of Cu2+ or trypsin in the cytosolic fraction from rabbit liver was examined. At concentrations ranging from 0.25 to 2.0 microM, allopurinol, which is generally considered to be a XO inhibitor, suppressed the XD activity (41.5-93.4% inhibition) in addition to the XO activity (28.6-88.4% inhibition) under basal conditions, without the addition of Cu2+ or trypsin. In contrast, NEM (100-400 microM) inhibited the XO activity (35.7-85.7% inhibition) without affecting the XD activity. Also, NEM inhibited the Cu2+- and trypsin-induced XO activities, but did not affect the XD activity at the same concentration range. These results demonstrate that NEM can be a selective inhibitor of XO activity in rabbit liver.

Synthesis, from cellobiose, of a trisaccharide closely related to the GlcNAc----GlcA----GlcN segment of the antithrombin-binding sequence of heparin.

O-(2-Deoxy-2-sulfamido-6-O-sulfo-alpha-D-glucopyranosyl)-(1----4)- O-(beta-D- glucopyranosyluronic acid)-(1----4)-1,6-anhydro-2-deoxy-2-sulfamido-6-O-sulfo-beta-D-gl ucopyranose pentasodium salt (14) was synthesized as a heparin-related oligosaccharide. The glycosyl acceptor (derived from cellobiose) and a glycosyl donor, 6-O-acetyl-2-azido-3,4-di-O-benzyl-2-deoxy-alpha-D-glucopyranosyl bromide, were coupled in the presence of mercuric bromide and molecular sieves 4A to afford a 69% yield of fully protected trisaccharide, namely, O-(6-O-acetyl-2-azido-3,4-di-O-benzyl-2-deoxy-alpha-D-glucopyranosyl)-(1 ----4)- O-(methyl 2,3-di-O-benzyl-beta-D-glucopyranosyluronate)-(1----4)-3-O-acetyl- 1,6-anhydro-2 - azido-2-deoxy-beta-D-glucopyranose (10), which was converted into the partially sulfated trisaccharide 14. Compound 10 also underwent acetolysis to afford the glycosyl acetate, for further elongation of the glycosyl chain.

The validity of the thread test for evaluating nasal secretion in nasal allergy.

The volume of nasal secretion is so small that it is difficult to measure. We placed a thread on the anterior surface of the inferior turbinate to measure the volume of nasal secretion in normal subjects and patients with nasal allergy. In 24 normal subjects, the average nasal secretion amounted to 1.09 microliter/min. In 48 patients with nasal allergy, the volume of nasal secretion (1.94 microliter/min) decreased significantly after nasal spraying: the subjective symptoms of sneezing, rhinorrhea, nasal obstruction, inconvenience in daily life and the objective signs such as watery rhinorrhea abated. The thread test results showed a good correlation with the above items in a quantitative analysis. The thread test is a simple way to measure a small volume of nasal secretion, and a useful indicator of the severity of watery rhinorrhea in patients with nasal allergy.

The validity of the thread test for evaluating nasal secretion in nasal allergy.

The volume of nasal secretion is so small that it is difficult to measure. We placed a thread on the anterior surface of the inferior turbinate to measure the volume of nasal secretion in normal subjects and patients with nasal allergy. In 24 normal subjects, the average nasal secretion amounted to 1.09 wVmin. In 48 patients with nasal allergy, the volume of nasal secretion (1.94 ul/min) decreased significantly after nasal spraying; the subjective symptoms of sneezing, rhinorrhea, nasal obstruction, inconvenience in daily life and the objective signs such as watery rhinorrhea abated. The thread test results showed a good correlation with the above items in a quantitative analysis. The thread test is a simple way to measure a small volume of nasal secretion, and a useful indicator of the severity of watery rhinorrhea in patients with nasal allergy.

Whole-body retention and tissue distribution of 60Co in rats after oral administration of freshwater fish contaminated with 60Co.

The purpose of this report is to compare the whole-body retention and tissue distribution in rats of 60Co administered by gavage as inorganic 60CoCl2 or in a form incorporated into freshwater fish. Orizias latipes were placed in vessels containing 21. of tap water with radioactive cobalt. Periodically thereafter the fish were sacrificed, homogenized, and administered to rats via a stomach tube. Control groups of rats were given the radionuclide alone or together with a homogenate of nonradioactive fish. The whole-body retention and tissue distribution of the radionuclide were determined with an Armac counter. The results revealed that rats gavaged with 60Co incorporated into the fish retained much more 60Co than control rats. This trend was notable in rats given fish kept in radioactive solution for longer periods. Marked differences in tissue distribution of 60Co were also observed between rats given 60Co incorporated into fish and control rats.

Dendritic arbolization of large pyramidal neurons in the motor cortex of normal and reeler mutant mouse.

Reeler, an autosomal recessive mutation in mice, is characterized by abnormal positioning of the neurons in the cerebral cortex. We performed a descriptive analysis on the arborization of dendritic processes of large pyramidal neurons in the motor cortex (hindlimb area) of normal and reeler mice, as seen in the Golgi preparations. In the normal mouse, somata of large pyramidal neurons were located in the layer V, and their apical dendrites ascend vertically to the pial surfaces. Their basal dendrites proceed horizontally or inferiorly. In the reeler mouse, typical large pyramidal neurons with a normal (upright) apical dendrite and a variety of atypical large pyramidal neurons with a disoriented apical dendrite were radially scattered within the motor cortex. Typical large pyramidal neurons occupied the lower half of the motor cortex, whereas atypical large pyramidal neurons were predominantly observed in the upper half of the motor cortex. Atypical large pyramidal neurons were further divided into inverted, tumbled, V-shaped, bipolar and superficial polymorphic cells, as previously reported (Terashima et al., J. Comp. Neurol. 218:314-326, 1983). Superficial polymorphic cells localized in the layer of polymorphic cells and the layer of the large pyramidal cells were characterized by the extremely poor dendritic arborizations and the smooth surface of the dendrites, which suggests development of dendrites of these neurons was deranged by the reeler genetic locus.

[Pterion and epipteric bones in Japanese adults and fetuses, with special reference to their formation and variations].

The formation and variations of the pterion and epipteric bones were examined in total of 614 Japanese skulls. The materials used consisted of 258 skulls of Japanese fetuses ranging from the fourth to the ninth month, 20 skulls of Japanese juveniles from the third month to 17 years of age, and 336 skulls of Japanese adults from 20 to 89 years of age. For the skulls examined the incidence of ossification in the fetal sphenoidal fontanelle was 3.6% on each side, whereas epipteric bones were observed in more than 10% of the juvenile and adult pteria. Great variation was seen in the form of the adult pterion. The most common form was a sphenoparietal contact in which the pteria were classified into usual (306 pteria), high (119), low (21), and narrow (32) types Another form of this type, a frontal process of the temporal bone without contact with frontal bone, was found in five pteria. The form of frontotemporal contact is classified into two types: One is with a frontal process of the temporal bone (17 pteria), and another is a K-shaped contact referred to as "stellate" (four). The two types were observable in adult skulls of all ages, although the fused pteria and fusing epipteric bones were most often seen in cases over 40 years of age. The results suggest that the pterion formation has two phases, the first occurring before the occlusion of sphenoidal fontanelle, and the second starting after 40 years of age.