Tight regulation of transgene expression by tetracycline-dependent activator and repressor in brain.

Methods to temporally and spatially regulate gene mutations will provide a powerful strategy to investigate gene function in the brain. To develop these methods, we have established a tightly regulated system for transgene expression in the forebrain using both a tetracycline (Tc)-dependent transcription activator (rtTA) and a repressor (TetR-Kruppel-associated box). In this system, the repressor binds to the Tc-responsive element (TRE) in the absence of doxycycline (Dox), leading to the repression of leaky activation of TRE-mediated transcription caused by weak binding of rtTA to TRE. Upon Dox administration, only the activator binds to TRE and activates transcription. We tested this system in cultured cells by bicistronically expressing both the regulators using an internal ribosome entry site (IRES). In COS-1, HeLa and SHSY5Y cells, leaky transcription activation led by rtTA in the absence of Dox was repressed without decreasing the level of activated transcription in the presence of Dox. Using this system, transgenic mice were produced that express both the regulators using IRES in the forebrain under the control of the alphaCaMKII promoter and were bred with transgenic mice carrying the TRE-dependent reporter transgene. In reverse transcription-polymerase chain reaction and in situ hybridization analyses of the forebrain in adult double transgenic mice, the treatment of Dox induces reporter mRNA expression, which was not detected before the treatment and after the withdraw of Dox following the treatment. These results indicate that this system allows the tight regulation of transgene expression in a Dox-dependent fashion in the forebrain and will be useful in investigating gene function in the brain.

Surgical pathology of spinal schwannoma: has the nerve of its origin been preserved or already degenerated during tumor growth?

OBJECTIVE: This study was aimed to understand ultrastructural pathology of nerves of tumor origin of spinal schwannomas, which has not been reported so far, in order to understand the mechanism of the postoperative functional restoration after the nerve transection. METHODS: From 13 patients who underwent sacrifice of an affected nerve root at total removal of spinal schwannomas (C2 conus), the proximal (spinal cord side, n = 12) and distal (dorsal root ganglion side, n = 10) stumps of the nerves of the tumor origin were collected and examined by light and electron microscope, followed by morphometric analysis (n = 9). RESULTS: Almost all of affected nerves at both proximal and distal to the lesion were composed of well-preserved myelin sheath and axons with mild disturbance of endo- and perineurial structures at light microscopic level except one case, which showed severe fibrosis. Electron-microscopically, regenerated axons with thin myelin were found in part in the proximal and distal nerves with few macrophages in three cases. The area of nerves (mm2), density of myelinated axons (axons/mm2) and total number of myelinated axons in the proximal stump (0.552 +/- 0.430, 10,400 +/- 5,240 and 5,480 +/- 4,790) was approximately 70%, 80% and 60%, respectively, of those in the distal stump (0.765 +/- 0.333, 12,400 +/- 5,180 and 9,970 +/- 8,630). CONCLUSIONS: This data combined with no permanent deficits after nerve transection suggest that the nerves of tumor origin are in the processes of slowly progressed deterioration with repeated degeneration and regeneration/remyelination, and the postoperative rapid recovery from the transient neurological deficit may be explained by functional compensation by the adjacent non-affected nerves with slow tumor growth.

Microendophenotypes of psychiatric disorders: phenotypes of psychiatric disorders at the level of molecular dynamics, synapses, neurons, and neural circuits.

Psychiatric disorders are caused not only by genetic factors but also by complicated factors such as environmental ones. Moreover, environmental factors are rarely quantitated as biological and biochemical indicators, making it extremely difficult to understand the pathological conditions of psychiatric disorders as well as their underlying pathogenic mechanisms. Additionally, we have actually no other option but to perform biological studies on postmortem human brains that display features of psychiatric disorders, thereby resulting in a lack of experimental materials to characterize the basic biology of these disorders. From these backgrounds, animal, tissue, or cell models that can be used in basic research are indispensable to understand biologically the pathogenic mechanisms of psychiatric disorders. In this review, we discuss the importance of microendophenotypes of psychiatric disorders, i.e., phenotypes at the level of molecular dynamics, neurons, synapses, and neural circuits, as targets of basic research on these disorders.

Membranous ultrastructure of human arachnoid cells.

The ultrastructure of arachnoid cell membranes was investigated by conventional transmission EM and by freeze-fracture techniques in human arachnoid granulations. Arachnoid cells showed widespread membrane specialization in the granulations including the formation of desmosomes, gap junctions, tight junctions, intermediate junctions, hemidesmosome-like structures, and micropinocytotic vesicles. However, the extent of the specialization varied from portion to portion; this was clearly shown on freeze-fracture replicas. Numerous extracellular cisterns were separated by cytoplasmic bodies or slender processes, joined by these junctional complexes. Uncoated and coated vesicles were abundant along the surface of extracellular cisterns representing the pathway of CSF. Complexes of branching tight junctions were comprised of 1-50 particle strands, which formed elaborate meshworks accompanied by numerous gap junctions and desmosomes. Micropinocytotic vesicles were often concentrated in the arachnoid cell cluster up to 40 per microm2, which is equivalent to the concentration in brain capillary endothelial cells. The results of this study clearly suggest that arachnoid cells in arachnoid granulations are not only tightly adherent to form a firm structure for the passage of CSF, but that the arachnoid cells lining the CSF pathway show intense cell-cell communication and pinocytotic activity. This high transcellular activity probably reflects active transports or secretion of certain molecules by arachnoid cells.

Localization of E-cadherin in peripheral glia after nerve injury and repair.

Peripheral nerve injury results in histological and histochemical changes in neurons and glia. We have recently found that Ca(2+)-dependent cell adhesion molecule E-cadherin plays an important role in the selective fasciculation of a particular subset of unmyelinated sensory fibers. In the present immunohistochemical and immunoblot analyses, the temporal profile of the subcellular expression of this molecule in spinal nerves was examined after crushing, transecting, or ligaturing the sciatic nerve in mice with special attention paid to E-cadherin expression in glial cells. After axotomy of the sciatic nerve, distal axons of the proximal stump and the fibers of the distal stump degenerated, but E-cadherin was still detectable at the outer mesaxons of the myelinated axons as long as they remained morphologically intact. Subsequently, Schwann cells proliferated and migrated to form Schwann cell columns (Büngner's bands) as initial responses to denervation, and expressed E-cadherin at their site of contact with each other and later with sprouting axons. At the initial stage of myelin formation, slender processes of a single Schwann cell interdigitated with an enveloped axons, and expressed E-cadherin at the contact site elaborated by a single Schwann cell. Immunoblot analysis on day 7 revealed that E-cadherin was detected in both the proximal nerve segments and the regenerative distal segments, but was negative in the degenerative distal segments. On the basis of present data, it is suggested that E-cadherin might be involved in the stabilization of the peripheral glial network which provides the guidance of sprouting axons and myelination.

Pathways of fluid drainage from the brain--morphological aspects and immunological significance in rat and man.

There is firm physiological evidence for the lymphatic drainage of interstitial fluid and cerebrospinal fluid from the brains of rats, rabbits and cats. The object of this review, is to describe firstly the morphological aspects of lymphatic drainage pathways from the rat brain and secondly, to explore through scanning and transmission electron microscope techniques, the possibility of similar lymphatic drainage pathways in man. Interstitial and oedema fluid spreads diffusely through the white matter in the rat and appears to drain into the ventricular cerebrospinal fluid. In grey matter, however, tracers pass along perivascular spaces to the surface of the brain and into the cerebrospinal fluid. Paravascular compartments in the subarachnoid space follow the course of major arterial branches to the circle of Willis and thence along the ethmoidal arteries to the cribriform plate of the ethmoid bone. Particulate tracers, such as Indian ink, enter channels in the arachnoid beneath the olfactory bulbs and connect directly with nasal lymphatics through channels which pass through holes in the cribriform plate. Proteins and other solutes may also drain along other cranial nerves. Thus, there is a bulk flow pathway for interstitial and cerebrospinal fluid from the rat brain into cervical lymphatics. In man, it is probable that diffuse interstitial drainage of fluid from the white matter occurs in a similar way to that in the rat. Furthermore, the anatomical pathways exist by which bulk drainage of fluid could occur along perivascular spaces from the grey matter into perivascular spaces of the leptomeningeal arteries and thence into the cerebrospinal fluid (CSF).(ABSTRACT TRUNCATED AT 250 WORDS)

Isolation of a cDNA encoding a mouse TFIID subunit containing histone H4 homology.

A cDNA encoding a mouse transcription factor IID (TFIID) subunit, containing histone H4 homology, was cloned and sequenced. The predicted 678-amino-acid (aa) sequence of this molecule showed 97 and 41% identity to the human and Drosophila melanogaster homologues, respectively. Four putative direct repeats were found in the most highly conserved region in the central part of this protein.

Three distinct regions in a rat TFIID subunit containing histone H4 homology.

A cDNA encoding a rat transcription factor IID (TFIID) subunit (p80), with histone H4 homology, was isolated and sequenced. The deduced amino acid (aa) sequence predicts a 678-aa protein with 97% identity to the human and 42% to the Drosophila melanogaster (Dm) homologues. Homologies between three species indicate the presence of three distinct regions.

Isolation and characterization of a cDNA encoding a new type of human transcription elongation factor S-II.

We report the isolation of a cDNA encoding a new type of transcription factor S-II, termed h-SII-T1, from a human library. The mRNA corresponding to the clone is highly expressed in testis and ovary. Comparison of the deduced amino acid (aa) sequence with those of other S-II molecules shows that (i) the C-terminal zinc finger (Zf) domain is highly conserved, and (ii) the central segment is most similar to that of the rat testis-specific S-II. Further analyses of the hS-II-T1 aa sequence indicate that its N-terminal sequence exhibits similarity to eubacterial sigma 54. The significance of tissue-specific S-II molecules for the regulation of transcription elongation is discussed.

Synthesis and antiviral activity of sulfonamidobenzophenone oximes and sulfonamidobenzamides.

To find antiviral agents, various sulfonamidobenzophenone oximes (II) were synthesized from the appropriate m-sulfonamidobenzophenones by hydroxylamine reaction. The reaction products were generally obtained as syn/anti mixtures which were separable by fractional crystallization. The anti isomer had more potent antipoliovirus activity than the syn isomer. Various sulfonamidobenzamides (III) which were structurally related to II were synthesized by the reactions of amino-substituted benzamides with sulfuryl chloride or amines with (aminosulfonyl)benzoyl chloride. Antiviral activity was examined by the plaque-inhibition test. Compounds 5, 36, and 69 exhibited strong antipicornavirus activity. The structure-activity relationships are discussed.

Synthesis and oral antifungal activity of novel azolylpropanolones and related compounds.

To find orally active antifungal agents, novel imidazolyl- and 1,2,4-triazolylpropanolones I and related compounds II-IV were synthesized. Compounds I were derived from ketones V (method A), alpha-diketone IX (method B), alpha-hydroxy ketones X (method C), alpha-chloro ketone XII (method D), and enones VI (method E). Diols II, synthesized from I with NaBH4, were cyclized to five-membered cyclic compounds III by using N,N'-carbonyldiimidazole, thionyl chloride, N,N'-(thiocarbonyl)diimidazole, bromochloromethane, 2,2-dimethoxypropane, and cyclohexanone dimethyl ketal. Diols IV were synthesized from I by Grignard reaction (method F), hydroxymethylation of X (method G), and reaction of ketones XXI with 1-[(trimethylsily)methyl]-1,2,4-triazole (method H). Compounds I-IV were examined for their antifungal activities in vitro by evaluation of broth dilution MIC values against three species of fungi and the inhibitory effect on pseudomycelium of Candida albicans, and they were examined for oral efficacy in vivo against subacute systemic candidiasis in mice and superficial dermatophytosis in guinea pigs. Compounds 2, 12, 38, 39, and 92 exhibited strong oral antifungal activity. An asymmetric synthesis and the structure-activity relationships of the compounds examined are discussed.

Synthesis and antifungal activity of new 1-vinylimidazoles.

Carbonyl compounds I were subjected to an imidazole transfer reaction with N,N'-sulfinyldiimidazole or N,N'-carbonyldiimidazole to obtain the diimidazole II and the monoimidazole III. Various 1-vinylimidazoles IV, derived from o-hydroxyacetophenones by imidazole transfer reaction, were alkylated to furnish the title compounds V. The structure-activity relationships of these 1-vinylimidazole compounds V are described.

Synthesis and antifungal activity of a series of novel 1,2-disubstituted propenones.

To find an antifungal agent other than those of the imidazole and triazole series, a new class of 1,2-disubstituted propenones I and II was prepared and tested for antifungal activity. Comparison of the structure-activity relationships showed that the conjugated structure of carbonyl and exomethylene groups in I and II plays an important role in potent antifungal activity. However, it is noteworthy that compounds 53, 54, and 56, which have a hydroxymethyl or methoxymethyl group instead of an exo-methylene group in I, also showed potent activity. Although many compounds exhibited strong antifungal activity in vitro, none showed activity in vivo of oral efficacy against subacute systemic candidiasis in mice.

Synthesis and neuroleptic activity of N-[(1-ethyl-2-pyrrolidinyl)methyl]-2-methoxy-5-sulfonamidobenzamide s.

A series of some novel N-[(1-ethyl-2-pyrrolidinyl)methyl]benzamides involving replacement of the sulfamoyl group in sulpiride with a sulfonamido group was synthesized and tested for dopamine receptor blockade. In comparison with sulpiride, several compounds were considerably more potent than sulpiride as dopamine receptor blockers. The structure-activity relationships are discussed.

Synthesis and antiarrhythmic activity of new 1-[1-[2-[3-(alkylamino)-2-hydroxypropoxy]phenyl]vinyl]-1 H-imidazoles and related compounds.

Various 1-[1-[2-[3-(alkylamino)-2-hydroxypropoxy]phenyl]vinyl]-1 H-azoles were synthesized and investigated for beta-adrenoceptor-blocking and antiarrhythmic activities. Although no compounds showed more potent beta-blocking effects than propranolol in the isolated guinea pig right atria, many compounds exhibited significant antiarrhythmic effects against aconitine or ischemic arrhythmia in mice or dogs. 1-[2,5-Dichloro-6-[1-(1H-imidazol-1-yl)-ethenyl] phenoxy]-3-[(1-methylethyl)amino]-2-propanol hydrochloride (48) (711389-S) was selected as a candidate for clinical evaluation in man, since its antiarrhythmic effects were superior to those of quinidine, disopyramide, or propranolol. Asymmetric synthesis of (R)-(+)- and (S)-(-)-48 is described, and it is proven that there is no stereospecificity in the antiarrhythmic effect of 48.

Restricted expression of a member of the transcription elongation factor S-II family in testicular germ cells during and after meiosis.

Whether expression of transcription elongation factors is regulated during development has not been investigated, though genes encoding elongation factor S-II are transcribed in a tissue-specific manner. We investigated the expression profile of tissue-specific S-II during development using an isolated cDNA, termed mouse S-II-T1, whose transcripts are detected almost exclusively in testis. Three experiments were performed with various types of germ and somatic cells in testis to determine in which cells S-II-T1 is expressed. (1) Expression of S-II-T1 is markedly reduced in the testes of adult WBB6F1-W/Wv mutant mice which lack testicular germ cells, indicating its expression is specific to testicular germ cells. (2) The onset of mouse S-II-T1 mRNA appearance in testis is seen about 10-14 days after birth, which is consistent with the start of meiotic events, suggesting that S-II-T1 is not transcribed in premeiotic and early meiotic cells such as spermatogonia, leptotene spermatocytes, or zygotene spermatocytes. (3) Mouse S-II-T1 transcripts accumulate in meiotic pachytene spermatocytes and are detected in postmeiotic haploid cells such as round and elongated spermatids during spermatogenesis, as shown by fractionation of testicular germ cells at four different stages. These results indicate that expression of mouse S-II-T1 is restricted to testicular germ cells during and after meiosis in the course of spermatogenesis. This is the first report that expression of a transcription elongation factor in particular cells is regulated in a stage-specific manner in the course of development.

Expression of E-cadherin-catenin complex in human benign schwannomas.

The Ca2+-dependent cell adhesion molecule E-cadherin has been known to express in normal and reactive Schwann cells in rodents, and to play an important role in Schwann cell-Schwann cell adhesion and maintenance of peripheral nervous tissue architecture. However, little is known about expression of E-cadherin in schwannomas. The aim of the present study was to investigate the cellular expression and localization of E-cadherin, and its associated protein, alpha E-, alpha N- and beta-catenins in human schwannomas, which are supposed to derive from Schwann cells. We tested the hypothesis that these proteins might show an altered expression/distribution in schwannoma cells which correlates with their neoplastic behavior, including sparse cell-cell contact, as seen those in meningiomas and various carcinomas. In human schwannomas, however, E-cadherin, alpha E-catenin, and beta-catenin were detected by western blotting and immunohistochemistry, whereas alpha N-catenin was not. Immunoprecipitation using anti-E-cadherin antibody resulted in alpha E-catenin forming a complex with E-cadherin. SSCP analysis revealed no mutations in the transmembrane domain or in intracellular catenin-binding site of E-cadherin. These data suggest that the E-cadherin-alpha E-catenin complex is well preserved in human schwannoma cells, which is compatible with its benign behavior, and these molecules might be used as additional cell markers of Schwann cell-derived tumors.

Relationship of the biosynthesis of alpha-fetoprotein, albumin, hemopexin, and haptoglobin to the growth state of fetal rat hepatocyte cultures.

AFP and albumin are produced by arginine-synthesizing fetal rat hepatocytes in vitro. AFP and hemopexin production are coupled to hepatocellular proliferation, whereas albumin and haptoglobin production are not. During the cell cycle, AFP is synthesized prior to S and released prior to M. AFP may play a role in regulation of hepatocellular growth through estradiol binding and modulation of the intracellular concentration of lipoprotein (VLDL).

An electron microscopic study of cerebral vasospasm with resultant myonecrosis in cases of subarachnoid haemorrhage, meningitis and trans-sylvian surgery.

Electron microscopic data on the development of myonecrosis following cerebral vasospasm associated with subarachnoid haemorrhage, meningitis and trans-sylvian surgery are presented. The basic feature of myonecrosis was dissolution of myofilaments with resultant fine granular or filamentous material. The disintegrating cytoplasm often contained numerous glycogen granules, dense bodies, autophagic vacuoles and myelin-like membranous bodies. A well-developed sarcoplasmic reticulum was preserved despite myofilament dissolution, while mitochondria showed marked swelling. The nuclei showed either dilution of chromatin or pyknotic change. The basal lamina was remarkably thickened and maintained an irregular outline of the necrotic smooth muscle cells. Enlarged intercellular space contained abundant cellular debris, vesicular structures and connective tissue fibres. The pathogenesis of these changes is discussed.

Middle cerebral artery occlusion in the rat causes a biphasic production of immunoreactive interleukin-1beta in the cerebral cortex.

Interleukin-1beta (IL-1beta) has been implicated in the sequence of events leading to neuronal cell death. We have used an ultrasensitive, highly specific immunometric assay for rat IL-1beta to determine the pattern of protein expression in the rat cerebral cortex following middle cerebral artery occlusion. In the ipsilateral cerebral cortex there was a biphasic pattern of production with a rapid early phase of IL-1beta expression followed by a delayed phase at 4 h which was sustained for up to 72 h. There was a similar but smaller expression of IL-1beta in the contralateral cortex. Sham operated animals showed no IL-1beta expression. These results provide further evidence to show that IL-1beta is expressed in the rat cerebral cortex following cerebral ischaemia.