Identification of two Sox17 messenger RNA isoforms, with and without the high mobility group box region, and their differential expression in mouse spermatogenesis.

The different mRNA isoforms of the mouse Sox17 gene were isolated from adult mouse testis cDNAs. One form (referred to as form Sox17) encodes an Sry-related protein of 419 amino acids containing a single high mobility group box near the NH2-terminus, while the other form (referred to as form t-Sox17) shows a unique mRNA isoform of the Sox17 gene with a partial deletion of the HMG box region. Analysis of genomic DNA revealed that these two isoforms were produced at least by alternative splicing of the exon corresponding to the 5' untranslated region and NH2-terminal 102 amino acids. RNA analyses in the testis revealed that form Sox17 began at the pachytene spermatocyte stage and was highly accumulated in round spermatids. Protein analyses revealed that t-Sox17 isoforms, as well as Sox17 isoforms, were translated into the protein products in the testis, although the amount of t-Sox17 products is lower in comparison to the high accumulation of t-Sox17 mRNA. By the electrophoretic mobility-shift assay and the random selection assay using recombinant Sox17 and t-Sox17 proteins, Sox17 protein is a DNA-binding protein with a similar sequence specificity to Sry and the other members of Sox family proteins, while t-Sox17 shows no apparent DNA-binding activity. Moreover, by a cotransfection experiment using a luciferase reporter gene, Sox17 could stimulate transcription through its binding site, but t-Sox17 had little effect on reporter gene expression. Thus, these findings suggest that Sox17 may function as a transcriptional activator in the premeiotic germ cells, and that a splicing switch into t-Sox17 may lead to the loss of its function in the postmeiotic germ cells.

Structural and functional characterization of homo-oligomeric complexes of alpha and beta chaperonin subunits from the hyperthermophilic archaeum Thermococcus strain KS-1.

To elucidate the function of group II chaperonin, the gene for the chaperonin from the hyperthermophilic archaeum Thermococcus strain KS-1 was cloned and sequenced. Two distinct genes coding for chaperonin subunits, designated alpha and beta, were obtained, and their deduced amino acid sequences are highly homologous to those of group II chaperonins from other sources. The alpha and beta subunits were individually expressed in Escherichia coli. Both of the recombinant subunits assemble to constitute the homo-oligomeric double-ring complexes, which are prone to form large aggregates. The alpha aggregate is dissociated into the typical chaperonin ring complex by incubation in buffer containing 15% (v/v) methanol, while the beta aggregate cannot be dissociated. At high temperature, both of the recombinant complexes have weak ATPase activities. They are able to arrest refolding of a chemically denatured thermophilic enzyme in the absence of ATP, and refolding is resumed when ATP is supplemented. These results suggest that homo-oligomeric complexes of the archaeal chaperonin have activity.

Nrk: a murine X-linked NIK (Nck-interacting kinase)-related kinase gene expressed in skeletal muscle.

We report the cloning and expression pattern of a novel Ste20-type kinase gene, NIK-related kinase (Nrk), located on the mouse X chromosome. The full-length Nrk cDNA encodes a 1455-amino-acid polypeptide characterized by a N-terminal Ste20-type catalytic domain and a C-terminal regulatory domain characteristic of the group I GCK subfamily. The overall structure of the NRK protein is closely related to that of Nck-interacting kinase (Nik). In situ hybridization revealed that Nrk was predominantly expressed in skeletal muscle during mouse embryogenesis. Nrk gene expression was detected in the myotome at 10.5 dpc and, thereafter, was observed in developing skeletal musculature from 11.5 to 13.5 dpc. However, expression in skeletal muscle was not observed in adults.

Hypokalemic myopathy associated with 17 alpha-hydroxylase deficiency: a case report.

We studied a patient with hypokalemic myopathy associated with 17 alpha-hydroxylase deficiency. An 18-year-old high school student, who appeared to be a girl with poorly developed secondary sex characteristics, had generalized muscle weakness. The cause of muscle weakness proved to be hypokalemic myopathy confirmed by clinical findings and muscle biopsy. Endocrinologic study demonstrated 17 alpha-hydroxylase deficiency with male pseudohermaphroditism. The metabolic abnormality of this patient was corrected by the administration of glucocorticoid. The possibility of this rare disease has to be considered when we examine a patient who has hypokalemic myopathy associated with hypogonadism.

Biological activity of the unique variants of human chorionic gonadotropin in choriocarcinoma tissue.

Human chorionic gonadotropin (hCG) in sera and placentae from normal pregnant women separated into 7 variants on analysis by an isoelectric focusing technique and determined by radioimmunoassay. The pIs ranged from 3.9 to 7.0. Three additional acidic variants were found in the sera and tumor tissues of patients with choriocarcinoma with pIs of 3.2, 3.5 and 3.7. The biological activity of each variant was determined by measuring testosterone production by rat Leydig cells in vitro. The pI 4.1 fraction corresponding to placental hCG possessed the highest biological activity while those focusing further afield from pI 4.1 showed decreasing activities. All 3 tumor unique acidic variants possessed biological activity with the fraction focusing at pI 3.7 having the greatest potency.

The mouse putative pheromone receptor was specifically activated by stimulation with male mouse urine.

To detect the biological activity of mammalian putative pheromone receptors (V1Rs and V2Rs), the mouse V1R gene was introduced into a primary culture of vomeronasal cells using the adenovirus expression system, and the response of these cells to mouse urine was analyzed by calcium imaging. These cells specifically responded to male but not female mouse urine. This response was attenuated by pertussis toxin, a specific inhibitor of G-protein G(ialpha)/G(oalpha) coupling from receptors. Our findings indicate that a putative pheromone receptor was specifically activated by mouse urine, a major source of mouse pheromones, and suggest that G(i)/G(o) are functionally coupled with the receptor.

Effects of extracellular matrix on differentiation of mouse fetal gonads in the absence of mesonephros in vitro.

The influence of mesonephric tissues and the extracellular matrix on mouse gonadal differentiation was examined in vitro. Gonadal ridges, with or without the adjacent mesonephric region, were removed from mouse embryos on day 12 post coitum (p.c.), and cultured in the presence or absence of reconstituted basement membrane (matrigel) for 5 days. Culturing control undifferentiated testes with mesonephric tissues induced normal testicular differentiation. When testes without mesonephric tissues were cultured in the absence of matrigel, testicular cord formation was not observed in the explants. Sertoli cells were irregularly arranged in the testicular parenchyma, and no continuous basal lamina was formed around the Sertoli cells. However, when testes without mesonephric tissues were embedded in matrigel and cultured for 5 days, the Sertoli cells were organized into testicular cord-like structures. The Sertoli cells positioned at the base of the cord-like structures were closely connected to the matrigel at their basal surface, and showed a polarized distribution of vimentin filaments in their basal cytoplasm. Leydig cells, on the other hand, were differentiated in all testicular explants. In all ovarian explants, germ cells normally entered meiotic prophase. Therefore, these findings indicate that the extracellular matrix permits testicular differentiation in the absence of the mesonephros, and that removal of mesonephric tissues leads to developmental failure of cord formation because the components of the extracellular matrix around pre-Sertoli cells are incomplete.

Electron microscopic studies of bacteriophage phi X174 intact and "eclipsing' particles, and the genome by the staining, and shadowing method.

Bacteriophage phi X174 particles were observed with a new method, a combination of staining and shadowing. Previously we used this technique to obtain clear visualization of the shape of cytoplasmic-polyhedrosis virus, especially the spikes of this particle. The phi X174 particle was observed to be an icosahedral particle of 25 nm in diameter carrying a spike at each vertex. The spike was a pentagonal frustum with a base of 9 nm and a height of 6 nm. When the phage was treated with calcium on the supporting film, the genome extruded from the particle. With the staining and shadowing method, it was clearly observed that the spike was the extruding channel of the genome. The genome extrusion occurred not only a single spike of the particle but also at two or more spikes. This implied that all 12 spikes of the particle had the same abilities to act as the channel of genome extrusion. The released genomes showed various thicknesses and shapes such as bending, looping, or branching, which were thought to reflect to compact form of DNA in a virus particle. The similarly between the spikes of bacteriophage phi X174 and those of cytoplasmic-polyhedrosis virus from silk worm was also discussed.

Demonstration of stepwise coiling of nucleoprotein in adenovirus core by application of critical point drying.

Mild destruction of a virus particle to observe the organized structure of the nucleoprotein complex in a virion was achieved by application of the critical point drying method. Adenovirus type 12 (ad12) virions have been treated by this method after the particles had been fixed with glutaraldehyde on an electron microscope grid. With 15 min prefixation, the capsids (shells) and the cores were in various stages of unfolding. The core was unfolded in the filamentous structure. The thickness of these filaments was 6.7, 13.3, +23 nm, or more. Some pictures showed that the thicker filaments consisted of super-coiling of two thinner filaments, for example two 6.7-nm filaments coiled up to give the 13.3-nm filaments. This suggests that the nucleoprotein complex of a circular double-stranded DNA and inner proteins of ad12 virus was folded in a stepwise fashion to produce the compacted form of the core.

Intracellular vesicular stomatitis virus nucleocapsids and virions visualized by surface spreading.

Spreading of cells on a solution surface could visualize vesicular stomatitis virus nucleocapsids and virions in infected cells easily and clearly without the need for any purification. Characteristic structures observed by the spreading of the infected cells are described and discussed.

A new method for extracting circular and supercoiled genome segments from cytoplasmic polyhedrosis virus.

The genome of a cytoplasmic polyhedrosis virus (CPV) consisting of 10 segments was extracted by a new two-step extraction method. The results from polyacrylamide gel electrophoresis, phenol and diepoxybutane treatment and electron microscopic examination indicated the extracted genome segments to have circular and supercoiled structures as genome-protein(s) complexes. Multiple cycles of the transcription of each segmented genome of CPV may take place on the circular structure of the segment.

Transcriptional regulatory region of Brn-2 required for its expression in developing olfactory epithelial cells.

Brain-2 is a class III POU transcription factor expressed in developing nervous system. In this study, we have examined the transcriptional regulatory region of Brn-2. Expression of Brn-2 is activated when P19 embryonal carcinoma cells are induced to differentiate into neural cells with retinoic acid (RA). In P19 cells, the 0.5 kb upstream region of Brn-2 was sufficient for the transcriptional activation during RA-induced differentiation. Deletion analysis of the 0.5 kb region located a proximal enhancer (between -422 and -379 with respect to the translational initiation codon), which was essential for the activation. By gel shift assay and methylation interference assay, a specific binding factor was detected that recognized a core sequence GAGCCAAT found within the proximal enhancer. To examine whether the 0.5 kb upstream region can function in embryos, transgenic mice were generated that contained LacZ gene driven by the 0.5 kb upstream region. In these transgenic mice, LacZ was expressed in developing olfactory epithelial cells between embryonic day 12.5 and 14.5. Immunostaining with an anti-Brain-2 antibody demonstrated the expression of Brain-2 in the olfactory epithelium (most likely olfactory receptor neurons) at similar developmental stages. These results suggest that the 0.5 kb upstream region of Brn-2 is sufficient for the expression in the developing olfactory cells and that the DNA binding factor recognizing the proximal enhancer may be involved in the olfactory cell specific expression.

Expression of brain-2 in the developing olfactory bulb.

The expression of Brain-2, a POU domain transcription factor, was examined in the developing olfactory bulb. Brain-2 was expressed mainly in the output neurons, mitral cell and tufted cells in the main olfactory bulb (MOB), and mitral/tufted cells (MT cells) in the accessory olfactory bulb (AOB). It was not expressed in granular cells in either the MOB or the AOB. Our results suggest that Brain-2 was specifically expressed in output neurons but not in interneurons in the developing olfactory bulb. Brain-2 may play a role in the development of these output neurons.

High-level expression of porcine muscle adenylate kinase in Escherichia coli: effects of the copy number of the gene and the translational initiation signals.

Porcine muscle adenylate kinase (ADK) was overproduced in Escherichia coli using the expression plasmid with double A-T-G codon at the translational starting site and the Shine-Dalgarno (SD) sequence 10 bp apart from the first A-T-G. We used the expression vectors pKK223-3 and pMK2. pMK2 is about 10-20 times larger in copy number than pK223-3. For both vectors, duplication of A-T-G was effective and the quantity of the expressed ADK from the double A-T-G plasmid was 2 approximately 4-fold more than that achieved when only one A-T-G was present. The amount of the produced ADK was maximum in the case of using pMK2 with double A-T-G. The overproduced ADK formed inclusion bodies in E. coli. It was solubilized in 6 M guanidine hydrochloride and refolded. Through two steps of column chromatography, ADK was purified. It has the same amino acid composition and grossly the same activity as that reported by Schirmer et al. (1970). Its amino acid sequence of the NH2-terminal region was identical with that deduced from the cDNA sequence including the NH2-terminal methionine.

Caffeic acid oligomers in Lithospermum erythrorhizon cell suspension cultures.

Lithospermum erythrorhizon cells cultured in pigment production (M-9) medium produced lithospermic acid B, a dimerized caffeic acid ester derivative, in quantities similar to the production of shikonin. The cells also produced a related dimer, (+)-rabdosiin. In Linsmaier-Skoog liquid medium, which suppresses shikonin production, both lithospermic acid B and (+)-rabdosiin were still formed. Lithospermic acid, a caffeic acid-rosmarinic acid conjugate, was isolated as a main constituent in Lithospermum hairy root cultures. In the aerial parts of L. erythrorhizon, the content of these phenylpropanoid oligomers was relatively low compared to that of rosmarinic acid.

Galloylglucoses and riccionidin A in Rhus javanica adventitious root cultures.

Adventitious root cultures of Rhus javanica L. produced large amounts of galloylglucoses (gallotannins) and an anthocyanidin, riccionidin A, formerly found only in liverworts. Production of both galloylglucoses and riccionidin A in the adventitious root culture system was suppressed by light. The Rhus root culture showed the highest productivity for those secondary metabolites in a modified Linsmaier-Skoog (LS) liquid medium containing 30 mM NH4+ and 30 mM NO3- as nitrogen sources in the presence of 10(-6) M 3-indoleacetic acid (IAA).

Production of macrocyclic ellagitannin oligomers by Oenothera laciniata callus cultures.

Callus cultures of Oenothera laciniata grown on LS agar medium supplemented with IAA and kinetin produced large amounts of the macrocyclic ellagitannin dimer, oenothein B, and a trimer, oenothein A, accompanied with related monomeric hydrolysable tannins. The content of the main compound oenothein B (65 mg/g dry wt) in calli cultured on modified LS medium containing 10 mM NH4+ and 5 mM NO3- was nearly two times higher than that in intact leaves.

Possible participation of a prostaglandin E1 analogue in the aggravation of diabetic nephropathy.

A relation between the progression of diabetic nephropathy and glomerular hyperfiltration has been speculated. We describe two cases of non-insulin-dependent diabetic males aged 55 and 59 years in whom diabetic nephropathy was aggravated during the administration of limaprost, a a prostaglandin E1 analogue with a vasodilatory action. We also observed a short-term effect of limaprost on renal hemodynamics in three cases with diabetic nephropathy. In case 1, one year after limaprost administration the serum albumin level fell from 3.6 to 2.6 g/dl and the serum creatinine level rose from 1.0 to 1.6 mg/dl. In case 2, 9 months after limaprost administration the serum albumin level fell from 3.6 to 2.9 g/dl and the serum creatinine level rose from 1.8 to 2.3 mg/dl. In the latter stages of limaprost administration, the downslopes of reciprocal serum creatinine against time appeared to be augmented in the two cases. After the 3-day administration of limaprost, the peripheral and renal blood flows, and the glomerular filtration rate (GFR) were observed to rise, but the filtration fraction (FF) and urinary protein output were elevated. Keeping in mind the pre-existing renal damage, the increases in GFR and FF suggested acceleration of compensatory glomerular hyperfiltration in less damaged surviving glomeruli. The sustained acceleration of hyperfiltration with long-term administration of limaprost as an exogenous vasodilatory prostaglandin was assumed to eventuate in the aggravation of diabetic nephropathy. Attention should be paid to drugs which increase GFR in patients with established diabetic nephropathy.

Stable transformation of Lithospermum erythrorhizon by Agrobacterium rhizogenes and shikonin production of the transformants.

Seedling hypocotyls of Lithospermum erythrorhizon were infected with Agrobacterium rhizogenes (strain 15834) harboring a binary vector with an intron-bearing the ß-glucuronidase (GUS) gene driven by cauliflower mosaic virus (CaMV) 35S promoter as well as the hygromycin phosphotransferase (HPT) gene as the selection marker. About 20% of the hairy roots isolated were hygromycin resistant and had co-integrated GUS and HPT genes in their Lithospermum genomic DNA. Because GUS activity was detected in almost all the hygromycin-resistant root tissues, the CaMV 35S promoter seems to be ubiquitously active in L. erythrorhizon hairy roots. In pigment production medium M9, the hairy root cultures had shikonin productivity similar to that of cell suspension cultures of Lithospermum. They also showed light-dependent inhibition of shikonin biosynthesis similar to that of Lithospermum cell cultures. These findings suggest that this hairy root system transformable with A. rhizogenes is a suitable model system for molecular characterization of shikonin biosynthesis via reverse genetics.

Shikonin derivative formation on the stem of cultured shoots in Lithospermum erythrorhizon.

LITHOSPERMUM ERYTHRORHIZON: , which are capable of producing red pigments, have been established. The red pigments were formed on the stems of L. erythrorhizon shoots cultured both on solid and in liquid media without phytohormones at 25 °C in the dark. Thin-layer chromatography, high-performance liquid chromatography and 1 H nuclear magnetic resonance analyses revealed that the red pigments which accumulated on the cultured shoots were shikonin derivatives. The effects of various basal media and phytohormones (indole-3-acetic acid, indole-3-butyric acid and kinetin) on the growth and the formation of shikonin derivatives were investigated. When the shoots were cultured on Murashige and Skoog solid medium, the addition of kinetin remarkably enhanced shikonin derivative accumulation in the shoots. However, these effects of kinetin were not observed in the liquid culture when cultured in Gamborg B5 medium. The maximum content of shikonin derivatives (2.3% as dry weight, ca. 1.5 mg/100 ml flask) was observed in the shoots cultured in phytohormone-free B5 liquid medium for 5 weeks.