Further studies on D-3-aminoisobutyrate-pyruvate aminotransferase

D-3-Aminoisobutyrate-pyruvate aminotransferase (EC 2.6.1.40, D-BAIB aminotransferase) participates in the metabolism of thymine. Recently we purified this enzyme from rat liver. We have studied D-BAIB aminotransferase further to clarify its physiological function. Among our findings were the following. (1) The enzyme activity was widely distributed in the organs of guinea pigs and rats. The kidney, liver, and lung showed high specific activities. (2) Using the livers of six vertebrates, differences between species were studied. Activity was detected in all species, the human liver showing the lowest activity among them. (3) Developmental study using rat liver showed that the activity was low at birth, increased sharply thereafter for 10 days, and then subsequently declined to the adult level. (4) Intraperitoneal injection of BAIB and beta-alanine in rats was performed to determine whether they induce activity of this aminotransferase. Only BAIB increased the activity of the aminotransferase in the liver significantly. (5) Subcellular distribution study of this aminotransferase in rat liver revealed that it is a mitochondrial enzyme.     

Tissue-specific regulation of a chicken delta-crystallin gene in mouse cells: involvement of the 5' end region.

A cloned delta-crystallin gene of the chicken is preferentially expressed in lens cells after introduction into various mouse tissues. The level of expression in the lens epithelium is 20 times higher than in fibroblasts. Taking advantage of this system, we attempted to define regulatory regions of the delta-crystallin gene using a variety of deletion and substitution mutants. The results indicate that tissue-specific regulation of the delta-crystallin gene is mediated by the 5' end region of the gene; sequences upstream from -93 are not required for expression and sequences downstream from +58 are not involved in tissue specificity. The high expression in lens cells requires 5' flanking sequences of 80-bp long from the cap site, whereas the low expression in fibroblasts requires an additional 12 bp upstream sequence. Expression of both types is lost in a mutant with only 51 bp of the 5' flanking sequence. Thus, fine deletion analysis demonstrated that expression in lens cells and expression in fibroblasts are distinct not only in level but in regulation.     

Assignment of Human Membrane-type Matrix Metalloproteinase-2 (MT2-MMP) Gene to 16q12 by FISH and PCR-based Human/Rodent Cell Hybrid Mapping Panel Analysis

A new group of matrixmetalloproteinase with a potential membranedomain was reported as membranetype matrixmetalloproteinases(MT-MMPs), and the gene coding for one of them, MT2-MMP, wasrecently cloned from a human lung cDNA library. To predict itsphysiological functions by the relation to the genetic disordersmapped on the chromosomes, the chromosomal location of the humanMT2-MMP gene was examined by fluorescence in situ hybridization(FISH) and PCR-based analysis with human/rodent hybrid cellmapping panels, and it was assigned to 16q12.     

High-Speed Video-Oculography for Measuring Three-Dimensional Rotation Vectors of Eye Movements in Mice

Background

The mouse is the most commonly used animal model in biomedical research because of recent advances in molecular genetic techniques. Studies related to eye movement in mice are common in fields such as ophthalmology relating to vision, neuro-otology relating to the vestibulo-ocular reflex (VOR), neurology relating to the cerebellum’s role in movement, and psychology relating to attention. Recording eye movements in mice, however, is technically difficult.

Methods

We developed a new algorithm for analyzing the three-dimensional (3D) rotation vector of eye movement in mice using high-speed video-oculography (VOG). The algorithm made it possible to analyze the gain and phase of VOR using the eye’s angular velocity around the axis of eye rotation.

Results

When mice were rotated at 0.5 Hz and 2.5 Hz around the earth’s vertical axis with their heads in a 30° nose-down position, the vertical components of their left eye movements were in phase with the horizontal components. The VOR gain was 0.42 at 0.5 Hz and 0.74 at 2.5 Hz, and the phase lead of the eye movement against the turntable was 16.1° at 0.5 Hz and 4.88° at 2.5 Hz.

Conclusions

To the best of our knowledge, this is the first report of this algorithm being used to calculate a 3D rotation vector of eye movement in mice using high-speed VOG. We developed a technique for analyzing the 3D rotation vector of eye movements in mice with a high-speed infrared CCD camera. We concluded that the technique is suitable for analyzing eye movements in mice. We also include a C++ source code that can calculate the 3D rotation vectors of the eye position from two-dimensional coordinates of the pupil and the iris freckle in the image to this article.     

Rapid and transient decrease of N-myc expression in retinoic acid-induced differentiation of OTF9 teratocarcinoma stem cells.

The level of expression of N-myc in mouse teratocarcinoma stem cells is very high. Previous studies have shown that N-myc expression significantly decreases when the stem cells are subjected to long-term induction for differentiation by retinoic acid (RA). We found that in a stem cell line, OTF9, a steep yet transient decrease of N-myc expression takes place much earlier, immediately after induction by RA. To examine whether this decrease is responsible for differentiation, we constructed a gene, miwNmyc, to express N-myc cDNA constitutively and transformed OTF9 cells with this gene construct. Transformants under the constitutive expression of miwNmyc differentiated normally, as judged by morphological changes and by modulation of c-myc, Hox1.1, and laminin B1 expression. Therefore, transient decrease of N-myc expression may be the consequence of RA-induced differentiation, even though it occurs very early in the process. Alternatively, in addition to N-myc decrease, there may be redundant mechanisms which lead to OTF9 differentiation after induction by RA, so that suppression of N-myc decrease is bypassed by at least one other mechanism.     

Electron microscopic observations on synapse-like contacts between pituicytes and different types of nerve fibers in the anuran pars nervosa

Summary Pituicytes of Rana pipiens could be classified into two types, pale and dense, according to their relative densities of cytoplasm and the populations of free ribosomes and cell organelles. An intermediate type of pituicyte was also recognized.Lipid droplet such as are typical in the cytoplasm of mammalian pituicytes, are not in the cytoplasm of either types of frog pituicyte. Both types have long cytoplasmic processes which run among the nerve fibers, and some of them end at the pericapillary space.Nerve endings making synapse-like contacts with the cell bodies or the processes of the pituicyte are frequent. According to the structures and sizes of granules and vesicles in the nerve endings, these endings are classified into one of three types: 1) A, which appears to be a peptidergic neuronal ending containing dense granules 1,200–2,000 Å in diameter and small clear vesicles 300–400 Å in diameter; 2) B, which appear to be monoaminergic endings containing cored vesicles 600–1,000 Å in diameter and small clear vesicles 300–500 Å in diameter; 3) C, which appear to be cholinergic endings containing only small clear vesicles. Type C endings are relatively rare. In the synaptic area the axonal membranes appose those of the pituicytes across a gap of about 200 Å and numerous presynaptic vesicles are clustered or accumulated near the presynaptic membranes.The author wish to express his hearty thanks Professor Dr. A. Gorbman, Zoology Department, University of Washington, Seattle, U.S.A. and Professor Dr. H. Fujita for their helpful advices and criticisms. The frog tissues were obtained and fixed in Professor A. Gorbman's laboratory supported by U.S.P.H.S. grant NS 04887.     

Neuroanatomy of the terminal (sixth abdominal) ganglion of the crayfish,Procambarus clarkii (Girard)

Summary We studied the neuroanatomy of the terminal (sixth abdominal) ganglion in the crayfish Procambarus clarkii with silver-impregnated sections and nickel fills. We describe the fiber tracts, commissures and neuropilar areas, and give the topological relationships of motoneurons and intersegmental interneurons with reference to their neuropilar landmark structures.All five anterior abdominal ganglia have an almost identical number of 600–700 neurons with a similar pattern of distribution. Each contains a single neuromere with a common plan of neuropil organization. In contrast, the terminal ganglion consists of two neuromeres which appear to be derived from the intrinsic sixth abdominal and telson ganglion. The basic organization of each neuromere parallels that of the third abdominal ganglion in the appearance and arrangement of fiber tracts and commissures, although some modifications occur. The fusion of two neuromeres is represented by the duplication of segmentally homologous neurons, MoGs and LGs, whose topological relationships to the neuropil structures are similar to those of the anterior ganglion.We also discuss the origin of the telson and its ganglion (the seventh abdominal neuromere), and dispute the classical theory that the telson derives from a postsegmental region.     

Extraadrenal expression of steroid 21-hydroxylase and 11β-hydroxylase by a benign testicular leydig cell tumor

We present an unusual case with bilateral testicular Leydig cell tumors displaying extraadrenal expression of steroid 21-hydroxylase and 11β-hydroxylase. Histological examination of a 38-yr-old man infertile due to azoospermia showed him to have bilateral testicular Leydig cell tumors. The in vitro steroidogenic potential of the tumors and their adjacent testicular tissue was evaluated using organ culture. Tumor tissue was found to secrete deoxycorticosterone (DOC), corticosterone (B) and cortisol, which are not produced in normal adult testis, into the medium, while testicular tissue adjacent to the tumors secreted a small amount of DOC and B. Northern blot analysis with cytochrome P-450_C21 complementary DNA (cDNA) and P-450_11β cDNA as probes revealed that the tumor contained a considerable amount of mRNA for P-450_C21 and P-450_11β, while the mRNAs were not detected in the testicular tissues adjacent to the tumors. It is suggested that the high local levels of estrogen and/or progesterone within the Leydig cell tumors and their adjacent testicular tissues induced extraadrenal expression of steroid 21-hydroxylase and 11β-hydroxylase by the tumors and their adjacent testicular tissues.     

Metallothionein in kidney and liver of the macular mouse as an animal model of Menkes' kinky hair disease

The tissue copper and metallothionein-Cu (MT-Cu) content of kidney and liver were measured in mutant (hemizygous macular male and homozygous macular female), heterozygous macular female and normal mouse. The tissue copper and MT-Cu contents in kidneys from 7-8 day mutants and heterozygotes were significantly greater than those of the normal kidney. Marked elevations in kidney copper and MT-Cu contents were also observed in the 8-9 week mutant (which achieved long-term survival with a single dose of subcutaneous copper administered at day 7) and in the heterozygote. The L-[35S]cystine incorporation experiments also revealed an abnormal synthesis of renal MT in the 8-9 week mutant and in the heterozygote. In contrast to kidney Cu levels, the tissue copper and MT-Cu contents of 7-8 day normal livers were extremely high, whereas the tissue copper and MT-Cu contents of mutant and heterozygote livers were extremely low. The tissue copper contents of livers of 8-9 week mutants and heterozygotes were slightly low compared to normal, and the MT-Cu contents of livers of the 8-9 week mouse were extremely low in all groups. In contrast to the changes in copper content, the changes in tissue zinc and MT-Zn contents in kidney and liver were slight in the 7-8 day and 8-9 week mouse.