A single amino acid substitution within the mature sequence of ornithine aminotransferase obstructs mitochondrial entry of the precursor.

We describe here evidence of congenital enzyme mistargeting induced not by abnormalities in the signal sequence. We examined the molecular mechanism of hereditary ornithine aminotransferase (OAT) deficiency causing gyrate atrophy of the choroid and retina (GACR). Nucleotide sequencing of OAT cDNA generated from a GACR patient's mRNA revealed a single base change from C to G at position 268, resulting in an amino acid substitution of neutral Gln(CAA) with negatively charged Glu(GAA) at position 90 (Q90E). Immunohistochemical and transient expression analyses suggested expression of a defective labile OAT in the patient's tissues. However, high-level expression and immunocytochemical analyses elucidated that Q90E OAT (the patient's OAT) was localized within the limits of cytoplasmic free ribosomes in precursor form without any mitochondrial entry, indicating that the patient's precursor OAT was synthesized and rapidly degraded because of accumulation in the cytosol. It is interesting that, although the mutation site (Q90E) in this GACR patient's OAT was within the coding sequence of the mature protein, the precursor exhibited loss of mitochondrial targeting function. These findings suggest that not only the signal sequence but a critical part of the mature sequence plays an essential role in mitochondrial entry of the OAT precursor protein.     

In vivo and in vitro studies on the appearance of LHRH neurons in the hypothalamus of perinatal rats

Summary Ontogenetic development of LHRH-containing neurons was studied by fluorescence and enzyme immunohistochemistry in rats. In in vitro studies, the tissues of the septal-chiasmatic and mediobasal hypothalamic areas of fetal rats on day 16.5 or 18.5 of gestation were trypsinized separately for dissociation of the neural cells, and cultured for several days. Immunopositive reaction against LHRH was first detected in nerve cells derived from both areas of the hypothalamus of the fetuses on days 16.5 and 18.5 of gestation, after 8 and 6 days culture, respectively. The cells were small, and seemed to be bipolar in morphology indicating an axon and arborized dendrites. Immunopositive material occurred in the cell soma as well as in the cellular processes. In in vivo studies, immunopositive material, possibly deposited in nerve fibers, appeared first in OVLT and simultaneously in the external layer of the median eminence of fetuses on day 20.5 of gestation. The immunoreactive fibers increased in number in both parts with development, especially after birth in the median eminence. No immunopositive material was detected within any neural cell bodies nor in the cytoplasm of any ependymal cells.This work was financed by the Ministry of Education, Japan. No. 257008. We would like to thank Dr. Katsuhiko Saito (Department of Surgery, Tokushima University) for his kind advice on the preparation of the antibody used for the immunofluorescence study.     

Production of L-aspartic acid from fumaric acid by a fumaric acid-assimilating obligate thermophile,Bacillus stearothermophilus KP 1041

Summary A fumaric acid-assimilating obligate thermophile having a high aspartase activity was isolated from soil. The isolate (KP 1041) that grew at 45 to 68 °C was assigned to a strain of Bacillus stearothermophilus. The cell suspensions produced L-aspartate from fumarate and ammonium ion, with the rapidest initial rate at 65 °C and pH 9.5. The Michaelis constant for fumarate was 0.2 M. The cellular aspartase was relatively stable for 18 h at and below 50 °C over a pH range 6.7–8.3 in the presence of ammonium fumarate; this substance protected the enzyme from heat inactivation. The best yield in L-aspartic acid production was achieved at 6 h incubation at 53 °C and pH 8.5, using 0.88 M fumarate, 3.1 M ammonium ion, and the cells at 53 mg dry weight per ml. In this case, 85% of fumarate added was converted into aspartic acid. The structure of the product was determined from its infrared spectrum, specific rotation, melting point and ultimate analysis.Presented at the Annual Meeting of the Agricultural Chemical Society of Japan, Yokohama, April 2, 1977     

Nerve Growth Factor-Induced Changes in the Structure of Sulfated Proteoglycans in PC 12 Pheochromocytoma Cells

Structural changes in proteoglycans (PGs) were examined during the neuritogenesis of PC12 cells induced by nerve growth factor (NGF). (1) A heparan sulfate (HS) PG and a chondroitin sulfate (CS) PG were synthesized by PC12 cells, irrespective of the presence of NGF or the duration of culture. PGs released from PC12 cells into the culture medium were mostly CSPGs. (2) In the absence of NGF, the apparent molecular mass of HSPG prepared from PC12 cells after 3 days of culture was in the range of 90-190 kDa for the intact form (Kav = 0.38 on Sepharose CL-6B), 12 kDa for HS, and 61 kDa for the core protein. In the presence of NGF, these values were 90-190 kDa, 10 kDa, and 51 kDa and 61 kDa, respectively. The intact forms of cell-associated CSPG had apparent molecular mass ranges of 120-150 kDa and 120-190 kDa (Kav = 0.38 and 0.34), with CSs of 15 kDa and 20 kDa in the presence and absence of NGF, respectively. The apparent molecular mass of the core protein of cell-associated CSPG was 92 kDa, irrespective of the presence of NGF. The molecular sizes of cell-associated PGs and their glycosaminoglycans remained unchanged during culture. (3) CSPGs released by PC12 cells into the culture medium were separated into two peaks (I and II) by column chromatography on DEAE-cellulose. The peak II fraction prepared from the medium with NGF after 3 days of culture consisted of CSPG with Kav = 0.22 on Sephacryl S-300 [40-84 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)].(ABSTRACT TRUNCATED AT 250 WORDS)     

Improved culture conditions for somatic embryogenesis from Asparagus officinalis L. using an aseptic ventilative filter

Summary Calli were induced from the crown of seedlings or lateral bud of young spears of Asparagus officinalis L. on Linsmaier and Skoog's (LS) solid-medium supplemented with 5 M 2,4-dichlorophenoxyacetic acid (2,4-D). Embryogenic callus was selected from induced calli and proliferated in LS liquid medium supplemented with 5 M 2,4-D. Non-vitrified somatic embryos were formed and efficiently developed into club-shaped embryos in LS hormone-free medium with 1 % gelrite in a culture vessel capped with an aseptic ventilative filter. Non-vitrified club-shaped embryos developed into normal plants when transferred to half-strength LS medium without hormones, and 0.8 % agar. Carbon dioxide concentration and moisture content inside the culture vessels were measured, and their effect on embryo development is discussed.     

Linear and circular dichroism of membranes from Rhodopseudomonas capsulata

Absorption, linear dichroism and circular dichroism spectra of Rhodopseudomonas capsulata (wild-type-St. Louis strain, mutant Y5 and mutant Ala⁺) are particularly sensitive to the nature of the light-harvesting bacteriochlorophyll-carotenoid-protein complexes. Evidence for exciton-type interactions is seen near 855 nm in the membranes from the wild-type and from mutant Y5, as well as in an isolated B-800 + 850 light-harvesting complex from mutant Y5. The strong circular dichroism that reflects these interactions is attenuated more than 10-fold in membranes from the Ala⁺ mutant, which lacks both B-800 + 850 and colored carotenoids and contains only the B-875 light-harvesting complex. These results lead to the conclusion that these two light-harvesting complexes have significantly different chromophore arrangements or local environments.     

Gas chromatographic method for the determination of urinary acetylpolyamines

A gas chromatographic method was developed for the determination of monoacetylputrescine, monoacetylcadaverine, N¹-acetylspermidine and N⁵-acetylspermidine in human urine. The amines were isolated from urine by silica gel column chromatography. 1, 10-Diaminodecane was used as internal standard. The amines were reacted with ethyl chloroformate in aqueous medium to four ethyloxycarbonyl derivatives prior to application to gas chromatography using a flame ionization detector. Separation and determination of the derivatives were carried out on a Uniport HP column (1.0 m) impregnated with 0.5% SP-1000 under temperature-programmed conditions. The monoacetylpolyamines could be measured accurately at the nanomole level. The method was used for the determination of the monoacetylpolyamines in urine of healthy volunteers. The values obtained were in the range of the published data.