Ultrastructure of neurofibrillary tangles in Alzheimer's disease

The ultrastructure of neurofibrillary tangles (NFT) was examined by electron microscopy. The fibrils of NFT seemed to consists of about eight protofilaments consisting of globular subunits; these protofilaments were helically wound in a longitudinal direction. The fibrils of NFT had hollow structures at their centers surrounded by the eight globular subunits. The subunits were tightly connected in the narrow parts of the fibril, but more loosely connected in the wider parts. From these findings, it seemed that the fibrils of NFT consist of a twisted tubule having periodical constrictions and is made up of eight helically wound protofilaments, forming globular subunits.     

Nucleotide sequence of the glutamine synthetase gene (glnA) and its upstream region from Bacillus cereus

We have determined the complete nucleotide sequence of a 2.4 kb chromosomal EcoT22I-NspV fragment, containing the Bacillus cereus glnA gene (structural gene of glutamine synthetase). The deduced amino acid sequence indicates that the glutamine synthetase subunit consists of 444 amino acid residues (50,063 Da). Comparisons are made with reported amino acid sequences of glutamine synthetases from other bacteria. Upstrem of glnA we found an open reading frame of 129 codons (ORF129) preceded by the consensus sequence for a typical promoter. Maxicell experiments showed two polypeptide bands, with molecular weights in good agreement with that of glutamine synthetase and that of ORF129, in addition to vector-coded protein. It is possible that the product of this open reading frame upstream of glnA has a regulatory role in glutamine synthetase expression.     

New methods to investigate ATP requirement for pre-mRNA splicing: inhibition by hexokinase/glucose or an ATP-binding site blocker

Addition of yeast hexokinase and glucose at various time points of the pre-mRNA splicing reaction rapidly depleted ATP and inhibited further progress of the reaction, indicating that ATP is required for both the first and second steps of splicing. ATP analogues, p-fluorosulfonylbenzoyl-5'-adenosine (FSBA) and 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl), which can modify amino acids at the ATP-binding site of a protein, inactivated the splicing activity of the nuclear extract. While the inactivation by the former was irreversible, the splicing activity was complemented by a Micrococcal nuclease-treated extract. This ATP analogue (FSBA) may be a useful tool for identification of ATP-dependent splicing factors.     

Alternative splicing mechanism in a cytochrome P-450 (P-450PB-1) gene generates the two mRNAs coding for proteins of different functions

Two mRNAs for P-450PB-1 and P-450PB-1(ps) are about 2 kilobase pairs long and have identical sequences with each other except for one short region of high variability (Kimura, H., Yoshioka, H., Sogawa, K., Sakai, Y., and Fujii-Kuriyama, Y. (1988) J. Biol. Chem. 263, 701-707). To clarify the origin of the short replacement block between the two mRNAs, we isolated several genomic clones containing relevant gene sequences. Sequence analysis of these genomic clones revealed that the two short segments specific for the two mRNAs are tandemly arranged in a genomic sequence and form exonic sequences equipped with AG and GT sequences on their 5' and 3' ends, respectively, and the putative consensus sequences for the lariat formation. The two short sequences lie between the two exonic sequences coding for the common part of the two mRNAs. Taken together with the structure of the related P-450(M-1) gene (Morishima, N., Yoshioka, H., Higashi, Y., Sogawa, K., and Fujii-Kuriyama, Y. (1987) Biochemistry 26, 8279-8285), all these results clearly demonstrate that the two mRNAs are generated from a single gene by alternative splicing at the eighth exons. The synthesis of the two mRNAs is regulated temporally in livers of male and female rats and brains of the female animals. One of the two mRNAs codes for a monooxygenase of P-450PB-1, and the other (P-450PB-1(ps) mRNA) lacks the sequence coding for the heme-binding site conserved among all species of P-450 molecules, and, therefore, it cannot function as a monooxygenase. The immunoblot analysis using an antibody specific for the 15-mer peptide uniquely encoded by P-450PB-1(ps) mRNA shows that the P-450PB-1(ps) peptide is synthesized at least in rat livers of both sexes in temporally regulated manners and is bound to the microsomal membranes. The function of this peptide remains to be seen.     

Particle size of airborne mouse crude and defined allergens

Laboratory animal allergy is a serious occupational diseases of many workers and scientists engaged in animal experimentation. Control measures depend upon characterization of allergens including airborne particles. This study measured the particle size of crude mouse urine and pelt aeroallergens generated in mouse housing rooms and compared them with mouse serum albumin, a defined major allergen. Allergens were detected by specific immunological methods. Most crude and defined allergens (74.5-86.4%) concentrated on a filter with a retention size greater than 7 microns. In distrubed air, allergen concentration increased 1.4 (albumin) to 5 (crude) fold and the proportion of small particles increased from 1.4% in calm air to 4.5% in distrubed air. This information on the generation and size distribution of aeroallergens will be important in the development of effective counter measures.     

Retinoic acid ambivalently regulates the expression of MyoD1 in the myogenic cells in the limb buds of the early developmental stages.

The expression of MyoD1 in myogenic cells located in the muscle prospective region of the limb bud at stage 20-22 was highly sensitive to retinoic acid. Unlike RAR-beta, the expression of MyoD1 mRNA in the muscle precursor cells was significantly increased by retinoic acid at lower concentrations (0.1-10 nM), but inhibited by it at higher concentrations (0.1-1 microM). The ambivalent modulation of MyoD1 expression suggested that MyoD1 expression is regulated by not only the retinoic acid receptor and its response element, but also by other factors. Retinoic acid may be involved in the differentiation of the myogenic cells during early development.     

Purification and partial characterization of two types of growth-inhibitory protein latently present in rabbit serum.

Normal rabbit serum contained two kinds of growth-inhibitory protein, GI-I and GI-II, in latent forms. These latent inhibitors were activated by incubation at 37 degrees C for 12 h, and their activation was lowered by inhibitors for serine, cysteine and metalloproteinases. Both growth inhibitors were highly purified in active forms by successive column chromatographies. GI-I showed a major protein band with an Mr of 18,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, while GI-II showed a major protein band with an Mr of 36,000. GI-I and GI-II half-inhibited the growth of rat tumorigenic cell line (RSV-BRL) at concentrations of 0.5 ng/ml and 10 ng/ml, excess concentrations. Of the 15 cell lines tested, GI-I specifically inhibited the growth of rodent and lagomorph cells, whereas GI-II nonspecifically inhibited the growth of all cell lines tested. Specificities for cell type and malignancy were not observed with either inhibitor. These growth inhibitors were stable to a reducing reagent and proteinase inhibitors, but labile to urea, acid, organic solvents, trypsin, plasmin and heating at 95 degrees C for 5 min. These properties suggested that both growth inhibitors might be distinct from known growth-inhibitory factors.