The 15 N-terminal amino acids of hexokinase II are not required for in vivo function: analysis of a truncated form of hexokinase II in Saccharomyces cerevisiae

The function of the N-terminal amino acids of Saccharomyces cerevisiae hexokinase II was studied in vivo using strains producing a form of hexokinase II lacking its first 15 amino acids (short form). This short form of hexokinase II was produced from a fusion between the promoter region of the PGK1 gene and the HXK2 coding sequence except the first 15 codons. As expected, the in vitro analysis of the short form protein by gel filtration chromatography indicates that the short protein does not form dimers under conditions where the wild-type protein dimerizes. Kinetic studies show that the enzymatic activities are very similar to wild-type behavior. The physiological experiments performed on the strains containing the fusion allele demonstrate that the short form of the enzyme is similar to the wild-type both in terms of phosphorylation of hexoses and glucose repression. We conclude that the N-terminal amino acids of hexokinase II are not required in vivo either for phosphorylation of hexoses or for glucose repression.     

Immunoelectron microscopy of tissues processed by rapid freezing and freeze-substitution fixation without chemical fixatives: application to catalase in rat liver hepatocytes

We report on the immunohistochemical demonstration of an enzyme at the electron microscopic level using specimens processed by rapid freezing and the freeze-substitution technique without the use of any chemical fixatives. Fresh rat liver tissue blocks were rapidly frozen by the metal contact method using liquid nitrogen, and were freeze-substituted with acetone without any chemical fixatives at -80 degrees C. Some of the freeze-substituted tissues were embedded in Lowicryl K4M at -20 degrees C; the others were returned to room temperature and embedded in Epok 812 at 60 degrees C. Ultra-thin sections were stained using anti-peroxisomal catalase antibody by the protein A-gold technique. The ultrastructure of the hepatocytes was very well preserved compared with that of conventionally processed tissues. The labeling for catalase was confined to peroxisomes. When the labeling density was compared among freeze-substituted tissues and conventionally processed tissues, that of freeze-substituted and Lowicryl K4M-embedded tissues was the most intense. These results show the usefulness of freeze-substituted tissues for immunohistochemical analysis of cell organelles.     

α1-Adrenergic Receptor Mediates Arachidonic Acid Release in Spinal Cord Neurons Independent of Inositol Phospholipid Turnover

The alpha 1-adrenergic receptor has been shown to mediate the release of arachidonic acid in FRTL5 thyroid cells and MDCK kidney cells. In primary cultures of spinal cord cells, norepinephrine stimulated release of arachidonic acid (from neurons only) and turnover of inositol phospholipids (from neurons and glia) via alpha 1-adrenergic receptors. These two responses were dissociated by treatment with phorbol ester and pertussis toxin, which inhibited production of inositol phosphates with no appreciable effect on release of arachidonic acid. Extracellular calcium was required for release of arachidonic acid, but not for production of inositol phosphates. The calcium channel blockers nifedipine and verapamil inhibited release of arachidonic acid only. However, 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8), a compound that blocks intracellular calcium release, diminished production of inositol phosphates, but had little effect on release of arachidonic acid. These results suggest that alpha 1-adrenergic receptors couple to release of arachidonic acid in primary cultures of spinal cord cells by a mechanism independent of activation of phospholipase C, possibly via the activation of phospholipase A2.     

Three separate mitochondrial DNA sequences are contiguous in human genomic DNA

We isolated from a HeLa genomic library 38 plaques that hybridized to total mitochondrial (mt) DNA isolated from human placenta. One clone (HLmt-17.8) hybridized to a 740 base-pair (12 S ribosomal RNA gene and displacement loop) mtDNA probe and was characterized in more detail. Within its 17.8 x 10(3) base-pair insert a 1.6 x 10(3) base-pair mtDNA fragment was similar to three non-sequential coding genes of human mtDNA, including a part of the 12 S ribosomal RNA (684-971), the cytochrome oxidase I (6553-7302), and two NADH dehydrogenase [ND4L/ND4] (10,606-11,159). The similarity to human mtDNA sequences was 92.0%, 92.3% and 92.4%, respectively, the highest degree of similarity to human mtDNA so far reported. This is also the first report of several adjacent mtDNA-like sequences in cellular chromosomes. The mtDNA-like sequences in HLmt-17.8 was found in the DNAs of human placenta, freshly isolated human leukocytes, foreskin and several human cell lines; but it was not present in other primates or lower organisms. The HLmt-17.8 mtDNA-like region appears to be a pseudogene that transferred into the nucleus in humans more recently than nine million years ago.     

伤害性刺激和非伤害性刺激对大鼠丘脑后核神经元电活动的影响

用微电极细胞外记录的方法,观察内脏痛、躯体痛和触觉刺激对大鼠丘脑后核(PO)中770个神经元电活动的影响,其中305(38.3％)个对伤害性刺激起反应,103(13.4％)个对触觉刺激起反应。对伤害性刺激反应的神经元中多数对躯体痛和内脏痛刺激均起反应且反应形式相同,少数反应不同或相反,对触觉刺激反应的神经元中多数也对两种伤害性刺激均起反应,只对触觉刺激反应的神经元很少。     

Modeling the acquisition of counting with an associative network

In the acquision of counting by children, there are three interesting phenomena (Fuson et al. 1982): (1) the number word sequence produced by children can be divided into three distinct portions, called the conventional, stable nonconventional, and unstable portions; (2) irregular number words such as fifteen are omitted more often than regular ones such as fourteen, sixteen, and seventeen; and (3) initially the number word sequence is in a recitation form, rather than in the form of an associative chain of separable serial elements. Our paper at first analyzes these phenomena from the viewpoint of associative memory by assuming the number word sequences are made up of many associative relationships between the number words. This assumption is not contradictory to the third phenomenon described above, because the associative relationships are not confined only to those between the serial number words. On the basis of these anaylses, an associative network model, HAPS proposed by one of the authors (Hirai 1983), is extended so that it can mimic some aspects of the learning of sequence which involves the above three phenomena. The learning and production of sequence by the network are simulated on a digital computer, and the results show that the three phenomena can be observed in the performance of the network.     

Nucleotide sequence and expression of cheF, an essential gene for chemotaxis in Bacillus subtilis.

The cheF gene, which is involved in chemotaxis in Bacillus subtilis, has been cloned, expressed, and sequenced. This gene is contained in a 0.7-kilobase PstI DNA fragment that was isolated from a lambda Charon 4A B. subtilis chromosomal DNA library. This fragment was subcloned into the expression vector pSI-1 and shown to complement the cheF mutation both for chemotaxis and for methanol production in response to the addition of attractants. Plasmid-encoded DNA expression in B. subtilis maxicells indicated that a membrane-associated polypeptide of 20-kilodaltons was expressed from this 0.7-kilobase DNA. The nucleotide sequence of this DNA fragment was determined, and an open reading frame capable of encoding a putative 175-amino-acid protein (Mr 20,002) was identified. In an effort to understand the function of the cheF protein, the dosage of the cheF gene product was varied by altering the concentration of IPTG (isopropyl-beta-D-thiogalactopyranoside) during growth. In the presence of high concentrations of IPTG, chemotaxis was inhibited and methanol production was impaired.     

Circulating immunologic complexes in blood sera of bronchial asthma patients treated with attenuated vaccine

The study included 20 patients (8 males and 12 females) with non-atopic bronchial asthma treated with attenuated vaccines. Circulating immune complexes were assayed with two methods prior to and after the treatment of each patient. No increase in serum circulating immune complexes was produced after the treatment. Clinical improvement was noted in 75% of treated patients. Circulating immune complexes were detected in the blood serum of three patients treated without an effect.