Complete genome sequence of an aerobic hyper-thermophilic crenarchaeon, Aeropyrum pernix K1.

The complete sequence of the genome of an aerobic hyper-thermophilic crenarchaeon, Aeropyrum pernix K1, which optimally grows at 95 degrees C, has been determined by the whole genome shotgun method with some modifications. The entire length of the genome was 1,669,695 bp. The authenticity of the entire sequence was supported by restriction analysis of long PCR products, which were directly amplified from the genomic DNA. As the potential protein-coding regions, a total of 2,694 open reading frames (ORFs) were assigned. By similarity search against public databases, 633 (23.5%) of the ORFs were related to genes with putative function and 523 (19.4%) to the sequences registered but with unknown function. All the genes in the TCA cycle except for that of alpha-ketoglutarate dehydrogenase were included, and instead of the alpha-ketoglutarate dehydrogenase gene, the genes coding for the two subunits of 2-oxoacid:ferredoxin oxidoreductase were identified. The remaining 1,538 ORFs (57.1%) did not show any significant similarity to the sequences in the databases. Sequence comparison among the assigned ORFs suggested that a considerable member of ORFs were generated by sequence duplication. The RNA genes identified were a single 16S-23S rRNA operon, two 5S rRNA genes and 47 tRNA genes including 14 genes with intron structures. All the assigned ORFs and RNA coding regions occupied 89.12% of the whole genome. The data presented in this paper are available on the internet homepage (http://www.mild.nite.go.jp).

Complete sequence and gene organization of the genome of a hyper-thermophilic archaebacterium, Pyrococcus horikoshii OT3.

The complete sequence of the genome of a hyper-thermophilic archaebacterium, Pyrococcus horikoshii OT3, has been determined by assembling the sequences of the physical map-based contigs of fosmid clones and of long polymerase chain reaction (PCR) products which were used for gap-filling. The entire length of the genome was 1,738,505 bp. The authenticity of the entire genome sequence was supported by restriction analysis of long PCR products, which were directly amplified from the genomic DNA. As the potential protein-coding regions, a total of 2061 open reading frames (ORFs) were assigned, and by similarity search against public databases, 406 (19.7%) were related to genes with putative function and 453 (22.0%) to the sequences registered but with unknown function. The remaining 1202 ORFs (58.3%) did not show any significant similarity to the sequences in the databases. Sequence comparison among the assigned ORFs in the genome provided evidence that a considerable number of ORFs were generated by sequence duplication. By similarity search, 11 ORFs were assumed to contain the intein elements. The RNA genes identified were a single 16S-23S rRNA operon, two 5S rRNA genes and 46 tRNA genes including two with the intron structure. All the assigned ORFs and RNA coding regions occupied 91.25% of the whole genome. The data presented in this paper are available on the internet at http:@www.nite.go.jp.

Complete genome sequence of an aerobic thermoacidophilic crenarchaeon, Sulfolobus tokodaii strain7.

The complete genomic sequence of an aerobic thermoacidophilic crenarchaeon, Sulfolobus tokodaii strain7 which optimally grows at 80 degrees C, at low pH, and under aerobic conditions, has been determined by the whole genome shotgun method with slight modifications. The genomic size was 2,694,756 bp long and the G + C content was 32.8%. The following RNA-coding genes were identified: a single 16S-23S rRNA cluster, one 5S rRNA gene and 46 tRNA genes (including 24 intron-containing tRNA genes). The repetitive sequences identified were SR-type repetitive sequences, long dispersed-type repetitive sequences and Tn-like repetitive elements. The genome contained 2826 potential protein-coding regions (open reading frames, ORFs). By similarity search against public databases, 911 (32.2%) ORFs were related to functional assigned genes, 921 (32.6%) were related to conserved ORFs of unknown function, 145 (5.1%) contained some motifs, and remaining 849 (30.0%) did not show any significant similarity to the registered sequences. The ORFs with functional assignments included the candidate genes involved in sulfide metabolism, the TCA cycle and the respiratory chain. Sequence comparison provided evidence suggesting the integration of plasmid, rearrangement of genomic structure, and duplication of genomic regions that may be responsible for the larger genomic size of the S. tokodaii strain7 genome. The genome contained eukaryote-type genes which were not identified in other archaea and lacked the CCA sequence in the tRNA genes. The result suggests that this strain is closer to eukaryotes among the archaea strains so far sequenced. The data presented in this paper are also available on the internet homepage (http://www.bio.nite.go.jp/E-home/genome_list-e.html/).

A complex of rab3A, SNAP-25, VAMP/synaptobrevin-2 and syntaxins in brain presynaptic terminals.

Two monoclonal antibodies (SPM-1 and SPM-2) immunoprecipitate brain N-type calcium channels. On immunoaffinity chromatography of digitonin extracts of bovine brain membranes on SPM-1- and SPM-2-Sepharose, proteins of 36 (syntaxins A and B), 28 and 19 kDa are specifically retained by both columns. Here we show that the 19 and 28 kDa bands contain VAMP/synaptobrevin-2, and rab3A/smg25A and SNAP-25, respectively. Since SPM-1 and SPM-2 recognize only syntaxins and the 28 kDa band (rab3A/sm25A and SNAP-25), respectively, the results indicate that all these proteins form a complex. Our results suggest tight linkage between the components involved in neurotransmitter release.

The role of gravity in chick embryogenesis.

Thirty fertilized chick eggs preincubated for 0, 7 and 10 days on earth (10 eggs each) were flown in the space shuttle 'Endeavour' and further incubated for 7 days under microgravity. Twenty out of thirty eggs (9/10 ten-day-old; 10/10 seven-day-old; 1/10 zero-day-old) were recovered alive after landing. The only living embryo of the zero-day-old group died 24 days after launch, and was comparable to a 16-day-old embryo. The high mortality of the 0-day-old eggs appeared to be related to the specific inner structure of the egg. Simulation experiments performed on earth indicated that when yolk stayed in the albumen for more than 2 days, most of the embryos died. The subtle difference in specific gravity between the yolk (1.029) and albumen (1.040) plays a critical role in early chick embryogenesis.

Spinocerebellar ataxia type 6. Molecular and clinical features of 35 Japanese patients including one homozygous for the CAG repeat expansion.

Spinocerebellar ataxia type 6 (SCA6) is a newly classified autosomal-dominant cerebellar ataxia (ADCA) associated with CAG repeat expansion. We screened 111 patients with cerebellar ataxia for the SCA6 mutation. Of these, 35 patients were found to have expanded CAG repeats in the SCA6 gene, indicating that second to SCA3, SCA6 is the most common ADCA in Japan. Expanded alleles ranged from 21 to 29 repeats, whereas normal alleles had seven to 17 repeats. There was no change in the CAG repeat length during meiosis. The age at onset was inversely correlated with the repeat length. The main clinical feature of the 35 patients with SCA6 was slowly progressive cerebellar ataxia; multisystem involvement was not common. The 35 patients included nine cases without apparent family history of cerebellar ataxia. The sporadic cases had smaller CAG repeats (21 or 22 repeats) and a later age at onset (64.9 +/- 4.9 years) than the other cases with established family history. We also identified one patient who was homozygous for the SCA6 repeat expansion. The homozygote showed an earlier age of onset and more severe clinical manifestations than her sister, a heterozygote carrying an expanded allele with the same repeat length as the homozygote. This finding suggests that the dosage of the CAG repeat expansion plays an important role in phenotypic expression in SCA6.

Turnover rates of the AMPA-type glutamate receptor GluR1 measured by transient gene expression.

Protein turnover rates have until now been measured by pulse chasing of the target protein after labeling it with radioactive amino acids. This procedure, however, requires severe amino acid depletion followed by specific immunoprecipitation of the target protein. In the present study, we assessed the turnover rates of an AMPA-type glutamate receptor, GluR1 (or GluRA), with the conventional method and a novel one using gene transfer, and compared both of them. GluR1 cDNA was introduced into PC12 cells and cultured rat hippocampal neurons by electroporation and lipofection, respectively. Expression of its mRNA was transient and had almost ceased 2 days in PC12 cells after transfection, while the receptor protein continued to be detectable by Western blotting for a week. When the levels of the receptor protein in PC12 cells were plotted on a semi-logarithmic scale, the decay curve appeared linear after 2 days: Its decay half time (tau 1/2) was calculated as 41 h. In contrast, the pulse chase experiment revealed that the decay half time was 2-4 h in PC12 cells although cell damage was seen during this procedure. The receptor decay speed was also measured in cultured hippocampal neurons using GluR1 cDNA attached to a tag sequence. Decay of the receptor protein was monitored by Western blotting probed by an anti-tag antibody: tau 1/2 was 52 h in hippocampal neurons, similar to that in PC12. These observations suggest that the transfection procedure is more sensitive and beneficial than the conventional pulse chasing method when measuring protein turnover rates in fragile neural cells.

Duration of catalepsy correlates with increased intrastriatal sulpiride.

To investigate the mechanism underlying sulpiride-induced catalepsy, we simultaneously examined cataleptic behavior and the kinetics of the dopamine receptor antagonist, sulpiride of dopamine, and the dopamine metabolite 3,4-dihydroxyphenylacetic acid (DOPAC), using in vivo voltammetry. After intrastriatal administration of sulpiride to freely moving rats, the levels increased, peaked at 20 min, and remained elevated for more than 3 h. Sulpiride-induced cataleptic behavior also continued for 3 h. Levels of DOPAC peaked 180 min after the injection and did not return to baseline within the experimental period. Thus, the time-course of cataleptic behavior correlated better with elevated extracellular levels of sulpiride than with that of DOPAC. These findings suggest that sulpiride induces catalepsy via a direct action.

The dopamine D2 receptor antagonist sulpiride causes long-lasting serotonin release.

The effects of the dopamine D2 receptor antagonist sulpiride on extracellular levels of serotonin (5-hydroxytryptamine, 5-HT) and the 5-HT metabolite 5-hydroxyindoleacetic acid (5-HIAA) were examined by using in vivo voltammetry. Sulpiride (1 or 3 mM, 2 microl over 24 min) was administered to freely moving rats via a cannula implanted in the striatum and 5-hydroxyindole levels were measured by using a carbon fiber voltammetry electrode implanted in the ipsilateral striatum. Six to 8 h after injection, 5-hydroxyindole levels increased 3-fold, peaked 1 to 2 days post-injection, and returned to normal levels within 2 to 4 days. These effects were suppressed by pretreatment with p-chlorophenylalanine. Two days after sulpiride injection, high-performance liquid chromatography of striatal homogenates revealed that although the 5-HT concentration was unchanged, the 5-HIAA concentration was increased significantly. These results suggest that the long-lasting elevation of 5-hydroxyindole concentrations was primarily due to increased 5-HT release.

The relationship between trinucleotide repeat length and phenotypic variation in Machado-Joseph disease.

Trinucleotide repeat expansion in the Machado-Joseph disease (MJD) gene has been found in 26 patients from 20 unrelated Japanese families. Expanded alleles had 68 to 84 repeats, whereas normal alleles had 14 to 37 repeats. The age of onset was inversely correlated with the repeat length. To evaluate in detail the relationship between the repeat length and clinical features, we subdivided the 26 patients into three groups on the basis of the repeat length (group 1, 78 repeats or more; group 2, 74 to 77 repeats; group 3, 73 repeats or less). Group 1 and group 2 had common features of spasticity, hyperreflexia, Babinski sign, bulging eyes, facial myokymia and extrapyramidal signs as well as cerebellar ataxia and ophthalmoplegia. It should be noted that group 1 showed more prominent pyramidal and extrapyramidal signs than group 2. In contrast, group 3 showed hypotonia, hyporeflexia and sensory disturbance in addition to cerebellar ataxia and ophthalmoplegia. These findings suggest that the repeat length plays an important role in phenotypic variation. DNA analysis for the MJD mutation was clearly useful for making an accurate diagnosis in patients without bulging eyes, facial myokymia, dystonia or marked spasticity.

Adriamycin and Cisplatin induce apoptosis in 2 lines of human gastric-cancer cells (mkn-28 and hsc-39).

Many anticancer agents induce an active process that leads to cell death, known as, apoptosis, in sensitive tumor cells. The fragmentation of DNA, an indicator of apoptosis, was analyzed in two different lines of human gastric cancer cells (HSC-39 and MKN-28) that had been exposed to adriamycin and cisplatin. The fragmentation of DNA was detected in HSC-39 cells (signet ring cell gastric carcinoma) after a 1-h incubation with 0.18 mu M and after a 6-h incubation with 0.09 mu M adriamycin, as well as after a 1-h incubation with 1.67 mu M cisplatin. However, in MKN-28 cells (moderately differentiated gastric adenocarcinoma), the fragmentation of DNA was not detected after a 6-h incubation with 0.18 mu M adriamycin or a 6-h incubation with 3.33 mu M cisplatin. The results suggest that signet ring cell gastric carcinoma is more sensitive to adriamycin and to cisplatin than moderately differentiated gastric adenocarcinoma.

Glucose, free fatty acid and ketone body metabolism in Duchenne muscular dystrophy.

We examined how the substances, especially glucose, free fatty acids (FFA) and ketone bodies, and hormones associated with energy metabolism change with the disease progress in Duchenne muscular dystrophy (DMD). Serum creatine kinase (CK) activity was used as an index of the stage of DMD, because this activity is exponentially decreases with the progress of the disease. The glucose concentration in DMD patients with CK activity of less than 1,000 U/l (low CK) was significantly lower than that in controls, although there was no significant difference between that in DMD patients with CK activity of more than 1,00 U/l (high CK) and that in controls. The FFA concentration in both high CK and low CK patients was significantly higher than that in controls. The FFA concentration in low CK patients tended to be higher than that in high CK patients. The ketone body concentration in low CK patients was significantly higher than that in controls and that in high CK patients. The [glucagon]:[insulin] ratio in low CK patients was significantly higher than that in controls and that in high CK patients. It was also observed in a correlational study that the glucose concentration decreased with the age and the decrease in CK activity, i.e., with the progress of DMD. The FFA and ketone body concentrations increased with the decrease in the glucose concentration. The decrease in the glucose concentration may be due to a caloric shortage and/or degenerated muscle, which cannot supply enough gluconeogenic substrates, such as alanine. The kinetics of insulin and glucagon in DMD may help to maintain the glucose metabolism. Increased concentrations of FFA and ketone bodies may be helpful in the advanced stage of DMD, as energy sources and as substrates, sparing muscle protein.

Initial transient phase of salivary secretion by the submandibular glands of rats under anoxic conditions.

Salivary secretion of the rat submandibular gland exhibits two phases upon administration of acetylcholine (ACh, 10(-6) M): an immediate initial transient phase of rapid secretion lasting 5 min followed by a longer steady phase of slower secretion. Application of an anoxic perfusate bubbled with 100% N2 for 10-20 min had no effect on the initial phase of secretion, but caused a marked decrease in secretion in the steady phase following stimulation with 10(-6) M ACh. After secretion under the anoxic condition, a recovery period without stimulation was performed for 30 min by perfusion with HEPES Ringer's solution bubbled with 100% O2 and containing various potassium concentrations. The initial secretion rate measured with application of an anoxic perfusate was markedly increased by the high-K+ recovery perfusate (25 mM) and decreased by the K(+)-free recovery perfusate. Administration of 10(-3) M ouabain in the normal perfusate resulted in inhibition of secretion during the steady phase similar to that seen under the anoxic condition, while the secretory rate during the initial phase remained unchanged. We concluded that the initial phase of secretion is relatively resistant to anoxia, and that oxygen supply and Na(+)-K+ pump activity were essential for maintaining the steady phase of secretion.

Nasal resistance measured by anterior rhinomanometry.

The technique and principle of the active anterior rhinomanometry were justified by human examination and model experiment. The resistance values obtained from anterior method did not always agree with those from posterior one. Causes of the disagreement were considered to be distortion of the nostrils, air leakage from the apparatus and resistive component in the nasopharynx. These possibilities were demonstrated and analyzed in model experiments. The anterior rhinomanometry was concluded as a precise and convenient method to assess the conductivity of the nasal cavities.

Computer analyses of Rohrer's coefficients.

In the field of respiratory physiology computers have been used for theoretical and automatic analyses of the test results. The purpose of this paper is to clarify whether computers are available in measurement of the nasal resistance. The nasal cavities, simulated by an acryl resin model, were ventilated at various volumes and speeds. The pressure and flow changes were recorded by a data recorder and analyzed by a minicomputer according to the prescribed situations. Rohrer's formula P = K1V+K2V2 and its modification R = K1+K2V were used for calculation of K1 and K2. The calculated values differed significantly when sampling was made on a particular part of the respiratory excursion or sampling time was extraordinarily prolonged. They were also different from the ordinary values when too slow or too rapid ventilatory speed was applied. It was concluded that more than twenty samplings were necessary at regular intervals in a normal inspiratory or expiratory phase. Too slow and too rapid respiration should be avoided because of functional limitations of the measuring apparatus.

Effects of glucose in drinking water on the changes in whole blood viscosity and plasma osmolality of broiler chickens during high temperature exposure.

This study was conducted to elucidate the influence of glucose in drinking water on whole blood viscosity and plasma osmolality of broilers during high temperature exposure. Two groups of birds, which had ad libitum access to either a 4% glucose-water solution (Group G) or tap water (Group W), were exposed simultaneously to 30 C from 0300 h for 12 h each day for 3 d. During the experimental period, Group G birds had greater metabolic energy intake and body weight gain than Group W. Hematocrit and whole blood viscosity decreased significantly at 30 C compared to controls at 20 C in Group W, whereas, in Group G, no changes were found for these two variables. Plasma osmolality also decreased at 30 C compared to 20 C in Group W, whereas no change was noted in this variable in Group G. However, at 20 C, plasma osmolality was significantly higher in Group W than in Group G, but no difference was observed between the two groups at 30 C. Plasma protein concentration decreased during exposure to 30 C in both groups, but the decrease tended to be greater in Group W than in Group G. These results suggest that glucose intake may alleviate the influence of heat stress on whole blood viscosity and plasma osmolality.

[A case of midbrain infarction with acute bilateral cerebellar ataxia].

An 81-year-old man was suffered from acute dysarthria and gait disturbance. Bilateral cerebellar ataxia and ataxic dysarthria were the only neurological findings. MRI images revealed an infarction in the lower and medial part of the midbrain. We consider that bilateral ataxia of the present case was caused by the lesion at the decussation of the superior cerebellar peduncle.

[A sporadic case of dentatorubral-pallidoluysian atrophy (DRPLA) having an elderly age of onset].

We reported a sporadic case of DRPLA that had an elderly age of onset. The patient was a 71-year-old woman. Her parents, sisters and a brother didn't have neurologic diseases. She had been well until the age of 68 years, when she noticed her unstable gait. On neurologic examination cerebellar ataxia and a tremor at the neck were noticed, but we were unable to differentiate her disease from the other types of spinocerebellar degeneration. An MRI of the cranium showed atrophy of the cerebellum, pons, brain stem and cerebrum, and a diffuse lesion of the cerebral white matter. These findings made us suspect her disease of DRPLA. When we analyzed the CAG repeat in the DRPLA gene, we found it expanded to 57. We thought that the elderly onset related to a relatively mild expansion.