Evaluation of essential and variable residues of nukacin ISK-1 by NNK scanning.

Nukacin ISK-1, a type-A(II) lantibiotic, comprises 27 amino acids with a distinct linear N-terminal and a globular C-terminal region. To identify the positional importance or redundancy of individual residues responsible for nukacin ISK-1 antimicrobial activity, we replaced the native codons of the parent peptide with NNK triplet oligonucleotides in order to generate a bank of nukacin ISK-1 variants. The bioactivity of each peptide variant was evaluated by colony overlay assay, and hence we identified three Lys residues (Lys1, Lys2 and Lys3) that provided electrostatic interactions with the target membrane and were significantly variable. The ring structure of nukacin ISK-1 was found to be crucially important as replacing the ring-forming residues caused a complete loss of bioactivity. In addition to the ring-forming residues, Gly5, His12, Asp13, Met16, Asn17 and Gln20 residues were found to be essential for antimicrobial activity; Val6, Ile7, Val10, Phe19, Phe21, Val22, Phe23 and Thr24 were relatively variable; and Ser4, Pro8, His15 and Ser27 were extensively variable relative to their positions. We obtained two variants, Asp13Glu and Val22Ile, which exhibited a twofold higher specific activity compared with the wild-type and are the first reported type-A(II) lantibiotic mutant peptides with increased potency.

Accumulation of NEDD8 in neuronal and glial inclusions of neurodegenerative disorders.

NEDD8 (neural precursor cell expressed, developmentally down-regulated 8) is a ubiquitin-like protein that controls vital biological events through its conjugation to members of the cullin family, which are components of certain ubiquitin E3 ligases. Recent studies have shown that NEDD8 is incorporated into Lewy bodies (LBs) in Parkinson's disease, Mallory bodies in alcoholic liver disease and Rosenthal fibres in astrocytoma. In order to examine whether NEDD8 plays a role in the formation of ubiquitinated inclusions, we performed immunohistochemical staining of brain tissue from patients with various neurodegenerative disorders, using an affinity-purified polyclonal antibody raised against NEDD8 that did not cross-react with ubiquitin. In LB disease, NEDD8 immunoreactivity was present in almost all of the LBs and Lewy neurites. Moreover, NEDD8 immunoreactivity was found in a variety of ubiquitinated inclusions, including neuronal and oligodendroglial inclusions in multiple system atrophy, neurofibrillary tangles in Alzheimer's disease, ubiquitinated inclusions in motor neurone disease, and intranuclear inclusions in triplet repeat diseases. These findings suggest that NEDD8 is involved in the formation of various ubiquitinated inclusions via the ubiquitin-proteasome system.

A quantitative investigation of neuronal cytoplasmic and intranuclear inclusions in the pontine and inferior olivary nuclei in multiple system atrophy.

Multiple system atrophy (MSA) is a sporadic neurodegenerative disease characterized by the presence of neuronal and oligodendroglial alpha-synuclein aggregates. To investigate the relationship between the occurrence of neuronal cytoplasmic and intranuclear inclusions (NCIs and NNIs, respectively) and the progression of neuronal degeneration, we performed a quantitative analysis of the pontine and inferior olivary nuclei based on 14 cases of MSA. alpha-Synuclein immunohistochemistry revealed that NCIs and NNIs were present in both brain nuclei in all the cases. The average incidence of NCIs in the pontine and inferior olivary nuclei was 9.1% and 25.8%, respectively, and that of NNIs was 9.2% and 9.0%, respectively. The number of NNIs was strongly correlated with that of neurones in the pontine and inferior olivary nuclei. Although the number of NCIs was not correlated with the neuronal population in both nuclei, the NCI count in patients with moderate MSA was higher than in patients with mild MSA. The NNI count was much higher than the NCI count in the pontine nucleus in four patients, and was the same in the olivary nucleus in three of the four patients. Moreover, the neuronal population in the NNI-predominant cases was significantly higher than in the NCI-predominant cases. These findings suggest that NCI formation is accelerated by the progression of the disease process, and that in MSA, NNI formation is an earlier phenomenon than NCI formation.

Anti-Hu associated paraneoplastic sensory neuronopathy with upper motor neurone involvement.

Paraneoplastic neurological syndrome is characterised by neuronal degeneration with lymphocytic infiltration in various regions of the central and peripheral nervous systems. Motor neurone symptoms may occur as a remote effect of malignancy, and have been considered because of the involvement of lower motor neurones. A case is reported of an 80 year old woman suffering from paraneoplastic sensory neuronopathy with anti-Hu antibody. Postmortem examination showed adenocarcinoma of the gall bladder and small cell carcinoma of the duodenum. Neuronal loss with lymphocytic infiltration was found in the dorsal root ganglia, brain stem, and cerebellum. Despite the absence of upper motor neurone signs, there was severe loss of Betz cells and degeneration of the bilateral pyramidal tracts. To our knowledge, this is the first demonstration of upper motor neurone involvement in anti-Hu associated paraneoplatic syndrome.

A hypopituitary patient who attained tall stature without growth hormone.

We describe an unusual patient with hypopituitarism who attained tall stature even without growth hormone (GH). A 37-year-old man was devoid of secondary sexual characteristics, but manifested tall stature with a eunuchoidal feature. Serum levels of GH, insulin-like growth factor-I, gonadotropins and testosterone were all below normal. GH secretion was not enhanced by any provocative stimulus. Adrenocorticotropic hormone increased after administration of corticotropin releasing hormone, but not after insulin-induced hypoglycemia. Thyrotropin increased in response to thyrotropin releasing hormone, but both free T3 and T4 did not rise. Magnetic resonance imaging disclosed a transected pituitary stalk. The present patient had hypopituitarism due to perinatal problems but had grown with the aid of non-GH growth-promoting factors, which suggests that man may be able to achieve statural growth even without GH.

[Isaacs' syndrome with abnormal F response and effects of double filtration plasmapheresis: a case report].

We reported a case of Isaacs' syndrome with abnormal F response detected electrophysiologically. A 14-year-old female was admitted to Hirosaki University Hospital with complaints of progressive myokymia and muscle cramp. PHT and CBZ were partially effective, but discontinued for drowsiness. A neurological examination revealed prominent myokymia and muscle cramp in the legs. The myokymia were worsened by exercise, bathing and diet. An electrophysiological examination showed characteristic F-response; high amplitude, long duration and increased number of phases. The epidural nerve block brought about a disappearance of the myokymia and an improvement of the abnormal features of F response. After repeated double filtration plasmapheresis, the myokymia and abnormal features of F response were remarkably reduced. Although Isaacs' syndrome is thought to have a hyperexcitability at the site of distal peripheral nerve, we suggested that the hyperexcitability might exist at the site of proximal region, and that immunological mechanisms underlie the cause of myokymia and unusual F-response in this case.

The primary structure of omega-amino acid:pyruvate aminotransferase.

The complete amino acid sequence of bacterial omega-amino acid:pyruvate aminotransferase (omega-APT) was determined from its primary structure. The enzyme protein was fragmented by CNBr cleavage, trypsin, and Staphylococcus aureus V8 digestions. The peptides were purified and sequenced by Edman degradation. omega-ATP is composed of four identical subunits of 449 amino acids each. The calculated molecular weight of the enzyme subunit is 48,738 and that of the enzyme tetramer is 194,952. No disulfide bonds or bound sugar molecules were found in the enzyme structure, although 6 cysteine residues were determined per enzyme subunit. Sequence homologies were found between an omega-aminotransferase, i.e. mammalian and yeast ornithine delta-aminotransferases, fungal gamma-aminobutyrate aminotransferase and 7,8-diaminoperalgonate aminotransferase, and 2,2-dialkylglycine decarboxylase. The enzyme structure is not homologous to those of aspartate aminotransferases (AspATs) including the enzymes of Escherichia coli and Sufolobus salfactaricus, though significant homology in the three-dimensional structures around the cofactor binding site has been found between omega-APT and AspATs (Watanabe, N., Sakabe, K., Sakabe, N., Higashi, T., Sasaki, K., Aibara, S., Morita, Y., Yonaha, K., Toyama, S., and Fukutani, H. (1989) J. Biochem. 105, 1-3).