The C-terminal region of the S component of staphylococcal leukocidin is essential for the biological activity of the toxin.

The Staphylococcal toxin leukocidin consists of two protein components, F and S. From a culture medium of Staphylococcus aureus RIMD 310925, we isolated a truncated form of S (LS2), of which the C-terminal 17-residue segment is missing. Unlike intact S, LS2, showed neither leukocytolytic activity in the presence of F nor affinity for monosialoganglioside GM1 (GM1). When excited at 280 nm, both S and LS2 exhibited intrinsic tryptophan fluorescence with an emission maximum at 318 nm. Upon binding to GM1, the emission maximum of S underwent a blue shift to 310 nm, whereas no change in fluorescence took place on mixing GM1 with LS2. We conclude that the C-terminal region of S is essential for its biological activity as well as for its binding to GM1 and that this binding is accompanied by a conformational change of the S protein.

The two Staphylococcal bi-component toxins, leukocidin and gamma-hemolysin, share one component in common.

Staphylococcal bi-component toxins, leukocidin and gamma-hemolysin, consist of two protein components, i.e. F and S for leukocidin and H gamma I and H gamma II for gamma-hemolysin. In this study we purified H gamma I and H gamma II to homogeneity from the culture medium of Staphylococcus aureus RIMD 310925 and compared their properties with those of F and S purified from the same source. The N-terminal 59- and C-terminal 2-residue amino acid sequences, apparent molecular mass, and isoelectric point of purified H gamma I were the same as those of F. In an Ouchterlony double diffusion test a fused line without spur was formed between F and H gamma I using either anti-F or anti-H gamma I antibodies. A synergistic action of F and H gamma II caused hemolysis of human red blood cells, and H gamma I acted synergistically with S to exhibit leukocidin activity. We conclude that the two toxins share one protein component (F = H gamma I) in common and leukocidin- and gamma-hemolysin-specific activities are determined by S and H gamma II, respectively. It is also reported that the N-terminal 58-residue sequence of H gamma II is 72% similar to the corresponding sequence of S.

Simultaneous induction of mitochondrial heat shock protein mRNAs in rat forebrain ischemia.

Several investigations have postulated evidence of the involvement of apoptosis in delayed neuronal death following brief periods of global cerebral ischemia. Apoptosis may be closely linked to mitochondrial dysfunction. Heat shock protein (HSP) 60 and HSP10 are mitochondrial matrix proteins induced by stress and form the chaperonin complex that is implicated in protein folding and assembly within the mitochondria. This study investigated the induction of these mitochondrial stress protein genes in the hippocampal CA1 region and less vulnerable regions following transient forebrain ischemia. In situ hybridization analysis revealed that the induction pattern of HSP60 mRNA was identical to that of HSP10 mRNA throughout the entire ischemic course. No changes occurred in the expression of both mRNAs after 2 min ischemia. Strong induction of both mRNAs occurred in the CA1 region after 10 min ischemia and persisted until 1 d after reperfusion. In contrast, induction of both mRNAs in the less vulnerable regions was terminated by 1 d after reperfusion. These results demonstrate that mitochondrial stress conditions persist concomitantly with cytosolic stress conditions in regions vulnerable to transient forebrain ischemia.

Induction of mitochondrial heat shock protein 60 and 10 mRNAs following transient focal cerebral ischemia in the rat.

Heat shock proteins (HSPs) 60 and 10 are stress-inducible mitochondrial matrix proteins that form a chaperonin complex that is important for mitochondrial protein folding and function. The effect of cerebral ischemia on mitochondrial HSPs is unclear. The topographical and chronological patterns of HSP60 and HSP10 messenger ribonucleic acid (mRNA) expression and induction were investigated in the rat focal cerebral ischemia model. Focal cerebral ischemia was produced by transient middle cerebral artery occlusion for 30 or 90 min. Expression of mRNAs was analyzed using reverse transcription-polymerase chain reaction (RT-PCR) and in situ hybridization. RT-PCR analysis showed that both HSP60 and HSP10 mRNA levels increased significantly in the ischemic cortex from 4 to 24 h of reperfusion after 30 min of occlusion. In situ hybridization analysis demonstrated significant induction of both mRNAs in the whole ischemic cortex after 30 min of occlusion and in the dorsomedial border (penumbra) of the ischemic cortex and ipsilateral hippocampus after 90 min of occlusion. Expression patterns and the timing of the induction of both HSP60 and HSP10 mRNAs were identical throughout the experiments. Simultaneous induction of the mRNAs for the mitochondrial chaperonins, HSP60 and HSP10, in various regions in focal cerebral ischemia demonstrates that mitochondrial stress conditions persist concomitantly with cytosolic stress conditions in focal cerebral ischemia.

Induction of Rheb mRNA following middle cerebral artery occlusion in the rat.

Rheb is a recently identified member of the Ras super-family and is an immediate early gene that is rapidly and transiently induced in the hippocampal granule cells by NMDA-dependent synaptic activity in the long term potentiation paradigm. The close homologies with Ras and its rapid inducibility strongly suggest that Rheb shares many biochemical and signaling properties with Ras. The present study investigated the effect of middle cerebral artery (MCA) occlusion on the expression of Rheb mRNA in the rat brain. In situ hybridization autoradiography showed that Rheb mRNA was induced in the extensive regions of cerebral cortex and medial striatum surrounding the ischemic region and bilateral hippocampal formation following MCA occlusion. The induction of Rheb mRNA in the cingulate cortex persisted prominently at 24 h of MCA occlusion. Although the Rheb mRNA induction in the medial striatum and hippocampal formation decreased after 8h of occlusion, it still remained significant at 24h of occlusion. The data suggest the possibility that Ras signaling pathways can be implicated in the cerebral ischemia-elicited events through NMDA receptor activation.

Activation of Arc gene, a dendritic immediate early gene, by middle cerebral artery occlusion in rat brain.

The effect of middle cerebral artery (MCA) occlusion on the activity-regulated cytoskeleton-associated protein (Arc) mRNA expression has been investigated using in situ hybridization. It was induced in the extensive regions of cerebral cortex, medial striatum, and distant areas such as the ipsilateral lateral septal nucleus, bilateral hippocampal formation and contralateral amygdala following MCA occlusion. In the hippocampal formation, it was induced in the granule cell layer and the stratum pyramidale at 1 h and in the molecular layer and in the stratum oriens and stratum radiatum bilaterally at 4 h. MK-801 pretreatment strongly attenuated the induction of Arc mRNA. The present results suggest that Arc may play an important role in the neuronal plasticity through NMDA activation following focal cerebral ischemia.

Ruptured suprasellar dermoid cyst presenting olfactory delusion (Eigengeruchs erlebnis).

A 24-year-old man sought treatment of a long-standing olfactory delusion (Eigengeruchs erlebnis) and associated psychotic symptoms, which disappeared after the removal of a suprasellar dermoid cyst. The pathophysiology of the phenomenon is discussed.

A case of early-onset benign occipital seizure susceptibility syndrome: decreased cerebral blood flow in the occipital region detected by interictal single photon emission computed tomography, corresponding to the epileptogenic focus.

Early-onset benign childhood occipital seizure susceptibility syndrome (EBOSS) recently described by Panayiotopoulos, is an early-onset variant of benign childhood epilepsy with occipital paroxysms. EBOSS is characterized by partial seizures that are predominantly manifested at night and associated with deviation of the eyes, vomiting and impairment of consciousness, but without ictal visual symptoms or postictal headache. The clinical features of our case were consistent with those of EBOSS, and we therefore diagnosed the patient as having a typical form of EBOSS. Neuroimaging by CT, MRI and MR angiography did not reveal a focal lesion. Interictal single photon emission computed tomography (SPECT) revealed decreased cerebral blood flow in the right occipital region corresponding to the epileptogenic focus shown on EEG. It remains unclear whether our finding on SPECT reflects secondary hypoperfusion due to minor morphological abnormality or immediate functional hypoperfusion. No reference to SPECT in a case of EBOSS has appeared in the literature to date. This report provides a better understanding of benign childhood epileptic syndromes with occipital spikes.

A macrocyclic antibiotic M-230B produced by Myxococcus xanthus. Isolation and characterization.

Myxococcus xanthus strain M516E produced at least three related antibiotics against Gram-positive and Gram-negative bacteria. From physico-chemical properties, a main component was identical to myxovirescin A and a second component, designated M-230B was found to be an antibiotic which is closely related to myxovirescin A. The structure of M-230B was determined from its physico-chemical properties, especially from 13C NMR spectrum as compared with that of myxovirescin A. The addition of alcohol, such as isobutyl alcohol, to the culture medium markedly stimulated production of the antibiotics.

Degradation of phospholipid in Pseudomonas aeruginosa induced by polymyxin B.

Effects of polymyxin B on the synthesis and degradation of lipid, ribonucleic acid (RNA) and protein in Pseudomonas aeruginosa were investigated. It was found that polymyxin B caused a marked degradation of the lipid fraction which was prelabeled with (3H-2)-glycerol. Thin-layer chromatographic analysis indicated that the main degraded lipids were phosphatidylethanolamine and phosphatidylglycerol, which constituted 80% and 15% of the total phospholipids of this organism, respectively. Polymyxin B also inhibited synthesis of RNA and protein in vivo. The severe inhibition of the uptake of labeled amino acids by polymyxin B indicated that the observed inhibition of RNA and protein synthesis possibly occurred at the level of substrate transports. The degradation of phospholipid might account for the defective membrane activities.

New polyenic antibiotics active against gram-positive and -negative bacteria. I. Isolation and purification of antibiotics produced by Gluconobacter sp. W-315.

A new antibiotic, tentatively named as AB-315, was isolated from the fermentation broth of Gluconobacter sp. W-315. The antibiotic consists of a mixture of chemically related compounds. These compounds showed similar profiles in UV absorbancy. The antibiotics were active against Gram-positive and -negative bacteria, slightly active against fungi but not against yeasts.

New polyenic antibiotics active against gram-positive and -negative bacteria. II. Screening of antibiotic producers and taxonomical properties of Gluconobacter sp. W-315.

Antibiotic producing bacteria were selected using a new screening method. Eight strains of antibiotic producing bacteria, which required a spent medium of fungi for antibiotic production, were isolated. One of them, a potent producer of antibacterial antibiotic, designated strain W-315, had following taxonomical characteristics; aerobic, Gram-negative, rod shaped and polar flagellated. Furthermore, the organism could grow under acidic conditions (pH 4.5) and had a GC content of 64.4 mole per cent. We concluded that the strain W-315 belonged to Gluconobacter sp. When this bacterium was inoculated into Czapek-Dox medium, bacterial growth and antibiotic production did not occur. The antibiotic production was also not observed even when poor growth was observed in Czapek-Dox medium supplemented with ammonium sulfate. The nutritional requirements for the antibiotic production were also discussed.

Mechanism of chloramphenicol resistance in Bacillus badius 211.

Six strains of chloramphenicol (CM)-resistant endospore-forming bacteria, which can grow in the presence of 100 microgram/ml of CM, were isolated and identified as Bacillus badius. Mechanism of CM-resistance in one of the isolated strains, Bacillus badius 211, was investigated. No inactivation of CM was demonstrated when the strain was grown in nutrient broth containing 100 microgram/ml of CM, as evidenced by paper-disc bioassay of CM in the growth medium. In accordance with this result, no CM acetylation activity was demonstrated either with the intact cells or with the crude extracts of the CM-resistant strain. Poly U- and Poly A-directed polyphenylalanine and polylysine syntheses by S--30 preparations of both CM-resistant and CM-sensitive strains of Bacillus badius were almost equally inhibited by CM. From these results, the mechanism of CM resistance in Bacillus badius 211 seems to be due to other unknown mechanism.

New polyenic antibiotics active against gram-positive and gram-negative bacteria. IV. Structural elucidation of enacyloxin IIa.

The chemical structure of a unique polyenic antibiotic enacyloxin IIa (former name: fr. 2) produced by Frateuria (formerly Gluconobacter) sp. W-315 has been determined by extensive spectroscopic studies, in particular by NMR spectral analysis. It has a novel non-lactonic structure involving 3,4-dihydroxycyclohexanecarboxylic acid with a chlorine-containing polyenic and polyhydroxy acyl side chain attached as an ester to the 3-hydroxyl substituent of the acid.

New polyenic antibiotics active against gram-positive and gram-negative bacteria. VIII. Construction of synthetic medium for production of mono-chloro-congeners of enacyloxins.

New antibiotics enacyloxins (ENXs) are a family of non-lactonic polyene antibiotics produced by Frateuria sp. W-315. For the production of antibiotics, we had to employ two-step fermentations, the first is the production of spent medium of Neurospora crassa and the second is the production of antibiotics by Frateuria. To simplify the production of antibiotics, systematic analyses have been done on the spent medium, and factors which affect the production of antibiotics characterized. From the above results, we constructed a new medium for antibiotic production. Moreover, we could get a new antibiotic named enacyloxin IIIa (1), C33H48O11NCl (m/z 669). 1 was deduced to be one of the congeners of enacyloxins because it was similar to ENX IIa or ENX IVa both in biological and physico-chemical properties. Chlorine of 1 could be replaced by bromine, biosynthetically, and the resultant bromine-containing antibiotic also showed an antibacterial activity comparable to 1.

The usefulness of 3D MR angiography in surgery for ruptured cerebral aneurysms.

BACKGROUND: We have used magnetic resonance angiography (MRA) in screening for unruptured cerebral aneurysms since 1993. The development of high-resolution magnetic resonance (MR) imaging has led to a remarkable improvement in image quality. Three-dimensional (3D) MRA can be used for surgical simulation. Here, we report on the usefulness of and problems associated with 3D MRA for the surgery of ruptured cerebral aneurysms. METHODS: Between June 1998 and June 2000, 106 patients with SAH diagnosed by 3D MRA underwent surgery. We compared 3D MRA images with operative findings and investigated the usefulness of this assessment tool. RESULTS: In 48 of 106 cases (45.3%), we were able to perform surgery based on 3D MRA alone. By using the 3D images, we could easily detect the relative location of the aneurysm, its neck and the surrounding arteries. The remaining cases required further examinations because of uncertainty of diagnosis or insufficient information. CONCLUSION: 3D MRA is a safe and useful procedure for the diagnosis and surgery of ruptured cerebral aneurysms. However, in approximately half of all cases, 3D computed tomographic angiography (CTA) or digital subtraction angiography (DSA) is required in addition for the planning of surgery. It is important to use 3D MRA for surgery only after taking sufficient consideration of certain limitations peculiar to MRA.

[Temporary placement of inferior vena cava filter prior to transcatheter arterial embolization (TAE) for hepatocellular carcinoma with IVC tumor thrombus--prevention of pulmonary tumor emboli after TAE].

A 66-year-old-man with a right huge hepatocellular carcinoma (HCC) extending into both the right portal vein and the right atrium underwent transcatheter arterial embolization (TAE) via the right hepatic artery. Prior to the TAE, a temporary inferior vena cava (IVC) filter was placed suprarenally for prevention of pulmonary tumor emboli. When we replaced the temporary IVC filter with a new one 7 days after the TAE, the filter which was pulled out of the IVC captured a fragment of the tumor thrombus. A histopathological specimen demonstrated only ghost cells. The patient has been followed at our outpatient clinic without any tumor thrombus or pulmonary infarction for 13 months after this procedure.