Trimeresurus flavoviridis (Habu snake) venom aspartate-49-phospholipase A2 (Asp-49-PLA2) was reacted at pH 9.0 with a 2-fold molar excess of 2,4,6-trinitrobenzenesulfonate in the absence of Ca2+ and two trinitrophenylated derivatives were isolated by HPLC. One was a derivative modified at Lys-11 and its activity was mostly retained. The other was a derivative modified at both Lys-11 and Lys-72 and its activity was 40% that of unmodified enzyme. Trinitrophenylation of Lys-72 appeared to bring about a conformational disorder at the lipid-water interface recognition site and thus a reduction of activity. When the enzyme was modified in the presence of Ca2+, activity decreased at a rate much faster than that in the absence of Ca2+ and Lys-69 came to be modified. These results suggested that conformational displacement of Asp-49-PLA2 of a local to global type occurs upon the binding of Ca2+. The derivative modified at Lys-69 had 28% activity and existed as a monomer. This supports a previous assumption that Lys-69 participates in dimerization of group II Asp-49-PLA2s [Brunie et al. (1985) J. Biol. Chem. 260, 9742-9749] and shows that dimerization is not necessarily essential for activity manifestation.

Amino Acid Sequence↗

PubMedDate not supplied

Discriminatory recognition of membrane phospholipids by lysine-49-phospholipases A2 from Trimeresurus flavoviridis venom.

Basic proteins I and II (BP-I and BP-II) isolated from Trimeresurus flavoviridis venom, which are classified into a group of lysine-49-phospholipases A2 (Lys-49-PLA2), exhibited only limited lipolytic activity for the mixed micelles of various phospholipids. Based on the finding that BP-II elicits a strong contraction of guinea pig ileum due to the release of arachidonic acid, BP-II together with BP-I has been tested for their interaction with artificial phospholipid bilayer membranes. The dye leakage experiments indicated that BP-II interacts strongly with liposomes of beta-arachidonoyl-gamma-stearoyl-L-alpha-phosphatidylcholine. The perturbation of liposomes was observed only in the Ca(2+)-containing buffer, and as demonstrated by HPLC analyses, accompanied by the release of arachidonic acid. The concentration of Ca2+ which gave a half maximal activity of BP-II was 3.0 x 10(-4) M, suggesting that the affinity of BP-II for Ca2+ is more than 10 times stronger than that of BP-II without liposomes. These observations clearly show that Lys-49-PLA2 of BP-II is the enzyme responsible for the hydrolysis of membrane phospholipids and that Ca2+ is essential for such enzymatic activity. The interaction of BP-I with liposomes was much weaker than BP-II BP-I and BP-II share a common sequence except for Asp-67 (BP-I) and Asn-67 (BP-II) in the aligned sequences. This implies that the amino acid at position 67 of Lys-49-PLA2s is the residue required for discriminatory recognition of phospholipid membranes.

Animals↗

PubMedDate not supplied

Trimeresurus flavoviridis venom gland phospholipase A2 isozymes genes have evolved via accelerated substitutions.

As a step towards understanding the structure and function of phospholipases A2 (PLA2s), five cDNAs encoding Trimeresurus flavoviridis venom gland PLA2 isozymes have been sequenced. They revealed that the 5' and 3' untranslated regions are much more homologous than the protein-coding regions and that base substitutions have occurred at similar rates for the three positions of codons in the protein-coding regions. Such novel findings are of great interest from the viewpoint of molecular evolution. To gain a further insight into this evolutional phenomenon, we have isolated and sequenced six T. flavoviridis PLA2 isozyme genes. Each gene consisted of four exons and three introns and encoded protein of 138 amino-acid residues, including the signal sequence of 16 amino-acid residues. The introns were much more homologous than the protein-coding regions of exons except for the signal peptide-coding region of the first exon. The absence of apparent functional role in the introns suggested that the protein-coding regions, except for the signal peptide-coding domains, have evolved at greater substitution rates than introns. The fact that the numbers of nucleotide substitutions per non-synonymous site are close to or larger than the numbers of nucleotide substitutions per synonymous site for relevant pairs of genes revealed that Darwinian-type accelerated substitutions have occurred in the protein-coding regions of exons. This is compatible with the presence of PLA2 species with diverse physiological activities in the venom.

Amino Acid Sequence↗

PubMedDate not supplied

Circadian variation of the urinary 6beta-hydroxycortisol to cortisol ratio that would reflect hepatic CYP3A activity.

OBJECTIVES: Chronopharmacokinetics of drugs metabolized by cytochrome P450 3A (CYP3A) has been reported recently; however, little is studied on intra-individual circadian variation in CYP3A activity in human. The aim of this study was to assess the intra-individual diurnal variation and day-to-day variation of the urinary 6beta-hydroxycortisol to cortisol ratio, a noninvasive index of human CYP3A activity. METHODS: Urine samples from ten healthy Japanese men were collected over four time intervals (0900 hours to 1300 hours, 1300 hours to 1700 hours, 1700 hours to 2100 hours and 2100 hours to 0900 hours) on days 1, 5 and 14 to verify diurnal variation, and 24-h urine was collected to study day-to-day variation over 2 weeks. Urinary 6beta-hydroxycortisol and cortisol were determined by means of high-performance liquid chromatography/atmospheric pressure chemical ionization-mass spectrometry. RESULTS: The ratio of urinary 6beta-hydroxycortisol to cortisol exhibited noteworthy diurnal variation intra-individually; 2.8-fold on average. However, day-do-day intra-individual variation of the ratio was not observed over 2 weeks; the coefficient of variation was 11.9 +/- 3.0%. CONCLUSION: The result indicates that imprudent use of random urine has a great risk of false evaluation in assessment of the 6beta-hydroxycortisol to cortisol ratio and that the ratio in 24-h urine samples provides a more robust measure of the inter-individual difference of this metabolic ratio, which to a certain but not complete extent represents the CYP3A activity.

Adult↗

PubMedDate not supplied

Histopathological study on cerebellar dysgenesis of shaking rat Kawasaki (SRK).

The cerebellum of shaking rat Kawasaki (SRK) was studied histochemically and immunocytochemically. The cerebellar cortex was characterized by a delay in the disappearance of the external granular layer, a narrow molecular layer, a narrow and cell sparse internal granular layer, and disarranged and heterotopically situated Purkinje cells with tortuous arborization and a large cell cluster in the depths of the cerebellum. Golgi-Cox staining revealed an abnormal ramification and polarity of ectopic Purkinje cells. Ultrastructurally, most spines of Purkinje cells in the depths remained naked. Based on these results, it is suggested that the genetically determined mechanisms responsible for the abnormal structure in the cerebellar cortex of SRK may include a migratory disorder of the Purkinje cells, and a decrease in the microneurons with a resulting decrease of synaptic contacts on the Purkinje cell soma and dendrite.

Animals↗

PubMedDate not supplied

Magnetic resonance imaging of intracranial venous angiomas.

Magnetic resonance imagings (MRI) were made of intracranial venous angioma (six angiographically proved, four presumed). All draining veins were identified as a linear or a small round structure with a flow void. The stellate configuration was observed in seven of the ten patients. In two of the ten, the associated intraparenchymal hematoma was evident. Increased intensity of adjacent parenchyma on T2-weighted images was detected in four of ten patients, and a decreased intensity on the T1-weighted images was noted in three of eight. Thus, MRI is a pertinent diagnostic modality for evaluating intracranial venous angioma. Angiography does not seem to be required for confirmation in patients with typical MR findings.

Brain↗

PubMedDate not supplied

MR imaging of middle cerebral artery occlusion without cerebral infarction.

Magnetic resonance (MR) images of 12 patients with angiographically proven middle cerebral artery (MCA) occlusion were analyzed, retrospectively. In three of the 12 patients, cerebral infarctions related to the MCA occlusions were not evident. Two of the three patients were cases of atherosclerotic occlusion and the remaining patient had an acute thromboembolism. In all of the occluded M1 portions of the MCA the flow void was absent and there were isointense linear structures, with or without a hyperintense component in the Sylvian vallecula, on T1-weighted images. For nine of the 12 patients, the absence of flow void in the ipsilateral Sylvian fissure was evident on the T2-weighted images. Therefore, even in cases with no evidence of a cerebral infarction, the presence of flow void in the Sylvian vallecula and Sylvian fissure must be searched for in routine reviews of MR images. If MR imaging can be obtained on an emergency basis, appropriate interventional therapy may be immediately initiated.

Adult↗

PubMedDate not supplied

CT and MRI manifestations of primary pulmonary plasmacytoma.

Primary pulmonary plasmacytoma (PPP) is rare and its radiographic appearances have been seldom described. We report on a patient with PPP presenting with a hilar mass on chest radiograph. The diagnosis was confirmed by immunohistochemical study of the resected specimen. The computed tomographic and magnetic resonance imaging findings are presented.

Contrast Media↗

PubMedDate not supplied

Accelerated evolution of snake venom phospholipase A2 isozymes for acquisition of diverse physiological functions.

The nucleotide sequences of two cDNAs and four genes encoding Trimeresurus gramineus venom gland phospholipase A2 (PLA2) isozymes were determined and compared internally and externally with those encoding Trimeresurus flavoviridis venom gland PLA2 isozymes. It was revealed that the protein-coding regions are much more diversified than the 5' and 3' untranslated regions (UTRs) and the introns except for the signal peptide domain. The numbers of nucleotide substitutions per site (KN) for the UTRs and the introns were approximately one-quarter of the numbers of nucleotide substitutions per synonymous site (KS) for the protein-coding regions and were at the same level as the KN value of T. gramineus and T. flavoviridis TATA box-binding protein (TBP) genes, indicating that the protein-coding regions of PLA2 isozyme genes are unusually variable and that the UTRs including the introns of venom gland PLA2 isozyme genes have evolved at similar rate to those of non-venomous genes. The numbers of nucleotide substitutions per non-synonymous site (KA) values were close to or larger than the KS values for the protein-coding regions in venom gland PLA2 isozyme genes, indicating that the protein-coding regions of snake venom gland PLA2 isozyme genes have evolved via accelerated evolution. Furthermore, the evolutionary trees derived from the combined sequences of the 5' and 3' UTRs and the signal peptide domain of cDNAs were in accord with the consequences from taxonomy. In contrast, the evolutionary trees from the mature protein-coding region sequences of cDNAs and from the amino acid sequences showed random patterns. Estimations of nucleotide divergence of genes and the phylogenetic analysis reveal that snake venom group IJ PLA2 isozyme genes have been evolving under adaptive pressure to acquire new physiological activities.

Amino Acid Sequence↗

PubMedDate not supplied

Noradrenergic DSP-4 lesions aggravate impairment of working memory produced by hippocampal muscarinic blockade in rats.

To clarify the interactions between hippocampal cholinergic and adrenergic systems in working memory function of rats, the effects of hippocampal muscarinic receptor blockade combined with noradrenaline depletion on this behavior were examined with a three-panel runway task. Intrahippocampal administration of the muscarinic receptor antagonist scopolamine at a dose of 3.2 micrograms/side significantly increased the number of errors (attempts to pass through two incorrect panels of the three panel-gates at four choice points) in the working memory task, whereas the 0.32 microgram/side dose of scopolamine did not affect working memory errors. Administration of the noradrenergic neurotoxin N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4) at 50 mg/kg IP caused a marked reduction in hippocampal noradrenaline concentration, but it had no effect on working memory errors. Intrahippocampal administration of 0.32 microgram/side scopolamine, the behaviorally ineffective dose in intact rats, significantly increased the number of working memory errors in the noradrenaline-depleted animals. These results suggest that hippocampal muscarinic/noradrenergic interactions are involved in neural processes mediating working memory function of rats.

Animals↗