Anoplin, a novel antimicrobial peptide from the venom of the solitary wasp Anoplius samariensis.

A novel antimicrobial peptide, anoplin, was purified from the venom of the solitary wasp Anoplius samariensis. The sequence was mostly analyzed by mass spectrometry, which was corroborated by solid-phase synthesis. Anoplin, composed of 10 amino acid residues, Gly-Leu-Leu-Lys-Arg-Ile-Lys-Thr-Leu-Leu-NH2, has a high homology to crabrolin and mastoparan-X, the mast cell degranulating peptides from social wasp venoms, and, therefore, can be predicted to adopt an amphipathic alpha-helix secondary structure. In fact, the circular dichroism (CD) spectra of anoplin in the presence of trifluoroethanol or sodium dodecyl sulfate showed a high content, up to 55%, of the alpha-helical conformation. A modeling study of anoplin based on its homology to mastoparan-X supported the CD results. Biological evaluation using the synthetic peptide revealed that this peptide exhibited potent activity in stimulating degranulation from rat peritoneal mast cells and broad-spectrum antimicrobial activity against both Gram-positive and Gram-negative bacteria. Therefore, this is the first antimicrobial component to be found in the solitary wasp venom and it may play a key role in preventing potential infection by microorganisms during prey consumption by their larvae. Moreover, this peptide is the smallest among the linear alpha-helical antimicrobial peptides hitherto found in nature, which is advantageous for chemical manipulation and medical application.

DNA sequencing using biotinylated dideoxynucleotides and mass spectrometry.

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MS) has been explored widely for DNA sequencing. The major requirement for this method is that the DNA sequencing fragments must be free from alkaline and alkaline earth salts as well as other contaminants for accurately measuring the masses of the DNA fragments. We report here the development of a novel MS DNA sequencing method that generates Sanger-sequencing fragments in one tube using biotinylated dideoxynucleotides. The DNA sequencing fragments that carry a biotin at the 3'-end are made free from salts and other components in the sequencing reaction by capture with streptavidin-coated magnetic beads. Only correctly terminated biotinylated DNA fragments are subsequently released and loaded onto a mass spectrometer to obtain accurate DNA sequencing data. Compared with gel electrophoresis-based sequencing systems, MS produces a very high resolution of DNA-sequencing fragments, fast separation on microsecond time scales, and completely eliminates the compressions associated with gel electrophoresis. The high resolution of MS allows accurate mutation and heterozygote detection. This optimized solid-phase DNA-sequencing chemistry plus future improvements in detector sensitivity for large DNA fragments in MS instrumentation will further improve MS for DNA sequencing.

Conflicting actions of parathyroid hormone-related protein and serum calcium as regulators of 25-hydroxyvitamin D(3)-1 alpha-hydroxylase expression in a nude rat model of humoral hypercalcemia of malignancy.

In patients with humoral hypercalcemia of malignancy (HHM), serum levels of 1,25-dihydroxyvitamin D (1,25(OH)(2)D) are generally low, although the pathophysiology of the impaired vitamin D metabolism is not fully understood. In the present study, we have investigated vitamin D metabolism in our newly developed rat model of HHM in which a human infantile fibrosarcoma producing parathyroid hormone-related protein (PTHrP), named OMC-1, was inoculated s.c. into athymic nude rats. In OMC-1-bearing rats, the serum concentration of 1,25(OH)(2)D was markedly reduced when the animals exhibited severe hypercalcemia (Ca > or =15 mg/dl), while it was rather elevated in those with mild hypercalcemia. To further examine whether serum Ca levels affect 1,25(OH)(2)D concentration, we administered bisphosphonate YM529 to OMC-1-bearing rats when they exhibited severe hypercalcemia. The restoration of the serum Ca level by administration of YM529 was accompanied by a marked increase in the 1,25(OH)(2)D level, suggesting that the serum Ca level itself plays an important role in the regulation of the 1,25(OH)(2)D level in these rats. On the other hand, when the OMC-1-bearing rats were treated with a neutralizing antibody against PTHrP, serum 1,25(OH)(2)D levels remained low despite the reduction in serum Ca levels. Expression of 25-hydroxyvitamin D-1 alpha-hydroxylase (1 alpha-hydroxylase) in kidney was decreased in OMC-1-bearing rats with severe hypercalcemia, and markedly enhanced after treatment with bisphosphonate. This enhancement in 1 alpha-hydroxylase expression was not observed after treatment with the antibody against PTHrP. These results suggest that PTHrP was responsible for the enhanced expression of 1 alpha-hydroxylase in YM529-treated rats, and that hypercalcemia played a role in reducing the serum 1,25(OH)(2)D level in OMC-1-bearing rats by suppressing the PTHrP-induced expression of the 1 alpha-hydroxylase gene.

Isolation and sequence determination of peptides in the venom of the spider wasp (Cyphononyx dorsalis) guided by matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry.

Micro-scale (sub-pmol) isolation and sequence determination of three peptides from the venom of the solitary spider wasp Cyphononyx dorsalis is described. We isolated two novel peptides Cd-125 and Cd-146 and a known peptide Thr(6)-bradykinin from only two venom sacs of solitary spider wasp Cyphononyx dorsalis without bioassay-guided fractionation, but instead guided by MALDI-TOF MS. The MALDI-TOF MS analysis of each fraction showed the purity and molecular weight of the components, which led to the isolation of the peptides virtually without loss of sample amount. The sequences of the novel peptides Cd-125 (Asp-Thr-Ala-Arg-Leu-Lys-Trp-His) and Cd-146 (Ser-Glu-Thr-Gly-Asn-Thr-Val-Thr-Val-Lys-Gly-Phe-Ser-Pro-Leu-Arg) were determined by Edman degradation together with mass spectrometry, and finally corroborated by solid-phase synthesis. The known peptide Thr(6)-bradykinin (Arg-Pro-Pro-Gly-Phe-Thr-Pro-Phe-Arg) was identified by comparison with the synthetic authentic specimen. This is the first example for any kinins to be found in Pompilidae wasp venoms. The procedure reported here can be applicable to studies on many other components of solitary wasp venoms with limited sample availability.

Nuclear transfer in cattle using colostrum-derived mammary gland epithelial cells and ear-derived fibroblast cells.

To assess the developmental potential of nuclear transfer embryos in cattle using mammary gland epithelial (MGE) cells derived from the colostrum, we compared the effectiveness of cloning using those cells and fibroblast cells derived from the ear. The fusion rate of the enucleated oocytes with fibroblast cells (75 +/- 4%) was significantly higher than that with MGE cells (56 +/- 7%, P<0.05). There were no significant differences in the cleavage rate (85 +/- 3% vs. 91+/- 2%) or in the developmental rate to the blastocyst stage (35 +/- 6% vs. 35 +/- 5%) using MGE cells vs. fibroblast cells as donor nuclei (P>0.05). After transfer of blastocysts derived from nuclear transfer embryos produced using MGE cells and fibroblast cells, 13% (4/31) and 16% (6/37) of recipient heifers were pregnant on Day 42 as assessed by ultrasonography, respectively. Two of the 4 and 4 of the 6 recipients of embryos with MGE cell- and fibroblast cell-derived nuclei, respectively, aborted within 150 days of pregnancy. Four live female calves were obtained from MGE cells or fibroblast cells. However, one died from internal hemorrhage of the arteria umbilicalis. The other three calves were normal and healthy. There were no differences in the pregnancy rate or calving rate when using MGE cells vs. fibroblast cells. Microsatellite DNA analyses confirmed that the cloned calves were genetically identical to the donor cows and different from the recipient heifers. We conclude that colostrum-derived MGE cells have the developmental potential to term by nuclear transfer, and the efficiency of development of those cloned embryos was the same as that of embryos obtained using fibroblast cells as donor nuclei, although there was a significant difference in the fusion rate. This method using MGE cells derived from colostrum, which is obtained easily and safely from live adult cows, is more advantageous for cloning with somatic cells.

Advantages of using nested collision induced dissociation/post-source decay with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry: sequencing of novel peptides from wasp venom.

We have examined the applicability of the 'nested' collision induced dissociation/post-source decay (CID/PSD) method to the sequencing of novel peptides from solitary wasps which have neurotoxic venom for paralyzing other insects. The CID/PSD spectrum of a ladder peptide derived from an exopeptidase digest was compared with that of the intact peptide. The mass peaks observed only in the CID/PSD spectrum of a ladder peptide were extracted as C-terminal fragment ions. Assignment of C-terminal fragment ions enabled calculation of N-terminal fragment masses, leading to differentiation between N-terminal fragment ions and internal fragment ions. This methodology allowed rapid and sensitive identification by removing ambiguity in the assignment of the fragment ions, and proved useful for sequencing unknown peptides, in particular those available as natural products with a limited supply.

The biosynthesis of A2E, a fluorophore of aging retina, involves the formation of the precursor, A2-PE, in the photoreceptor outer segment membrane.

The autofluorescent lipofuscin that accumulates in retinal pigment epithelial cells with age may contribute to an age-related decline in cell function. The major lipofuscin fluorophore, A2E, is a pyridinium bisretinoid. We previously proposed that the biogenesis of A2E involves the following: (i) formation of the Schiff base, N-retinylidene phosphatidylethanolamine from all-trans-retinal and phosphatidylethanolamine in the photoreceptor outer segment membrane; (ii) further reaction of N-retinylidene phosphatidylethanolamine with retinal to yield phosphatidylethanolamine-bisretinoid, A2-PE; (iii) hydrolysis of A2-PE to generate A2E. To provide evidence for this biogenic scheme, all-trans-retinal was reacted with dipalmitoyl-l-alpha-phosphatidylethanolamine to yield DP-A2-PE (A2-PE), as confirmed by UV, with mass spectrometry revealing the molecular ion at m/z 1222.9 (C(77)H(124)O(8)PN) accompanied by product ion at m/z 672.8, representing the phosphoryl-A2E fragment of A2-PE. In reaction mixtures of retinal and outer segments and in samples of Royal College of Surgeons rat retina containing outer segment membranous debris, A2-PE was detected as a series of high performance liquid chromatography peaks, each with UV similar to reference A2-PE. By mass spectrometry, A2-PE consisted of multiple peaks, representing fatty acids with different chain lengths, and the phosphoryl-A2E moiety, m/z 673. Incubation of the retinal/outer segment reaction mixture with phospholipase D generated A2E, as detected by high performance liquid chromatography, thus confirming A2-PE as the A2E precursor.

Sexing of porcine embryo by in situ hybridization using chromosome Y- and 1-specific DNA probes.

This study was carried out to determine if a rapid, simultaneous detection system using chromosome Y- and 1-bearing boar spermatozoa was applicable for sexing embryos. Porcine embryos were recovered from gilts and sows 4 to 6 d after mating, and whole embryos or biopsy cells were mounted on a glass slide with a small amount of fixative (methanol: acetic acid: distilled water = 9:1:4). The samples were then stained by means of a fluorescence in situ hybridization (FISH) procedure developed specifically for the detection of Y-bearing spermatozoa. Hybridization was performed using digoxigenin (dig)-labeled chromosome Y- specific DNA, and biotin-labeled chromosome 1-specific DNA sequences were detected as a signal of FITC and Texas Red on nucleus visualized DAPI-stain. Proportions of whole embryos labeled with chromosome 1-probe were 17 and 97% at the 3 to 16 and > or = 32 cell stage, respectively. Of the 93 biopsied embryos analyzed by FISH, 85 embryos (91%) could be accurately classified as male or female. Of the 65 biopsied embryos, 60 embryos (92%) had a clear blastocoele and a inner cell mass after 48 h of culture in vitro, and these embryos were evaluated as available embryos. One out of 4 recipient gilts which received sexed embryos at transfer farrowed 12 piglets of the expected sex. The results of this study demonstrated that porcine embryos at the > or = 32 cell stage can be sexed within 2 h using the FISH method. Moreover further development of the FISH technique could make it an effective tool for the study of early porcine embryos and for the control of porcine sex.

Molecular determinants of binding of a wasp toxin (PMTXs) and its analogs in the Na+ channels proteins.

The structural specificity of alpha-PMTX, a novel peptide toxin derived from wasp venom has been studied on the neuromuscular synapse in the walking leg of the lobster. alpha-PMTX is known to induce repetitive action potentials in the presynaptic axon due to sodium channel inactivation. We synthesized 29 analogs of alpha-PMTX by substituting one or two amino acids and compared threshold concentrations of these mutant toxins for inducing repetitive action potentials. In 13 amino acid residues of alpha-PMTX, Arg-1, Lys-3 and Lys-12 regulate the toxic activity because substitution of these basic amino acid residues with other amino acid residues greatly changed the potency. Determining the structure-activity relationships of PMTXs will help clarifying the molecular mechanism of sodium channel inactivation.

Structure and biological activities of eumenine mastoparan-AF (EMP-AF), a new mast cell degranulating peptide in the venom of the solitary wasp (Anterhynchium flavomarginatum micado).

A new mast cell degranulating peptide, eumenine mastoparan-AF (EMP-AF), was isolated from the venom of the solitary wasp Anterhynchium flavomarginatum micado, the most common eumenine wasp found in Japan. The structure was analyzed by FAB-MS/MS together with Edman degradation, which was corroborated by solid-phase synthesis. The sequence of EMP-AF, Ile-Asn-Leu-Leu-Lys-Ile-Ala-Lys-Gly-Ile-Ile-Lys-Ser-Leu-NH(2), was similar to that of mastoparan, a mast cell degranulating peptide from a hornet venom; tetradecapeptide with C-terminus amidated and rich in hydrophobic and basic amino acids. In fact, EMP-AF exhibited similar activity to mastoparan in stimulating degranulation from rat peritoneal mast cells and RBL-2H3 cells. It also showed significant hemolytic activity in human erythrocytes. Therefore, this is the first example that a mast cell degranulating peptide is found in the solitary wasp venom. Besides the degranulation and hemolytic activity, EMP-AF also affects on neuromuscular transmission in the lobster walking leg preparation. Three analogs EMP-AF-1 approximately 3 were snythesized and biologically tested together with EMP-AF, resulting in the importance of the C-terminal amide structure for biological activities.

Squamous cell carcinoma arising in a mature teratoma with metastasis to the urinary bladder.

We describe a patient with squamous cell carcinoma arising in a mature teratoma. Magnetic resonance (MR) images revealed a solid lobulated mass attached to the ovarian cyst containing a fat-fluid level. The solid component with extension into pelvic fat showed as hypointensity on T2-weighted MR images with good enhancement. A metastatic tumor to the urinary bladder was also demonstrated.

Nonconvulsive status epilepticus in a child with congenital bilateral perisylvian syndrome.

A 9-year-old male with congenital bilateral perisylvian syndrome is described. He had pseudobulbar palsy, mental retardation, and intractable epilepsy. Computed tomography and magnetic resonance images of the brain demonstrated bilateral perisylvian malformations and a diffuse pachygyric appearance. At 8 years of age, he had episodes of excessive drooling, fluctuating impairment of consciousness, unsteady sitting, and frequent head drop that lasted several days. The electroencephalogram demonstrated continuous diffuse slow spike and waves. These findings suggested atypical absence status epilepticus. Intravenous administration of diazepam resulted in transient improvement of clinical and electroencephalographic findings. Status epilepticus recurred within several minutes after diazepam administration. Although no patient has been reported to have a history of status epilepticus among those affected by this syndrome, it seems that atypical absence status can occur more frequently than expected, as seen in Lennox-Gastaut syndrome. After recognition and confirmation of nonconvulsive status epilepticus, immediate treatment must be attempted.

Usefulness of dual SPECT with Tc-99m pyrophosphate and Tl-201 to predict further events after acute myocardial infarction with single-vessel coronary artery disease.

PURPOSE: This study was undertaken to determine whether the findings of dual SPECT with Tc-99m pyrophosphate (PYP) and Tl-201 were predictive of further cardiac events after acute myocardial infarction. METHODS: The authors evaluated 88 patients with acute myocardial infarction who underwent dual SPECT for single-vessel coronary artery disease. RESULTS: Twenty-nine patients showed overlapping of Tc-99m PYP and Tl-201 in the same location (overlap-positive group), and 59 patients had no overlap (overlap-negative group). In patients in the overlap-positive group, the incidence of subsequent events was significantly higher than in patients in the overlap-negative group (P < 0.001). In the overlap-positive group, the number of overlap segments in patients with further events was significantly greater than that in patients without further events (P < 0.005). CONCLUSIONS: Areas with overlapping of Tc-99m PYP and Tl-201 may contain jeopardized myocardium. These results suggest that patients who have a Tc-99m PYP and Tl-201 overlap-negative scan are a low risk group, whereas patients who have more overlapping segments may require catheterization and revascularization. Thus simultaneous SPECT imaging with Tc-99m PYP and Tl-201 might be useful to identify patients with greater ischemic risk after acute myocardial infarction.

On the structure of endogenous ouabain.

The ouabain-like sodium pump inhibitor in mammals (so-called "endogenous ouabain") has been considered a subtle structural isomer of ouabain. Its structural investigation, however, has long been hindered by the paucity of sample material. Our recent purification of endogenous ouabain (3 micrograms) from bovine hypothalamus allowed the measurement of its 1H-NMR. The obtained spectrum as well as reexamination of past microscale structural studies on endogenous ouabain led us to identify the purified material as ouabain in an unusual manner. It turned out that the structural analysis had been complicated by a facile ouabain-borate complexation in borosilicate glassware. In retrospect, it is not surprising that the polyhydroxylated ouabain molecule serves as a polydentate ligand to inorganic species. In its physiological environment, ouabain may exist as some unknown complex. The chemical species giving rise to the reported biological activities of hypothalamic inhibitory factor preparations remain to be clarified.

Enhancement of antitumor effect of hyperthermia with glucose administration in murine mammary carcinoma.

Antitumor effects of hyperthermia are enhanced by lowering the pH in the tumor tissue with administration of glucose. This decreased pH in the tumor tissue with glucose administration was determined using mouse experimental tumors. 31P-MRS microelectrodes were used for the measurement of pH. By using these two measurement methods, time course change in the tumor tissue was determined in the controls and the groups treated with 6 g/kg of intraperitoneal glucose. The determination of pH with 31P-MRS was calculated from the chemical shift of the peak of creatine phosphate (Pcr) and that of inorganic phosphate (Pi). Following glucose administration, the tumor tissue showed a decrease of 0.3 pH units with the microelectrode method, but did not show any significant decrease in pH with the MRS determination. This finding suggested that 31P-MRS showed intracellular pH (pHi) due to the localization of Pi and that the microelectrode indicated interstitial or extracellular pH (pHe). The ATP/Pi ratio obtained in tumor tissue 24 h after heat treatment (with, without glucose) was correlated with tumor inhibition.

In-111 labeled leukocyte scintigraphy in patients with suspected inflammation after failed antibiotic therapy.

In-111 labeled leukocyte scintigraphy (In-111 WBC scan) was performed in 16 patients with inflammation suspected on the basis of laboratory findings, symptoms, and diagnostic imaging, but who had failed antibiotic therapy. In-111 WBC scans revealed an abnormal focus of radiotracer activity (positive scans) in five of 16 patients. No correlation was found between the peripheral WBC count and accumulation of In-111 WBC. Inflammatory disease suspected on the basis of the CRP level should be considered when In-111 WBC scanning results in negative findings. Our results indicated that In-111 WBC scanning has low sensitivity after antibiotic therapy. Selection of patients on the basis of persistent elevation of CRP may be valuable.

Isolation and structure of pompilidotoxins, novel peptide neurotoxins in solitary wasp venoms.

Novel peptide neurotoxins, alpha- and beta-pompilidotoxins (alpha- and beta-PMTXs), were purified from the venoms of the solitary wasps Anoplius samariensis and Batozonellus maculifrons. Their structures were analyzed mostly by MALDI-TOF-MS, which were corroborated by solid-phase synthesis. alpha-PMTX, with 13 amino acid residues and the sequence of Arg-Ile-Lys-Ile-Gly-Leu-Phe-Gln-Asp-Leu-Ser-Lys-Leu-NH2, greatly potentiates synaptic transmission of lobster leg muscle by the presynaptic mechanisms. beta-PMTX, in which the lysine residue at 12 position of alpha-PMTX was replaced with arginine, was more potent than alpha-PMTX.