The molecular mechanism of the termination of insect diapause, part 1: A timer protein, TIME-EA4, in the diapause eggs of the silkworm Bombyx mori is a metallo-glycoprotein.

TIME-EA4 is an ATPase that measures time intervals, functioning as a diapause duration clock in diapause eggs of the silkworm, Bombyx mori. Characterization of the primary and higher structures of the TIME-EA4 would be desirable to clarify the mechanism by which the protein measures the time intervals. In our current studies, the whole sequence of TIME-EA4 has been established as that of a metallo-glycoprotein by combinational means involving peptide sequence analysis, nano-HPLC-ESI-Q-TOF-MS and MS/MS, and cDNA dictation.

Determination of a sugar chain and its linkage site on a glycoprotein TIME-EA4 from silkworm diapause eggs by means of LC-ESI-Q-TOF-MS and MS/MS.

The electrospray ionization (ESI)-tandem quadrupole/orthogonal-acceleration time-of-flight (Q-TOF) mass spectrometer combined with the nano-HPLC system was utilized to determine the glycosylation site and the glycan structure in glycoprotein TIME-EA4 (EA4) from Bombyx diapause eggs. LC-MS analysis of EA4 and deglycosylated EA4 indicated that the carbohydrate moiety of EA4 has the mass of 730.58 Da.

Extensive investigations on oxidized amino acid residues in H(2)O(2)-treated Cu,Zn-SOd protein with LC-ESI-Q-TOF-MS, MS/MS for the determination of the copper-binding site.

The ESI (electrospray ionization)-Q-TOF (tandem quadrupole/orthogonal-acceleration time-of-flight) mass spectrometer combined with the nano-HPLC (high performance liquid chromatography) system was utilized to pinpoint the Cu-binding site in Cu,Zn-SOD (superoxide dismutase) protein. Cu,Zn-SOD was treated with hydrogen peroxide, intended to specifically oxidize histidine residues coordinated to the copper ion as a mass spectrometric probe. The oxidized Cu,Zn-SOD was then fragmented with the successive treatment of endoproteinase Asp-N and DTT (dithiothreitol).

7,8-Dihydropterin-6-carboxylic acid as light emitter of luminous millipede, Luminodesmus sequoiae.

A luminous millipede. Luminodesmus sequoiae, emits light centered at a wavelength of 500 nm. To determine the light emitter of this bioluminescent system, fluorescent compounds were isolated from pulverized cuticles.

Elucidation of high micro-heterogeneity of an acidic-neutral trichotoxin mixture from Trichoderma harzianum by electrospray ionization quadrupole time-of-flight mass spectrometry.

The high micro-heterogeneity of an acidic-neutral trichotoxin mixture from T. harzianum, PC01, was elucidated using a modern tandem mass spectrometer equipped with an electrospray ionization source, a hybrid quadrupole-orthogonal accelerator and a reflectron time-of-flight analyzer. The trichotoxins appeared predominantly in six possible doubly charged pseudo molecular ions with three different adducts (H, Na and K) as [M + 2H](2+), [M + H + Na](2+), [M + H + K](2+), [M + 2Na](2+), [M + Na + K](2+) and [M + 2K](2+).

Carbohydrate moiety of time-interval measuring enzyme regulates time measurement through Its interaction with time-holding peptide PIN.

An ATPase called EA4 seems to measure time as a diapause-duration timer in the seasonal cycle of the silkworm, Bombyx mori. A peptide named PIN seems to regulate the time measurement of EA4. We characterize the EA4 as the first step to analyse its interaction with PIN.

High incorporation of L-amino acids to cereulide, an emetic toxin from Bacillus cereus.

Cereulide is a principal toxin causing emetic syndrome produced by Bacillus cereus. This paper deals with biosynthetic studies on this unusual cyclic depsipeptide toxin from 13C labeled L-amino acid precursors (Val, Leu, Ala) upon cultivation in synthetic media. The analyses were made at atomic level of the constituent amino- or oxy-acids through NMR and ESI-MS/MS spectroscopic methods on cereulide and its hydrolysate dipeptides.

Silkworm diapause hormone, structure-activity relationships indispensable role of C-terminal amide.

To determine the structure-activity relationships of the silkworm diapause hormone, a series of peptide analogs having different chain lengths starting from the parent C-terminus and analogs having identical sequences with free acid C-termini were chemically synthesized by solid-phase Fmoc methodology and were further purified by HPLC. Bioassay showed that the analogs with free acid C-termini were non active. The retained activities of those shorter chains were shown only with the amidated C-terminal analogs among which the potency depended on the length of the chain.

A novel dodecadepsipeptide, cereulide, isolated from Bacillus cereus causes vacuole formation in HEp-2 cells.

A HEp-2 cell-vacuolation factor was extracted and purified from the culture supernatant of a Bacillus cereus strain which caused emetic-syndrome food poisoning. The final preparation was chemically pure, and the toxin was named as cereulide. Mass spectrometry, NMR studies and chemical degradation revealed that the cereulide is a cyclic dodecadepsipeptide, (D-O-Leu-D-Ala-L-O-Val-L-Val)3, which is closely related to the potassium ionophore, valinomycin.

Plasma amino acid patterns in normal Thais and in patients with chronic renal failure.

The effect of chronic renal failure (CRF) on the pattern of plasma free amino acid concentrations was studied in 22 healthy controls (group 1); 43 CRF patients of which serum creatinine levels were 2-4.9 mg/dl (group 2, n = 11), 5-10 mg/dl (group 3, n = 10), more than 10 mg/dl (group 4, n = 9), and chronically hemodialysed patients (group 5, n = 13). In all renal failure groups, plasma concentrations of eight free essential amino acids-isoleucine, leucine, lysine, methionine, threonine, tryptophan, tyrosine and valine and those of three non-essential amino acids-alanine, glutamate and serine were significantly lower than those in controls.