Prepubertal exposure to T-2 toxin advances pubertal onset and development in female rats via promoting the onset of hypothalamic-pituitary-gonadal axis function.

T-2 toxin, a naturally produced Type A trichothecene mycotoxin, has been shown to damage the reproductive and developmental functions in livestocks. However, whether T-2 toxin can disturb the pubertal onset and development following prepubertal exposure remains unclear. To clarify this point, infantile female Sprague-Dawley rats were given a daily intragastric administration of vehicle or T-2 toxin at a dose of 375 µg/kg body weight for 5 consecutive days from postnatal day (PND) 15-19 (PND15-PND19). The days of vaginal opening, first diestrus, and first estrus in regular estrous cycle were advanced following T-2 toxin treatment, indicating prepubertal exposure to T-2 toxin induced the advancement of puberty onset. The relative weights of uterus and ovaries and the incidence of corpora lutea were all increased in T-2 toxin-treated rats; serum hormone levels of luteinizing hormone and estradiol and the messenger RNA expressions of gonadotropin-releasing hormone (GnRH) and GnRH receptor also displayed marked increases following exposure to T-2 toxin, all of which were well consistent with the manifestations of the advanced puberty onset. In conclusion, the present study reveals that prepubertal exposure to a high level of T-2 toxin promotes puberty onset in infantile female rats by advancing the initiation of hypothalamic-pituitary-gonadal axis function in female rats.

Constitutive expression of human angiostatin in Pichia pastoris by high-density cell culture.

A high-density cell culture method to produce human angiostatin has been successfully established by constitutive expression of the protein in Pichia pastoris. The fermentation was carried out in a 20 l bioreactor with a 10 l working volume, using a high-density cell culture method by continuously feeding with 50% glycerol-0.8% PTM4 to the growing culture for 60 h at 30 degrees C. Dissolved oxygen level was maintained at 25-30% and pH was controlled at 5 by the addition of 7 M NH4OH. Angiostatin was constitutively expressed during the fermentation by linking its expression to the P. pastoris constitutive GAP promoter (pGAP). But after 36 h of fermentation, the peak biomass growth was 305 as measured by absorption of 600 nm, while the peak angiostatin expression was 176 mg/l. Similar to the product expressed from inducible system [24], angiostatin produced from constitutive system also inhibited the angiogenesis on the CAM and suppressed the growth of B16 melanoma in C57BL/6J mouse. The above results suggest that GAP promoter is more efficient than AOX1 promoter for the expression of angiostatin in P. pastoris by shake flask culture or high-density cell fermentation and is likely to be an alternative to AOX1 promoter in large-scale expression of angiostatin and other heterologous proteins.

[Inhibition of platelet function by L-arginine.L-aspartate salt].

The effects of L-arginine.L-aspartate salt (DR) on platelet aggregation, adhesion and release were investigated. Platelet aggregation induced by adenosine 5 -diphosphate (ADP) was significantly inhibited (P<0.01) by intravenous injection of DR (15 mg/kg) in rats or oral administration (15 mg/kg) in rabbits, the inhibitory effect on rabbit platelet aggregation lasting for more than 8 h (P<0.01). Platelet aggregation induced by ADP, collagen or thrombin in rats was all markedly inhibited by 7.5, 15 or 30 mg/kg of DR (bid for 3.5 d, ig, P<0.01). Platelet adhesion to foreign objects was inhibited by 30 mg/kg of DR (ig). Bleeding time in rat tails was prolonged by 30 mg/kg of DR (P<0.05). Furthermore, PGI(2) released from the vascular wall was increased in DR-treated rats (P<0.05), however, TXA(2) released from platelets was not affected. These data demonstrate the inhibitory effect of DR on platelet function, suggesting that its action target may be different from that of acetylsalicylic acid, and that the increase of PGI(2) release may be responsible partly for this effect. It is suggested that DR may probably be used as a new agent for regulating platelet function.

Molecular mechanism of apoptosis induced by ricin in HeLa cells.

AIM: To study the morphological changes and molecular mechanism of HeLa cell apoptosis induced by ricin. METHODS: HeLa cells were coincubated with ricin 0.05 mumol.L-1 for 1, 2, 3, 6, 12, 18, and 24 h, then scanning electron microscopy (SEM), transmission electron microscopy (TEM), Western blot cell cycle, cell cytotoxicity, and cell viability were assayed. RESULTS: The typical apoptosis was induced by ricin 0.05 mumol.L-1 and necrotic cells increased after being cultured with ricin 0.05 mumol.L-1 for more than 12 h. The apoptotic cells mainly showed cytoplasmic membrane blebbing, chromatin condensation and fragmentation, and crescentic nuclear and membrane bound apoptotic bodies formation. No detectable levels of p53, Bax, Bcl-2 and the subunit p20 of interleukin-1 beta-converting enzyme (ICE) were found by Western blot, but the active subunit p17 of 32-kDa putative cysteine protease (CPP32) was detected at 3, 6, and 9 h after ricin treatment. The activity of CPP32 in HeLa cells increased 4 to 5 folds after being treated with ricin 0.05 mumol.L-1 and reached the peak at 6 h of treatment. There was no significant difference of ICE activity between the ricin treated cells and control cells. The percentage of G2/M cells increased from 13.9% +/- 0.5% to 33.2% +/- 0.5% after 24 h of ricin 0.05 mumol.L-1 treatment. CONCLUSION: CPP32 but not ICE was involved in the ricin-induced apoptosis in HeLa cells. Ricin 0.05 mumol.L-1 had no effect on the G0/G1 phase of cell cycle, but induced G2/M arrest.

Synthesis and characterization of the human elastin W4 sequence.

Following the nomenclature of Sandberg, the W4 sequence of human elastin, [sequence: see text], has been synthesized by solid-phase methods and characterized by carbon-13 nuclear magnetic resonance, amino-acid analysis, mass spectra and elemental analysis. This sequence was then polymerized to greater than 50 kDa as determined by retention in 50 kDa molecular weight cut-off dialysis tubing. It has been successfully cross-linked by gamma-irradiation (20 Mrad) to form an elastomeric matrix, designated as X20-poly(W4). Physical characterizations such as stress/strain, thermolelasticity, acid-base titration and inverse temperature transition studies have been carried out on this elastomer, which is homologous to the striking, poly(VPGVG), W4 sequence of bovine and porcine elastins. These results are compared with previous results on the polypentapeptide of elastin, (VPGVG)n, and it has been demonstrated that X20-poly(W4) also is a dominantly entropic elastomer. Finally, the working model for the structure of this human elastin sequence was derived computationally using molecular mechanics and dynamics calculations. Thus the human W4 sequence appears to be structurally and functionally equivalent to the bovine and porcine W4 sequences in spite of the less regular repeating pentamer sequence.

Electro-chemical transduction in elastic protein-based polymers: a model for an energy conversion step of oxidative phosphorylation.

A pair of functional moieties, the carboxyl of an aspartic acid (Asp, D) residue and an N-methyl nicotinamide (NMeN) formed on amide linkage to the epsilon-amino group of the lysine (Lys, K) residue, are coupled to perform energy conversion by means of controlling the transition temperature, Tt, of a common hydrophobic folding and assembly domain within the polytricosapeptide, poly[GDGFP GVGVP GVGVP GFGVP GVGVP GVGK(NMeN)P]. The input of electrochemical energy in the form of the reduction of nicotinamide results in a reduction-induced increase in pKa by 2.5 pH units which represents the performance of the chemical work of picking up a proton. The primary structure and the structures of the oxidized and reduced states are verified by two-dimensional nuclear magnetic resonance. Thus electrochemical transduction, the conversion of electrochemical energy into chemical energy, has been demonstrated for the first time in a designed, synthetic protein-based polymer.

Hydrophobicity-induced pK shifts in elastin protein-based polymers.

Three polypentapeptides--poly[0.8(GVGVP), 0.2(GEGVP)], poly[0.8(GVGIP), 0.2(GEGIP)], and poly[0.75(GFGVP), 0.25(GEGVP)]--all analogues of the polypentapeptide of elastin--(Val1-Pro2-Gly3-Val4-Gly5)n or poly(VPGVG)--have been prepared to determine the effect of changing the hydrophobicity, i.e., Val1----Ile1 (I) and Val4----Phe4 (F), on the pKa and the temperature dependence of pKa of the Glu (E) residue. Shifts in pKa as large as 1.7 units are observed and the temperature dependence is much steeper for the structure-dependent proximity of the more hydrophobic Ile1 residues to the Glu4 residue. Even though this system is dominated by the inverse temperature transition of hydrophobically driven folding on raising the temperature, the effect of adding 0.15 N NaCl is to suppress the hydrophobicity-induced pKa shift.

The synthesis of protective fragments of alpha-human atrial natriuretic polypeptide (alpha-hANP).

In this paper the synthesis of alpha-human atrial natriuretic polypeptide fragments (positions, 1-4, 5-10, 11-16, 17-22, 23-28) by solution method is reported. In the synthesis of some peptides, the results from mixed anhydride procedure and active ester procedure are compared. In the synthesis of other peptides, the effects of reaction temperature, basicity of catalyst, and reaction time on the specific rotations and yields of these compounds are observed. The sequence of amino acids of all the protective polypeptides is confirmed by FAB-MS.