N3-methyl-mafosfamide as a chemically stable, alternative prodrug of mafosfamide

The presence of an alkyl substituent at N3 in the oxazaphosphorine ring stabilizes N-substituted 4-(alkylthio)cyclophosphamides from spontaneous decomposition. Based on this finding, N3-methyl-mafosfamide was synthesized and examined as a chemically stable, biooxidative prodrug of mafosfamide. This prodrug was stable in aqueous buffer (pH 7.4, 37 degrees C) and underwent N-demethylation in a time dependent manner when incubated with rat hepatic microsomes. N3-Methyl-mafosfamide was 10-fold more cytotoxic in vitro than cyclophosphamide against mouse embryo Balb/c 3T3 cells (LC50 = 3.6 microM). Preliminary in vivo antitumor evaluation against L1210 leukemia in mice showed that this prodrug was active [Increase of life span (ILS) > 29%].     

Red cell volume determination using a stable isotope of chromium

OBJECTIVE: To validate and improve a method of red cell volume determination by use of a stable isotope of chromium. METHODS: Twelve subjects were sequentially injected with red blood cells labeled with a stable isotope of chromium (53Cr) and red blood cells labeled with radioisotopic chromium (51Cr). Measurement of 53Cr dilution was by gas chromatography/mass spectrometry. Measurement of 51Cr dilution was by gamma counter. RESULTS: Comparison of the two methods led to results that differed on average by 34.5 +/- 45.0 mL (1.8 +/- 2.2%), 0.3 to 3.2%, 95% confidence interval. CONCLUSION: Measurement of red cell volume by use of a stable isotope of chromium is accurate, with potential applications including measurement in pregnant women and children or other groups in whom exposure to radioisotopes is undesirable.     

Characterization of a human alpha1-antitrypsin variant that is as stable as ovalbumin

The metastability of inhibitory serpins (serine proteinase inhibitors) is thought to play a key role in the facile conformational switch and the insertion of the reactive center loop into the central beta-sheet, A-sheet, during the formation of a stable complex between a serpin and its target proteinase. We have examined the folding and inhibitory activity of a very stable variant of human alpha1-antitrypsin, a prototype inhibitory serpin. A combination of seven stabilizing single amino acid substitutions of alpha1-antitrypsin, designated Multi-7, increased the midpoint of the unfolding transition to almost that of ovalbumin, a non-inhibitory but more stable serpin. Compared with the wild-type alpha1-antitrypsin, Multi-7 retarded the opening of A-sheet significantly, as revealed by the retarded unfolding and latency conversion of the native state. Surprisingly, Multi-7 alpha1-antitrypsin could form a stable complex with a target elastase with the same kinetic parameters and the stoichiometry of inhibition as the wild type, indicating that enhanced A-sheet closure conferred by Multi-7 does not affect the complex formation. It may be that the stability increase of Multi-7 alpha1-antitrypsin is not sufficient to influence the rate of loop insertion during the complex formation.     

Examination of the compaction properties of a 1:1 acetaminophen:microcrystalline cellulose mixture using precompression and main compression

The compaction properties of a 1:1 acetaminophen and microcrystalline cellulose (MCC) mixture have been studied using a compaction simulator to make tablets by single compression and by a combination of precompression and main compression. The tensile strengths of the tablets and the energies involved in the compressions were determined. The tensile strengths of the tablets increased with increases in single compression pressure from 80 to 400 MPa and as the total applied pressure increased from 80 MPa up to around 400 MPa when combinations of precompression and main compression pressures were used. The tablet porosity decreased with increase in main compression pressure while the tablet tensile strengths increased. At minimum tablet porosity, further increase in main compression pressure could no longer result in increase in tablet strengths. Tablets compressed with combinations of precompression and main compression were stronger (2.15 +/- 0.02 to 3.99 +/- 0.1 MPa) than those produced with single compression (0.73 +/- 0.01 to 3.09 +/- 0.05 MPa). The total gross energies of compression increased with an increase in pressure of both the precompression and main compression. The elastic energies during main compression increased with an increase in precompression pressure as the tablet exhibited greater elastic deformation and reduced plasticity on second compression. The increase in elastic energies during main compression may also be because elastic energy is recoverable and is independent of precompression. As the precompression pressure increased, the minimum tablet porosity was attained; hence, the plastic energy during main compression became smaller while the elastic energy increased. Thus, a combination of low precompression and main compression pressures of 160/80 MPa or 80/160 MPa are more advantageous in the tableting of the 1:1 acetaminophen:MCC than a high single compression pressure of 320 or 400 MPa.     

稳定生石灰配比提高烧结矿质量

本文对二烧车间生石灰系统的搅笼改皮带前后的烧结矿质量进行了分析,着重说明了稳定生石灰配比是提高烧结矿质量的重要条件.     

Organophosphorus hydrolase is a remarkably stable enzyme that unfolds through a homodimeric intermediate

Organophosphorus hydrolase (OPH, EC 8.1.3.1) is a homodimeric enzyme that catalyzes the hydrolysis of organophosphorus pesticides and nerve agents. We have analyzed the urea- and guanidinium chloride-induced equilibrium unfolding of OPH as monitored by far-ultraviolet circular dichroism and intrinsic tryptophan fluorescence. These spectral methods, which monitor primarily the disruption of protein secondary structure and tertiary structure, respectively, reveal biphasic unfolding transitions with evidence for an intermediate form of OPH. By investigating the protein concentration dependence of the unfolding curves, it is clear that the second transition involves dissociation of the monomeric polypeptide chains and that the intermediate is clearly dimeric. The dimeric intermediate form of OPH is devoid of enzymatic activity, yet clearly behaves as a partially folded, dimeric protein by gel filtration. Therefore, we propose an unfolding mechanism in which the native dimer converts to an inactive, well-populated dimeric intermediate which finally dissociates and completely unfolds to individual monomeric polypeptides. The denaturant-induced unfolding data are described well by a three-state mechanism with delta G for the interconversion between the native homodimer (N2) and the inactive dimeric intermediate (I2) of 4.3 kcal/mol while the overall standard state stability of the native homodimer relative to the unfolded monomers (2U) is more than 40 kcal/mol. Thus, OPH is a remarkably stable protein that folds through an inactive, dimeric intermediate and will serve as a good model system for investigating the energetics of protein association and folding in a system where we can clearly resolve these two steps.     

Modifier theory of meiotic drive: is Mendelian segregation stable?

1. Certain sugars were transported across the buccal mucosa by a carrier-mediated mechanism. 2. The metabolic loss of sugars from the mouth in a 5 min test period was negligible. 3. The buccal mucosal transport process was stereospecific for D-glucose and L-arabinose. 4. The absorption of D-glucose, galactose and 3-O-methyl-D-glucose was at least partly dependent on the presence of sodium ions in the luminal fluid. 5. The transport of D-glucose, was inhibited by galactose and 3-O-methyl-D-glucose, suggesting at least one common carrier system.     

Prolonged tamoxifen exposure selects a breast cancer cell clone that is stable in vitro and in vivo

The effects of long-term tamoxifen exposure on cell growth and cell cycle kinetics were compared between oestrogen receptor (ER)-positive (MCF-7) and ER-negative (MDA-MB-231) cell lines. In the MCF-7 cell line, prolonged tamoxifen exposure (0.5 mumol/l for > 100 days) blocked cells in G0-G1 of the cell cycle, and slowed the doubling time of cells from 30 to 59 h. These effects corresponded to an increase in the cellular accumulation of tamoxifen over time [mean area under concentration curve (AUC) = 77.92 mumoles/10(6)/cells/day]. In contrast, in the MDA-MB-231 cell line, long-term tamoxifen exposure had no obvious effect on the doubling time, and reduced cellular tamoxifen accumulation (mean AUC = 50.50 mumoles/10(6)/cells/day) compared to the MCF-7 cells. Flow cytometric analysis of MDA-MB-231 cells demonstrated that a new tetraploid clone emerged following 56 days of tamoxifen exposure. Inoculation of the MDA-MB-231 tetraploid clone and MDA-MB-231 wildtype cells into the opposite flanks of athymic nude mice resulted in the rapid growth of tetraploid tumours. The tetraploid tumours maintained their ploidy following tamoxifen treatment for nine consecutive serial transplantations. Histological examination of the fifth transplant generation xenografts revealed that the tetraploid tumour had a 25-30 times greater mass, area of haemorrhage and necrosis, a slightly higher mitotic index and was more anaplastic than the control neoplasm. The control wildtype MDA-MB-231 tumours maintained a stable ploidy following tamoxifen treatment until the eighth and ninth transplantation, when a tetraploid population appeared, suggesting that tamoxifen treatment may select for this clone in vivo. These studies suggest that prolonged tamoxifen exposure may select for new, stable, fast growing cell clones in vitro as well as in vivo.     

An intercalated and thermally stable FAPY adduct of aflatoxin B1 in a DNA duplex: structural refinement from 1H NMR

The structure of a formamidopyrimidine (FAPY) adduct arising from imidazole ring opening of the initially formed trans-8, 9-dihydro-8-(N7-guanyl)-9-hydroxyaflatoxin B1 adduct under basic conditions and positioned in the 5'-d(CTATFAPYGATTCA)-3'*5'-d(TGAATCATAG)-3' oligodeoxynucleotide was determined. The FAPY adduct may be a major progenitor of aflatoxin B1-induced mutations in DNA. The freshly prepared sample showed biphasic melting, with transitions at 28 and 56 degreesC. NMR initially showed multiple subspectra. Over a period of several days at 4 degreesC, the sample converted to a single species with a Tm of 56 degreesC, 15 degrees C greater than the unmodified duplex. The deoxyribose was in the beta configuration about the anomeric carbon, evidenced by NOEs between FAPYG5 H3', H2', H2", and H1'. FAPY formation resulted in the loss of the guanine H8 proton, and the introduction of the formyl proton, which showed NOEs to FAPYG5 H1' and A6 N6Ha. A total of 31 NOEs from AFB1 to DNA protons were observed, mostly to the 5'-neighboring base, T4 in the modified strand. Sequential NOEs were interrupted between T4 and FAPYG5 in the modified strand, between C16 and A17 in the complementary strand, and between T4 N3H and FAPYG5 N1H. An NOE between FAPYG5 N1H and C16 N4H showed intact hydrogen bonding at FAPYG5*C16. Upfield chemical shifts were observed for T4 H6 and A17 H8. Molecular dynamics calculations converged with pairwise rmsd differences of <0.9 A. The sixth root residual was 8.7 x 10(-2). The AFB1 moiety intercalated from the major groove between FAPYG5 and T4*A17, and stacked with T4 and FAPYG5 and partially stacked with A17. The base step between T4*A17 and FAPYG5*C16 was increased from 3.4 to 7 A. The duplex unwound by about 15 degrees. The FAPY formyl group was positioned to form a hydrogen bond with A6 N6Ha. Strong stacking involving the AFB1 moiety, and this hydrogen bond explains the thermal stabilization of four base pairs by this adduct, and may be a significant factor in its processing.     

Cardiorespiratory and anesthetic effects of propofol and thiopental in dogs

OBJECTIVE: To compare cardiorespiratory and anesthesia effects of IV administered propofol and thiopental in dogs. ANIMALS: 6 healthy mixed-breed dogs. PROCEDURE: Each dog was anesthetized with isoflurane, then a thermistor catheter was inserted in the pulmonary artery. After a minimum of 2.5 hours of recovery, a catheter was placed in a cephalic vein for administration of lactated Ringer's solution and drugs. Propofol (8 mg/kg of body weight) or thiopental (19.4 mg/kg) was administered to each dog in a randomized crossover design study. All dogs were intubated and allowed to breathe 100% oxygen spontaneously. Heart rate and rhythm; systolic, diastolic, and mean arterial blood pressures; respiratory rate; end-tidal carbon dioxide concentration; tidal volume; and reflexes (toe web pinch, palpebral response, and jaw tone) were measured before and every 2 minutes for the first 10 minutes, then at 15, 30, and 60 minutes after drug administration. Cardiac output was determined at 0, 2, 6, 10, 15, 30, and 60 minutes, and blood samples were collected at 0, 2, 10, and 30 minutes. Time to endotracheal extubation, head lift, and ability to sit sternally and walk unaided were recorded. RESULTS: 3 of 6 dogs in each group were apneic after drug administration. Reflexes were decreased similarly for both anesthetic agents, but were not completely lost. Time to sternal position and walking unaided were significantly shorter in response to propofol. CONCLUSION: Anesthesia was rapid; however, respiratory depression and apnea were major adverse effects associated with propofol and thiopental. Propofol has the advantage of inducing rapid, coordinated anesthesia recovery.     

DNA binding properties of a chemically synthesized DNA binding domain of hRFX1

The RFX DNA binding domain (DBD) is a novel highly conserved motif belonging to a large number of dimeric DNA binding proteins which have diverse regulatory functions in eukaryotic organisms, ranging from yeasts to human. To characterize this novel motif, solid phase synthesis of a 76mer polypeptide corresponding to the DBD of human hRFX1 (hRFX1/DBD), a prototypical member of the RFX family, has been optimized to yield large quantities (approximately 90 mg) of pure compound. Preliminary two-dimensional1H NMR experiments suggested the presence of helical regions in this sequence in agreement with previously reported secondary structure predictions. In gel mobility shift assays, this synthetic peptide was shown to bind in a cooperative manner the 23mer duplex oligodeoxynucleotide corresponding to the binding site of hRFX1, with a 2:1 stoichoimetry due to an inverse repeat present in the 23mer. The stoichiometry of this complex was reduced to 1:1 by decreasing the length of the DNA sequence to a 13mer oligonucleotide containing a single half-site. Surface plasmon resonance measurements were achieved using this 5'-biotylinated 13mer oligonucleotide immobilized on an avidin-coated sensor chip. Using this method an association constant (K a = 4 x 10(5)/M/s), a dissociation constant (K d = 6 x 10(-2)/s) and an equilibrium dissociation constant (K D = 153 nM) were determined for binding of hRFX1/DBD to the double-stranded 13mer oligonucleotide. In the presence of hRFX1/DBD the melting temperature of the 13mer DNA was increased by 16 degreesC, illustrating stabilization of the double-stranded conformation induced by the peptide.     

HIV-1: is Nef a PAK animal?

HIV-1 Nef is clearly essential for efficient viral replication in vivo, but it has been difficult to determine why. Recent evidence that Nef specifically activates a PAK-dependent signalling cascade may be the first step in defining the mechanism of action of this enigmatic viral protein.     

Us9, a stable lysine-less herpes simplex virus 1 protein, is ubiquitinated before packaging into virions and associates with proteasomes

The US9 gene of herpes simplex virus 1 encodes a virion tegument protein with a predicted Mr of 10,000. Earlier studies have shown that the gene is not essential for viral replication in cells in culture. We report that (i) US9 forms in denaturing polyacrylamide gels multiple overlapping bands ranging in Mr from 12,000 to 25,000; (ii) the protein recovered from infected cells or purified virions reacts with anti-ubiquitin antibodies; (iii) autoradiographic images of US9 protein immunoprecipitated from cells infected with [35S]methionine-labeled virus indicate that the protein is stable for at least 4 h after entry into cells (the protein was also stable for at least 4 h after a 1-h labeling interval 12 h after infection); (iv) antibody to subunit 12 of proteasomes pulls down US9 protein from herpes simplex virus-infected cell lysates; and (v) the US9 gene is highly conserved among the members of the alpha subfamily of herpes viruses, and the US9 gene product lacks lysines. We conclude that US9 is a lysine-less, ubiquitinated protein that interacts with the ubiquitin-dependent pathway for degradation of proteins and that this function may be initiated at the time of entry of the virus into the cell.