Stabilization of recombinant Drosophila acetylcholinesterase

OBJECTIVE: To estimate the early death-rate in HIV infected injectors whose HIV infection was during the injection-related HIV outbreak in Lothian region in Scotland in 1983-1984, which was coincident with Hepatitis B transmissions. SETTING: Regional Virus Laboratory in Edinburgh. SAMPLES: Sera from 1983-1984, originally received for Hepatitis B surface antigen testing, from individuals aged 15-55 years who were positive for Hepatitis B surface antigen in 1983-1984 (group A: census) or tested negative but were at high risk for blood-borne virus transmission according to their reason for testing (group B: 50% sample). METHODS: Survival status of individuals in groups A and B who had not been diagnosed with HIV disease by the end of December 1995 was checked against the deaths' records of the Registrar General for Scotland. Stored sera from 1983-1984 for patients who had died early (that is: in 1983-1984) were tested anonymously for HIV and Hepatitis C antibodies; and prior to testing, causes of death were scored by RPB according to the likelihood of their being HIV or drugs related. RESULTS: Three early deaths were found in group A patients who were not known to be HIV infected. None of the deaths was likely to be HIV-related; the sera were not tested in order not to risk deductive disclosure. Twenty-four early deaths were found in group B patients who were not known to be HIV-infected, five of whom were both HIV and Hepatitis C antibody positive, and one other was HIV antibody negative but Hepatitis C positive. Reclassification after unlinked anonymous testing and multiplying up of the group B results (to account for 50% sample) gave the early death rate (that is: in 1983-1986) as 15/155 (10%) for HIV-infected drug users (95% CI: 6%-13%). CONCLUSION: Injection-related outbreaks of HIV infection in Lothian in 1983-1984 and at Glenochil Prison in 1993 were each associated with substantial--estimated 10%--early death-rate in HIV-infected injectors. Both HIV outbreaks were coincident with Hepatitis B transmissions, which may be relevant. Further investigations of the death-rate within 2 years of HIV infection are warranted in other exposure categories than injection-related and for injectors who have been immunized against Hepatitis B.     

Poly(L-lysine)-graft-dextran copolymer is a novel stabilizer of triplex DNA (I): stabilization of poly(dA).2poly(dT) triplex

Comb-type polylysine copolymer having grafted hydrophilic side chains was newly designed as a novel stabilizer of triplex DNAs. The comb-type copolymer elevated melting temperature of poly(dA).2poly(dT) triplex by 50 degrees C without affecting reversibility, melting and reassociation, of the triplex in buffer with physiological salt concentrations. The stabilizing effect of the copolymer was greater than spermine. Our results indicate that the molecular designing of polycation with comb-type structure is a successful strategy for creating an effective triplex stabilizer.     

Stabilization of active recombinant retroviruses in an amorphous dry state with trehalose

1. The objective of this investigation was to determine quantitatively whether experimental epilepsy is associated with a change in the pharmacodynamics of benzodiazepines in vivo. For that purpose the pharmacodynamics of midazolam were quantified by an integrated pharmacokinetic-pharmacodynamic approach in three different models of experimental epilepsy: amygdala kindling, cortical stimulation and genetic absence epilepsy. 2. The time course of the EEG effect was determined in conjunction with the decline of drug concentrations after intravenous administration of 10 mg kg(-1) midazolam. The pharmacokinetics of midazolam were most adequately described by a bi-exponential equation. No influence of epilepsy on the pharmacokinetics of midazolam was observed. 3. The increase in beta activity (11.5-30 Hz) of the EEG as derived by Fast Fourier Transformation analysis was used as pharmacodynamic endpoint. For each individual rat the increase in beta activity was directly related to the concentration in blood on the basis of the sigmoidal Emax pharmacodynamic model. In all three models a significant reduction in the maximal effect was observed, in amygdala kindling 28%, in the cortical stimulation model 49% and in genetic absence epilepsy 37%. No differences in the other pharmacodynamic parameters, E0 EC50,u and Hill factor, were observed. 4. It is inferred that in three different models of epilepsy there is a similar change in GABAergic functioning which is associated with a significant reduction in the intrinsic activity of midazolam in vivo. These models provide therefore a useful basis for further studies on the mechanism of epilepsy-induced changes in pharmacodynamics of anti-epileptic drugs.     

Bactericidal action of 4,4''-(alpha,omega-polymethylenedithio)bis-(1-alkylpyridinium iodide)s

BACKGROUND: Patients with intestinal disease are at risk of developing selenium deficiency due to impaired intestinal absorption. The aim of the present study was to evaluate selenium status and to identify predictive factors of selenium depletion in patients with gastrointestinal disease. METHODS: The concentration of selenium and the activity of glutathione peroxidase in plasma and erythrocytes were measured by fluorometry and by spectrophotometry. Eighty-six patients with Crohn's disease, 40 patients with ulcerative colitis, and 39 patients with various other gastrointestinal diseases were studied. Twenty-seven patients (16%) received home parenteral nutrition. Stool mass, faecal fat, and vitamin B12 absorption were analysed in 100 patients. RESULTS: The plasma selenium concentration was decreased in 85% of the patients receiving supplementary parenteral nutrition and in 20% of the patients receiving oral nutrition, among them in 26% of the patients with Crohn's disease. Almost all patients with ulcerative colitis had normal selenium levels. A statistically significant correlation was found between plasma selenium and vitamin B12 absorption, stool mass, faecal fat excretion, body mass index, P-albumin, P-zinc, and the length of the remaining small bowel. Stepwise regression analyses showed that the strongest predictors of selenium deficiency were stool mass, vitamin B12 absorption, and the length of the small-bowel resection. CONCLUSION: Selenium deficiency is common in patients with severe gastrointestinal disorders. The deficiency is mainly related to malabsorption, and a low selenium level was almost invariably present in patients who needed parenteral supplementation due to gut failure.     

Thermodynamic, kinetic, and conformational properties of a parallel intermolecular DNA triplex containing 5' and 3' junctions

The interaction of the 11-mer oligodeoxypyrimidine d(TCTTCTUTCCT) with the 17 bp duplex d(CGCTAGAAGAAAGGACG).d(CGTCCUTTCTTCTAGCG) in forming an intermolecular DNA triplex has been examined in solution by surface plasmon resonance (SPR), UV thermal denaturation, circular dichroism (CD), and NMR methods. Thermodynamic data were also acquired for the shorter 15 bp target duplex d(CGCTAGAAGAAAGGA). d(TCCUTTCTTCTAGCG), which forms a 3' flush-ended parallel triplex. CD titrations at pH 5 gave a triplex --> (duplex + strand) dissociation constant Kd of 0.5 microM at 15 degreesC and approximately 2 microM at 25 degreesC for both the 11-15.15 and 11-17.17 systems, in agreement with analysis of the UV melting data and a direct calorimetric measurement. In contrast, the "apparent" Kd value determined by SPR was 10-20-fold smaller. The rate constant for dissociation (kd) of the third strand from the triplex was found to be approximately 0.0002 s-1 at 25 degreesC by SPR. The rate constant for exchange between the triplex and duplex states determined by NMR was approximately 2 s-1 at 40 degreesC. The dissociation kinetics measured by SPR are considerably underestimated, which largely accounts for the poor estimation of Kd using this technique. Extensive 1H NMR assignments were obtained for both the 17 bp DNA duplex and the triplex. Large changes in chemical shifts were observed in the purine strand of the host duplex, but only small shift changes were induced in the complementary pyrimidine strand. Dramatic differences in shifts were observed for the G and A residues, especially in the minor groove, consistent with only small, localized conformational changes in the underlying duplex. The magnitude of the shift changes decreased to baseline within one base of the 3' triplex-duplex junction and over two to three bases at the 5' junction. Chemical shift changes at the 5' junction suggest small conformational anomalies at this site. COSY and NOESY spectra indicate that the nucleotides are in the "S" domain in both the triplex and duplex states. These data rule out major conformation changes at the triplex-duplex boundaries. NOEs between pyrimidines in the third strand and those in the duplex showed proximity for these bases in the major groove, which could be ascribed to buckling of the Hoogsteen bases out of the plane of the Watson-Crick base pairs.     

Stabilization of a proteolytically sensitive cytoplasmic recombinant protein during transition to downstream processing

The influence of aeration and glucose feeding on the stability of recombinant protein A in Escherichia coli during the transition period from a fed-batch cultivation to downstream processing was studied. Neither interruption of the feeding under aerobic conditions nor anaerobic conditions in presence of glucose could stabilize protein A completely and the intracellular ATP pool did not decrease to less than 0.75-1 mM by this treatment. On the other hand, the absence of both oxygen and glucose resulted in a decrease of the ATP pool to less than 0.5 mM and almost complete stabilization of protein A. The decrease of ATP was more severe when sulfite was used instead of nitrogen gas to create anaerobic conditions in presence of glucose. This also resulted in nearly complete stabilization of protein A, which might be explained by an inhibiting effect of sodium sulfite on fermentation. Therefore, protein stabilization and decrease of the ATP pool were correlated in experiments in vivo. The concentrations of ADP and AMP increased during starvation and may also play a role in stabilization of the protein in vivo. ATP may be a limiting factor of proteolysis also during further steps of downstream processing. Its concentration decreases by 80-90% during harvesting and centrifugation of biomass and even further during disruption of cells. However, neither addition nor regeneration of ATP in cell disintegrate was enough to restore degradation of protein A, indicating that an additional factor limits proteolysis in vitro.     

Stabilization of 10-hydroxycamptothecin in poly(lactide-co-glycolide) microsphere delivery vehicles

PURPOSE: The purpose of this study was to investigate the potential of poly(lactide-co-glycolide) (PLGA) microspheres to stabilize and deliver the analogue of camptothecin, 10-hydroxycamptothecin (10-HCPT). METHODS: 10-HCPT was encapsulated in PLGA 50:50 microspheres by using an oil-in-water emulsion-solvent evaporation method. The influence of encapsulation conditions (i.e., polymer molecular weight (Mw), polymer concentration, and carrier solvent composition) on the release of 10-HCPT from microspheres at 37 degrees C under perfect sink conditions was examined. Analysis of the drug stability in the microspheres was performed by two methods: i) by extraction of 10-HCPT from microspheres and ii) by sampling release media before lactone--carboxylate conversion could take place. RESULTS: Microspheres made of low Mw polymer (inherent viscosity 0.15 dl/g) exhibited more continuous drug release than those prepared from polymers of higher Mw (i.v. = 0.58 and 1.07 dl/g). In addition, a high polymer concentration and the presence of cosolvent in the carrier solution to dissolve 10-HCPT were both necessary in the microsphere preparation in order to eliminate a large initial burst of the released 10-HCPT. An optimal microsphere formulation released 10-HCPT slowly and continuously for over two months with a relatively small initial burst of the released drug. Both analytical methods used to assess the stability of 10-HCPT revealed that the unreleased camptothecin analogue in the microspheres remained in its active lactone form (> 95%) over the entire 2-month duration of study. CONCLUSIONS: PLGA carriers such as those described here may be clinically useful to stabilize and deliver camptothecins for the treatment of cancer.     

Phosphorylation of recombinant N-syndecan (syndecan 3) core protein

The cytoplasmic domain of the syndecan family of heparan sulfate proteoglycans is punctuated by the presence of four regularly spaced tyrosine residues. In this report, we explore the possibility of whether the four tyrosine residues in the cytoplasmic domain of N-syndecan (Syndecan 3) are potential substrates for phosphorylation by a tyrosine kinase. Bacterially expressed elk kinase was used to phosphorylate a series of bacterially expressed N-syndecan fusion proteins. Our results clearly demonstrate that the tyrosine residues in the cytoplasmic domain of N-syndecan can be phosphorylated by a tyrosine-specific kinase, and that all four tyrosine residues are capable of being phosphorylated.     

Acute band-shaped keratopathy after intraocular fibrinolysis with recombinant tissue plasminogen activator (rt-PA)

The study was planned to determine the proportion of parents that wish to know the sex of their fetus at the 20-week anomaly scan, and to investigate our ability to diagnose correctly the sex of the fetus when undertaken as part of a routine scan. A total of 472 patients gave their informed consent. An attempt was made to identify the genitalia as part of the routine scan. No extra time was allowed to determine the sex of the fetus. Altogether 353 (74.7%) women wanted to know the sex, of which four (0.9%) wanted to know but did not want their partners to know. In 50 (10.6%) cases, it was not possible to determine the fetal sex in the time allowed. When the sex was identified, it was correct in 408 (96.7%) cases, and incorrect in 14 (3.3%) cases. Where the parents wanted to know the sex of the fetus, 24 (6.8%) scans were inconclusive, 319 (97%) were correctly identified, and ten (3%) were incorrectly identified (six male, four female). There were no terminations of pregnancy. The majority of prospective parents wish to know the sex of their child, and, in most cases, it is possible to determine the fetal sex at the time of the routine anomaly scan. During the time allowed, the fetal sex was undetermined in one in ten cases, and 3% were sexed incorrectly. If parents wish to know the gender of their fetus, it would appear reasonable to provide this information, provided that the parents are aware of the failure and error rates of sex identification using ultrasound.     

Structure-function relationship of recombinant follicle stimulating hormone (Puregon)

After separation by means of preparative isoelectrofocusing, the isohormones of a Chinese hamster ovary (CHO)-derived recombinant follicle stimulating hormone (rFSH, Puregon) were characterized with respect to structural and functional features. A carbohydrate analysis revealed that rFSH isohormones with a low isoelectric point (pl) have a high sialic acid/galactose ratio and are rich in tri- and tetra-antennary N-linked carbohydrate chains in comparison with the high pl isohormones. The relative basic isohormones exhibit receptor binding activity and intrinsic bioactivity 2-3-fold higher than the relative acidic isohormones. However, due to their lower clearance rate these acidic isohormones displayed a 20-fold higher in-vivo bioactivity in the rat. A comparison of the isohormone profile of rFSH and urinary FSH (Metrodin) revealed that rFSH contains about 2-fold more basic isohormones with pl > or = 4.7 and 2-fold less acidic isohormones with pl < 4.1. In-vitro studies showed that the receptor binding affinity and intrinsic bioactivity of both FSH preparations are similar. Also the in-vivo efficacy and the pharmacokinetic behaviour of rFSH and urinary FSH in the rat were similar, which is not surprising since both preparations were compared in terms of in-vivo bioactivity calibrated in the rat Steelman-Pohley assay. However, in dogs the bioavailability of rFSH was lower than that of urinary FSH, which is in agreement with the higher percentage of relative basic isohormones in rFSH. This suggests that the pharmacokinetic behaviour of FSH in rats and dogs is different, which is supported by the much longer elimination half-life of rFSH and urinary FSH in dogs (27.9 and 30.4 h respectively) compared with rats (11.4 and 10.4 h respectively) for rFSH and urinary FSH respectively. The observed differences in pharmacokinetic behaviour in dogs and rats indicate that the rat Steelman-Pohley assay might not be a valid model for the prediction of the FSH bioactivity in species other than rat.     

A 3rd Generation Advanced High-Strength Steel (AHSS) Produced by Dual Stabilization Heat Treatment (DSHT)

A 3rd generation advanced high-strength steel containing, in wt pct, 0.3 C, 4.0 Mn, 1.5 Al, 2.1 Si, and 0.5 Cr has been produced using a dual stabilization heat treatment—a five stage thermal processing schedule compatible with continuous galvanized steel production. In excess of 30 vol pct retained austenite containing at least 0.80 wt pct C was achieved with this alloy, which had tensile strengths up to 1650 MPa and tensile elongations around 20 pct.     

Characterization of recombinant human fibroblast growth factor (FGF)-10 reveals functional similarities with keratinocyte growth factor (FGF-7)

A newly identified member of the fibroblast growth factor (FGF) family, designated FGF-10, is expressed during development and preferentially in adult lung. The predicted FGF-10 protein is most related to keratinocyte growth factor (KGF, or FGF-7). The latter is unique among FGFs in that it binds and signals only through the FGF receptor (FGFR2b) isoform KGF receptor (KGFR) expressed specifically by epithelial cells. In order to examine the biological and biochemical properties of human FGF-10, we isolated the cDNA and expressed its encoded protein in bacteria. The recombinant protein (rFGF-10) was a potent mitogen for Balb/MK mouse epidermal keratinocytes with activity detectable at 0.1 nM and maximal at around 5 nM. Within this concentration range, FGF-10 did not stimulate DNA synthesis in NIH/3T3 mouse fibroblasts. rFGF-10 bound the KGFR with high affinity comparable to that of KGF, and did not bind detectably to either the FGFR1c (Flg) or FGFR2c (Bek) receptor isoforms. The mitogenic activity of FGF-10 could be distinguished from that of KGF by its different sensitivity to heparin and lack of neutralization by a KGF monoclonal antibody. These results indicate that FGF-10 and KGF have similar receptor binding properties and target cell specificities, but are differentially regulated by components of the extracellular matrix.     

Propagating structure of Alzheimer's beta-amyloid(10-35) is parallel beta-sheet with residues in exact register

The pathognomonic plaques of Alzheimer's disease are composed primarily of the 39- to 43-aa beta-amyloid (Abeta) peptide. Crosslinking of Abeta peptides by tissue transglutaminase (tTg) indicates that Gln15 of one peptide is proximate to Lys16 of another in aggregated Abeta. Here we report how the fibril structure is resolved by mapping interstrand distances in this core region of the Abeta peptide chain with solid-state NMR. Isotopic substitution provides the source points for measuring distances in aggregated Abeta. Peptides containing a single carbonyl 13C label at Gln15, Lys16, Leu17, or Val18 were synthesized and evaluated by NMR dipolar recoupling methods for the measurement of interpeptide distances to a resolution of 0.2 A. Analysis of these data establish that this central core of Abeta consists of a parallel beta-sheet structure in which identical residues on adjacent chains are aligned directly, i. e., in register. Our data, in conjunction with existing structural data, establish that the Abeta fibril is a hydrogen-bonded, parallel beta-sheet defining the long axis of the Abeta fibril propagation.     

Treatment of non-renal anemia with human recombinant erythropoietin (rHU-EPO

Four cases of Rett syndrome were ascertained among 19,060 girls born between 1978 and 1990 in a small, defined area of Northern Tuscany (Italy) (prevalence rate of 2.1 per 10,000). A fifth girl with a reported clinical picture of Rett syndrome, born in 1978 and deceased at age 13, was also found. One of the four Rett syndrome cases had a healthy female dizygote twin. Family tree studies going back as far as the 17th century were performed. A number of common ancestors were found in different generations leading to a single family tree encompassing all four Rett syndrome cases. In addition, a Rett girl with preserved speech, born in 1974, was found as part of this family tree. These observations confirm the role of genetic factors in the etiology of Rett syndrome and support the hypothesis that Rett syndrome is a clinically variable phenotype.     

Stabilization of Organic Soils with Fly Ash

The effectiveness of fly ash use in the stabilization of organic soils and the factors that are likely to affect the degree of stabilization were studied. Unconfined compression and resilient modulus tests were conducted on organic soil–fly ash mixtures and untreated soil specimens. The unconfined compressive strength of organic soils can be increased using fly ash, but the amount of increase depends on the type of soil and characteristics of the fly ash. Resilient moduli of the slightly organic and organic soils can also be significantly improved. The increases in strength and stiffness are attributed primarily to cementing caused by pozzolanic reactions, although the reduction in water content resulting from the addition of dry fly ash solid also contributes to strength gain. The pozzolonic effect appears to diminish as the water content decreases. The significant characteristics of fly ash that affect the increase in unconfined compressive strength and resilient modulus include CaO content and CaO/SiO2 ratio [or CaO/(SiO2+Al2O3) ratio]. Soil organic content is a detrimental characteristic for stabilization. Increase in organic content of soil indicates that strength of the soil–fly ash mixture decreases exponentially. For most of the soil–fly ash mixtures tested, unconfined compressive strength and resilient modulus increased when fly ash percentage was increased.     

Stabilization of Arsenite Wastes with Prior Oxidation

The stabilization of arsenite hazardous waste is more difficult than arsenate waste. Oxidation of arsenite was investigated as a pretreatment before stabilization of the resulting arsenate. The effectiveness of air oxidation was limited and depended on the amount of water present. Hydrogen peroxide oxidation was effective but limited by reactions with calcium hydroxide. After oxidation, the resulting arsenate waste was effectively stabilized using ferric sulfate.     

Structure of d(GT)n.d(GA)n sequences: formation of parallel stranded duplex DNA

Alternating polypurine sequences exhibit remarkable polymorphism. In this study, we report that dGA.dGT sequences form parallel stranded duplex DNA at neutral pH. Using two model hairpins, 3'-d(GT)3-5'5'-T4(AG)3-3' (I) and 3'-d(GT)4-5'5'-T4(AG)4-3' (II), containing 5'5' linkages which direct parallel strand formation, we systematically explored the spectroscopic and thermodynamic properties of parallel stranded d(GA)n.d(GT)n. The parallel stranded hairpins are remarkably stable structures with TM's of 41.5 and 47.5 degreesC (in 0.4 M NaCl) for the shorter and longer hairpins, respectively. The van't Hoff enthalpies of 80.7 and 114 kJ mol-1 are relatively low but are comparable to a parallel stranded d(GA)n duplex. On the basis of the spectroscopic and electrophoretic characteristics, we conclude that parallel strand formation is not restricted to hairpin systems, but also readily occurs in unconstrained dimeric duplexes with the appropriate sequence homologies. Both melting curves and electrophoretic analyses of parallel stranded heteroduplexes in which the sequence enforces specific base pairing demonstrate that G-G and A-T base pairs are formed in d(GA)n.d(GT)n segments.     

Vibrational normal modes and dynamical stability of DNA triplex poly(dA). 2poly(dT): S-type structure is more stable and in better agreement with observations in solution

A normal-mode and statistical mechanical calculation was carried out to determine the vibrational normal modes, contribution of internal fluctuations to the free energy, and hydrogen bond disruption of DNA triplex poly(dA).2poly(dT). The calculation was performed on both the x-ray fiber diffraction model with a N-type sugar conformation, and a newly proposed model with a S-type sugar conformation. Our calculated normal modes for the S-type structure are in better agreement with observed IR spectra for samples in D2O solution. We also find that the contribution of internal fluctuations to free energy, premelting hydrogen bond disruption probability, and hydrogen bond melting temperatures for the Hoogsteen and Watson-Crick hydrogen bonds all show that the S-type structure is dynamically more stable than the N-type structure in a nominal solution environment. Therefore our calculation supports experimental findings that the triplex d(T)n.d(A)nd(T)n most likely adopts a S-type sugar conformation in solution or at high humidity. Our calculations, however, do not preclude the possibility of an N-type conformation at lower humidities.     

Solution structure of an intramolecular DNA triplex containing 5-(1-propynyl)-2'-deoxyuridine residues in the third strand

Incorporation of the modified base 5-(1-propynl)-2'-deoxyuridine (propynylU) in the third strand of a triplex leads to enhanced triplex stabilization. To investigate effects of the propyne nucleotide on triplex structure and the factors underlying the increased stability, we have determined the solution structure of the intramolecular DNA pyrmidine-purine-pyrimdine d(AGAGAGAA-(EG)6-TTCTCTCT-(EG)6-PCPCPCPP) (PDD-EG), which contains 5-(1-propynl)-2'-deoxyuridine (P) in the third strand and hexakis(ethylene glycol) linkers [(EG)6]. The structure was calculated using X-PLOR with distance and dihedral angle restraints obtained from two-dimensional NMR experiments and refined with the direct relaxation matrix method. The structures show that the extended aromatic electron cloud of the propynylU nucleotide stacks well over the 5'-neighboring nucleotides, resulting in increased stabilization. The propynylU nucleotides also affect the overall structure of the triple helix. A comparison of the structure to that of the nonmodified intramolecular DNA triplex of the same sequence, d(AGAGAGAA-(EG)6-TTCTCTCT-(EG)6-TCTCTCTT) (DDD-EG), shows that PDD-EG has a more A-DNA like X displacement and inclination than DDD-EG yet still maintains predominantly S-type sugar puckers as found in DDD-EG and other DNA triplexes.