Modification of the in vivo four-point loading model for studying mechanically induced bone adaptation

We modified the noninvasive, in vivo technique for strain application in the tibiae of rats (Turner et al,, Bone 12:73-79, 1991), The original model applies four-point bending to right tibiae via an open-loop, stepper-motor-driven spring linkage, Depending on the magnitude of applied load, the model produces new bone formation at periosteal (Ps) or endocortical surfaces (Ec.S). Due to the spring linkage, however, the range of frequencies at which loads can be applied is limited. The modified system replaces this design with an electromagnetic vibrator. A load transducer in series with the loading points allows calibration, the loaders' position to be adjusted, and cyclic loading completed under load central as a closed servo-loop. Two experiments were conducted to validate the modified system: (1) a strain gauge was applied to the lateral surface of the right tibia of 5 adult female rats and strains measured at applied loads from 10 to 60 N; and (2) the bone formation response was determined in 28 adult female Sprague-Dawley rats. Loading was applied as a haversine wave with a frequency of 2 Hz for 18 sec, every second day for 10 days. Peak bending loads mere applied at 33, 40, 52, and 64 N, and a sham-loading group tr as included at 64 N, Strains in the tibiae were linear between 10 and 60 N, and the average peak strain at the Ps.S at 60 N was 2664 +/- 250 microstrain, consistent with the results of Turner's group. Lamellar bone formation was stimulated at the Ec.S by applied bending, but not by sham loading. Bending strains above a loading threshold of 40 N increased Ec Lamellar hone formation rate, bone forming surface, and mineral apposition rate with a dose response similar to that reported by Turner et al, (J Bone Miner Res 9:87-97, 1994). We conclude that the modified loading system offers precision for applied loads of between 0 and 70 N, versatility in the selection of loading rates up to 20 Hz, and a reproducible bone formation response in the rat tibia, Adjustment of the loader also enables study of mechanical usage in murine tibia, an advantage with respect to the increasing variety of transgenic strains available in bone and mineral research. (Bone 23:307-310; 1998) (C) 1998 by Elsevier Science Inc. All rights reserved.

Acute intermittent porphyria: the in vitro expression of mutant hydroxymethylbilane synthase

Acute intermittent porphyria (AIP) is an inborn error of haem biosynthesis caused by a variety of mutations in the gene coding for hydroxymethylbilane synthase (HMB-S). The entire coding sequence of this gene, from each of three South African AIP patients, was therefore screened for mutations using chemical cleavage mismatch (CCM) analysis and any changes detected characterized by DNA sequencing. Three single base changes were identified; a G(77) to A in exon 3, a C-346 to T in exon 8 and a G(518) to A in exon 10. These missense mutations, previously reported to be present in other populations, are known to be responsible for the structurally deleterious amino acid replacements R26H, R116W and R173Q, respectively. The in vitro expression of the enzymes containing these mutations and the subsequent measurement of their specific activities revealed a reduction to approximately 4% of normal activity. (C) 1997 Academic Press Limited.

Species and regional variations in the effectiveness of antivenom against the in Vitro neurotoxicity of death adder (Acanthophis) venoms

Although viperlike in appearance and habit, death adders belong to the Elapidae family of snakes. Systemic envenomation represents a serious medical problem with antivenom, which is raised against Acanthophis antarcticus venom, representing the primary treatment. This study focused on the major Acanthophis variants from Australia and islands in the Indo-Pacific region. Venoms were profiled using liquid chromatography-mass spectrometry, and analyzed for in vitro neurotoxicity (0.3-10 mug/ml), as well as the effectiveness of antivenom. (1-5 units/ml; 10 min prior to the addition of 10 mug/ml venom). The following death adder venoms were examined: A. antarcticus (from separate populations in New South Wales, Queensland, South Australia, and Western Australia), A. hawkei, A. praelongus, A. pyrrhus, A. rugosus, A. wellsi, and venom from an unnamed species from the Indonesian island of Seram. All venoms abolished indirect twitches of the chick isolated biventer cervicis nerve-muscle preparation in a dose-dependent manner. In addition, all venoms blocked responses to exogenous acetylcholine (1 m-M) and carbachol (20 muM), but not KCl (40 mM), suggesting postsynaptic neurotoxicity. Death adder antivenom (1 unit/ml) prevented the neurotoxic effects of A. pyrrhus, A. praelongus, and A. hawkei venoms, although it was markedly less effective against venoms from A. antarcticus (NSW, SA, WA), A. rugosus, A. wellsi, and A. sp. Scram. However, at 5 units/ml, antivenom was effective against all venoms tested. Death adder venoms, including those from A. antarcticus geographic variants, differed not only in their venom composition but also in their neurotoxic activity and susceptibility to antivenom. For the first time toxicological aspects of A. hawkei, A. wellsi, A. rugosus, and A. sp. Seram venoms were studied. (C) 2001 Academic Press.

Cross-reactivity of Sydney funnel-web spider antivenom: neutralization of the in vitro toxicity of other Australian funnel-web (Atrax and Hadronyche) spider venoms

Australian funnel-web spiders are recognized as one of the most venomous spiders to humans world-wide. Funnel-web spider antivenom (FWS AV) reverses clinical effects of envenomation from the bite of Atrax robustus and a small number of related Hadronyche species. This study assessed the in vitro efficacy of FWS AV in neutralization of the effects of funnel-web spider venoms, collected from various locations along the eastern seaboard of Australia, in an isolated chick biventer cervicis nerve-muscle preparation. Venoms were separated by SDS-PAGE electrophoresis to compare protein composition and transblotted for Western blotting and incubation with FWS AV. SDS-PAGE of venoms revealed similar low and high molecular weight protein bands. Western blotting with FWS AV showed similar antivenom binding with protein bands in all the venoms tested. Male funnel-web spider venoms (7/7) and female venoms (5110) produced muscle contracture and fasciculation when applied to the nerve-muscle preparation. Venom effects were reversed by subsequent application of FWS AV or prevented by pretreatment of the preparation with antivenom. FWS AV appears to reverse the in vitro toxicity of a number of funnel-web spider venoms from the eastern seaboard of Australia. FWS AV should be effective in the treatment of envenomation from most, if not all, species of Australian funnel-web spiders. (C) 2001 Elsevier Science Ltd. All rights reserved.

Determination of the effect of lipophilicity on the in vitro permeability and tissue reservoir characteristics of topically applied solutes in human skin layers

In order to establish the relationship between solute lipophilicity and skin penetration (including flux and concentration behavior), we examined the in vitro penetration and membrane concentration of a series of homologous alcohols (C2-C10) applied topically in aqueous solutions to human epidermal, full-thickness, and dermal membranes. The partitioning/distribution of each alcohol between the donor solution, stratum corneum, viable epidermis, dermis, and receptor phase compartments was determined during the penetration process and separately to isolated samples of each tissue type. Maximum flux and permeability coefficients are compared for each membrane and estimates of alcohol diffusivity are made based on flux/concentration data and also the related tissue resistance (the reciprocal of permeability coefficient) for each membrane type. The permeability coefficient increased with increasing lipophilicity to alcohol C8 (octanol) with no further increase for C10 (decanol). Log vehicle:stratum corneum partition coefficients were related to logP , and the concentration of alcohols in each of the tissue layers appeared to increase with lipophilicity. No difference was measured in the diffusivity of smaller more polar alcohols in the three membranes; however, the larger more lipophilic solutes showed slower diffusivity values. The study showed that the dermis may be a much more lipophilic environment than originally believed and that distribution of smaller nonionized solutes into local tissues below a site of topical application may be estimated based on knowledge of their lipophilicity alone.

Comparison of the in vitro neuromuscular activity of venom from three Australian snakes (Hoplocephalus stephensi, Austrelaps superbus and Notechis scutatus): Efficacy of tiger snake antivenom

1. Tiger snake antivenom, raised against Notechis scutatus venom, is indicated not only for the treatment of envenomation by this snake, but also that of the copperhead (Austrelaps superbus ) and Stephen's banded snake (Hoplocephalus stephensi ). The present study compared the neuromuscular pharmacology of venom from these snakes and the in vitro efficacy of tiger snake antivenom. 2. In chick biventer cervicis muscle and mouse phrenic nerve diaphragm preparations, all venoms (3-10 mug/mL) produced inhibition of indirect twitches. In the biventer muscle, venoms (10 mug/mL) inhibited responses to acetylcholine (1 mmol/L) and carbachol (20 mumol/L), but not KCl (40 mmol/L). The prior (10 min) administration of 1 unit/mL antivenom markedly attenuated the neurotoxic effects of A. superbus and N. scutatus venoms (10 mug/mL), but was less effective against H. stephensi venom (10 mug/mL); 5 units/mL antivenom attenuated the neurotoxic activity of all venoms. 3. Administration of 5 units/mL antivenom at t(90) partially reversed, over a period of 3 h, the inhibition of twitches produced by N. scutatus (10 mug/mL; 41% recovery), A. superbus (10 mug/mL; 25% recovery) and H. stephensi (10 mug/mL; 50% recovery) venoms. All venoms (10-100 mug/mL) also displayed signs of in vitro myotoxicity. 4. The results of the present study indicate that all three venoms contain neurotoxic activity that is effectively attenuated by tiger snake antivenom.

On systems thinking, systems biology and the in Silico Plant

The recent summary report of a Department of Energy Workshop on Plant Systems Biology (P.V. Minorsky [2003] Plant Physiol 132: 404-409) offered a welcomed advocacy for systems analysis as essential in understanding plant development, growth, and production. The goal of the Workshop was to consider methods for relating the results of molecular research to real-world challenges in plant production for increased food supplies, alternative energy sources, and environmental improvement. The rather surprising feature of this report, however, was that the Workshop largely overlooked the rich history of plant systems analysis extending over nearly 40 years (Sinclair and Seligman, 1996) that has considered exactly those challenges targeted by the Workshop. Past systems research has explored and incorporated biochemical and physiological knowledge into plant simulation models from a number of perspectives. The research has resulted in considerable understanding and insight about how to simulate plant systems and the relative contribution of various factors in influencing plant production. These past activities have contributed directly to research focused on solving the problems of increasing biomass production and crop yields. These modeling approaches are also now providing an avenue to enhance integration of molecular genetic technologies in plant improvement (Hammer et al., 2002).

Ability of some plant extracts, traditionally used to treat ciguatera fish poisoning, to prevent the in vitro neurotoxicity produced by sodium channel activators

The effects of 31 plant extracts, which most are traditionally used to treat ciguatera fish poisoning in the Pacific area, were Studied on the cytotoxicity of mouse neuroblastoma cells produced by ouabain, veratridine and/or brevetoxin-3 or Pacific ciguatoxin-1. The cell viability was determined using a quantitative colorimetric method. A marked cytotoxicity of seven of the 31 plant extracts studied, was observed. Despite this, these plant extracts were suspected to contain active compound(s) against the cytotoxicity produced by brevetoxin (2 extracts), brevetoxin, ouabain and/or veratridine (3 extracts), or only against that of ouabain and/or veratridine (2 extracts). Among the 24 plant extracts that exhibited by themselves no cytotoxicity, 22 were active against the effect of brevetoxin or against that of both veratridine and brevetoxin. similar results were obtained when the seven most active plant extracts were reassayed using ciguatoxin instead of brevetoxin. In conclusion, the present work reports the first activity assessment of some plant extracts, achieved in vitro on a quite large scale. The fact that 27 plant extracts were found to exert, in vitro, a protective effect against the action of ciguatoxin and/or brevetoxin, paves the way for finding new active compounds to treat ciguatera fish poisoning, provided these compounds also reverse the effects of sodium channel activators. (c) 2005 Elsevier Ltd. All rights reserved.

The in-vitro bioactivity of mesoporous bioactive glasses

Ordered mesoporous bioactive glasses (MBGs) with different compositions were prepared by using nonionic block copolymer surfactants as structure-directing agents through an evaporation-induced self-assembly process. Their in-vitro bioactivities were studied in detail by electron microscopy, Fourier-transform infrared spectroscopy, and inductively coupled plasma (ICP) atomic emission spectroscopy. The ICP element analysis results were further calculated in terms of the total consumption of Ca and P, Delta[Ca]/Delta[P] ratios, and ionic activity product (IP) of hydroxyapatite. Through the above analysis, it is clear that MBGs show a different structure-bioactivity correlation compared to conventional sol-gel-derivcd BGs. The in vitro bioactivity of MBGs is dependent on the Si/Ca ratio in the network when the other material parameters such as the mesostructure and texture properties (pore size, pore volume) are controlled. MBG 80S15C with relatively lower calcium content exhibits the best in vitro bioactivity, in contrast to conventional sol-gel-derived BGs where usually higher calcium percentage BGs (e.g. 60S35C) show better bioactivity. Calcination temperature is another important factor that influences the in vitro bioactivity. According to our results, MBGs calcined at 973 K may possess the best in vitro bioactivity. The influences of the composition and calcination temperature upon bioactivity are explained in terms of the unique structures of MBGs. (c) 2006 Elsevier Ltd. All rights reserved.

The in vitro effect of different PRP concentrations on osteoblasts and fibroblasts

Objectives:The aim of this study was to assess the biological rationale for the use of platelet-rich plasma (PRP) by evaluating the effect of different concentrations of PRP on osteoblasts (OB) and fibroblasts (FB) function in vitro. Materials and methods:PRP was obtained from volunteer donors using standard protocols. Primary human cultures of oral FBs and OBs were exposed to both activated and non-activated plasma as well as various concentrations of PRP (2.5 x, 3.5 x and max (4.2-5.5 x)). Cell proliferation was evaluated after 24 and 72 h using an MTT proliferation assay. Production of osteocalcin (OCN), osteoprotegerin (OPG) and transforming growth factor beta 1 (TGF-beta 1) was evaluated in OB after 24 and 72 h. Statistical analysis was performed using one-way ANOVA. Results:PRP-stimulated cell proliferation in both OBs and FBs. The effect of different PRP concentrations on cell proliferation was most notable at 72 h. The maximum effect was achieved with a concentration of 2.5 x, with higher concentrations resulting in a reduction of cell proliferation. Upregulation of OCN levels and downregulation of OPG levels were noted with increasing PRP concentrations at both 24 and 72 h. TGF-beta 1 levels were stimulated by increasing concentrations of PRP, with the increased levels being maintained at 72 h. Conclusions:PRP preparations exert a dose-specific effect on oral FBs and OBs. Optimal results were observed at a platelet concentration of 2.5 x, which was approximately half of the maximal concentrate that could be obtained. Increased concentrations resulted in a reduction in proliferation and a suboptimal effect on OB function. Hence, different PRP concentrations may have an impact on the results that can be obtained in vivo.

Acupressure for the in-patient treatment of nausea and vomiting in early pregnancy: A randomized control trial

Objective: The purpose of this study was to evaluate the efficacy of acupressure at the P6 point for the in-patient treatment of severe nausea and vomiting in early pregnancy. Study design: This was a prospective single-blind randomized control trial that involved 80 patients with nausea and vomiting plus ketonuria before 14 weeks of gestation. Results: There was no difference between length of stay, amount of medication, or fluid required between the acupressure and placebo groups, although acupressure reduced the number of patients who stayed >= 4 nights in the hospital. Acupressure was well tolerated and not associated with an increase in perinatal morbidity or death. Conclusion: The use of acupressure Lit the P6 point does not reduce the amount of antiemetric medication that is required, the requirement for intravenous fluid, and median duration of inpatient stay more than the use of placebo. A small reduction was seen in the number of women who required >= 4 days in the hospital. (c) 2006 Mosby, Inc. All rights reserved.

Analysis of the Pasteurella multocida outer membrane sub-proteome and its response to the in vivo environment of the natural host

This study describes the identification of outer membrane proteins (OMPs) of the bacterial pathogen Pasteurella multocida and an analysis of how the expression of these proteins changes during infection of the natural host. We analysed the sarcosine-insoluble membrane fractions, which are highly enriched for OMPs, from bacteria grown under a range of conditions. Initially, the OMP-containing fractions were resolved by 2-DE and the proteins identified by MALDI-TOF MS. In addition, the OMP-containing fractions were separated by 1-D SDS-PAGE and protein identifications were made using nano LC MS/MS. Using these two methods a total of 35 proteins was identified from samples obtained from organisms grown in rich culture medium. Six of the proteins were identified only by 2-DE MALDI-TOF MS, whilst 17 proteins were identified only by 1-D LC MS/MS. We then analysed the OMPs from P. multocida which had been isolated from the bloodstream of infected chickens (a natural host) or grown in iron-depleted medium. Three proteins were found to be significantly up-regulated during growth in vivo and one of these (Pm0803) was also up-regulated during growth in iron-depleted medium. After bioinformatic analysis of the protein matches, it was predicted that over one third of the combined OMPs predicted by the bioinformatics sub-cellular localisation tools PSORTB and Proteome Analyst, had been identified during this study. This is the first comprehensive proteomic analysis of the P. multocida outer membrane and the first proteomic analysis of how a bacterial pathogen modifies its outer membrane proteome during infection.