Transdermal Pharmacology of Small Molecule Cyclic C5a Antagonists

Overproduction or underregulation of the proinflammatory complement component C5a has been implicated in numerous immune and inflammatory conditions. Therefore, targeting the C5a receptor (C5aR) has become an innovative strategy for antiinflammatory drug development. The novel cyclic peptide C5aR antagonist, AcF-[OP(D-Cha)WR] (PMX53), attenuates injury in numerous animal models of inflammation following intravenous, subcutaneous, intraperitoneal, and oral administration. In the present study the transdermal pharmacology of PMX53 and three analogs designed with increased lipophilicity, hydrocinnamate-[OP(D-Cha)WCit] (PMX200), AcF-[OP(D-Cha)WCit] (PMX201) and hydrocinnamate-[OP(D-Cha)WR] (PMX205), have been examined in order to assess their transdermal permeability and inhibitory effect on C5a-mediated lipopolysaccharide (LPS)-induced systemic responses. In the rat, PMX53, PMX201, and PMX205, were bioavailable following topical dermal administration (10 mg/50 cm(2) site/rat). All analogs functionally antagonized neutropenia and hypotension induced by systemic challenge with LPS (I mg/kg i.v.). Interestingly, PMX200 attenuated LPS-induced neutropenia more effectively than other analogs, despite undetectable (< 5 ng/ml) circulating levels following topical administration. In conclusion, we have demonstrated that cyclic peptide C5aR antagonists can penetrate transdermally sufficiently to have systemic effects. However, increasing lipophilicity in these compounds did not result in increased blood levels. Nonetheless, topical application of C5aR antagonists produced circulating levels of the drugs that antagonized the LPS-induced systemic responses of neutropenia and hypotension. This suggests that these small-molecule C5aR antagonists may be developed for topical administration for the treatment of local and systemic inflammatory conditions in the human and veterinary pharmaceutical markets.

Receptor for Fc on the surfaces of schistosomes

Schistosoma mansoni masks its surface with adsorbed host proteins including erythrocyte antigens, immunoglobulins, major histocompatibility complex class I, and beta (2)-microglobulin (beta (2)m), presumably as a means of avoiding host immune responses, How this is accomplished has not been explained. To identify surface receptors for host proteins, we biotinylated the tegument of live S, mansoni adults and mechanically transformed schistosomula and then removed the parasite surface with detergent, Incubation of biotinylated schistosome surface extracts witt l human immunoglobulin G (IgG) Fc-Sepharose resulted in purification of a 97-kDa protein that was subsequently identified as paramyosin (Pmy), using antiserum specific for recombinant Pmy, Fc also bound recombinant S. mansoni Pmy and native S. japonicum Pmy, Antiserum to Pmy decreased the binding of Pmy to Fc-Sepharose, and no proteins bound after removal of Pmy from extracts. Fluoresceinated human Fe bound to the surface, vestigial penetration glands, and nascent oral cavity of mechanically transformed schistosomula, and rabbit anti-Pmy Fab fragments ablated the binding of Fc to the schistosome surface, Pmy coprecipitated with host IgG from parasite surface extracts, indicating that complexes formed on the parasite surface as well as in vitro. Binding of Pmy to Fe was not inhibited by soluble protein A, suggesting that Pmy does not bind to the region between the CH2 and CH3 domains used by many other Fc-binding proteins. beta (2)m did not bind to the schistosome Fc receptor (Pmy), a finding that contradicts reports from earlier workers but did bind to a heteromultimer of labeled schistosomula surface proteins, This is the first report of the molecular identity of a schistosome Fc receptor; moreover it demonstrates an additional aspect of the unusual and multifunctional properties of Pmy from schistosomes and other parasitic flatworms.

Bcl-X-L translocation in renal tubular epithelial cells in vitro protects distal cells from oxidative stress

Background. The molecular pathogenesis of different sensitivities of the renal proximal and distal tubular cell populations to ischemic injury, including ischemia-reperfusion (IR)-induced oxidative stress, is not well-defined. An in vitro model of oxidative stress was used to compare the survival of distal [Madin-Darby canine kidney (MDCK)] and proximal [human kidney-2 (HK-2)] renal tubular epithelial cells, and to analyze for links between induced cell death and expression and localization of selected members of the Bcl-2 gene family (anti-apoptotic Bcl-2 and Bcl-X-L, pro-apoptotic Bax and Bad), Methods. Cells were treated with 1 mmol/L hydrogen peroxide (H2O2) Or were grown in control medium for 24 hours. Cell death (apoptosis) was quantitated using defined morphological criteria. DNA gel electrophoresis was used for biochemical identification. Protein expression levels and cellular localization of the selected Bcl-2 family proteins were analyzed (West ern immunoblots, densitometry, immunoelectron microscopy). Results. Apoptosis was minimal in control cultures and was greatest in treated proximal cell cultures (16.93 +/- 4.18% apoptosis) compared with treated distal cell cultures (2.28 +/- 0.85% apoptosis, P < 0.001). Endogenous expression of Bcl-X-L and Bax, but not Bcl-2 or Bad, was identified in control distal cells, Bcl-X-L and Bax had nonsignificant increases (P > 0.05) in these cells. Bcl-2, Bax, and Bcl-X-L, but not Bad, were endogenously expressed in control proximal cells. Bcl-X-L was significantly decreased in treated proximal cultures (P < 0.05), with Bas and Bcl-2 having nonsignificant increases (P > 0.05). Immunoelectron microscopy localization indicated that control and treated hut surviving proximal cells had similar cytosolic and membrane localization of the Bcl-2 proteins. In comparison, surviving cells in the treated distal cultures showed translocation of Bcl-X-L from cytosol to the mitochondria after treatment with H2O2, a result that was confirmed using cell fractionation and analysis of Bcl-XL expression levels of the membrane and cytosol proteins. Bax remained distributed evenly throughout the surviving distal cells, without particular attachment to any cellular organelle. Conclusion. The results indicate that in this in vitro model, the increased survival of distal compared with proximal tubular cells after oxidative stress is best explained by the decreased expression of anti-apoptotic Bcl-X-L in proximal cells, as well as translocation of Bcl-X-L protein to mitochondria within the surviving distal cells.

Diffusion modeling of percutaneous absorption kinetics: 2. Finite vehicle volume and solvent deposited solids

The diffusion model for percutaneous absorption is developed for the specific case of delivery to the skin being limited by the application of a finite amount of solute. Two cases are considered; in the first, there is an application of a finite donor (vehicle) volume, and in the second, there are solvent-deposited solids and a thin vehicle with a high partition coefficient. In both cases, the potential effect of an interfacial resistance at the stratum corneum surface is also considered. As in the previous paper, which was concerned with the application of a constant donor concentration, clearance limitations due to the viable eqidermis, the in vitro sampling rate, or perfusion rate in vivo are included. Numerical inversion of the Laplace domain solutions was used for simulations of solute flux and cumulative amount absorbed and to model specific examples of percutaneous absorption of solvent-deposited solids. It was concluded that numerical inversions of the Laplace domain solutions for a diffusion model of the percutaneous absorption, using standard scientific software (such as SCIENTIST, MicroMath Scientific software) on modern personal computers, is a practical alternative to computation of infinite series solutions. Limits of the Laplace domain solutions were used to define the moments of the flux-time profiles for finite donor volumes and the slope of the terminal log flux-time profile. The mean transit time could be related to the diffusion time through stratum corneum, viable epidermal, and donor diffusion layer resistances and clearance from the receptor phase. Approximate expressions for the time to reach maximum flux (peak time) and maximum flux were also derived. The model was then validated using reported amount-time and flux-time profiles for finite doses applied to the skin. It was concluded that for very small donor phase volume or for very large stratum corneum-vehicle partitioning coefficients (e.g., for solvent deposited solids), the flux and amount of solute absorbed are affected by receptor conditions to a lesser extent than is obvious for a constant donor constant donor concentrations. (C) 2001 Wiley-Liss, Inc. and the American Pharmaceutical Association J Pharm Sci 90:504-520, 2001.

Antagonism of the two-needle pine stem rust fungi Cronartium flaccidum and Peridermium pini by Cladosporium tenuissimum in vitro and in planta

Selected isolates of Cladosporium tenuissimum were tested for their ability to inhibit in vitro aeciospore germination of the two-needle pine stem rusts Cronartium flaccidum and Peridermium pini and to suppress disease development in planta. The antagonistic fungus displayed a number of disease-suppressive mechanisms. Aeciospore germination on water agar slides was reduced at 12, 18, and 24 h when a conidial suspension (1.5 x 10(7) conidia per ml) of the Cladosporium tenuissimum isolates was added. When the aeciospores were incubated in same-strength conidial suspensions for 1, 11, 21, and 31 days, viability was reduced at 20 and 4 degreesC. Light and scanning electron microscopy showed that rust spores were directly parasitized by Cladosporium tenuissimum and that the antagonist had evolved several strategies to breach the spore wail and gain access to the underlying tissues. Penetration occurred with or without appressoria. The hyperparasite exerted a mechanical force to destroy the spore structures (spinules, cell wall) by direct contact, penetrated the aeciospores and subsequently proliferated within them. However, an enzymatic action could also be involved. This was shown by the dissolution of the host tell wall that comes in contact with the mycelium of the mycoparasite, by the lack of indentation in the host wall at the contact site, and by the minimal swelling at the infecting hyphal tip. Culture filtrates of the hyperparasite inhibited germination of rust propagules. A compound purified from the filtrates was characterized by chemical and spectroscopic analysis as cladosporol, a known beta -1,3-glucan biosynthesis inhibitor. Conidia of Cladosporium tenuissimum reduced rust development on new infected pine seedlings over 2 years under greenhouse conditions. Because the fungus is an aggressive mycoparasite, produces fungicidal metabolites, and can survive and multiply in forest ecosystems without rusts, it seems a promising agent for the biological control of pine stem rusts in Europe.

The nutritive value of Rhodes grass (Chloris gayana) when treated with CaO, NaOH or a microbial inoculant and offered to dairy heifers as big-bale silage

A series of laboratory and animal studies examined the use of chemical and biological agents to enhance the digestibility of Rhodes grass (grass) cut at 60 (young) and 100 (mature) days of regrowth and ensiled as big round bales. The treatments included an untreated control (C), a microbial inoculant (I), NaOH, CaO and NaOH plus inoculant (NaOH + I). Inoculant was grown anaerobically, using a starter culture of rumen fluid from cattle given Rhodes grass. Treatments C, 1, NaOH, NaOH + I, were offered separately to twelve dairy heifers, in a 3 X 4 randomized complete block design, repeated twice for each grass silage. C and I had substantial mould growth, compared with no visible mould in NaOH or NaOH + 1. CaO treatment was effective in preventing mould growth, but had little effect on the chemical composition and in sacco digestibility of mature grass silage. NaOH reduced NDF content and increased in sacco digestibility (P < 0.05) but not the in vivo digestibility (P > 0.05) of both mature- and young-grass silage. The effects of other treatments on nutritive value were non-significant at both stages of maturity. NaOH increased the intake of mature-grass silage by 24-26% (P < 0.05), but had little effect on the intake of young-grass silage (P > 0.05). Treatment I consistently reduced grass silage intake (P < 005) for young-grass silage. The findings of these studies show that treating mature Rhodes grass with NaOH will improve its nutritive value and reduce mould growth in conserved herbage. However none of the treatments in this study had any consistently positive effects on the in vivo nutritive value or storage quality of young-grass silage.

Copolymers obtained by the radiation-induced grafting of styrene onto poly(tetrafluoroethylene-co-perfluoropropylvinyl ether) substrates. 1. Preparation and structural investigation

A study has been made to investigate the radiation grafting of styrene onto poly(tetrafluoroethylene-co-perfluoropropylvinyl ether) (PFA) substrates, using the simultaneous irradiation method. Two PFA polymers of different comonomer perfluoropropyl vinyl ether (PPVE) content and degree of crystallinity were used. Effects of grafting conditions such as monomer concentrations, type of solvent, dose rate, and irradiation dose on the grafting yield were investigated. Of the six different solvents used, the most efficient in terms of increasing grafting yield were dichloromethane, benzene, and methanol. The degree of grafting increased with increasing radiation dose up to 500 kGy, stabilizing above this dose. However, the grafting yield decreased with an increase in the dose rate. The grafting of styrene onto the PFA substrates was confirmed by FTIR-ATR and micro-Raman spectroscopy, The increase in the overall grafting yield was accompanied by a proportional increase in the penetration depth of the grafts into the substrate.

The copolymerization of N-vinyl-2-pyrrolidone with 2-hydroxyethyl methacrylate

The bulk free radical copolymerization of 2-hydroxyethyl methacrylate (HEMA) with N-vinyl-2-pyrrolidone (VP) was carried out to low conversions at 50 degreesC, using benzoyl peroxide (BPO) as initiator. The compositions of the copolymers; were determined using C-13 NMR spectroscopy. The conversion of monomers to polymers was studied using FT-NIR spectroscopy in order to predict the extent of conversion of monomer to polymer. From model fits to the composition data, a statistical F-test revealed that die penultimate model describes die copolymerization better than die terminal model. Reactivity ratios were calculated by using a non-linear least squares analysis (NLLS) and r(H) = 8.18 and r(V) = 0.097 were found to be the best fit values of the reactivity ratios for the terminal model and r(HH) = 12.0, r(VH) = 2.20, r(VV) = 0.12 and r(HV) = 0.03 for the penultimate model. Predictions were made for changes in compositions as a function of conversion based upon the terminal and penultimate models.

Grafted fluoropolymers as supports for solid-phase organic chemistry: Preparation and characterization

Solid-phase organic chemistry has rapidly expanded in the last decade, and, as a consequence, so has the need for the development of supports that can withstand the extreme conditions required to facilitate some reactions. The authors here prepare a thermally stable, grafted fluoropolymer support (see Figure for an example) in three solvents, and found that the penetration of the graft was greatest in dichloromethane.

Numerical investigations of flow and energy fields near a thermoacoustic couple

The flow field and the energy transport near thermoacoustic couples are simulated using a 2D full Navier-Stokes solver. The thermoacoustic couple plate is maintained at a constant temperature; plate lengths, which are short and long compared with the particle displacement lengths of the acoustic standing waves, are tested. Also investigated are the effects of plate spacing and the amplitude of the standing wave. Results are examined in the form of energy vectors, particle paths, and overall entropy generation rates. These show that a net heat-pumping effect appears only near the edges of thermoacoustic couple plates, within about a particle displacement distance from the ends. A heat-pumping effect can be seen even on the shortest plates tested when the plate spacing exceeds the thermal penetration depth. It is observed that energy dissipation near the plate increases quadratically as the plate spacing is reduced. The results also indicate that there may be a larger scale vortical motion outside the plates which disappears as the plate spacing is reduced. (C) 2002 Acoustical Society of America.

Virulence of Streptococcus pneumoniae: PsaA mutants are hypersensitive to oxidative stress

psaA encodes a 37-kDa pneumococcal lipoprotein which is part of an ABC Mn(II) transport complex. Streptococcus pneumoniae D39 psaA mutants have previously been shown to be significantly less virulent than wild-type D39, but the mechanism underlying the attenuation has not been resolved. In this study, we have shown that psaA and psaD mutants are highly sensitive to oxidative stress, i.e., to superoxide and hydrogen peroxide, which might explain why they are less virulent than the wild-type strain. Our investigations revealed altered expression of the key oxidative-stress response enzymes superoxide dismutase and NADH oxidase in psaA and psaD mutants, suggesting that PsaA and PsaD may play important roles in the regulation of expression of oxidative-stress response enzymes and intracellular redox homeostasis.

Combined chemical separation of Lu, Hf, Sm, Nd, and REEs from a single rock digest: Precise and accurate isotope Determinations of Lu-Hf and Sm-Nd using multicollector-ICPMS

A combined procedure for separating Lu, Hf, Sm, Nd, and rare earth elements (REEs) from a single sample digest is presented. The procedure consists of the following five steps: (1) sample dissolution via sodium peroxide sintering; (2) separation of the high field strength elements from the REEs and other matrix elements by a HF-free anion-exchange column procedure; (3) purification of Hf on a cation-exchange resin; (4) separation of REEs from other matrix elements by cation exchange; (5) Lu, Sm, and Nd separation from the other REEs by reversed-phase ion chromatography. Analytical reproducibilities of Sm-Nd and Lu-Hf isotope systematics are demonstrated for standard solutions and international rock reference materials. Results show overall good reproducibilities for Sm-Nd systematics independent of the rock type analyzed. For the Lu-Hf systematics, the reproducibility of the parent/daughter ratio is much better for JB-1 (basalt) than for two analyzed felsic crustal rocks (DR-N and an Archaean granitoid). It is demonstrated that this poorer reproducibility of the Lu/Hf ratio is truly caused by sample heterogeneity; thus, results are geologically reasonable.

The 2000 Ogura Lecture: Olfactory neural cells: An untapped diagnostic and therapeutic resource

Objective. This is an over-view of the cellular biology of upper nasal mucosal cells that have special characteristics that enable them to be used to diagnose and study congenital neurological diseases and to aid neural repair. Study Design: After mapping the distribution of neural cells in the upper nose, the authors' investigations moved to the use of olfactory neurones to diagnose neurological diseases of development, especially schizophrenia. Olfactory-ensheating glial cells (OEGs) from the cranial cavity promote axonal penetration of the central nervous system and aid spinal cord repair in rodents. The authors sought to isolate these cells from the more accessible upper nasal cavity in rats and in humans and prove they could likewise promote neural regeneration, making these cells suitable for human spinal repair investigations. Methods: The schizophrenia-diagnosis aspect of the study entailed the biopsy of the olfactory areas of 10 schizophrenic patients and 10 control subjects. The tissue samples were sliced and grown in culture medium. The ease of cell attachment to fibronectin (artificial epithelial basement membrane), as well as the mitotic and apoptotic indices, was studied in the presence and absence of dopamine in those cell cultures. The neural repair part of the study entailed a harvesting and insertion of first rat olfactory lamina propria rich in OEGs between cut ends of the spinal cords and then later the microinjection of an OEG-rich suspension into rat spinal cords previously transected by open laminectomy. Further studies were done in which OEG insertion was performed up to 1 month after rat cord transection and also in monkeys. Results: Schizophrenic patients' olfactory tissues do not easily attach to basement membrane compared with control subjects, adding evidence to the theory that cell wall anomalies are part of the schizophrenic lesion of neurones. Schizophrenic patient cell cultures had higher mitotic and apoptotic indices compared with control subjects. The addition of dopamine altered these indices enough to allow accurate differentiation of schizophrenics from control patients, leading to, possibly for the first time, an early objective diagnosis of schizophrenia and possible assessment of preventive strategies. OEGs from the nose were shown to be as effective as those from the olfactory bulb in promoting axonal growth across transected spinal cords even when added I month after injury in the rat. These otherwise paraplegic rats grew motor and proprioceptive and fine touch fibers with corresponding behavioral improvement. Conclusions. The tissues of the olfactory mucosa are readily available to the otolaryngologist. Being surface cells, they must regenerate (called neurogenesis). Biopsy of this area and amplification of cells in culture gives the scientist a window to the developing brain, including early diagnosis of schizophrenia. The Holy Grail of neurological disease is the cure of traumatic paraplegia and OEGs from the nose promote that repair. The otolaryngologist may become the necessary partner of the neurophysiologist and spinal surgeon to take the laboratory potential of paraplegic cure into the day-to-day realm of clinical reality.

Porous solids from layered clays by combined pillaring and templating approaches

The pore structure formation in bentonite, pillared with a mixed sol of silicon and titanium hydroxides and treated subsequently with quaternary ammonium surfactants, is investigated. The surfactant micelles act as a template, similar to their role in MCM41 synthesis. Because both the surfactant micelles and the sol particles are positively charged, it is greatly favorable for them to form meso-phase assembles in the galleries between the clay layers that bear negative charges. Besides, the sol particles do not bond the clay layers strongly as other kinds of pillar precursors do, so that the treatment with surfactants can result in radical structure changes in sol-pillared clays. This allows us to tailor the pore structure of these porous clays by choice of surfactant. The surfactant treatment also results in profound increases in porosity and improvement in thermal stability. Therefore, the product porous clays have great potential to be Used to deal with large molecules or at high operating temperatures. We also found that titanium in these samples is highly dispersed in the silica matrix rather than existing in the form of small particles of pure titania. Such highly dispersed Ti active centers may offer excellent activities for catalytic oxidation reactions such as alkanes into alcohols and ketones.

Therapeutic potential of selective superoxide dismutase mimetics

Selective superoxide dismutase (SOD) mimetics are potentially useful in pathological conditions in which there is an overproduction of the superoxide anion O-2.(-). These pathological conditions include inflammation, ischemia/reperfusion, shock, various cardiovascular disorders, amyotrophic lateral sclerosis (ALS) and other neurodegenerative disorders. A major step forward in this field was the development of small-molecule selective SOD mimetics that penetrate cell membranes, These selective SOD mimetics catalytically remove O-2.(-) without interfering with nitric oxide (NO), peroxynitrite (ONOO-) or other radicals such as hydroxyl radical or hydrogen peroxide (H2O2). These selective SOD mimetics (SC-52608, SC-55858, M-40403 and M-40401) have been shown to have benefits in animal models of inflammation, ischemia/reperfusion, shock, thrombosis and diabetes. The next challenge with selective SOD mimetics is to develop therapeutic potential into therapeutic agents.