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The solid state structure of dimethylcadmium, a classic organometallic compound with a long history, has remained elusive for almost a century. X-ray crystallography and density functional theory reveal similar phase behaviour as in dimethylzinc. The high temperature tetragonal phase, α-Me2Cd, exhibits two-dimensional disorder, while the low temperature monoclinic phase, β-Me2Cd, is ordered. Both phases contain linearly coordinated cadmium atoms. While the methyl groups are staggered in the α-phase, they are eclipsed in the β-phase.

Many years of work have been invested in the identification of potent and selective COX-2 inhibitors for the treatment of chronic inflammatory pain. One issue faced by workers is the balance between the lipophilicity required for potent enzyme inhibition and the physical properties necessary for drug absorption and distribution in vivo. Frequently approaches to reduce lipophilicity through introduction of polar functionality is hampered by highly challenging chemistry to prepare key molecules. We have complemented traditional synthetic chemistry with a biotransformations approach which efficiently provided access to an array of key target molecules.

On the basis of a mechanistic understanding of the toxicity of the 4-aminoquinoline amodiaquine (1b), three series of amodiaquine analogues have been prepared where the 4-aminophenol "metabolic alert" has been modified by replacement of the 4'-hydroxy group with a hydrogen, fluorine, or chlorine atom. Following antimalarial assessment and studies on mechanism of action, two candidates were selected for detailed ADME studies and in vitro and in vivo toxicological assessment. 4'-Fluoro-N-tert-butylamodiaquine (2k) was subsequently identified as a candidate for further development studies based on potent activity versus chloroquine-sensitive and resistant parasites, moderate to excellent oral bioavailability, low toxicity in in vitro studies, and an acceptable safety profile.

N-tert-Butyl isoquine (4) (GSK369796) is a 4-aminoquinoline drug candidate selected and developed as part of a public-private partnership between academics at Liverpool, MMV, and GSK pharmaceuticals. This molecule was rationally designed based on chemical, toxicological, pharmacokinetic, and pharmacodynamic considerations and was selected based on excellent activity against Plasmodium falciparum in vitro and rodent malaria parasites in vivo. The optimized chemistry delivered this novel synthetic quinoline in a two-step procedure from cheap and readily available starting materials. The molecule has a full industry standard preclinical development program allowing first into humans to proceed. Employing chloroquine (1) and amodiaquine (2) as comparator molecules in the preclinical plan, the first preclinical dossier of pharmacokinetic, toxicity, and safety pharmacology has also been established for the 4-aminoquinoline antimalarial class. These studies have revealed preclinical liabilities that have never translated into the human experience. This has resulted in the availability of critical information to other drug development teams interested in developing antimalarials within this class.

Undifferentiated rat pheochromocytoma (PC12) cells extend neurites when cultured in the presence of nerve growth factor (NGF). Extracellular guanosine synergistically enhances NGF-dependent neurite outgrowth. We investigated the mechanism by which guanosine enhances NGF-dependent neurite outgrowth. Guanosine administration to PC12 cells significantly increased guanosine 3',5'-cyclic monophosphate (cGMP) within the first 24 h whereas addition of soluble guanylate cyclase (sGC) inhibitors abolished guanosine-induced enhancement of NGF-dependent neurite outgrowth. sGC may be activated either by nitric oxide (NO) or by carbon monoxide (CO). [Formula: see text]-Nitro-L-: arginine methyl ester (L-: NAME), a non-isozyme selective inhibitor of nitric oxide synthase (NOS), had no effect on neurite outgrowth induced by guanosine. Neither nNOS (the constitutive isoform), nor iNOS (the inducible isoform) were expressed in undifferentiated PC12 cells, or under these treatment conditions. These data imply that NO does not mediate the neuritogenic effect of guanosine. Zinc protoporphyrin-IX, an inhibitor of heme oxygenase (HO), reduced guanosine-dependent neurite outgrowth but did not attenuate the effect of NGF. The addition of guanosine plus NGF significantly increased the expression of HO-1, the inducible isozyme of HO, after 12 h. These data demonstrate that guanosine enhances NGF-dependent neurite outgrowth by first activating the constitutive isozyme HO-2, and then by inducing the expression of HO-1, the enzymes responsible for CO synthesis, thus stimulating sGC and increasing intracellular cGMP.

Nitric oxide (NO), a diffusible and unstable gas, has been implicated in inter- and intra-cellular communication in the nervous system. NO also plays a role in neural development, plasticity and alterations of synaptic function such as long-term potentiation and long-term depression (Gally et al.: Proc NY Acad Sci, 87: 354-355, 1990; Zhuo et al.: Science 260:1946-1950, 1993; Schuman and Madison.: Science 254:1503-1506, 1991; Bruhwyler et al.: Neurosci Biobehav Rev 17:373-384, 1993) some of which likely involve growth and remodelling of neurites. Some actions of NO are mediated directly by protein modification (e.g., nitrosylation) and others by activation of soluble guanylyl cyclase (soluble GC), which increases intracellular levels of guanosine 3',5'-cyclic monophosphate (cGMP). NO is synthesized by the enzyme nitric oxide synthase (NOS), which is induced by treatment of CNS neurons (Holtzman et al.: Neurobiol Disease 1:51-60, 1994) or pheochromocytoma PC12 cells (Hirsch et al.: Curr Biol 3:749-754, 1993) with NGF. NO has been proposed to mediate some of the effects of NGF on PC12 cells by inhibiting cell division (Peunova and Enikolopov: Nature 374:68-73, 1995). In addition, NO can substitute for NGF by delaying the death of trophic factor-deprived PC12 cells through a mechanism that does not involve a cytostatic action (Farinelli et al.: J Neurosci 16:2325-2334, 1996). We investigated whether NO stimulated neurite outgrowth from hippocampal neurons and PC12 cells. Primary cultures of E17 mouse hippocampal neurons co-cultured with neopallial astrocytes were exposed to the NO donors sodium nitrite (100 microM) or sodium nitroprusside (100 nM). After 48 hr, NO donor-treated cultures contained a greater proportion of cells bearing neurites and neurites that were much longer than those found in control cultures. In cultures of PC12 cells, NO donors also enhanced the neuritogenic effects of NGF. The proportion of PC12 cells with neurites 48 hr after exposure to NO donors sodium nitrite (100 microM-10mM) or sodium nitroprusside (100 nM-1 micro M) plus 2.5S nerve growth factor (NGF) was approximately twice the proportion of cells with neurites in sister cultures grown in NGF alone. Neither of the NO donors elicited neurites from the PC12 cells in the absence of NGF. The effects of the NO donors were likely mediated by release of NO since their effects were antagonized by addition of hemoglobin, which avidly binds NO, to the culture medium. The enhancement by NO of NGF-mediated neurite outgrowth in PC12 cells appeared to occur through a cGMP-dependent mechanism. The NO donors stimulated a prompt increase in intracellular cGMP in PC12 cells. Moreover their action was mimicked by addition of the membrane-permeant cGMP analogs 8-Bromo-cGMP (8-Br-cGMP) and para (chlorophenylthio)-cGMP (pCPT-cGMP) to the culture medium and by atrial natriuretic factor which stimulates particulate guanylyl cyclase. The neuritogenic activity of the NO donors was inhibited by LY83583 and methylene blue, inhibitors of guanylyl cyclase. These data imply that NO may act alone or with other growth factors to regulate synapse formation and maintenance by stimulating neurite outgrowth.

AIT-082 is a novel, metabolically stable, derivative of the purine hypoxanthine. Addition of AIT-082 to cultured PC12 cells enhanced significantly nerve growth factor (NGF)-mediated neurite outgrowth from PC12 cells. These results suggest a cellular mechanism, the enhancement of NGF-action, that might account for the ability of AIT-082 to restore age-induced working memory deficits in mice.

Oligosaccharide profiles were obtained for chimeric mouse-human antibodies corresponding to each of the human IgG subclasses 1-4, and mouse IgG2b antibodies each expressed in the mouse J558L cell line. These antibodies have specificity for the NIP hapten and form a matched set of IgGs. An IgG4 chimeric antibody (B72.3) produced in the chinese hamster ovary (CHO-K1) cell line was also analysed for carbohydrate. Additionally aglycosylated mutants of this IgG4 (B72.3) and anti-NIP mouse IgG2b were analysed. The total lack of carbohydrate found in the aglycosylated site-directed mutants human chimeric IgG4 B72.3 (Asn 297-->Gln) and mouse IgG2b (Asn 297-->Ala) demonstrates that there are no N-glycosylation sites other than Asn 297. Therefore glycosylation profiles for all the IgGs analysed reflect carbohydrate attached to this site. Factors such as cell type (A), template direction by the IgG heavy chains (B) and culture conditions (C) are shown to influence IgG glycosylation profiles. (A) The anti-NIP IgG antibodies expressed by the J558L cell line may have one or two Gal (alpha 1-->3) Gal residues per oligosaccharide unit, indicative of the presence of (alpha 1-->3) galactosyl transferase in the J558L mouse cell line. (B) The galactosylation profiles obtained for the IgG heavy chains, in particular the preference for galactosylation of the Man (alpha 1-->6) arm rather than the Man (alpha 1-->3) arm, contrary to the beta-galactosyltransferase specificity, suggest that the polypeptide chain may act as a template to influence the extent of galactosylation and hence the proportions of each oligosaccharide incorporated. The IgG2 antibody does not display this galactosylation preference. (C) The extent of galactosylation appears to be influenced by the growth conditions, with the highest levels of galactosylation being found for IgG produced by cells grown in still cultures, rather than cells grown as ascites or in hollow fibre bioreactors. It is concluded that though the profile of glycosylation is controlled predominantly by the glycosylation activity of the cell in which the IgG is expressed, differences between the IgG heavy chain templates of the various subclasses and culture conditions can also influence glycosylation.

Oligosaccharide profiles for monoclonal chimeric mouse-human and mouse IgG2b antibodies produced in the mouse J558L cell line have been determined. These chimeric antibodies share the same mouse light chain and VH region and have a single, complex, oligosaccharide moiety attached to residue 297 of the heavy chain. Since the panel of chimeric antibodies included each of the human IgG subclasses and a set of IgG3 proteins having single residue differences, it was possible to evaluate the contributions of the protein template, the cell line, and the culture conditions to glycosylation. The study shows that the J558L cell line glycosylates human heavy chains with oligosaccharides characteristic of mouse immunoglobulins. This includes a galactose alpha 1-->3 galactose structure not found in human IgG and for which most humans have naturally occurring "anti-Gal" antibodies. The extent of galactosylation was very dependent on the culture conditions and was maximal for still culture. The findings have significance for the production of antibodies for in vivo administration.

Intraperitoneal and intracerebral injections of methyl beta-carboline-3-carboxylate (beta CCM) and intracerebral injections of RO 15-1788 were given to rats. The performance of the rats in the social interaction test was measured to determine if changes in social interaction induced by beta CCM were mediated in part by the nucleus raphé dorsalis (NRD). Intraperitoneal injections of beta CCM, 2 and 4 mg kg-1, reduced social interaction. Intracerebral microinjections of beta CCM (10-0.1 ng in 0.5 microliter) into the NRD reduced social interaction. Injections outside the NRD did not have this effect. Intracerebral microinjections of RO 15-1788 (1 ng in 0.5 microliters) into the NRD had no effect when given alone, but blocked the reduction in social interaction caused by intracerebral or intraperitoneal injections of beta CCM. No effect was observed when R 15-1788 was microinjected into sites outside the NRD. Changes in social interaction may reflect changes in anxiety. The NRD may be one of the important sites for the expression of the anxiogenic actions of beta CCM.

Skin testing with antigens from Histoplasma capsulatum, Mycobacterium tuberculosis (PPD-S), and atypical mycobacteria (PPD-B, PPD-G, PPD-Y, and PPD-platy) was carried out among six population groups in the Solomon Islands between 1968 and 1972. There was no positive reaction to histoplasmin among any of the groups, suggesting that histoplasmosis is not endemic in the Solomon Islands. There were significant numbers of tuberculin reactors among each group. Largest mean reactions to PPD-S were present among the Lau and Ulawa, in whom reactions to PPD-S were larger than those to any other antigen tested. Thus significant infection with M. tuberculosis appears to occur in these populations. This was corroborated by radiologic survey. Among the Lau, large reactions to PPD-G and PPD-Y were also elicited, raising the possibility of multiple infection. Among the Aita, Baegu, Nagovisi, and Ontong Java, PPD-G elicited the largest reactions. PPD-G produced the second largest reactions among the Lau and Ulawa. PPD-S elicited the largest or second largest reaction among 4 of the 6 groups. A notable exception was the Aita, in whom PPD-S elicited the smallest mean reaction. The Aita also had the lowest prevalence of radiologic findings consistent with tuberculosis. These observations suggest that M. tuberculosis has been introduced into the Solomon Islands from outside sources, a hypothesis which may explain the variability in prevalence of tuberculosis-like disease demonstrated by chest film among the six groups. Genetic differences may also play a role in this variability. The study also demonstrated a high prevalence of "baseline" sensitivity to the atypical mycobacteria among the Solomon Islanders. This sensitivity may confer some immunity to infection with M; tuberculosis, but this protection is far from complete.

1. At 3 min after an intravenous injection of radioactive amino acids into the rat, the bulk of radioactivity associated with liver polyribosomes can be interpreted as growing peptides. 2. In an attempt to identify the rate-limiting step of protein synthesis in vivo and in vitro, use was made of the action of puromycin at 0 degrees C, in releasing growing peptides only from the donor site, to study the distribution of growing peptides between the donor and acceptor sites. 3. Evidence is presented that all growing peptides in a population of liver polyribosomes labelled in vivo are similarly distributed between the donor and acceptor sites, and that the proportion released by puromycin is not an artifact of methodology. 4. The proportion released by puromycin is about 50% for both liver and muscle polyribosomes labelled in vivo, suggesting that neither the availability nor binding of aminoacyl-tRNA nor peptide bond synthesis nor translocation can limit the rate of protein synthesis in vivo. Attempts to alter this by starvation, hypophysectomy, growth hormone, alloxan, insulin and partial hepatectomy were unsuccessful. 5. Growing peptides on liver polyribosomes labelled in a cell-free system in vitro or by incubating hemidiaphragms in vitro were largely in the donor site, suggesting that either the availability or binding of aminoacyl-tRNA, or peptide bond synthesis, must be rate limiting in vitro and that the rate-limiting step differs from that in vivo. 6. Neither in vivo nor in the hemidiaphragm system in vitro was a correlation found between the proportion of growing peptides in the donor site and changes in the rate of incorporation of radioactivity into protein. This could indicate that the intracellular concentration of amino acids or aminoacyl-tRNA limits the rate of protein synthesis and that the increased incorporation results from a rise to a higher but still suboptimum concentration.