The design and discovery of novel amide CCR5 antagonists

The synthesis of a range of novel amine-containing structures and their primary potency as inhibitors of HIV-1 fusion via blocking of the CCR5 receptor is described. The development of the medicinal chemistry strategy and SAR’s which led to the identification of the piperidine amide compounds 33 and 36 as excellent leads for further evaluation is described, along with key physicochemical data which highlighted their lead potential.     

Use of a rat Y chromosome probe to determine the long-term survival of glial cells transplanted into areas of CNS demyelination

A lack of suitable markers for cells which undergo division following transplantation has hindered studies assessing the long-term survival of glial cell grafts in the CNS. A probe specific to the rat Y chromosome was used to identify male glial cells grafted into an area of ethidium bromide-induced demyelination in syngeneic adult female rat spinal cord 4 weeks, 6 months and 12 months post-transplantation. At all time points there was extensive oligodendrocyte remyelination of transplanted lesions, and graft-derived cells were present within the lesion up to 12 months post-transplantation. In order to demonstrate graft-derived oligodendrocytes in the remyelinated region at 6 and 12 months, double-labelling studies were performed using the oligodendrocyte-specific antibodies carbonic anhydrase II or phosphatidyl ethanolamine-binding protein in combination with the Y chromosome probe. It was found that the majority of oligodendrocytes in the transplanted region were graft-derived. Graft-mediated remyelination was associated with a reduction in myelin sheath thickness and increase in nodal frequency similar to that observed in spontaneous remyelination, suggesting that, like axons remyelinated spontaneously, axons remyelinated by grafted cells will be capable of secure conduction. An alteration in the immunoreactivity of oligodendrocytes from carbonic anhydrase II-negative in the unlesioned dorsal funiculus to carbonic anhydrase II-positive in the remyelinated dorsal funiculus was considered to reflect a reduction in the amount of myelin supported by each oligodendrocyte, leading to the proposal that carbonic anhydrase II immunoreactivity may provide a means of identifying areas of remyelination in normally carbonic anhydrase II-negative white matter tracts.     

Synergistic iron reduction and citrate dissimilation by Shewanella alga and Aeromonas veronii

Two bacterial isolates from Great Bay Estuary, New Hampshire, in co-culture carried out anaerobic dissimilation of citric acid with Fe(III) as the terminal electron acceptor. Neither isolate oxidized citrate with Fe(III) anaerobically in axenic culture. The Fe(III) reducer, Shewanella alga strain BrY, did not grow anaerobically with citrate as an energy source. The citrate utilizer, Aeromonas veronii, did not reduce iron axenically with a variety of electron donors including citrate. The onset of iron reduction by the co-culture occurred after initiation of citrate dissimilation and just prior to initiation of growth by either organism (as measured by viable plate counts). Anaerobic culture growth rates and final cell densities of each bacterial strain were greater in co-culture than in axenic cultures. By 48 h of growth, the co-culture had consumed 27 mM citrate as compared with 12 mM dissimilated by the axenic culture of A. veronii. By 48 h the co-culture produced half as much formate (6 mM) and twice as much acetate (40 mM) as did A. veronii grown axenically (12 mM and 20 mM, respectively). Formate produced from citrate by A. veronii appeared to have supported growth and Fe(III) reduction by S. alga.Although not obligatory, nutrient coupling between these two organisms illustrates that fermentative (A. veronii-type) organisms can convert organic compounds such as citrate to those used as substrates by dissimilatory Fe(III) reducers, including S. alga. This synergism broadens the range of substrates available for iron reduction, stimulates the extent and rate of organic electron donor degradation (and that of iron reduction) and enhances the growth of each participant. Received: 11 December 1995 / Accepted: 19 June 1996     

The catabolism of mammalian glycoproteins. Comparison of the storage products in bovine, feline and human mannosidosis.

Analysis of the neutral urinary oligosaccharides in bovine, feline and human mannosidosis by thin-layer and gel-permeation chromatography has shown that the patterns of stored oligosaccharides in the three species are different. In bovine and feline mannosidosis the most abundant urinary oligosaccharide is also the most abundant in the tissues of each species. The predominant oligosaccharides were purified by a combination of gel-filtration, ion-exchange and thin-layer chromatography and shown to contain only mannose and N-acetylglucosamine by g.l.c. and g.l.c.--mass spectrometry. The probable composition and size of each oligosaccharide were predicted from its chromatographic properties, sugar composition and the known structure of asparagine-linked oligosaccharides. The bovine and feline oligosaccharides belonged to a homologous series of general composition Mann (GlcNAc)2, whereas the human oligosaccharides belong to a different series, MannGlcNAc. These structures suggest that lysosomal endohexosaminidase is not present in bovine and feline tissues. The predominant feline storage product, Man3(GlcNAc)2, was the expected storage product from the catabolism of complex asparagine-linked glycans. In contrast, the predominant bovine oligosaccharide, Man2(GlcNAc)2, probably lacks one of the alpha-linked mannose residues in the core region. A similar situation occurs in human mannosidosis. It is predicted that in these species either that the residual mutant alpha-D-mannosidase retains activity towards one of the core alpha-linked mannose residues or that another form of lysosomal alpha-D-mannosidase that is unaffected in these disorders occurs. It is concluded that the differences in storage products are due to differences in the catabolic pathways of glycoproteins among the species.     

Biochemical studies on a case of feline mannosidosis.

Evidence is presented for the biochemical diagnosis of the first case of feline mannosidosis. A marked deficiency of acidic alpha-D-mannosidase in the brain, kidney and liver and excessive excretion of mannose-rich oligosaccharides in the urine were found in a kitten suffering from a nervous disorder. Residual acidic alpha-D-mannosidase, ranging from 2 to 5.5% of the normal activity, was observed in the tissues of the affected kitten. It has similar kinetic and physicochemical properties to the normal activity. The amount of mannose in the urine of the affected kitten was 19-fold greater than in a comparable control, and the molar ratio of mannose to N-acetylglucosamine was approx. 6 : 1. High concentrations of neutral oligosaccharides were detected in the urine. The predominant oligosaccharide appeared to be a hexasaccharide. The biochemical features of bovine, feline and human mannosidosis are compared, and it is concluded that feline mannosidosis may be a useful animal model for studying the human disease.     

Recovery from monocular deprivation in the monkey. III. Reversal of anatomical effects in the visual cortex

Transneuronal autoradiography was used to study the effects of visual deprivation on the ocular dominance stripes in layer IVc of the striated cortex of Erythrocebus patas (Old World) monkeys. The animals were studied after: (a) 21-28 days of monocular deprivation starting at, or within, a few days of birth; (b) the same treatment followed by a further 3, 6, 15 or 126 days of monocular vision through both eyes (reopening). One other monkey was monocularly deprived from birth to 1890 days. In most cases the behaviour of the ocular dominance stripes formed by the initially closed eye was studied. After 24 days of monocular deprivation from birth, the input from the normal eye was distributed uniformly within layer IVc, with no periodicity evident. After 21 days of deprivation, the deprived eye's input formed narrow stripes occupying about 38% of layer IVc in the operculum. Seven months of monocular deprivation reduced this to about 29%. Opening the closed eye after the deprivation produced no change in the area innervated: when periods of 15 or 96 days of binocular vision followed the deprivation, the areas innervated by the initially deprived eye were 26 and 30% respectively. However, in both cases the deprived eye's input formed blobs and spots, rather than uniformly narrow stripes. In contrast to reopening, reverse suturing increased the fraction of layer IVc occupied by input form the initially deprived eye. In the operculum, the effects of reverse suturing appeared to be fully developed after only 6 days of reversal: the initially deprived eye's stripes having expanded to occupy about 50% of layer IVc. A further 9 days' reversal produced little change in this. In the visual cortex in the calcarine fissure, the effect of the initial deprivation ws more severe, and the expansion induced by reverse suturing more pronounced. The initial deprivation caused the stripes to shrink to occupy 24% of layer IVc; after 6 days of reverse sulture the proportion increased to 52%, while after 15 days of reverse suture about 88% of IVc was occupied. These results show that reverse suturing can cause fresh growth of afferent axons in regions of layer IVc from which they had been at least partially removed, either by the normal process of segregation, or as a consequence of monocular deprivation. Taken in conjuction with the findings of the accompanying two papers (Blackemore et al...     

Iron reduction byAquaspirillum magnetotacticum

Iron reductase activity in cell extracts ofAquaspirillum magnetotacticum strain MS-1 (wild type) or nonmagnetotactic mutant strain NM-1A was located primarily in the periplasm. Cytoplasm contained 20%–35% and membrane fractions 3% of total iron reductase activity detected. Iron reduction was reversibly inhibited by oxygen, required -NADH, and was enhanced by flavins. Reduced disulfide bonds and uncomplexed sulfhydryl groups were necessary for reductase activity. Respiratory inhibitors did not appreciably affect iron reductase activity. Iron complexed with quinic acid, dihydroxybenzoic acid, acetohydroxamic acid, citric acid, or deferrioxamine B was reduced by soluble iron reductases of strain MS-1.     

First-trimester prenatal diagnosis of Tay-Sachs disease.

The prenatal diagnosis of Tay-Sachs disease was made in two at-risk fetuses by the analysis of chorionic villi obtained at 9 and 11 menstrual weeks, respectively. The diagnoses were based on the absence of beta-hexosaminidase A activity as determined by: (1) specific enzyme assays, (2) anion-exchange chromatography, and (3) cellulose acetate gel electrophoresis. The enzymatic diagnoses were confirmed on fetal tissue as well as by ultrastructural demonstration of the first-trimester fetal neuropathology. Optimal assay conditions for beta-hexosaminidase A in chorionic villi were established for the prenatal diagnosis of Tay-Sachs disease. Importantly, it was noted that a small amount of decidua or maternal blood could lead to misdiagnosis. Thus, extreme care must be taken in the preparation of chorionic villi for Tay-Sachs as well as other prenatal metabolic diagnoses.     

The use of optical emission spectroscopy for human 15N tracer studies.

Optical emission spectroscopy is a convenient method for determing ¹⁵N specific activity in a variety of metabolites following the administration of an ¹⁵N-labeled amino acid to humans. The only disadvantage, compared to the more conventional isotope ratio mass spectrometer analytical system is that more tracer is needed to give accurate results. When an ¹⁵N-labeled amino acid is used as the ¹⁵N carrier, the amount of ¹⁵N is not above a tracer dose. A suitable procedure is described for performing such studies using [¹⁵N]glycine as a tracer. As an experimental example, [¹⁵N]glycine was infused into a subject at a rate of 35 mg of N/hr for 8 hr. After 3 hr a meal was consumed by the subject. The urinary urea-¹⁵N, ammonia-¹⁵N and amino acid-¹⁵N profiles were determined.     

Effects of Polychlorinated Biphenyls on Macromolecular Synthesis by a Heterotrophic Marine Bacterium

Growth rates and final cell yields of a polychlorinated biphenyl (PCB)-sensitive pseudomonad isolated from the open ocean were reduced in a dose-dependent manner by 10 to 100 μg of Aroclor 1254 per liter, a commercial mixture of PCB isomers added to its culture medium. Effects on growth rates were detected within 1 h (approximately one doubling time) of treatment. By 4 h posttreatment, the amounts of deoxyribonucleic acid and ribonucleic acid per cell in exponentially growing populations treated with sublethal doses of Aroclor were detectably lower than in appropriate controls. Corresponding cell protein values were slightly higher than in controls. Selective degradation of cell proteins or nucleic acids was not detected in cells whose growth was totally suppressed for 4 h by PCBs. Cells whose growth rate was inhibited 20 to 50% by Aroclor synthesized protein at normal rates for periods in excess of 5 h from the time the chlorinated hydrocarbons were added. In contrast, rates per cell of adenine uptake and adenine incorporation into deoxyribonucleic acid and total nucleic acids by the cells treated with PCBs were significantly lower than in control cells. Intracellular adenine pools of cells whose growth was inhibited to 20% of the control rate by PCBs were 30% smaller and appeared to require a longer interval to equilibrate than those of untreated cells. This may indicate impaired transport and/or efflux of this nucleic acid precursor through the membrane of affected cells. Inhibition of nucleic acid synthesis in this sensitive bacterium by PCBs could explain the observed inhibitory effects of the chlorinated hydrocarbons on its growth.