Gillian Robin Bushell

Neurogenesis in adult human

THIS report describes neurogenesis in the adult human olfactory epithelium in vitro. Olfactory epithelium was collected at autopsy and by biopsy, and grown in serum-free medium. Basic fibroblast growth factor induced the differentiation of bipolar cells which were immunopositive for several neuronal proteins but not glial proteins. [3H]thymidine autoradiography confirmed that these neurones were born in vitro. The results demonstrate that the adult human olfactory epithelium retains the capacity for neurogenesis and neuronal differentiation, at least until the age of 72 years. It is now possible to examine neurones and neurogenesis in biopsies from patients with disorders that may involve a neurodevelopmental or neurodegenerative aetiology such as schizophrenia, bipolar disorder and Alzheimers disease.

The 140/130/105 kilodalton protein complex in the rhoptries of Plasmodium falciparum consists of discrete polypeptides

Four monoclonal antibodies (MAbs) recognise an antigen localised in the rhoptries of Plasmodium falciparum merozoites using both indirect immunofluorescence assay and immunoelectron microscopy with immunogold labeling. All MAbs immunoprecipitated bands at 140, 130 and 105 kDa from [35S]methionine-labeled parasites; however, one MAb immunoblotted only the 130 kDa protein and another MAb immunoblotted the 105 kDa protein. The affinity purified antigen complex consisted of proteins of 140, 130, 105 and 98 kDa. The individual proteins were subjected to peptide mapping with Staphylococcus aureus V8 protease; the 98 kDa protein was a degradation product of the 105 kDa protein and the 140, 130, and 105 kDa proteins were found to be unrelated. The antigen complex was synthesised at the mid trophozoite stage and was considered to be soluble as judged by release from mature schizonts by freeze/thaw lysis. One of the MAbs inhibited parasite growth and/or merozoite invasion of erythrocytes, in vitro, to a small but significant extent.

Colloidal metallic gold is not bio-inert

Abstract.Metallic gold (Au°) is a likely biotransformation product of monovalent gold, Au(I) whenever it is dissociated from in vivo ligands, Au° being formed either by bioreduction or by spontaneous dismutation (with co-production of trivalent gold). This review discusses the preparation and some biologically relevant properties of colloidal metallic gold (CMG) in its nano-particulate form. Tyndall’s purple, a well characterised preparation of CMG, shows potent anti-arthritic activity in rats, approximately 103 times that of sodium aurothiomalate (Myocrysin). Even more remarkable is its broader spectrum of action in rats compared to this classic DMARD.

Nanogold-pharmaceutics (i) the use of colloidal gold to treat experimentally-induced arthritis in rat models; (ii) characterization of the gold in Swarna bhasma, a microparticulate used in traditional Indian medicine

Nanosized gold particles (27 +/- 3 nm) have been proven to be effective in ameliorating the symptoms of mycobacterial-, collagen- and pristane-induced arthritis in rat models. This contrasts with the drug sodium aurothiomalate that was only effective against mycobacterial-induced arthritis but not to the same extent as Au0. Gold in the traditional Indian Ayurvedic medicine, Swarna bhasma (gold ash), has been characterized as globular particles of gold with an average size of 56-57 nm.

Imaging and Force-Distance Analysis of Human Fibroblasts In Vitro by Atomic Force Microscopy

The structure of human fibroblasts have been characterised in vitro by atomic force microscopy (AFM) operated in the imaging or in the force versus distance (F-d) modes. The choice of cell substrate is important to ensure good adhesion. Of greater significance in the context of AFM analysis, is the observation that the substrate affects the imaging conditions for in vitro analysis of live cells. For instance, very rarely will glass coverslips lead to acceptable outcomes (i.e., resolved cytoskeletal structure). Activated tissue culture dishes, on the other hand, promote conditions that routinely result in good quality images. Those conditions are then unaffected by adoption of relatively high force loadings (more than 10 nN), large fields of view (100 x 100 microm2) and high scan speeds (up to ca. 200 microm/sec), all of which exceed values recommended in the literature. Plasma membranes are fragile in the context of AFM analysis (F-d analysis gives an equivalent Youngs Modulus of ca. 5 kPa). However, the present work suggests that fragility per se need not be a problem, rather it is the adhesive interactions with the tip, which under some circumstances may exceed 20 nN, that are the source of poor imaging conditions. The present results, being supported by a qualitative model, suggest that the activated substrate acts as a preferential scavenger of cellular debris thus preventing the tip from biofouling, and will therefore promote low adhesion between tip and membrane. Good imaging conditions provide non-destructive in vitro information about cytoskeletal structure and dynamics, as shown in two examples concerned with cytochalasin treatment and with the MTT assay.

Cloning and characterisation of cDNA clones encoding two Babesia bovis proteins with homologous amino- and carboxy-terminal domains

A dextran sulphate protein (DSP) fraction derived from Babesia bovis has previously been shown to induce a protective immune response in cattle. A B. bovis cDNA library was screened with both the complete anti-DSP serum and a subfraction of the anti-DSP serum affinity purified on a native B. bovis protein of approx. 80 kDa. cDNA clones encoding two different B. bovis proteins were identified. The product of one gene, Bv80, has a single divergent copy of a sequence of 149 amino acids (approx. 30% amino acid identity) in both the amino- and carboxy-terminal domains. These domains are separated by an array of short variant repeat sequences rich in proline and glutamic acid. The product of the other gene, BvVAl (homologous to the previously described 225-kDa B. bovis protein)[19], is predicted to have a single divergent copy of a sequence of 170-171 amino acids (approx. 35% amino acid identity) in both the amino- and carboxy-terminal domains. These domains are also separated by an array of repeats. The 73-amino acid repeat unit of this array is composed of a number of variant derivatives of shorter repeat units. Detailed analysis of genomic clones flanking two alleles of the gene encoding BvVAl/225 kDa identified further members of a multi-gene family. This region of the genome of B. bovis has been subject to a large number of amplification processes.

Jun, Fos and Krox in the thalamus after C-fiber stimulation: coincident-input-dependent expression, expression across somatotopic boundaries, and nucleolar translocation

Expression of the inducible transcription factors Jun, Fos and Krox is commonly used to map neurons in the brain that are activated by sensory inputs. However, some neurons known to be electrically excited by such inputs do not always express these factors. In particular, stimulation of hindlimb sensory nerve C-fibers induces expression of c-Fos in the medial thalamus (the mediodorsal, intermediodorsal, centrolateral and centromedial), but not in the lateral thalamus (the ventroposterolateral, ventroposteromedial and posterior group). We hypothesized that c-Fos expression might only occur in these lateral areas after more complex stimulation patterns, or that only other transcription factors can be induced in these areas by such stimuli. Thus we examined the effects of single, repeated and coincident C-fiber inputs on expression of six inducible transcription factors in the medial, lateral and reticular thalamus of the rat. A weak C-fiber input caused by noxious mechanical stimulation of the skin of one hindpaw did not induce expression of c-Fos, FosB, Krox-20 or Krox-24; but it did reduce the basal expressions of c-Jun and JunD in both the medial and lateral areas. An intense input produced by electrical stimulation of all the C-fibers in one sciatic nerve also failed to induce expression of c-Fos, FosB, Krox-20 or Krox-24 in the medial or lateral areas. However, in the medial thalamus it increased c-Jun and reduced the basal expression of JunD, whereas in the lateral thalamus it had no effect on c-Jun but again reduced the basal expression of JunD. With repeated stimulation, i.e. when the noxious stimulus was applied to the contralateral hindpaw 6 h after the sciatic stimulation, there was again no induction of c-Fos, FosB or Krox-20 in the medial thalamus; but there was an increase in c-Jun and Krox-24, and a decrease in JunD levels. In the lateral thalamus the repeated stimulation again failed to induce c-Fos, but the expressions of FosB, c-Jun and Krox-24 were increased, and that of JunD was again reduced. With coincident stimulation, i.e. when a stimulus was applied to each hindpaw simultaneously, c-Fos and Krox-24 remained absent; but there was a marked induction of FosB and Krox-20, a strong repression of c-Jun, and no effect or a reduction of the basal levels of JunD. This coincident stimulation also caused FosB to appear in the nucleolus of many thalamic neurons. MK-801, but not L-NAME, blocked all these changes. In summary, noxious stimulation affects the expression of all transcription factors in the medial, lateral and reticular thalamus in a complex manner depending upon the inducible transcription factor considered, the thalamic nucleus, and the stimulation paradigm. The expression of some transcription factors uniquely after simultaneous inputs suggests they act as coincidence detectors at the gene level.

Olfactory neuronal cell lines generated by retroviral insertion of the n-myc oncogene display different developmental phenotypes

Being genetically homogeneous, clonal cell lines are potentially important for investigating many aspects of cellular differentiation. We describe here the creation of clonal cell lines by immortalization of neuronal precursor cells from the adult mouse olfactory epithelium. Unlike neurons elsewhere in the vertebrate nervous system, the olfactory sensory neuron can be replaced throughout the lifespan of the animal. However, little is known about the molecular aspects of olfactory neurogenesis. Continuous cell lines were generated by retroviral transduction of the n‐myc proto‐oncogene into the mitotically active basal cells of the olfactory epithelium which give rise to the sensory neuron. Twenty‐one clonal cell lines were produced which could be divided into three distinct morphological classes: one with flat, epithelial‐like cells only; another with round, flat, and bipolar cells; and a third with large flat and large bipolar cells. These morphological classes had different patterns of intermediate filament expression, as shown by immunocytochemistry and immunoblot analysis. All cells in all cell lines expressed the intermediate filament protein vimentin. Most bipolar cells, but not other cell types, expressed neurofilament protein and in one morphological class the bipolar cells co‐expressed neurofilament and glial fibrillary acidic protein. Several cell lines expressed mRNA for OMP, a marker of mature olfactory sensory neurons, and GOLF, a guanine nucleotide binding protein involved in olfactory sensory transduction. It is concluded that these cell lines were immortalized from sensory neuron precursors late in the lineage pathway. Other cell lines appear to have been immortalized at earlier stages in the lineage pathway. These cell lines therefore provide useful tools for the investigation of neuronal differentiation and sensory transduction in the olfactory epithelium.

Imaging and nano-dissection of tobacco mosaic virus by atomic force microscopy

Tobacco mosaic virus (TMV) has been deposited on freshly cleaved mica substrates. The topography was investigated by contact, non‐contact and lateral‐force microscopy under ambient conditions in air. The results were in accord with known dimensions of TMV (i.e. 18 nm in diameter and 300 nm in length). However, convolution of tip shape with TMV morphology resulted in an apparent width of 80–140 nm in the lateral plane, a factor of 4–7 greater than the known diameter. Other artefacts ‐ broadening and double images ‐ were observed and ascribed to tip anomalies.

Aurocyanide, dicyano-aurate (I), a pharmacologically active metabolite of medicinal gold complexes

Abstract.The aurocyanide anion, Au(CN)2−, is a human metabolite of several anti-rheumatic gold complexes containing monovalent gold (I) bound to a sulphur ligand. This article reviews some of the chemical and pharmacological properties of this intriguing metabolite, and reports its anti-arthritic and anti-inflammatory activity in rats. Au(CN)2− is generated from the therapeutic gold complexes by small amounts of hydrogen cyanide, HCN, produced from thiocyanate, SCN−, by myeloperoxidase (MPO) an enzyme in neutrophils which normally produces hypochlorite, OCl−. Thus, Au(CN)2− is formed at sites of inflammation where activated neutrophils are present. This includes atherosclerotic lesions as well as inflamed joints. MPO also oxidises Au(CN)2− to Au(III) complexes such as Au(CN)4−.Au(CN)2− is normally a very stable monovalent gold complex. In a biological context, only low concentrations are ever present at both extracellular and intracellular sites. However, Au(CN)2− produced locally may facilitate the cellular uptake and hence the therapeutic and toxic effects of gold drugs. Au(CN)2− may also be involved in a redox cycle where Au(CN)2− is oxidised to Au(CN)4− which is, in turn, reduced back to Au(CN)2− by endogenous thiols. There are still many questions to be resolved concerning Au(CN)2− including its intrinsic toxicity and the extent to which it may contribute to the overall anti-arthritic activities of the gold-thiolates from which it is formed in vivo.