The use of and search for drugs and dietary supplements derived from plants have accelerated in recent years. Ethnopharmacologists, botanists, microbiologists, and natural-products chemists are combing the Earth for phytochemicals and “leads” which could be developed for treatment of infectious diseases. While 25 to 50% of current pharmaceuticals are derived from plants, none are used as antimicrobials. Traditional healers have long used plants to prevent or cure infectious conditions; Western medicine is trying to duplicate their successes. Plants are rich in a wide variety of secondary metabolites, such as tannins, terpenoids, alkaloids, and flavonoids, which have been found in vitro to have antimicrobial properties. This review attempts to summarize the current status of botanical screening efforts, as well as in vivo studies of their effectiveness and toxicity. The structure and antimicrobial properties of phytochemicals are also addressed. Since many of these compounds are currently available as unregulated botanical preparations and their use by the public is increasing rapidly, clinicians need to consider the consequences of patients self-medicating with these preparations.

Plant Products as Antimicrobial Agents

Rapid progress in deciphering the biological mechanism of Alzheimer's disease (AD) has arisen from the application of molecular and cell biology to this complex disorder of the limbic and association cortices. In turn, new insights into fundamental aspects of protein biology have resulted from research on the disease. This beneficial interplay between basic and applied cell biology is well illustrated by advances in understanding the genotype-to-phenotype relationships of familial Alzheimer's disease. All four genes definitively linked to inherited forms of the disease to date have been shown to increase the production and/or deposition of amyloid β-protein in the brain. In particular, evidence that the presenilin proteins, mutations in which cause the most aggressive form of inherited AD, lead to altered intramembranous cleavage of the β-amyloid precursor protein by the protease called γ-secretase has spurred progress toward novel therapeutics. The finding that presenilin itself may be the long-sought γ-...

Alzheimer's Disease: Genes, Proteins, and Therapy

Matrix metalloproteinases (MMPs) are a family of nine or more highly homologous Zn(++)-endopeptidases that collectively cleave most if not all of the constituents of the extracellular matrix. The present review discusses in detail the primary structures and the overlapping yet distinct substrate specificities of MMPs as well as the mode of activation of the unique MMP precursors. The regulation of MMP activity at the transcriptional level and at the extracellular level (precursor activation, inhibition of activated, mature enzymes) is also discussed. A final segment of the review details the current knowledge of the involvement of MMP in specific developmental or pathological conditions, including human periodontal diseases.

Matrix Metalloproteinases: A Review

Mutations in the genes for amyloid precursor protein (APP) and presenilins (PS1, PS2) increase production of β-amyloid 42 (Aβ42) and cause familial Alzheimer's disease (FAD). Transgenic mice that express FAD mutant APP and PS1 overproduce Aβ42 and exhibit amyloid plaque pathology similar to that found in AD, but most transgenic models develop plaques slowly. To accelerate plaque development and investigate the effects of very high cerebral Aβ42 levels, we generated APP/PS1 double transgenic mice that coexpress five FAD mutations (5XFAD mice) and additively increase Aβ42 production. 5XFAD mice generate Aβ42 almost exclusively and rapidly accumulate massive cerebral Aβ42 levels. Amyloid deposition (and gliosis) begins at 2 months and reaches a very large burden, especially in subiculum and deep cortical layers. Intraneuronal Aβ42 accumulates in 5XFAD brain starting at 1.5 months of age (before plaques form), is aggregated (as determined by thioflavin S staining), and occurs within neuron soma and neurites. Some amyloid deposits originate within morphologically abnormal neuron soma that contain intraneuronal Aβ. Synaptic markers synaptophysin, syntaxin, and postsynaptic density-95 decrease with age in 5XFAD brain, and large pyramidal neurons in cortical layer 5 and subiculum are lost. In addition, levels of the activation subunit of cyclin-dependent kinase 5, p25, are elevated significantly at 9 months in 5XFAD brain, although an upward trend is observed by 3 months of age, before significant neurodegeneration or neuron loss. Finally, 5XFAD mice have impaired memory in the Y-maze. Thus, 5XFAD mice rapidly recapitulate major features of AD amyloid pathology and may be useful models of intraneuronal Aβ42-induced neurodegeneration and amyloid plaque formation.

Intraneuronal beta-amyloid aggregates, neurodegeneration, and neuron loss in transgenic mice with five familial Alzheimer's disease mutations: potential factors in amyloid plaque formation.

Antimicrobial host defense peptides are produced by all complex organisms as well as some microbes and have diverse and complex antimicrobial activities. Collectively these peptides demonstrate a broad range of antiviral and antibacterial activities and modes of action, and it is important to distinguish between direct microbicidal and indirect activities against such pathogens. The structural requirements of peptides for antiviral and antibacterial activities are evaluated in light of the diverse set of primary and secondary structures described for host defense peptides. Peptides with antifungal and antiparasitic activities are discussed in less detail, although the broad-spectrum activities of such peptides indicate that they are important host defense molecules. Knowledge regarding the relationship between peptide structure and function as well as their mechanism of action is being applied in the design of antimicrobial peptide variants as potential novel therapeutic agents.

Peptide Antimicrobial Agents

Genomic cloning of the mouse LDL receptor related protein/α2-macroglobulin receptor gene

Alpha-Macroglobulins (AM) are protease inhibitors with important roles in inflammation and in immunomodulation that behave as acute-phase proteins in many experimental models. In the present work the levels of AM in the plasma of outbred Swiss albino mice acutely infected with Trypanosoma cruzi were studied. The results showed that increased levels of AM were present in the majority of the infected mice and that AM levels increased independently of the rise in parasitaemia. There was a high degree of heterogeneity in the intensity of the modulation of AM levels as well as in the kinetics of AM synthesis. This heterogeneity was related neither with the intensity of infection nor with the sex of the host. No correlation between AM levels and survival to the acute phase could be observed in the outbred mice. The consequence of such a heterogeneity is unclear, although AM as immunoregulatory molecules could play a role in the development of the symptoms of the chronic phase of Chagas' disease.

Heterogeneity in the synthesis of alpha-macroglobulins in outbred Swiss albino mice acutely infected with Trypanosoma cruzi.

In contrast to established cell lines, normal human skin fibroblasts spread on their own fibronectin. The present investigation has examined whether human fibroblasts, like established cell lines, would be capable of spreading on substrata coated with proteins with different reactivity towards the cell surface. Coverslips were coated with human serum, fibronectin, alpha 2 macroglobulin-trypsin, a polyspecific anti-fibroblast antibody and a polyspecific anti-calf-serum antibody. The attachment of the cells to these substrate was of the same extent. Spreading was examined qualitatively using phase, interference contrast and reflection contrast optics on live cells, as well as scanning electron microscopy on fixed cells. To quantitate the maximum degree of cell spreading a semi-automated system was used, which measured the cell perimeter on a large number of cells. The distributions of the degree of cell spreading on the five substrata were compared statistically. The qualitative and quantitative differences observed on the various substrata could be further differentiated by adding various amines to the cells during 60 min spreading or during a 30 min preincubation before spreading. No strict correlation could be found between the effect of the amines on attachment or on spreading and their presumed effects on cellular transglutaminases. The results clearly indicate that the spreading of human fibroblasts can be modulated by the nature of the substratum and that, by using quantitative methods, these differences in behaviour can be measured accurately.

Qualitative and quantitative differences in spreading of human fibroblasts on various protein coats. Modulation by treatment of the cells with amines

Uncharged derivatives of thioflavin-T have known in vitro and in vivo affinity for amyloid β. We synthesized and evaluated two derivatives with a fluorine-18 labelled fluoropropoxy substituent either at the 6-position or at the 2′-position of the 2-(4′-aminophenyl)-1,3-benzothiazole core with the aim to get suitable radiotracers to perform amyloid plaque imaging. The fluorine-18 labelled compounds were obtained by nucleophilic substitution of the corresponding tosyl precursors with [18F]fluoride with a radiochemical yield of 50%, yielding 6-(3′′-[18F]fluoropropoxy)-2-(4′-aminophenyl)-1,3-benzothiazole ([18F]2) and 2-[2′-(3′′-[18F]fluoropropoxy)-4′-aminophenyl]-1,3-benzothiazole ([18F]3) with a specific activity between 33 and 51 GBq/µmol. The identity of the radiolabelled compounds was confirmed using radio-LC-MS and by comparing retention times on RP-HPLC. Biodistribution studies in healthy mice showed for both compounds a relatively high initial brain uptake, which was significantly higher for [18F]2 than for [18F]3 (4.5% ID/g versus 3.0% ID/g, p<0.05). Wash-out from control brain was faster for [18F]3. In vitro binding affinity tests using human AD brain homogenates revealed that only compound 2 has affinity for fibrillar amyloid β (Ki=14.5 nM). This was confirmed by the incubation of transgenic APP mouse brain sections with the cold compounds, where 3 did not stain any structure whereas 2 stained amyloid plaques present in APP mouse brain. These data suggest that [18F]2 may be a useful tracer for in vivo visualization of fibrillar amyloid β. Copyright © 2009 John Wiley & Sons, Ltd.

Synthesis and evaluation of two fluorine-18 labelled phenylbenzothiazoles as potential in vivo tracers for amyloid plaque imaging

Fibronectins are large glycoproteins (440 kDa), present in many tissues and in most body fluids. The soluble form, usually referred to as “plasma fibronectin”, is present in rather high concentrations (300 μg/ml) in blood (Mosesson and Umfleet, 1970). The insoluble, multimeric form, called “cellular fibronectin”, is found in connective tissue and basement membranes and is organized in short fibrils, in amorphous granules, or in thick cross-striated fibrils consisting of collagen I and II decorated with fibronectin (Fyrand, 1979; Couchman et al., 1979; Fleishmajer and Timpl, 1984). Fibronectin containing matrices are particularly prominent in vivo at sites of tissue remodelling and cell migration (e.g., wound repair, neural crest cell migration). In vitro, fibronectin localizes in extracellular fibrillar structures and in the basement membranes of most cultured cells (Yamada, 1978; Hedman et al., 1978). In these structures, fibronectin rather than collagen is the major structural component (Chen et al., 1978). Extensive accumulation of collagen fibrils appears to be a subsequent event and the distribution seems to depend on the integrity of the fibronectin matrix as shown by inhibition studies with anti-fibronectin antibodies (Mc Donald et al., 1982).

F. Van Leuven

Papers

Genomic cloning of the mouse LDL receptor related protein/α2-macroglobulin receptor gene

Heterogeneity in the synthesis of alpha-macroglobulins in outbred Swiss albino mice acutely infected with Trypanosoma cruzi.

Qualitative and quantitative differences in spreading of human fibroblasts on various protein coats. Modulation by treatment of the cells with amines

Synthesis and evaluation of two fluorine-18 labelled phenylbenzothiazoles as potential in vivo tracers for amyloid plaque imaging

Fibronectin-Mediated Cellular (Inter)Actions