Marine metagenomics: strategies for the discovery of novel enzymes with biotechnological applications from marine environments

Metagenomic based strategies have previously been successfully employed as powerful tools to isolate and identify enzymes with novel biocatalytic activities from the unculturable component of microbial communities from various terrestrial environmental niches. Both sequence based and function based screening approaches have been employed to identify genes encoding novel biocatalytic activities and metabolic pathways from metagenomic libraries. While much of the focus to date has centred on terrestrial based microbial ecosystems, it is clear that the marine environment has enormous microbial biodiversity that remains largely unstudied. Marine microbes are both extremely abundant and diverse; the environments they occupy likewise consist of very diverse niches. As culture-dependent methods have thus far resulted in the isolation of only a tiny percentage of the marine microbiota the application of metagenomic strategies holds great potential to study and exploit the enormous microbial biodiversity which is present within these marine environments.     

Strain persistence of invasive Candida albicans in chronic hyperplastic candidosis that underwent malignant change

Objectives: The aim of this study was to assess persistence and tissue invasion of Candida albicans strains isolated from a 65 year‐old patient with chronic hyperplastic candidosis (CHC), that subsequently developed into squamous cell carcinoma (SCC). Materials and Methods: C. albicans (n=7) were recovered from the oral cavity of the patient over seven years. Confirmation of CHC and SCC in this patient was achieved by histopathological examination of incisional biopsy tissue. DNA fingerprinting was performed on the seven isolates from the CHC patient together with a further eight isolates from patients with normal oral mucosa (n=2), chronic atrophic candidosis (n=1), SCC (n=1) and CHC (n=4). Genotyping involved the use of inter‐repeat PCR using the eukaryotic repeat primer 1251. Characterisation of the tissue invasive abilities of the isolates was achieved by infecting a commercially available reconstituted human oral epithelium (RHE; SkinEthic, Nice, France). After 24 h. C. albicans tissue invasion was assessed by histopathological examination. Results: DNA fingerprinting demonstrated strain persistence of C. albicans in the CHC patient over a seven year period despite provision of systemic antifungal therapy. The strain of C. albicans isolated from this patient was categorised as a high invader within the RHE compared to other isolates. Conclusions: Candidal strain persistence was evident in a patient with CHC over seven years. This persistence may be due to incomplete eradication from the oral cavity following antifungal therapy or subsequent recolonisation from other body sites or separate exogenous sources. The demonstration of enhanced in vitro tissue invasion by this particular strain may, in part, explain the progression to carcinoma.     

A modified PAS stain combined with immunofluorescence for quantitative analyses of glycogen in muscle sections

Simultaneous analyses of glycogen in sections with other subcellular constituents within the same section will provide detailed information on glycogen deposition and the processes involved. To date, staining protocols for quantitative glycogen analyses together with immunofluorescence in the same section are lacking. We aimed to: (1) optimise PAS staining for combination with immunofluorescence, (2) perform quantitative glycogen analyses in tissue sections, (3) evaluate the effect of section thickness on PAS-derived data and (4) examine if semiquantitative glycogen data were convertible to genuine glycogen values. Conventional PAS was successfully modified for combined use with immunofluorescence. Transmitted light microscopic examination of glycogen was successfully followed by semiquantification of glycogen using microdensitometry. Semiquantitative data correlated perfectly with glycogen content measured biochemically in the same sample (r²=0.993, P<0.001). Using a calibration curve (r²=0.945, P<0.001) derived from a custom-made external standard with incremental glycogen content, we converted the semiquantitative data to genuine glycogen values. The converted semiquantitative data were comparable with the glycogen values assessed biochemically (P=0.786). In addition we showed that for valid comparison of glycogen content between sections, thickness should remain constant. In conclusion, the novel protocol permits the combined use of PAS with immunofluorescence and shows valid conversion of data obtained by microdensitometry to genuine glycogen data.     

Different metabolic adaptation of heart and skeletal muscles to moderate-intensity treadmill training in the rat

The effect was investigated of treadmill training of moderate intensity on the fatty acid-binding protein (FABP) content in relation to parameters of oxidative and glycolytic metabolism. To this end, the cytoplasmic FABP content and the activity of beta-hydroxyacyl-coenzyme A dehydrogenase (HAD), citrate synthase (CS), and 6-phosphofructokinase (PFK) were measured in heart, fast-twitch extensor digitorum longus (EDL) and slow-twitch soleus muscles (SOL) of male Wistar rats. To investigate the influence of the amount of training (defined as the product of exercise duration, intensity and frequency), two training groups were created that differed in training frequency (HF, high frequency 5 days x week(-1), n = 9; LF, low frequency 2 days x week(-1), n = 9; the exercise being 20 m x min(-1) for 2 h with no gradient, over 6 weeks) and compared with SC, sedentary controls (n = 7). In heart muscle, the cytoplasmic FABP content was 34% higher in HF than in SC but was the same as in LF. The CS and HAD activities were no different in the three groups, suggesting that the capacity to oxidize fatty acids (FA) was not affected by training. The PFK activity was higher (43%) in HF, suggesting a shift towards carbohydrate utilization. The FABP content and HAD activity did not change in SOL and EDL after training whereas the CS activity increased (27%) in SOL and decreased (21%) in EDL in both training groups. In addition, PFK activity in EDL was much higher (113%) in the HF than in SC group. The HF training was associated with a fine-tuning of FA availability and use in heart muscle, and with a more efficient energy production. It is suggested therefore that cytoplasmic FABP could be an early marker of muscle adaptation to training in heart but not in skeletal muscle. The training reinforced the metabolic profile of the skeletal muscles, in particular that of the fast-twitch glycolytic muscle. We concluded that a large amount of training is needed when the effect on both oxidative and glycolytic parameters is to be studied.     

C-terminal domain of insulin-like growth factor (IGF) binding protein-6: structure and interaction with IGF-II

IGFs are important mediators of growth. IGF binding proteins (IGFBPs) 1-6 regulate IGF actions and have IGF-independent actions. The C-terminal domains of IGFBPs contribute to high-affinity IGF binding and modulation of IGF actions and confer some IGF-independent properties, but understanding how they achieve this has been constrained by the lack of a three-dimensional structure. We therefore determined the solution structure of the C-domain of IGFBP-6 using nuclear magnetic resonance (NMR). The domain consists of a thyroglobulin type 1 fold comprising an alpha-helix followed by a loop, a three-stranded antiparallel beta-sheet incorporating a second loop, and finally a disulfide-bonded flexible third loop. The IGF-II binding site on the C-domain was identified by examining NMR spectral changes upon complex formation. It consists of a largely hydrophobic surface patch involving the alpha-helix, the first beta-strand, and the first and second loops. The site was confirmed by mutagenesis of several residues, which resulted in decreased IGF binding affinity. The IGF-II binding site lies adjacent to surfaces likely to be involved in glycosaminoglycan binding of IGFBPs, which might explain their decreased IGF affinity when bound to glycosaminoglycans, and nuclear localization. Our structure provides a framework for understanding the roles of IGFBP C-domains in modulating IGF actions and conferring IGF-independent actions, as well as ultimately for the development of therapeutic IGF inhibitors for diseases including cancer.     

Immunologic studies with LFA-1- and Mo1-deficient lymphocytes from a patient with recurrent bacterial infections

A patient and his parents, deficient for lymphocyte function associated antigen-1 (LFA-1) and Mo1 (OKM1), were studied with respect to leukocyte surface marker expression and functional properties. The patient had a history of severe recurrent bacterial infections. Two siblings had already died of bacterial infections. The patient's granulocytes, monocytes, and lymphocytes expressed low but detectable amounts (less than or equal to 10%) of LFA-1 and Mo1. Intracellularly, LFA-1 and Mo1 (OKM1) were detectable and LFA-1 expression was enhanced on patient T cells stimulated with phytohemagglutinin. Granulocytes and monocytes of both the patient's parents expressed markedly decreased amounts of LFA-1 and Mo1. Lymphocytes of the mother expressed 40 to 60% of the amount of LFA-1 expressed on control lymphocytes, but his father's lymphocytes showed a normal LFA-1 expression. Granulocytes of the patient and of his deceased sister showed normal phagocytosis, but they had a dysfunction in the activation of the oxidative metabolism. Functional activities mediated by patient T cells were all normal. Moreover, all lymphocyte functions, including killer (K), natural killer (NK), cytotoxic T cell activity, helper activity for in vitro immunoglobulin (Ig) production by normal B cells, and PHA-induced proliferation were inhibitable by anti-LFA-1 monoclonal antibodies. K and NK activity mediated by patient leukocytes was 100-fold more sensitive to the inhibiting effect of anti-LFA-1 antibody than K and NK activity of normal donor leukocytes. Thus, although the amount of LFA-1 expressed was strongly reduced, it was still sufficient and required for the functional activity exhibited by patient T cells. The major functional defect observed with leukocytes of the patient and his father was an apparent B cell defect. B cells of the father and of the patient failed to produce Ig in the pokeweed mitogen (PWM)-driven system. The B cells of patient and of his father only produced Ig when cultured with T cells of the father, and not with normal donor T cells or T cells of the mother, in the presence of exogenous interleukin 2 (IL 2). In addition, the father's B cells produced Ig when cocultivated with patient T cells in the IL 2-driven system. This restriction of helper T cell activity is noteworthy because PWM- and IL 2-driven Ig synthesis by normal lymphocytes show no histocompatibility requirements between cooperating T and non-T cell populations.(ABSTRACT TRUNCATED AT 400 WORDS)     

Distribution of GNAQ and GNA11 Mutation Signatures in Uveal Melanoma Points to a Light Dependent Mutation Mechanism

Uveal melanomas (UM) originate from melanocytes in the interior wall of the eye, namely from the iris, ciliary body and the choroid with marked differences in light exposure (from dark anterior to illuminated posterior). In contrast to UV radiation, focused or converging visible light readily reaches the retina and can damage DNA which possibly contributes to UM development. In this report choroidal, ciliochoroidal and iridociliary melanomas were analyzed for GNAQ and GNA11 mutations which were subsequently correlated to the location of tumor origin. Hotspot mutations in GNAQ and GNA11 can be divided in A>T and in A>C mutation signatures. The GNAQ A626C mutation (Q209P) was almost exclusively observed in choroidal melanomas from the illuminated posterior side. On the other hand, ciliochoroidal UM from the dark anterior side with mostly A>T mutations were clearly associated with light-colored eyes. Combined these data suggest a light and a pigment dependent etiology in UM development.     

Structure, dynamics and heparin binding of the C-terminal domain of insulin-like growth factor-binding protein-2 (IGFBP-2)

Insulin-like growth factor-binding protein-2 (IGFBP-2) is the largest member of a family of six proteins (IGFBP-1 to 6) that bind insulin-like growth factors I and II (IGF-I/II) with high affinity. In addition to regulating IGF actions, IGFBPs have IGF-independent functions. The C-terminal domains of IGFBPs contribute to high-affinity IGF binding, and confer binding specificity and have overlapping but variable interactions with many other molecules. Using nuclear magnetic resonance (NMR) spectroscopy, we have determined the solution structure of the C-terminal domain of IGFBP-2 (C-BP-2) and analysed its backbone dynamics based on 15N relaxation parameters. C-BP-2 has a thyroglobulin type 1 fold consisting of an alpha-helix, a three-stranded anti-parallel beta-sheet and three flexible loops. Compared to C-BP-6 and C-BP-1, structural differences that may affect IGF binding and underlie other functional differences were found. C-BP-2 has a longer disordered loop I, and an extended C-terminal tail, which is unstructured and very mobile. The length of the helix is identical with that of C-BP-6 but shorter than that of C-BP-1. Reduced spectral density mapping analysis showed that C-BP-2 possesses significant rapid motion in the loops and termini, and may undergo slower conformational or chemical exchange in the structured core and loop II. An RGD motif is located in a solvent-exposed turn. A pH-dependent heparin-binding site on C-BP-2 has been identified. Protonation of two histidine residues, His271 and His228, seems to be important for this binding, which occurs at slightly acidic pH (6.0) and is more significant at pH 5.5, but is largely suppressed at pH 7.4. Possible preferential binding of IGFBP-2 and its C- domain fragments to glycosaminoglycans in the acidic extracellular matrix (ECM) of tumours may be related to their roles in cancer.