Enhanced stress tolerance in Escherichia coli and Nicotiana tabacum expressing a betaine aldehyde dehydrogenase/choline dehydrogenase fusion protein

In Escherichia coli the osmoprotective compound glycine betaine is produced from choline by two enzymes; choline dehydrogenase (CDH) oxidizes choline to betaine aldehyde and then further on to glycine betaine, while betaine aldehyde dehydrogenase (BADH) facilitates the conversion of betaine aldehyde to glycine betaine. To evaluate the importance of BADH, a BADH/CDH fusion enzyme was constructed and expressed in E. coli and in Nicotiana tabacum. The fusion enzyme displayed both enzyme activities, and a coupled reaction could be measured. The enzyme was characterized regarding molecular weight and the dependence of the enzyme activities on environmental factors (salt, pH, and poly(ethylene glycol) addition). At high choline concentrations, E. coli cells expressing BADH/CDH were able to grow to higher final densities and to accumulate more glycine betaine than cells expressing CDH only. The intracellular glycine betaine levels were almost 5-fold higher for BADH/CDH when product concentration was related to CDH activity. Also, after culturing the cells at high NaCl concentrations, more glycine betaine was accumulated. On medium containing 20 mM choline, transgenic tobacco plants expressing BADH/CDH grew considerably faster than vector-transformed control plants.     

SHIP-deficient mice are severely osteoporotic due to increased numbers of hyper-resorptive osteoclasts

The hematopoietic-restricted protein Src homology 2-containing inositol-5-phosphatase (SHIP) blunts phosphatidylinositol-3-kinase-initiated signaling by dephosphorylating its major substrate, phosphatidylinositol-3,4,5-trisphosphate. As SHIP(-/-) mice contain increased numbers of osteoclast precursors, that is, macrophages, we examined bones from these animals and found that osteoclast number is increased two-fold. This increased number is due to the prolonged life span of these cells and to hypersensitivity of precursors to macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor-kappa B ligand (RANKL). Similar to pagetic osteoclasts, SHIP(-/-) osteoclasts are enlarged, containing upwards of 100 nuclei, and exhibit enhanced resorptive activity. Moreover, as in Paget disease, serum levels of interleukin-6 are markedly increased in SHIP(-/-) mice. Consistent with accelerated resorptive activity, 3D trabecular volume fraction, trabecular thickness, number and connectivity density of SHIP(-/-) long bones are reduced, resulting in a 22% loss of bone-mineral density and a 49% decrease in fracture energy. Thus, SHIP negatively regulates osteoclast formation and function and the absence of this enzyme results in severe osteoporosis.     

Evaluation of redox indicators and the use of digital scanners and spectrophotometer for quantification of microbial growth in microplates

The growth indicators 2,3,5-triphenyltetrazolium chloride (TTC), 2-[4-iodophenyl]-3-[4-dinitrophenyl]-5-phenyltetrazolium chloride (INT), 2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide inner salt (XTT), 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), and resazurin were tested for their ability to indicate bacterial growth/growth inhibition. Two reading devices were evaluated and compared, a microplate spectrophotometer and a digital flatbed scanner. The bacteria used in the study were cultivated in 96-wells microplates and readings were made after 24 h. The scanned pictures were analysed with a software developed in-house to generate numerical values. It was found that resazurin was difficult to use since it shifts between three colours. MTT and TTC had a high correlation between the spectrophotometer data and the data from the scanned images. The reproducibility was similar for both reading devices. In no case was there a need to resuspend the pellets before reading. Both the XTT and INT showed lower correlations.It is concluded that bacterial growth/growth inhibition can be easily and reproducibly measured from microplate cultivations with a flatbed scanner or with a microplate spectrophotometer.     

Preparation of partially 2H/13C-labelled RNA for NMR studies. Stereo-specific deuteration of the H5" in nucleotides

An effective in vitro enzymatic synthesis is described for the production of nucleoside triphosphates (NTPs) which are stereo-specifically deuterated on the H5" position with high selectivity (>98%), and which can have a variety of different labels (13C, 15N, 2H) in other positions. The NTPs can subsequently be employed in the enzymatic synthesis of RNAs using T7 polymerase from a DNA template. The stereo-specific deuteration of the H5" immediately provides the stereo-specific assignment of H5' resonances in NMR spectra, giving access to important structural parameters. Stereo-chemical H-exchange was used to convert commercially available 1,2,3,4,5,6,6-2H-1,2,3,4,5,6-13C-D-glucose (d7-13C6-D-glucose) into [1,2,3,4,5,6(R)-2H-1,2,3,4,5,6-13C]-D-glucose (d6-13C6-D-glucose). [1',3',4',5"-2H-1',2',3',4',5'-13C]GTP (d4-13C5-GTP) was then produced from d6-13C6-D-glucose and guanine base via in vitro enzymatic synthesis employing enzymes from the pentose-phosphate, nucleotide biosynthesis and salvage pathways. The overall yield was approximately 60 mg NTP per 1 g glucose, comparable with the yield of NTPs isolated from Escherichia coli grown on enriched media. The d4-13C5-GTP, together with in vitro synthesised d5-UTP, d5-CTP and non-labelled ATP, were used in the synthesis of a 31 nt RNA derived from the primer binding site of hepatitis B virus genomic RNA. (13C,1H) hetero-nuclear multiple-quantum spectra of the specifically deuterated sample and of a non-deuterated uniformly 13C/15N-labelled sample demonstrates the reduced spectral crowding and line width narrowing compared with 13C-labelled non-deuterated RNA.     

Secretion of MUC5AC mucin from pancreatic cancer cells in response to forskolin and VIP

Bisphosphonates are potent antiresorptive drugs commonly employed in the treatment of metabolic bone diseases. Despite their frequent use, the mechanisms of bisphosphonates on bone cells have largely remained unclear. Receptor activator of nuclear factor-kappaB ligand (RANKL) is essential for osteoclast formation and activation, whereas osteoprotegerin (OPG) neutralizes RANKL. Various osteotropic drugs have been demonstrated to modulate osteoblastic production of RANKL and OPG. In this study, we assessed the effects of the bisphosphonates pamidronate (PAM) and zoledronic acid (ZOL) on OPG mRNA steady-state levels (by semiquantitative RT-PCR) and protein production (by ELISA) in primary human osteoblasts (hOB). PAM increased OPG mRNA levels and protein secretion by hOB by up to 2- to 3-fold in a dose-dependent fashion with a maximum effect at 10(-6) M (P < 0.001) after 72 h. Similarly, ZOL enhanced OPG gene expression and protein secretion by hOB in a dose-dependent fashion with a maximum effect at 10(-8) M after 72 h, consistent with the higher biological potency of ZOL. Time course experiments indicated a stimulatory effect of PAM and ZOL on osteoblastic OPG protein secretion by 6-fold, respectively (P < 0.001). Pretreatment with PAM and ZOL prevented the inhibitory effects of the glucocorticoid dexamethasone on OPG mRNA and protein production. Analysis of cellular markers of osteoblastic differentiation revealed that PAM and ZOL induced type I collagen secretion and alkaline phosphatase activity by 2- and 4-fold, respectively (P < 0.0001 by ANOVA). In conclusion, our data suggest that bisphosphonates modulate OPG production by normal human osteoblasts, which may contribute to the inhibition of osteoclastic bone resorption. Since, OPG production increases with osteoblastic cell maturation, enhancement of OPG by bisphosphonates could be related to their stimulatory effects on osteoblastic differentiation.     

Human alpha-fetoprotein binds to primary macrophages

We have previously reported that alpha-fetoprotein (AFP) inhibits infection of human monocyte-derived macrophages (MDM) by R5-HIV-1 strains and that a peptide mimicking the clade B HIV-1 gp120 consensus V3 domain (V3Cs) binds to CCR5. We demonstrate here that AFP binds high- and low-affinity binding sites of MDM, characterized, respectively, by 5.15 and 100nM K(d) values. Heat denaturation or neuraminidase treatment of AFP inhibits this binding, suggesting the involvement of protein-protein and lectin-carbohydrate interactions. Moreover, AFP displaces V3Cs binding to MDM. In addition, MIP-1beta, the most specific CCR5 ligand, displaces AFP binding to MDM (IC(50)=4.3nM). Finally, we demonstrate that AFP binds to a ligand of HIV-gp120 V3Cs domain, CCR5, expressed by MDM and by HeLa cells expressing CCR5. Such binding is not observed in the presence of HeLa cells lacking CCR5. The present results provide strong evidence that AFP directly binds to CCR5 expressed by human primary macrophages and by transfected CCR5+ HeLa cells.