Significance of pantothenate for glucose fermentation by Oenococcus oeni and for suppression of the erythritol and acetate production

The heterofermentative lactic acid bacterium Oenococcus oeni requires pantothenic acid for growth. In the presence of sufficient pantothenic acid, glucose was converted by heterolactic fermentation stoichiometrically to lactate, ethanol and CO2. Under pantothenic acid limitation, substantial amounts of erythritol, acetate and glycerol were produced by growing and resting bacteria. Production of erythritol and glycerol was required to compensate for the decreasing ethanol production and to enable the synthesis of acetate. In ribose fermentation, there were no shifts in the fermentation pattern in response to pantothenate supply. In the presence of pantothenate, growing O. oeni contained at least 10.2 microM HSCoA, whereas the HSCoA content was tenfold lower after growth in pantothenate-depleted media. HSCoA and acetyl-CoA are cosubstrates of phosphotransacetylase and acetaldehyde dehydrogenase from the ethanol pathway. Both enzymes were found with activities commensurate with their function in ethanol production during heterolactic fermentation. From the kinetic data of the enzymes and the HSCoA and acetyl-CoA contents, it can be calculated that, under pantothenate limitation, phosphotransacetylase, and in particular acetaldehyde dehydrogenase activities become limiting due to low levels of the cosubstrates. Thus HSCoA deficiency represents the major limiting factor in heterolactic fermentation of glucose under pantothenate deficiency and the reason for the shift to erythritol, acetate, and glycerol fermentation.     

NMR experiments for the sign determination of homonuclear scalar and residual dipolar couplings

A modified version of the JHH-TOCSY experiment, `signed COSY', is presented that allows the determination of the sign of residual dipolar ¹H-¹H coupling constants with respect to the sign of one-bond ¹H-X coupling constants in linear three-spin systems X-¹H-¹H, where X = ¹³C or ¹⁵N. In contrast to the original JHH-TOCSY experiments, the signs of J _HH couplings may be determined for CH₂-CH₂ moieties and for uniformly ¹³C/¹⁵N-labelled samples. In addition, sensitivity is enhanced, diagonal peaks are suppressed and cross peaks are observed only between directly coupled protons, as in a COSY spectrum.     

Heteronuclear correlation experiments for the determination of one-bond coupling constants

Spin-state selective experiments, HSQC-/ and CT-HMQC-/, are proposed for the simple and rapid measurement of scalar one-bond coupling constants in two-dimensional,¹ H-detected ¹⁵N-¹H or¹³ C-¹H correlation experiments based on HSQC and HMQC schemes. Pairs of subspectra are obtained, containing either the high-field or the low-field component of the doublet representing the one-bond coupling constant. The subspectral editing procedure retains the full sensitivity of HSQC and HMQC spectra recorded without heteronuclear decoupling during data acquisition, with a spectral resolution similar to that of decoupled spectra.     

HMQC and HSQC experiments with water flip-back optimized for large proteins

An HMQC experiment is proposed, dubbed FHMQC, where water flip-back is achieved by a single water-selective pulse preceding the basic HMQC pulse sequence. The scheme is demonstrated with a ¹⁵N, ¹H-HMQC spectrum of uniformly ¹⁵N/²H-labelled S. aureus DNA gyrase B with a molecular weight of 45 kDa for the unlabelled protein. The sensitivity of the experiment is improved compared to that of an FHSQC spectrum. It is further shown that the original FHSQC experiment can be shortened by the use of bipolar gradients. Relaxation times of different ¹⁵N magnetizations and coherences were measured. The new FHMQC scheme is implemented in 3D NOESY-¹⁵N-HMQC and 3D¹⁵ N-HMQC-NOESY-¹⁵N-HMQC pulse sequences which are demonstrated with a 24 kDa fragment of uniformly ¹⁵N/¹³C/²H-labelled S. aureus DNA gyrase B.     

OMEC II: A new ovine mammary epithelial cell line

We have established three independent ovine mammary epithelial cell lines which arose from primary cultures of ovine mammary epithelial cells by spontaneous immortalization. One of them, OMEC II, was characterised in greater detail. The cells grow rapidly on plastic dishes in medium containing 10% FCS without any requirement for additional growth factors or hormones. Immunofluorescence staining of this cell line showed expression of cytokeratin (46 kDa) and ZO-1, a tight-junction associated protein, but negative immunostaining for an anti-vimentin antibody. In confluent cell monolayers ‘domes’ became visible indicating the development of a polarised phenotype and the ability of directed secretion. When grown in collagen gels typical ducts with end-buds were observed. Treatment with lactogenic hormones increased the frequency of dome formation, but no expression of β-lactoglobulin was found. To our knowledge this is the first report on an ovine mammary epithelial cell line.     

Large-Scale Purification of r28M: A Bispecific scFv Antibody Targeting Human Melanoma Produced in Transgenic Cattle

Background

30 years ago, the potential of bispecific antibodies to engage cytotoxic T cells for the lysis of cancer cells was discovered. Today a variety of bispecific antibodies against diverse cell surface structures have been developed, the majority of them produced in mammalian cell culture systems. Beside the r28M, described here, no such bispecific antibody is known to be expressed by transgenic livestock, although various biologicals for medical needs are already harvested—mostly from the milk—of these transgenics. In this study we investigated the large-scale purification and biological activity of the bispecific antibody r28M, expressed in the blood of transgenic cattle. This tandem single-chain variable fragment antibody is designed to target human CD28 and the melanoma/glioblastoma-associated cell surface chondroitin sulfate proteoglycan 4 (CSPG4).

Results

With the described optimized purification protocol an average yield of 30 mg enriched r28M fraction out of 2 liters bovine plasma could be obtained. Separation of this enriched fraction by size exclusion chromatography into monomers, dimers and aggregates and further testing regarding the biological activity revealed the monomer fraction as being the most appropriate one to continue working with. The detailed characterization of the antibody’s activity confirmed its high specificity to induce the killing of CSPG4 positive cells. In addition, first insights into tumor cell death pathways mediated by r28M-activated peripheral blood mononuclear cells were gained. In consideration of possible applications in vivo we also tested the effect of the addition of different excipients to r28M.

Conclusion

Summing up, we managed to purify monomeric r28M from bovine plasma in a large-scale preparation and could prove that its biological activity is unaffected and still highly specific and thus, might be applicable for the treatment of melanoma.     

A Peptide to Reduce Pulmonary Edema in a Rat Model of Lung Transplantation

Background

Despite significant advances in organ preservation, surgical techniques and perioperative care, primary graft dysfunction is a serious medical problem in transplantation medicine in general and a specific problem in patients undergoing lung transplantation. As a result, patients develop lung edema, causing reduced tissue oxygenation capacity, reduced lung compliance and increased requirements for mechanical ventilatory support. Yet, there is no effective strategy available to protect the grafted organ from stress reactions induced by ischemia/reperfusion and by the surgical procedure itself.

Methods

We assessed the effect of a cingulin-derived peptide, XIB13 or a random peptide in an established rat model of allogeneic lung transplantation. Donor lungs and recipients received therapeutic peptide at the time of transplantation and outcome was analyzed 100min and 28 days post grafting.

Results

XIB13 improved blood oxygenation and reduced vascular leak 100min post grafting. Even after 28 days, lung edema was significantly reduced by XIB13 and lungs had reduced fibrotic or necrotic zones. Moreover, the induction of an allogeneic T cell response was delayed indicating a reduced antigen exchange between the donor and the host.

Conclusions

In summary, we provide a new tool to strengthen endothelial barrier function thereby improving outcomes in lung transplantation.     

Structure of the Ni(II) complex of Escherichia coli peptide deformylase and suggestions on deformylase activities depending on different metal(II) centres

Crystal structures of polypeptide deformylase (PDF) of Escherichia coli with nickel(II) replacing the native iron(II) have been solved with chloride and formate as metal ligands. The chloro complex is a model for the correct protonation state of the hydrolytic hydroxo ligand and the protonated status of the Glu133 side chain as part of the hydrolytic mechanism. The ambiguity that recently some PDFs have been identified with Zn²⁺ ion as the active-site centre whereas others are only active with Fe²⁺ (or Co²⁺, Ni²⁺) is discussed with respect to Lewis acid criteria of the metal ion and substrate activation by the CD loop.