Assignment of interchain disulfide bonds in platelet-derived growth factor (PDGF) and evidence for agonist activity of monomeric PDGF.

Platelet-derived growth factor (PDGF) is a dimeric factor stabilized by disulfide bonds. Using an approach involving partial reduction of PDGF, we have identified the 2nd and 4th cysteine residues in the PDGF chains as the cysteine residues forming interchain disulfide bonds. Analysis of PDGF mutants in which the 2nd and 4th cysteine residues were mutated to serine residues revealed that the disulfide bonds are arranged in a cross-wise manner, with the 2nd cysteine residue in one chain being linked to the 4th cysteine residue in the other. A PDGF B-chain mutant, in which both the 2nd and 4th cysteine residues were substituted with serine residues, migrated as a monomer in sodium dodecyl sulfate gel electrophoresis and retained receptor binding activity. When analyzed in receptor dimerization and autophosphorylation assays, this mutant showed agonistic activity. Thus, structural information has been obtained that will allow the large scale production of properly folded monomeric PDGF, as well as design of specific PDGF heterodimers.     

Endotoxin and staphylococcus aureus enterotoxin a induce different patterns of cytokines

The effects of Staphylococcus aureus enterotoxin A (SEA) and lipopolysaccharide (LPS) in cytokine production were assessed at the single cell level in cells obtained from healthy blood donors. Cytokine production was studied with UV-microscopy of fixed and permeabilized cells stained with cytokine specific monoclonal antibodies. The cytokines evaluated included tumour necrosis factor (TNF)-α, interleukin (IL)-1α, IL-1β, IL-6, IL-8, IL-10, IL-2, IL-4, interferon (IFN)-γ and TNF-β. LPS exhibited marked production of IL-1α, IL-1β, TNF-α, IL-6 and IL-8. After LPS stimulation IL-1α, IL-1β, TNF-α and IL-8 were the dominating products, all peaking at or before 4 hours after cell stimulation. In addition, IL-10 production was evident after 12 hours of cell stimulation. The T-lymphocyte-derived cytokines TNF-β, IL-2, IFN-γ and IL-4 were never detected in the cultures. All cytokine production, except IL-8, was downregulated at 96 hours.In contrast, peak production of IL-1α, IL-1β and IL-8, which were the dominant products, occurred after 12 hours in the SEA-stimulated cultures. Further, a significant T-lymphocyte production of TNF-β, TNF-α, IFN-γ and IL-2 was found with peak production 12–48 hours after initiation. Only low amounts of IL-6 were evident.The two types of cytokine pattern and kinetics found may correspond to the different clinical conditions after invasive Gram-negative Escherichia coli vs Gram-positive Staphylococcus aureus infections in humans, with a much more rapid onset of disease after E. coli infections. The data may also imply that different immunomodulating approaches should be considered in life-threatening diseases with the two microbacterial agents.     

Tartrate-resistant acid ATPase as a cytochemical marker for osteoclasts

We present a modified histochemical method for staining osteoclasts and adjacent mononuclear cells which takes advantage of the recently described substrate specificity for ATP of osteoclastic acid phosphatase. Staining of osteoclasts using ATP as substrate exhibits by light microscopy the same tartrate resistance as conventional acidic phosphatases, without the bone surface staining seen with other substrates. This feature, coupled with specific staining of fewer vicinal mononuclear cells, makes this method potentially useful for studying osteoclast ontogeny and function.     

Model-based design and control of a small-scale integrated continuous end-to-end mAb platform

A continuous integrated bioprocess available from the earliest stages of process development allows for an easier, more efficient and faster development and characterization of an integrated process as well as production of small-scale drug candidates. The process presented in this article is a proof-of-concept of a continuous end-to-end monoclonal antibody production platform at a very small scale based on a 200 ml alternating tangential flow filtration perfusion bioreactor, integrated with the purification process with a model-based design and control. The downstream process, consisting of a periodic twin-column protein A capture, a virus inactivation, a CEX column and an AEX column, was compactly implemented in a single chromatography system, with a purification time of less than 4 hr. Monoclonal antibodies were produced for 17 days in a high cell density perfusion culture of CHO cells with titers up to 1.0 mg/ml. A digital twin of the downstream process was created by modelling all the chromatography steps. These models were used for real-time decision making by the implementation of control strategies to automatize and optimize the operation of the process. A consistent glycosylation pattern of the purified product was ensured by the steady state operation of the process. Regarding the removal of impurities, at least a 4-log reduction in the HCP levels was achieved. The recovery yield was up to 60%, and a maximum productivity of 0.8 mg/ml/day of purified product was obtained.     

Molecular engineering of a thermostable carbohydrate-binding module

Structure–function studies are frequently practiced on the very diverse group of natural carbohydrate-binding modules in order to understand the target recognition of these proteins. We have taken a step further in the study of carbohydrate-binding modules and created variants with novel binding properties by molecular engineering of one such molecule of known 3D-structure. A combinatorial library was created from the sequence encoding a thermostable carbohydrate-binding module, CBM4-2 from a Rhodothermus marinus xylanase, and the phage-display technology was successfully used for selection of variants with specificity towards different carbohydrate polymers (birchwood xylan, Avicel?, ivory nut mannan and recently also xyloglucan), as well as towards a glycoprotein (human IgG4). Our work not only generated a number of binders with properties that would suite a range of biotechnological applications, but analysis the selected binders also helped us to identify residues important for their specificities.     

The Redox State of Glutathione,Cysteine and Homocysteine in the Extracellular Fluid in the Skin

Glutathione, the most abundant low-molecular weight thiol in the skin, has been shown to protect the skin from both photobiological and chemical injury. The thiols, glutathione in particular, have also been shown to be crucially involved in defence against contact allergens. Since the levels of extracellular thiol concentrations are important determinants of intracellular thiol status, we have compared the normal concentrations and the redox status of the main low-molecular weight thiol components in the extracellular fluid at the dermo-epidermal junction with the corresponding plasma levels. In their sulfhydryl form, all three thiols, i.e. glutathione, cysteine and homocysteine, were more abundant in experimental skin blister fluid than in plasma, as were the free disulfides of glutathione and homocysteine, whereas the free disulfides of cysteine were about the same in blister fluid and in plasma. Protein mixed disulfide levels were higher in plasma than in blister fluid. The present results provide information concerning the extracellular defence in the skin.