Detection and reduction of microaggregates in insulin preparations

Insulin is an important biotherapeutic protein, and it is also a model protein used to study amyloid diseases, such as Alzheimer's and Parkinson's. The preparation of the protein can lead to small amounts of aggregate in the solution, which in turn may lead to irreproducible in vitro results. Using several pre‐treatment methods, we have determined that pH cycling and diafiltration of the insulin removes microaggregates that may be present in the solution. These microaggregates were not detectable with traditional biochemical methods, but using small‐angle neutron scattering, we were able to show that pH cycling reduces the radius of gyration of the insulin. Diafiltration removes the aggregates by size and pH cycling dissolves the aggregates by adjusting the pH through the pI of the protein. Pre‐treating the insulin with either pH cycling or diafiltration allowed reproducible kinetics of fibrillation for the insulin protein. Microaggregates are a common problem in protein production, formulation, and preparation; here we show that they are the main cause for inconsistent behavior and how pH cycling and diafiltration can mitigate this problem. Biotechnol. Bioeng. 2011; 108:237–241. © 2010 Wiley Periodicals, Inc.     

Isolating toxic insulin amyloid reactive species that lack β-sheets and have wide pH stability

Amyloid diseases, including Alzheimer's disease, are characterized by aggregation of normally functioning proteins or peptides into ordered, β-sheet rich fibrils. Most of the theories on amyloid toxicity focus on the nuclei or oligomers in the fibril formation process. The nuclei and oligomers are transient species, making their full characterization difficult. We have isolated toxic protein species that act like an oligomer and may provide the first evidence of a stable reactive species created by disaggregation of amyloid fibrils. This reactive species was isolated by dissolving amyloid fibrils at high pH and it has a mass >100 kDa and a diameter of 48 ± 15 nm. It seeds the formation of fibrils in a dose dependent manner, but using circular dichroism and deep ultraviolet resonance Raman spectroscopy, the reactive species was found to not have a β-sheet rich structure. We hypothesize that the reactive species does not decompose at high pH and maintains its structure in solution. The remaining disaggregated insulin, excluding the toxic reactive species that elongated the fibrils, returned to native structured insulin. This is the first time, to our knowledge, that a stable reactive species of an amyloid reaction has been separated and characterized by disaggregation of amyloid fibrils.     

The AAA motor complex of subunits CobS and CobT of cobaltochelatase visualized by single particle electron microscopy

Cobalamins belong to the tetrapyrrole family of prosthetic groups. The presence of a metal ion is a key feature of these compounds. In the oxygen-dependent (aerobic) cobalamin biosynthetic pathway, cobalt is inserted into a ring-contracted tetrapyrrole called hydrogenobyrinic acid a,c-diamide (HBAD) by a cobaltochelatase that is constituted by three subunits, CobN, CobS and CobT, with molecular masses of 137, 37 and 71 kDa, respectively. Based on the similarities with magnesium chelatase, cobaltochelatase has been suggested to belong to the AAA⁺ superfamily of proteins. In this paper we present the cloning of the Brucella melitensis cobN, cobS and cobT, the purification of the encoded protein products, and a single-particle reconstruction of the macromolecular assembly formed between CobS and CobT from negatively stained electron microscopy images of the complex. The results show for the first time that subunits CobS and CobT form a chaperone-like complex, characteristic for the AAA⁺ class of proteins. The molecules are arranged in a two-tiered ring structure with the six subunits in each ring organized as a trimer of dimers. The similarity between this structure and that of magnesium chelatase, as well as analysis of the amino acid sequences confirms the suggested evolutionary relationship between the two enzymes.     

Phloem loading in two Scrophulariaceae species. What can drive symplastic flow via plasmodesmata?

To determine the driving forces for symplastic sugar flux between mesophyll and phloem, gradients of sugar concentrations and osmotic pressure were studied in leaf tissues of two Scrophulariaceae species, Alonsoa meridionalis and Asarina barclaiana. A. meridionalis has a typical symplastic configuration of minor-vein phloem, i.e. intermediary companion cells with highly developed plasmodesmal connections to bundle-sheath cells. In A. barclaiana, two types of companion cells, modified intermediary cells and transfer cells, were found in minor-vein phloem, giving this species the potential to have a complex phloem-loading mode. We identified all phloem-transported carbohydrates in both species and analyzed the levels of carbohydrates in chloroplasts, vacuoles, and cytoplasm of mesophyll cells by nonaqueous fractionation. Osmotic pressure was measured in single epidermal and mesophyll cells and in whole leaves and compared with calculated values for phloem sap. In A. meridionalis, a 2-fold concentration gradient for sucrose between mesophyll and phloem was found. In A. barclaiana, the major transported carbohydrates, sucrose and antirrhinoside, were present in the phloem in 22- and 6-fold higher concentrations, respectively, than in the cytoplasm of mesophyll cells. The data show that diffusion of sugars along their concentration gradients is unlikely to be the major mechanism for symplastic phloem loading if this were to occur in these species. We conclude that in both A. meridionalis and A. barclaiana, apoplastic phloem loading is an indispensable mechanism and that symplastic entrance of solutes into the phloem may occur by mass flow. The conditions favoring symplastic mass flow into the phloem are discussed.     

Identification of trimeric peptides that bind porcine parvovirus from mixtures containing human blood plasma

Virus contamination in human therapeutics is of growing concern as more therapeutic products from animal or human sources come into the market. All biopharmaceutical processes are required to have at least two distinct viral clearance steps to remove viruses. Most of these steps work well for enveloped viruses and large viruses, whether enveloped or not. That leaves a class of small non-enveloped viruses, like parvoviruses and hepatitis A, which are not easily removed by these typical steps. In this study, we report the identification of trimeric peptides that bind specifically to porcine parvovirus (PPV) and their potential use to remove this virus from process solutions. All of the trimeric peptides isolated completely removed all detectable PPV from buffer in the first nine column volumes, corresponding to a clearance of 4.5-5.5 log of infectious virus. When the virus was spiked into a more complex matrix consisting of 7.5% human blood plasma, one of the trimers, WRW, was able to remove all detectable PPV in the first three column volumes, after which human blood plasma began to interfere with the binding of the virus to the peptide resin. These trimer resins removed considerably more virus than weak ion exchange resins. The results of this work indicate that small peptide ligand resins have the potential to be used in virus removal processes where removal of contaminating virus is necessary to ensure product safety.     

Microfacies, biostratigraphy, and geochemistry of the hemipelagic Barremian–Aptian in north-central Tunisia: Influence of the OAE 1a on the southern Tethys margin

Upper Barremian–lower Upper Aptian hemipelagic deposits of the Hamada Formation in the Djebel Serdj area, north-central Tunisia were studied in detail with regard to microfacies, biostratigraphy, δ¹³C stratigraphy, and geochemistry. Our data provide insights into the palaeoenvironmental evolution and sea-level fluctuations of the Tunisian shelf. The unusually expanded deposits consist of mud-, wacke-, and packstones which reflect mid- and outer-ramp depositional environments. Planktonic foraminifer and δ¹³C stratigraphy allowed us to establish a detailed time-frame and the recognition of the Lower Aptian Oceanic Anoxic Event 1a (OAE 1a) as well as time-equivalent deposits of shallow-marine carbonate-platform drowning. Based on our microfacies studies, we subdivide the studied sections into four genetic intervals: a pre-OAE 1a interval, an OAE 1a and platform-drowning-equivalent interval, and a post-platform-drowning interval. We present a 3rd-order sea-level curve for the Tunisian shelf, deriving from the results of our microfacies studies.

Deposits of the OAE 1a in the sections investigated are characterised by bioclastic wacke- and packstones with high abundances of poorly preserved radiolarians and moderately to well preserved planktonic foraminifers, suggesting a transgression and an eutrophication of the upper water column. Scarceness of benthic macrofossils, low abundances of small benthic foraminifers and ostracods possibly suggest dysoxic conditions at the seafloor. Mudstones of the platform-drowning time-equivalent deposits, directly overlying the OAE 1a, are partly showing a pronounced drop in carbonate content and are scarce of macrofossils.