Stabilizing Additives Added during Cell Lysis Aid in the Solubilization of Recombinant Proteins

Insoluble recombinant proteins are a major issue for both structural genomics and enzymology research. Greater than 30% of recombinant proteins expressed in Escherichia coli (E. coli) appear to be insoluble. The prevailing view is that insolubly expressed proteins cannot be easily solubilized, and are usually sequestered into inclusion bodies. However, we hypothesize that small molecules added during the cell lysis stage can yield soluble protein from insoluble protein previously screened without additives or ligands. We present a novel screening method that utilized 144 additive conditions to increase the solubility of recombinant proteins expressed in E. coli. These selected additives are natural ligands, detergents, salts, buffers, and chemicals that have been shown to increase the stability of proteins in vivo. We present the methods used for this additive solubility screen and detailed results for 41 potential drug target recombinant proteins from infectious organisms. Increased solubility was observed for 80% of the recombinant proteins during the primary and secondary screening of lysis with the additives; that is 33 of 41 target proteins had increased solubility compared with no additive controls. Eleven additives (trehalose, glycine betaine, mannitol, L-Arginine, potassium citrate, CuCl₂, proline, xylitol, NDSB 201, CTAB and K₂PO₄) solubilized more than one of the 41 proteins; these additives can be easily screened to increase protein solubility. Large-scale purifications were attempted for 15 of the proteins using the additives identified and eight (40%) were prepared for crystallization trials during the first purification attempt. Thus, this protocol allowed us to recover about a third of seemingly insoluble proteins for crystallography and structure determination. If recombinant proteins are required in smaller quantities or less purity, the final success rate may be even higher.     

Altered Regulation of Akt Signaling with Murine Cerebral Malaria,Effects on Long-Term Neuro-Cognitive Function,Restoration with Lithium Treatment

Neurological and cognitive impairment persist in more than 20% of cerebral malaria (CM) patients long after successful anti-parasitic treatment. We recently reported that long term memory and motor coordination deficits are also present in our experimental cerebral malaria model (ECM). We also documented, in a murine model, a lack of obvious pathology or inflammation after parasite elimination, suggesting that the long-term negative neurological outcomes result from potentially reversible biochemical and physiological changes in brains of ECM mice, subsequent to acute ischemic and inflammatory processes. Here, we demonstrate for the first time that acute ECM results in significantly reduced activation of protein kinase B (PKB or Akt) leading to decreased Akt phosphorylation and inhibition of the glycogen kinase synthase (GSK3β) in the brains of mice infected with Plasmodium berghei ANKA (PbA) compared to uninfected controls and to mice infected with the non-neurotrophic P. berghei NK65 (PbN). Though Akt activation improved to control levels after chloroquine treatment in PbA-infected mice, the addition of lithium chloride, a compound which inhibits GSK3β activity and stimulates Akt activation, induced a modest, but significant activation of Akt in the brains of infected mice when compared to uninfected controls treated with chloroquine with and without lithium. In addition, lithium significantly reversed the long-term spatial and visual memory impairment as well as the motor coordination deficits which persisted after successful anti-parasitic treatment. GSK3β inhibition was significantly increased after chloroquine treatment, both in lithium and non-lithium treated PbA-infected mice. These data indicate that acute ECM is associated with abnormalities in cell survival pathways that result in neuronal damage. Regulation of Akt/GSK3β with lithium reduces neuronal degeneration and may have neuroprotective effects in ECM. Aberrant regulation of Akt/GSK3β signaling likely underlies long-term neurological sequelae observed in ECM and may yield adjunctive therapeutic targets for the management of CM.     

The RenTg Mice: A Powerful Tool to Study Renin-Dependent Chronic Kidney Disease

Background

Several studies have shown that activation of the renin-angiotensin system may lead to hypertension, a major risk factor for the development of chronic kidney disease (CKD). The existing hypertension-induced CDK mouse models are quite fast and consequently away from the human pathology. Thus, there is an urgent need for a mouse model that can be used to delineate the pathogenic process leading to progressive renal disease. The objective of this study was dual: to investigate whether mice overexpressing renin could mimic the kinetics and the physiopathological characteristics of hypertension-induced renal disease and to identify cellular and/or molecular events characterizing the different steps of the progression of CKD.

Methodology/Principal Findings

We used a novel transgenic strain, the RenTg mice harboring a genetically clamped renin transgene. At 3 months, heterozygous mice are hypertensive and slightly albuminuric. The expression of adhesion markers such as vascular cell adhesion molecule-1 and platelet endothelial cell adhesion molecule-1 are increased in the renal vasculature indicating initiation of endothelial dysfunction. At 5 months, perivascular and periglomerular infiltrations of macrophages are observed. These early renal vascular events are followed at 8 months by leukocyte invasion, decreased expression of nephrin, increased expression of KIM-1, a typical protein of tubular cell stress, and of several pro-fibrotic agents of the TGFβ family. At 12 months, mice display characteristic structural alterations of hypertensive renal disease such as glomerular ischemia, glomerulo- and nephroangio-sclerosis, mesangial expansion and tubular dilation.

Conclusions/Significance

The RenTg strain develops CKD progressively. In this model, endothelial dysfunction is an early event preceding the structural and fibrotic alterations which ultimately lead to the development of CKD. This model can provide new insights into the mechanisms of chronic renal failure and help to identify new targets for arresting and/or reversing the development of the disease.     

Molecular titration and ultrasensitivity in regulatory networks

Protein sequestration occurs when an active protein is sequestered by a repressor into an inactive complex. Using mathematical and computational modeling, we show how this regulatory mechanism (called “molecular titration”) can generate ultrasensitive or “all-or-none” responses that are equivalent to highly cooperative processes. The ultrasensitive nature of the input-output response is mainly determined by two parameters: the dimer dissociation constant and the repressor concentration. Because in vivo concentrations are tunable through a variety of mechanisms, molecular titration represents a flexible mechanism for generating ultrasensitivity. Using physiological parameters, we report how details of in vivo protein degradation affect the strength of the ultrasensitivity at steady state. Given that developmental systems often transduce signals into cell-fate decisions on timescales incompatible with steady state, we further examine whether molecular titration can produce ultrasensitive responses within physiologically relevant time intervals. Using Drosophila somatic sex determination as a developmental paradigm, we demonstrate that molecular titration can generate ultrasensitivity on timescales compatible with most cell-fate decisions. Gene duplication followed by loss-of-function mutations can create dominant negatives that titrate and compete with the original protein. Dominant negatives are abundant in gene regulatory circuits, and our results suggest that molecular titration might be generating an ultrasensitive response in these networks.     

Biome-level biogeography of streambed microbiota

A field study was conducted to determine the microbial community structures of streambed sediments across diverse geographic and climatic areas. Sediment samples were collected from three adjacent headwater forest streams within three biomes, eastern deciduous (Pennsylvania), southeastern coniferous (New Jersey), and tropical evergreen (Guanacaste, Costa Rica), to assess whether there is biome control of stream microbial community structure. Bacterial abundance, microbial biomass, and bacterial and microbial community structures were determined using classical, biochemical, and molecular methods. Microbial biomass, determined using phospholipid phosphate, was significantly greater in the southeastern coniferous biome, likely due to the smaller grain size, higher organic content, and lower levels of physical disturbance of these sediments. Microbial community structure was determined using phospholipid fatty acid (PLFA) profiles and bacterial community structure from terminal restriction fragment length polymorphism and edited (microeukaryotic PLFAs removed) PLFA profiles. Principal component analysis (PCA) was used to investigate patterns in total microbial community structure. The first principal component separated streams based on the importance of phototrophic microeukaryotes within the community, while the second separated southeastern coniferous streams from all others based on increased abundance of fungal PLFAs. PCA also indicated that within- and among-stream variations were small for tropical evergreen streams and large for southeastern coniferous streams. A similar analysis of bacterial community structure indicated that streams within biomes had similar community structures, while each biome possessed a unique streambed community, indicating strong within-biome control of stream bacterial community structure.     

Alterations in the testis of hormone sensitive lipase-deficient mice is associated with decreased sperm counts, sperm motility, and fertility

Hormone-sensitive lipase (HSL, Lipe, E.C.3.1.1.3) functions as a triglyceride and cholesteryl esterase, supplying fatty acids, and cholesterol to cells. Gene-targeted HSL-deficient (HSL(-/-)) mice reveal abnormal spermatids and are infertile at 24 weeks after birth. The purpose of this study was to follow the evolution of spermatid abnormalities as HSL(-/-) mice age, characterize sperm motility in older HSL(-/-) mice, and determine if mice expressing a human testicular HSL transgene (HSL(-/-)ttg) produce normal motile sperm. In situ hybridization indicated that HSL is expressed exclusively in steps 5-16 spermatids, but not in Sertoli cells. In HSL(-/-) mice, abnormalities were evident in step 16 spermatids at 5 weeks after birth, with defects progressively increasing in spermatids with age. The defects included multinucleation of spermatids, abnormal shapes and a reduction of elongating spermatids. In older HSL(-/-) mice, sperm counts appeared reduced by 42%, but this value was lower because samples were compromised by the presence of small degenerating germ cells in addition to sperm, both of which appeared of similar size and density. Sperm motility was dramatically reduced with only 11% classified as motile in HSL(-/-) mice compared to 76-78% of sperm in wild-type and HSL(-/-)ttg mice. Sperm morphology, counts, and motility were normal in HSL(-/-)ttg mice, as was their fertility. Collectively, the data indicate that HSL deficiency results in abnormal spermatid development with defects arising at 5 weeks of age and progressively increasing at later ages. HSL(-/-) mice also show a dramatic reduction in sperm counts and motility and are infertile.     

Bioengineering a humanized acne microenvironment model: proteomics analysis of host responses to Propionibacterium acnes infection in vivo

Acne is a human disease of the sebaceous hair follicle. Unlike humans, most animals produce little or no triglycerides in hair follicles to harbor Propionibacterium acnes a fact that has encumbered the development of novel treatments for acne lesions. Although genetic mutant mice with acne-like skins have been used for screening anti-acne drugs, the mice generally have deficits in immune system that turns out to be inappropriate to generate antibodies for developing acne vaccines. Here, we employed a bioengineering approach using a tissue chamber integrated with a dermis-based cell-trapped system (DBCTS) to mimic the in vivo microenvironment of acne lesions. Human sebocyte cell lines were grown in DBCTS as a scaffold and inserted into a perforated tissue chamber. After implantation of a tissue chamber bearing human sebocytes into ICR mice, P. acnes or PBS was injected into a tissue chamber to induce host immune response. Infiltrated cells such as neutrophils and macrophages were detectable in tissue chamber fluids. In addition, a proinflammatory cytokine macrophage-inflammatory protein-2 (MIP-2) was elevated after P. acnes injection. In tissue chamber fluids, 13 proteins including secreted proteins and cell matrix derived from mouse, human cells or P. acnes were identified by proteomics using isotope-coded protein label (ICPL) coupled to nano-LC-MS analysis. After P. acnes infection, four proteins including fibrinogen, alpha polypeptide, fibrinogen beta chain, S100A9, and serine protease inhibitor A3K showed altered concentrations in the mimicked acne microenvironment. The bioengineered acne model thus provides an in vivo microenvironment to study the interaction of host with P. acnes and offers a unique set-up for screening novel anti-acne drugs and vaccines.     

Mice deficient in the CXCR2 ligand, CXCL1 (KC/GRO-alpha), exhibit increased susceptibility to dextran sodium sulfate (DSS)-induced colitis

The role of TLRs and MyD88 in the maintenance of gut integrity in response to dextran sodium sulfate (DSS)-induced colitis was demonstrated recently and led to the conclusion that the innate immune response to luminal commensal flora provides necessary signals that facilitate epithelial repair and permits a return to homeostasis after colonic injury. In this report, we demonstrate that a deficit in a single neutrophil chemokine, CXCL1/KC, also results in a greatly exaggerated response to DSS. Mice with a targeted mutation in the gene that encodes this chemokine responded to 2.5% DSS in their drinking water with significant weight loss, bloody stools, and a complete loss of gut integrity in the proximal and distal colon, accompanied by a predominantly mononuclear infiltrate, with few detectable neutrophils. In contrast, CXCL1/KC(- /-) and wild-type C57BL/6J mice provided water showed no signs of inflammation and, at this concentration of DSS, wild-type mice showed only minimal histopathology, but significantly more infiltrating neutrophils. This finding implies that neutrophil infiltration induced by CXCL1/KC is an essential component of the intestinal response to inflammatory stimuli as well as the ability of the intestine to restore mucosal barrier integrity.     

Characterization of 25 microsatellite loci in bowhead whales (Balaena mysticetus)

Bowhead whales (Balaena mysticetus) experienced a severe demographic population bottleneck caused by commercial whaling that ceased in 1914. Aboriginal subsistence whale harvests have continued and are managed by the International Whaling Commission. In an effort to provide management advice for bowhead whales, 25 microsatellite loci were isolated from genomic DNA libraries. This panel of markers will be utilized to analyse stock structure hypotheses of current bowhead whale populations.