The contribution of cell death to medium-released fractions of cell cultures

Summary Cell death was estimated by prelabeling primary chick embryo skeletal muscle cell cultures with [³H]thymidine and by subsequently measuring the release of label into complete culture medium or serum-and embryo-extract-free medium for a 6 h period. Cultures of the established muscle cell line L6 and the fibroblastic cell line 3T3 were used for comparative purposes. Comparison of the nigrosin exclusion test with the thymidine release test shows that the former underestimates cell death because it measures only the instantaneously occurring cell death. The [³H]thymidine release test estimates the cumulative amount of cell death. From cumulative cell death estimates it is calculated that 12.0 and 17.8% of the³H-fucosylated medium-released fractions from primary cell cultures are the result of cell death contamination when release occurs in complete or macromolecule-free media, respectively. High speed centrifugation is shown to eliminate most contamination from cell death. Evidence is presented that the absence of macromolecules in the culture medium has little effect on the release process. Contamination of the released fraction resulting from cell death is much less in the established cell lines than in the primary cells. It is concluded that the release process can be studied in primary muscle cell cultures and especially in established cell lines if adequate precautions are taken and if corrections for cell death contamination are taken into account. This research benefited from use of the Cell Culture Facility supported by National Cancer Institute Grant CA 14733. This research was supported by a Muscular Dystrophy Association Grant to Dr. Heinz Herrmann and by American Cancer Society Grant RDP 8 and was submitted in partial fulfillmant of a Ph.D. degree at the University of Connecticut (T. C. Doetschman).     

Rapid expression and purification of 100 nmol quantities of active protein using cell-free protein synthesis

Two strategies for ATP regeneration during cell-free protein synthesis were applied to the large-scale production and single-column purification of active chloramphenicol acetyl transferase (CAT). Fed-batch reactions were performed on a 5-10 mL scale, approximately 2 orders of magnitude greater than the typical reaction volume. The pyruvate oxidase system produced 104 nmol of active CAT in a 5 mL reaction over the course of 5 h. The PANOx system produced 261 +/- 42 nmol, about 7 mg, of active CAT in a 10 mL reaction over the course of 4 h. The reaction product was purified to apparent homogeneity with approximately 70% yield by a simple affinity chromatography adsorption and elution. To our knowledge, this is the largest amount of actively expressed protein to be reported in a simple, fed-batch cell-free protein synthesis reaction.     

Borrelia burgdorferi bb0426 encodes a 2'-deoxyribosyltransferase that plays a central role in purine salvage

Borrelia burgdorferi is an obligate parasite with a limited genome that severely narrows its metabolic and biosynthetic capabilities. Thus survival of this spirochaete in an arthropod vector and mammalian host requires that it can scavenge amino acids, fatty acids and nucleosides from a blood meal or various host tissues. Additionally, the utilization of ribonucleotides for DNA synthesis is further complicated by the lack of a ribonucleotide reductase for the conversion of nucleoside-5'-diphosphates to deoxynucleosides-5'-diphosphates. The data presented here demonstrate that B. burgdorferi must rely on host-derived sources of purine bases, deoxypurines and deoxypyrimidines for the synthesis of DNA. However, if deoxyguanosine (dGuo) is limited in host tissue, the enzymatic activities of a 2'-deoxyribosyltransferase (DRTase, encoded by bb0426 ), IMP dehydrogenase (GuaB) and GMP synthase (GuaA) catalyse the multistep conversion of hypoxanthine (Hyp) to dGMP for DNA synthesis. This pathway provides additional biochemical flexibility for B. burgdorferi when it colonizes and infects different host tissues.     

Assisted peptide folding by surface pattern recognition

Natively disordered proteins belong to a unique class of biomolecules whose function is related to their flexibility and their ability to adopt desired conformations upon binding to substrates. In some cases these proteins can bind multiple partners, which can lead to distinct structures and promiscuity in functions. In other words, the capacity to recognize molecular patterns on the substrate is often essential for the folding and function of intrinsically disordered proteins. Biomolecular pattern recognition is extremely relevant both in vivo (e.g., for oligomerization, immune response, induced folding, substrate binding, and molecular switches) and in vitro (e.g., for biosensing, catalysis, chromatography, and implantation). Here, we use a minimalist computational model system to investigate how polar/nonpolar patterns on a surface can induce the folding of an otherwise unstructured peptide. We show that a model peptide that exists in the bulk as a molten globular state consisting of many interconverting structures can fold into either a helix-coil-helix or an extended helix structure in the presence of a complementary designed patterned surface at low hydrophobicity (3.7%) or a uniform surface at high hydrophobicity (50%). However, we find that a carefully chosen surface pattern can bind to and catalyze the folding of a natively unfolded protein much more readily or effectively than a surface with a noncomplementary or uniform distribution of hydrophobic residues.     

Relative stability of de novo four-helix bundle proteins: insights from coarse grained molecular simulations

We use a recently developed coarse-grained computational model to investigate the relative stability of two different sets of de novo designed four-helix bundle proteins. Our simulations suggest a possible explanation for the experimentally observed increase in stability of the four-helix bundles with increasing sequence length. In details, we show that both short subsequences composed only by polar residues and additional nonpolar residues inserted, via different point mutations in ad hoc positions, seem to play a significant role in stabilizing the four-helix bundle conformation in the longer sequences. Finally, we propose an additional mutation that rescues a short amino acid sequence that would otherwise adopt a compact misfolded state. Our work suggests that simple computational models can be used as a complementary tool in the design process of de novo proteins.     

Binding between the integrin alphaXbeta2 (CD11c/CD18) and heparin

The interactions between cell surface receptors and sulfated glucosamineglycans serve ubiquitous roles in cell adhesion and receptor signaling. Heparin, a highly sulfated polymer of uronic acids and glucosamine, binds strongly to the integrin receptor alphaXbeta2 (p150,95, CD11c/CD18). Here, we analyze the structural motifs within heparin that constitute high affinity binding sites for the I domain of integrin alphaXbeta2. Heparin oligomers with chain lengths of 10 saccharide residues or higher provide strong inhibition of the binding by the alphaX I domain to the complement fragment iC3b. By contrast, smaller oligomers or the synthetic heparinoid fondaparinux were not able to block the binding. Semipurified heparin oligomers with 12 saccharide residues identified the fully sulfated species as the most potent antagonist of iC3b, with a 1.3 microM affinity for the alphaX I domain. In studies of direct binding by the alphaX I domain to immobilized heparin, we found that the interaction is conformationally regulated and requires Mg2+. Furthermore, the fully sulfated heparin fragment induced conformational change in the ectodomain of the alphaXbeta2 receptor, also demonstrating allosteric linkage between heparin binding and integrin conformation.     

The critical role of the linear plasmid lp36 in the infectious cycle of Borrelia burgdorferi

Borrelia burgdorferi, the aetiological agent of Lyme disease, follows a life cycle that involves passage between the tick vector and the mammalian host. To investigate the role of the 36 kb linear plasmid, lp36 (also designated the B. burgdorferi K plasmid), in the infectious cycle of B. burgdorferi, we examined a clone lacking this plasmid, but containing all other plasmids known to be required for infectivity. Our results indicated that lp36 was not required for spirochete survival in the tick, but the clone lacking lp36 demonstrated low infectivity in the mammal. Restoration of lp36 to the mutant strain confirmed that the infectivity defect was due to loss of lp36. Moreover, spirochetes lacking lp36 exhibited a nearly 4-log increase in ID(50) relative to the isogenic lp36(+) clone. The infectivity defect of lp36-minus spirochetes was localized, in part, to loss of the bbk17 (adeC) gene, which encodes an adenine deaminase. This work establishes a vital role for lp36 in the infectious cycle of B. burgdorferi and identifies the bbk17 gene as a component of this plasmid that contributes to mammalian infectivity.