Diffusion coefficient of fluorescein-labeled tubulin in the cytoplasm of embryonic cells of a sea urchin: video image analysis of fluorescence redistribution after photobleaching

The diffusion coefficient of tubulin has been measured in the cytoplasm of eggs and embryos of the sea urchin Lytechinus variegatus. We have used brain tubulin, conjugated to dichlorotriazinyl-aminofluorescein, to inject eggs and embryos. The resulting distributions of fluorescence were perturbed by bleaching with a microbeam of light from the 488-nm line of an argon ion laser. Fluorescence redistribution after photobleaching was monitored with a sensitive video camera and photography of the television-generated image. With standard photometric methods, we have calibrated this recording system and measured the rates of fluorescence redistribution for tubulin, conjugated to dichlorotriazinyl-aminofluorescein, not incorporated into the mitotic spindle. The diffusion coefficient (D) was calculated from these data using Fick's second law of diffusion and a digital method for analysis of the photometric curves. We have tested our method by determining D for bovine serum albumin (BSA) under conditions where the value is already known and by measuring D for fluorescein-labeled BSA in sea urchin eggs with a standard apparatus for monitoring fluorescence redistribution after photobleaching. The values agree to within experimental error. Dcytoplasmtubulin = 5.9 +/- 2.2 X 10(-8) cm2/s; DcytoplasmBSA = 8.6 +/- 2.0 X 10(-8) cm2/s. Because DH2OBSA = 68 X 10(-8) cm2/s, these data suggest that the viscosity of sea urchin cytoplasm for protein is about eight times that of water and that most of the tubulin of the sea urchin cytoplasm exists as a dimer or small oligomer, which is unbound to structures that would impede its diffusion. Values and limitations of our method are discussed, and we draw attention to both the variations in D for single proteins in different cells and the importance of D for the upper limit to the rates of polymerization reactions.     

Microwave thawing of frozen kidneys: A theoretically based experimentally-effective design

Equipment was designed and fabricated for uniformly thawing frozen canine kidneys using single-frequency electromagnetic radiation. Complete and uniform warming of frozen kidneys from ?70 to +14 °C over periods ranging from 1.5 to 4.5 min was achieved without “cooking” or experiencing thermal runaway. Dielectric measurements of renal slices (medulla and cortex) were performed as a function of temperature at a frequency of 918 MHz for a Me₂SO cryoprotectant concentration of 5% (0.7 M). Results of these measurements were then employed as an input to analytical computer models which were used to predict the internal field intensities and power distribution results for both frozen and thawed kidneys. From these predictions, a 918-MHz EM illuminator for thawing canine kidneys was designed and fabricated. Twenty-seven kidneys were thawed using this illumination system. Of these, excellent uniformity of thawing was achieved for 17 kidneys, good uniformity for 8 kidneys, and for only 2 kidneys was thawing uniformity fair to poor.     

Functional preservation of the mammalian kidney. IV. Functional effects of perfusion with dimethyl sulfoxide (DMSO) at normothermia

Rabbit kidneys were perfused at 37 °C with various concentrations of DMSO in a K⁺-Mg²⁺-rich perfusate. The effects of DMSO on various functional parameters of the rabbit kidney perfused for 60 min were compared with the functional effects of perfusion without DMSO under the same conditions. DMSO produced deviations in vascular resistance and perfusate flow rate from control values. In kidneys perfused with 1.4 and 2.8 m DMSO these vascular changes resulted in changes in GFR at relatively unchanged filtration fractions. The closely parallel relationship between changes in GFR and urine flow rate in all groups indicates that perfusion per se or perfusion with DMSO may shift the regulation of urine flow rate from tubular reabsorption, which obtains in the in vivo situation, to glomerular filtration. This view was supported by the relatively unchanged parameters of Na⁺ reabsorption and fractional water excretion during perfusion with all concentrations of DMSO. Additionally, DMSO perfusion resulted in significantly greater weight gains than those observed in kidneys perfused without DMSO, and significantly depressed clearances of PAH, with 2.1 and 2.8 m DMSO.     

Buffer capacity of biologics—from buffer salts to buffering by antibodies

Controlling pH is essential for a variety of biopharmaceutical process steps. The chemical stability of biologics such as monoclonal antibodies is pH‐dependent and slightly acidic conditions are favorable for stability in a number of cases. Since control of pH is widely provided by added buffer salts, the current study summarizes the buffer characteristics of acetate, citrate, histidine, succinate, and phosphate buffers. Experimentally derived values largely coincide with values calculated from a model that had been proposed in 1922 by van Slyke. As high concentrated protein formulations become more and more prevalent for biologics, the self‐buffering potential of proteins becomes of relevance. The current study provides information on buffer characteristics for pH ranges down to 4.0 and up to 8.0 and shows that a monoclonal antibody at 50 mg/mL exhibits similar buffer capacity as 6 mM citrate or 14 mM histidine (pH 5.0–6.0). Buffer capacity of antibody solutions scales linearly with protein concentration up to more than 200 mg/mL. At a protein concentration of 220 mg/mL, the buffer capacity resembles the buffer capacity of 30 mM citrate or 50 mM histidine (pH 5.0–6.0). The buffer capacity of monoclonal antibodies is practically identical at the process relevant temperatures 5, 25, and 40°C. Changes in ionic strength of ΔI=0.15, in contrast, can alter the buffer capacity up to 35%. In conclusion, due to efficient self‐buffering by antibodies in the pH range of favored chemical stability, conventional buffer excipients could be dispensable for pH stabilization of high concentrated protein solutions. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29: 480–492, 2013     

Potent inhibition of werner and bloom helicases by DNA minor groove binding drugs

Maintenance of genomic integrity is vital to all organisms. A number of human genetic disorders, including Werner Syndrome, Bloom Syndrome and Rothmund–Thomson Syndrome, exhibit genomic instability with some phenotypic characteristics of premature aging and cancer predisposition. Presumably the aberrant cellular and clinical phenotypes in these disorders arise from defects in important DNA metabolic pathways such as replication, recombination or repair. These syndromes are all characterized by defects in a member of the RecQ family of DNA helicases. To obtain a better understanding of how these enzymes function in DNA metabolic pathways that directly influence chromosomal integrity, we have examined the effects of non-covalent DNA modifications on the catalytic activities of purified Werner (WRN) and Bloom (BLM) DNA helicases. A panel of DNA-binding ligands displaying unique properties for interacting with double helical DNA was tested for their effects on the unwinding activity of WRN and BLM helicases on a partial duplex DNA substrate. The levels of inhibition by a number of these compounds were distinct from previously reported values for viral, prokaryotic and eukaryotic helicases. The results demonstrate that BLM and WRN proteins exhibit similar sensitivity profiles to these DNA-binding ligands and are most potently inhibited by the structurally related minor groove binders distamycin A and netropsin (K_i ≤1 µM). The distinct inhibition of WRN and BLM helicases by the minor groove binders suggest that these helicases unwind double-stranded DNA by a related mechanism.     

Leucine aminopeptidase is not essential for trimming peptides in the cytosol or generating epitopes for MHC class I antigen presentation

To detect viral infections and tumors, CD8+ T lymphocytes monitor cells for the presence of antigenic peptides bound to MHC class I molecules. The majority of MHC class I-presented peptides are generated from the cleavage of cellular and viral proteins by the ubiquitin-proteasome pathway. Many of the oligopeptides produced by this process are too long to stably bind to MHC class I molecules and require further trimming for presentation. Leucine aminopeptidase (LAP) is an IFN-inducible cytosolic aminopeptidase that can trim precursor peptides to mature epitopes and has been thought to play an important role in Ag presentation. To examine the role of LAP in generating MHC class I peptides in vivo, we generated LAP-deficient mice and LAP-deficient cell lines. These mutant mice and cells are viable and grow normally. The trimming of peptides in LAP-deficient cells is not reduced under basal conditions or after stimulation with IFN. Similarly, there is no reduction in presentation of peptides from precursor or full-length Ag constructs or in the overall supply of peptides from cellular proteins to MHC class I molecules even after stimulation with IFN. After viral infection, LAP-deficient mice generate normal CTL responses to seven epitopes from three different viruses. These data demonstrate that LAP is not an essential enzyme for generating most MHC class I-presented peptides and reveal redundancy in the function of cellular aminopeptidases.     

A distributed chemosensory circuit for oxygen preference in C. elegans

The nematode Caenorhabditis elegans has complex, naturally variable behavioral responses to environmental oxygen, food, and other animals. C. elegans detects oxygen through soluble guanylate cyclase homologs (sGCs) and responds to it differently depending on the activity of the neuropeptide receptor NPR-1: npr-1(lf) and naturally isolated npr-1(215F) animals avoid high oxygen and aggregate in the presence of food; npr-1(215V) animals do not. We show here that hyperoxia avoidance integrates food with npr-1 activity through neuromodulation of a distributed oxygen-sensing network. Hyperoxia avoidance is stimulated by sGC-expressing oxygen-sensing neurons, nociceptive neurons, and ADF sensory neurons. In npr-1(215V) animals, the switch from weak aerotaxis on food to strong aerotaxis in its absence requires close regulation of the neurotransmitter serotonin in the ADF neurons; high levels of ADF serotonin promote hyperoxia avoidance. In npr-1(lf) animals, food regulation is masked by increased activity of the oxygen-sensing neurons. Hyperoxia avoidance is also regulated by the neuronal TGF-β homolog DAF-7, a secreted mediator of crowding and stress responses. DAF-7 inhibits serotonin synthesis in ADF, suggesting that ADF serotonin is a convergence point for regulation of hyperoxia avoidance. Coalitions of neurons that promote and repress hyperoxia avoidance generate a subtle and flexible response to environmental oxygen.