pH Condition in temperature shift cultivation enhances cell longevity and specific hMab productivity in CHO culture

Controlling cell proliferation during cell culturing is an effective way to improve the production yield in mammalian cell culture. We examined the effect of temperature shifts (TS) under pH control conditions in Chinese hamster ovary cells. When we shifted the culture temperature from 37 °C to 31 °C before a stationary phase at pH 6.8 (TS/pH 6.8), cell viability remained high, and the final human monoclonal antibody (hMab) concentration increased to 2.3 times that in the culture remaining at 37 °C. However, there were no significant effects on the cell viability or production yield with the same TS at pH 7.0 (TS/pH 7.0). The average specific hMab productivity and mRNA level of TS/pH 7.0 were the same as that of TS/pH 6.8. The control of cell growth by the TS or the addition of rapamycin was effective in the maintenance of cell viability, but there was no significant increase of the average specific hMab productivity in the culture where cell proliferation was controlled with rapamycin. The hMab mRNA concentration decreased to 55%–65% at a 37 °C culture with the addition of actinomycin D. In contrast, actinomycin D did not affect the mRNA level in the TS culture. This result suggested that the increase in the mRNA level in the TS condition was caused by an increase in mRNA stability. In this study, we show that TS can produce two unrelated effects: a prolongation of cell longevity and an improvement in mRNA stability.     

Uncertainty in positioning ion chamber at reference depth for various water phantoms

Background

Uncertainty in the calibration of high-energy radiation sources is dependent on user and equipment type.

A Novel Myosin-like Protein (Myocilin) Expressed in the Connecting Cilium of the Photoreceptor: Molecular Cloning, Tissue Expression, and Chromosomal Mapping

We have isolated a human cDNA clone encoding a novel acidic protein of MW 55,000 that we designated “myocilin” since it has homology to myosin and is localized preferentially in the ciliary rootlet and basal body of the connecting cilium of photoreceptor cells. The deduced amino acid sequence of human myocilin showed significant homologies with nonmuscle myosin ofDictyostelium discoideumin the N-terminal region and also with olfactomedin of bullfrog in the C-terminal region. Myocilin contained a leucine zipper-like motif similar to that seen in kinectin and other cytoskeletal proteins. These findings suggest that myocilin is a novel cytoskeletal protein involved in the morphogenesis of ciliated neuroepithelium such as photoreceptor cells. The myocilin gene (MYOC) was mapped to human chromosome 1q23–q24 by fluorescencein situhybridization.     

Quantifying and monitoring functional photosystem II and the stoichiometry of the two photosystems in leaf segments: approaches and approximations

Given its unique function in light-induced water oxidation and its susceptibility to photoinactivation during photosynthesis, photosystem II (PS II) is often the focus of studies of photosynthetic structure and function, particularly in environmental stress conditions. Here we review four approaches for quantifying or monitoring PS II functionality or the stoichiometry of the two photosystems in leaf segments, scrutinizing the approximations in each approach. (1) Chlorophyll fluorescence parameters are convenient to derive, but the information-rich signal suffers from the localized nature of its detection in leaf tissue. (2) The gross O(2) yield per single-turnover flash in CO(2)-enriched air is a more direct measurement of the functional content, assuming that each functional PS II evolves one O(2) molecule after four flashes. However, the gross O(2) yield per single-turnover flash (multiplied by four) could over-estimate the content of functional PS II if mitochondrial respiration is lower in flash illumination than in darkness. (3) The cumulative delivery of electrons from PS II to P700(+) (oxidized primary donor in PS I) after a flash is added to steady background far-red light is a whole-tissue measurement, such that a single linear correlation with functional PS II applies to leaves of all plant species investigated so far. However, the magnitude obtained in a simple analysis (with the signal normalized to the maximum photo-oxidizable P700 signal), which should equal the ratio of PS II to PS I centers, was too small to match the independently-obtained photosystem stoichiometry. Further, an under-estimation of functional PS II content could occur if some electrons were intercepted before reaching PS I. (4) The electrochromic signal from leaf segments appears to reliably quantify the photosystem stoichiometry, either by progressively photoinactivating PS II or suppressing PS I via photo-oxidation of a known fraction of the P700 with steady far-red light. Together, these approaches have the potential for quantitatively probing PS II in vivo in leaf segments, with prospects for application of the latter two approaches in the field.     

Magenta pigment produced by fungus

A fungus producing magenta was isolated from cellulosic material by visual observation on Czapek's agar media and the product was conventionally analyzed. The fungal strain that produced magenta pigment was closely related to Phoma herbarum. The type of fibers added to Czapek's medium influenced which pigments were produced. Mycelia attached to the surface of nylon-6 and excreted magenta pigment into the fibers. The pigment structure was partially determined. This is the first report of the production of magenta pigment by a microorganism specifically in the presence of nylon-6 fibers, via an unknown mechanism. This phenomenon raises the question of why and how the fungus disperses the pigment inside the fiber and suggests that fabrics can be dyed using microorganisms.     

Rapid quantification of periodontitis-related bacteria using a novel modification of Invader PLUS technologies

The Invader PLUS technology is a sensitive, rapid method for the detection and quantification of nucleic acid. While the original technology is based on the amplification by polymerase chain reaction (PCR) of the target sequence followed by its detection using the Invader technology, the current modification allows simultaneous PCR amplification and Invader reaction. The PCR primers and the Invader probes are designed to operate at the same temperature. This allows simpler design and faster results. This technology has been applied for the quantification of six periodontitis-related bacteria (Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia, Toreponema denticola, Tannerella forsythensis and Fusobacterium nucleatum). Direct comparison of this modified Invader PLUS with real-time PCR demonstrated similar linear range. Furthermore, testing of 64 volunteers showed a good correlation between both technologies with correlation factors r2 spanning between 0.827 and 0.987. We demonstrated here that the proposed improvement of the Invader PLUS allows the detection and quantification of DNA sequences using a simple design and protocol that can be implemented in clinical testing.     

Las21 participates in extracellular/cell surface phenomena in Saccharomyces cerevisiae

Las21 (Yj1062W) is a member of the major facilitator super family, possessing multimembrane spanning domains. The LAS21 gene was identified as a responsible gene for a Saccharomyces cerevisiae mutan which shows sensitivity to a local anestheticum, tetracaine. The null las21 mutant (las21 delta) is viable but shows temperature sensitive growth. We found, in addition to this phenotype, that the las21 delta strain shows a number of defects; mating deficiency, calcofluor resistance, and formation of Zymolyase sensitive spores. Temperature sensitive growth of the las21 delta mutant was found to be suppressed by 0.1 M MgSO4. Two multicopy suppressors were obtained. They are ECM33 (YBR078W) and PIR2/HSP150 (YJR159W) both have some roles in an extracellular function. The common features of the suppressors, genetic and physiological, of the las21 delta mutation suggest that Las21 participates in a global activity of extracellular phenomena. The las 21 phenotypes are consistent with the idea that Las21/Gpi7 acts in metabolism of glycosylphosphatidylinositol.     

Low pKa lysine residues at the active site of sarcosine oxidase from Corynebacterium sp. U-96

Sarcosine oxidase from Corynebacterium sp. U-96 is inactivated by iodoacetamide with the modification of two specific residues. Comparing the amino acid sequence and mass spectra of the peptide fragments containing the modified residues with those from the native enzyme, the modified residues were identified to be lysine. The pKa of these residues were estimated to be 8.5 and 6.7 from the pH dependence of inactivation in the presence and absence of the competitive inhibitor, acetate. These estimated pKa values are much lower than that of the epsilon-amino group of lysine residue. There may be unique microenvironments around these residues that activate their -amino groups to be susceptible to iodoacetamide. A possible role of the lysine residue with pKa 6.7 is discussed.