Dynamic aspects of pressure and temperature-stabilized intermediates of outer surface protein A

Structural characterization of alternatively folded and partially disordered protein conformations remains challenging. Outer surface protein A (OspA) is a pivotal protein in Borrelia infection, which is the etiological agent of Lyme disease. OspA exists in equilibrium with intermediate conformations, in which the central and the C-terminal regions of the protein have lower stabilities than the N-terminal. Here, we characterize pressure- and temperature-stabilized intermediates of OspA by nuclear magnetic resonance spectroscopy combined with paramagnetic relaxation enhancement (PRE). We found that although the C-terminal region of the intermediate was partially disordered, it retains weak specific contact with the N-terminal region, owing to a twist of the central β-sheet and increased flexibility in the polypeptide chain. The disordered C-terminal region of the pressure-stabilized intermediate was more compact than that of the temperature-stabilized form. Further, molecular dynamics simulation demonstrated that temperature-induced disordering of the β-sheet was initiated at the C-terminal region and continued through to the central region. An ensemble of simulation snapshots qualitatively described the PRE data from the intermediate and indicated that the intermediate structures of OspA may expose tick receptor-binding sites more readily than does the basic folded conformation.     

Quantitative measurement of damage caused by 1064-nm wavelength optical trapping of Escherichia coli cells using on-chip single cell cultivation system

We quantitatively examined the possible damage to the growth and cell division ability of Escherichia coli caused by 1064-nm optical trapping. Using the synchronous behavior of two sister E. coli cells, the growth and interdivision times between those two cells, one of which was trapped by optical tweezers, the other was not irradiated, were compared using an on-chip single cell cultivation system. Cell growth stopped during the optical trapping period, even with the smallest irradiated power on the trapped cells. Moreover, the damage to the cell's growth and interdivision period was proportional to the total irradiated energy (work) on the cell, i.e., irradiation time multiplied by irradiation power. The division ability was more easily affected by a smaller energy, 0.36 J, which was 30% smaller than the energy that adversely affected growth, 0.54 J. The results indicate that the damage caused by optical trapping can be estimated from the total energy applied to cells, and furthermore, that the use of optical trapping for manipulating cells might cause damage to cell division and growth mechanisms, even at wavelengths under 1064 nm, if the total irradiation energy is excessive.     

Optimal lineage principle for age-structured populations

We present a formulation of branching and aging processes that allows age distributions along lineages to be studied within populations, and provides a new interpretation of classical results in the theory of aging. We establish a variational principle for the stable age distribution along lineages. Using this optimal lineage principle, we show that the response of a population's growth rate to age-specific changes in mortality and fecundity--a key quantity that was first calculated by Hamilton--is given directly by the age distribution along lineages. We apply our method also to the Bellman-Harris process, in which both mother and progeny are rejuvenated at each reproduction event, and show that this process can be mapped to the classic aging process such that age statistics in the population and along lineages are identical. Our approach provides both a theoretical framework for understanding the statistics of aging in a population, and a new method of analytical calculations for populations with age structure. We discuss generalizations for populations with multiple phenotypes, and more complex aging processes. We also provide a first experimental test of our theory applied to bacterial populations growing in a microfluidics device.     

Quantitative evaluation of cell-to-cell communication effects in cell group class using on-chip individual-cell-based cultivation system

Cell-to-cell communication is considered to underlie the coordinated behavior and the multicellularity of cell group class, which cannot be explained only by the knowledge of lower class of life system from molecule to individual cell, because they are determined by at least two different ways: diffusible chemical signals and their direct physical contacts. We show in this paper a new method of individual-cell-based cell observation that can estimate the role of cell-to-cell communication, diffusible chemical signals, and physical contacts as separated properties, by applying an on-chip individual-cell-based cultivation system. The exchange of stationary phase medium on isolated individual Escherichia coli from exponential phase medium and the control of physical contacts indicated that the cell-to-cell direct contact did not affect the growth rate; only the communication through diffusible signals affects the growth rates as Hill's equation manner.     

Distinct Expression and Localization of Diacylglycerol Kinase Isozymes in Rat Retina

Recent studies have revealed that phosphoinositide (PI) signaling molecules are expressed in mammalian retinas, suggesting their importance in its signal transduction. We previously showed that diacylglycerol kinase (DGK) isozymes are expressed in distinct patterns in rat retina at the mRNA level. However, little is known about the nature and morphological aspects of DGKs in the retina. For this study, we performed immunohistochemical analyses to investigate in the retina the expression and localization of DGK isozymes at the protein level. Here, we show that both DGKβ and DGKι localize in the outer plexiform layer, within which photoreceptor cells make contact with bipolar and horizontal cells. These isozymes exhibit distinct subcellular localization patterns: DGKι localizes to the synaptic area of bipolar cells in a punctate manner, whereas DGKβ distributes diffusely in the subsynaptic and dendritic regions of bipolar and horizontal cells. However, punctate labeling for DGKϵ is evident in the outer limiting membrane. DGKζ and DGKα localize predominantly to the nucleus of ganglion cells. These findings show distinct expression and localization of DGK isozymes in the retina, suggesting a different role of each isozyme.     

Release of ciliary neurotrophic factor from cultured astrocytes and its modulation by cytokines

CNTF rescues various types of lesioned neurons in vivo, and it needs to be released from astrocytes into the extracellular space to have the effect. However, direct evidence for CNTF release has not been unequivocally demonstrated. We hypothesized that the rapid sequestration by CNTF receptor present on cultured astrocytes might be the cause of the inability to detect CNTF released into astrocyte-conditioned medium (ACM). Therefore, we measured CNTF immunoreactivity in medium conditioned by astrocytes treated with phosphatidylinositol-specific phospholipase C (PI-PLC) which was used to prevent released CNTF from binding to the CNTF receptor, since PI-PLC cleaves glycosyl-phosphatidylinositol anchor of CNTFR, the unique component involved in CNTF binding. CNTF was not detectable in untreated ACM, but was detectable in PI-PLC-treated ACM. These results together with the evidence that PI-PLC treatment did not have a toxic effect on astrocytes prove the fact that CNTF can be released from astrocytes without cell lysis. Subsequently, the effect of cytokines such as IL-1, TNF-, and EGF on CNTF release was examined. These cytokines increased CNTF protein levels in ACMs without increasing CNTF protein levels in astrocyte-extracts, indicating that they enhanced CNTF release from astrocytes.     

Asymmetric hydrolysis of α-aminonitriles to optically active amino acids by a nitrilase of Rhodococcus rhodochrous PA-34

Summary Rhodococcus rhodochrous PA-34 isolated from soil as a propionitrile-utilizing microorganism, hydrolysed several -aminonitriles to optically active amino acids. The hydrolysis of -aminonitriles was found to be catalysed by a nitrilase. The characteristics of the purified enzyme revealed that this is a new nitrilase as it has a molecular mass of 45 kDa and acts as a monomer. The optimum pH and temperature for the activity of the purified enzyme were 7.5 and 35° C, respectively. Thiol-specific reagents caused inhibition whereas chelators did not significantly alter the activity of this enzyme. The amino acids produced were of L-form, except for alanine. In the case of leucine production from -aminoisocapronitrile, the enantiomeric ratio of L-leucine to D-leucine was about 60.     

Role of the N-terminal hydrophobic residues of DGKε in targeting the endoplasmic reticulum

The endoplasmic reticulum (ER), comprised of an interconnected membrane network, is a site of phospholipid and protein synthesis. The diacylglycerol kinase (DGK) enzyme family catalyzes phosphorylation of diacylglycerol to phosphatidic acid. Both of these lipids are known not only to serve as second messengers but also to represent intermediate precursors of lipids of various kinds. The DGK family is targeted to distinct subcellular sites in cDNA-transfected and native cells. Of DGKs, DGKε localizes primarily to the ER, suggesting that this isozyme plays a role in this organelle. Using experiments with various deletion and substitution mutants, this study examined the molecular mechanism of how DGKε is targeted to the ER. Results demonstrate that the N-terminal hydrophobic sequence 20–40 plays a necessary role in targeting of DGKε to the ER. This hydrophobic amino acid segment is predicted to adopt an α-helix structure, in which Leu22, L25, and L29 are present in mutual proximity, forming a hydrophobic patch. When these hydrophobic Leu residues were replaced with hydrophilic amino acid Gln, the mutant fragment designated DGKε (20–40/L22Q,L25Q,L29Q) exhibits diffuse distribution in the cytoplasm. Moreover, full-length DGKε containing these substitutions, DGKε (L22Q,L25Q,L29Q), is shown to distribute diffusely in the cytoplasm. These results indicate that the N-terminal hydrophobic residues play a key role in DGKε targeting to the ER membrane. Functionally, knockdown or deletion of DGKε affects the unfolding protein response pathways, thereby rendering the cells susceptible to apoptosis, to some degree, under ER stress conditions.     

In Situ Hybridization Analysis of the Expression of Futsch,Tau, and MESK2 Homologues in the Brain of the European Honeybee (Apis mellifera L.)

Background

The importance of visual sense in Hymenopteran social behavior is suggested by the existence of a Hymenopteran insect-specific neural circuit related to visual processing and the fact that worker honeybee brain changes morphologically according to its foraging experience. To analyze molecular and neural bases that underlie the visual abilities of the honeybees, we used a cDNA microarray to search for gene(s) expressed in a neural cell-type preferential manner in a visual center of the honeybee brain, the optic lobes (OLs).

Methodology/Principal Findings

Expression analysis of candidate genes using in situ hybridization revealed two genes expressed in a neural cell-type preferential manner in the OLs. One is a homologue of Drosophila futsch, which encodes a microtubule-associated protein and is preferentially expressed in the monopolar cells in the lamina of the OLs. The gene for another microtubule-associated protein, tau, which functionally overlaps with futsch, was also preferentially expressed in the monopolar cells, strongly suggesting the functional importance of these two microtubule-associated proteins in monopolar cells. The other gene encoded a homologue of Misexpression Suppressor of Dominant-negative Kinase Suppressor of Ras 2 (MESK2), which might activate Ras/MAPK-signaling in Drosophila. MESK2 was expressed preferentially in a subclass of neurons located in the ventral region between the lamina and medulla neuropil in the OLs, suggesting that this subclass is a novel OL neuron type characterized by MESK2-expression. These three genes exhibited similar expression patterns in the worker, drone, and queen brains, suggesting that they function similarly irrespective of the honeybee sex or caste.

Conclusions

Here we identified genes that are expressed in a monopolar cell (Amfutsch and Amtau) or ventral medulla-preferential manner (AmMESK2) in insect OLs. These genes may aid in visualizing neurites of monopolar cells and ventral medulla cells, as well as in analyzing the function of these neurons.