Evaluation of the influence of the internal aqueous solvent structure on electrostatic interactions at the protein-solvent interface by nonlocal continuum electrostatic approach

The dielectric properties of the polar solvent on the protein-solvent interface at small intercharge distances are still poorly explored. To deconvolute this problem and to evaluate the pair-wise electrostatic interaction (PEI) energies of the point charges located at the protein-solvent interface we used a nonlocal (NL) electrostatic approach along with a static NL dielectric response function of water. The influence of the aqueous solvent microstructure (determined by a strong nonelectrostatic correlation effect between water dipoles within the orientational Debye polarization mode) on electrostatic interactions at the interface was studied in our work. It was shown that the PEI energies can be significantly higher than the energies evaluated by the classical (local) consideration, treating water molecules as belonging to the bulk solvent with a high dielectric constant. Our analysis points to the existence of a rather extended, effective low-dielectric interfacial water shell on the protein surface. The main dielectric properties of this shell (effective thickness together with distance- and orientation-dependent dielectric permittivity function) were evaluated. The dramatic role of this shell was demonstrated when estimating the protein association rate constants.     

Metal-binding sites at the active site of restriction endonuclease BamHI can conform to a one-ion mechanism

The number of metal ions required for phosphoryl transfer in restriction endonucleases is still an unresolved question in molecular biology. The two Ca(2+) and Mn(2+) ions observed in the pre- and post-reactive complexes of BamHI conform to the classical two-metal ion choreography. We probed the Mg(2+) cofactor positions at the active site of BamHI by molecular dynamics simulations with one and two metal ions present and identified several catalytically relevant sites. These can mark the pathway of a single ion during catalysis, suggesting its critical role, while a regulatory function is proposed for a possible second ion.     

A multicomponent analysis of amino acid transport systems in human lymphocytes. 1. Kinetic parameters of the A and L systems and pathways of uptake of naturally occurring amino acids in blood lymphocytes

We have determined the kinetic parameters of natural and system-specific synthetic amino acid transport by human blood lymphocytes, using a multi-component computer analysis that separates carrier-mediated uptake from diffusion. These studies were initiated in order to provide the basis for studies of human blood T and B lymphocytes and malignant lymphocytes. Methylaminoisobutyric acid (methyl-AIB) and 2-amino-2-carboxy-bicyclo (2,2,1) heptane (BCH) uptakes into lymphocytes were measured as prototypes of A- and L-system amino acid transport. The Michaelis constant for methyl-AIB uptake was 540 microM; the maximal velocity of uptake was 28 mumol/L cell water/min, and the diffusion coefficient was .004 min-1. In contrast, the Michaelis constant for BCH uptake was 63 microM; the maximal velocity was 969 mumol/L cell water/min, and the diffusion coefficient was .141 min-1. The transport of the naturally occurring amino acids, alanine, proline, and leucine was defined by studies of: (1) competitive inhibition with the system-specific synthetic amino acids, methyl-AIB and BCH, (2) the effect of the transcellular sodium gradient on transport, and (3) evaluation of the time-dependent increase of transport in amino acid-deficient medium (adaptation). Alanine was transported principally (approximately 70%) by the ASC-system, and leucine was transported principally (70%) by the L-system in lymphocytes. The analysis of proline transport was more complex because of a large component of uptake by diffusion even at low amino acid concentrations. Taken together, the kinetics of sodium-sensitive uptake and the results of competitive inhibition studies indicated that proline was transported by the A-system (30%), the ASC system (30%), and also by the L-system (15%).     

UmuD and RecA directly modulate the mutagenic potential of the Y family DNA polymerase DinB

DinB is the only translesion Y family DNA polymerase conserved among bacteria, archaea, and eukaryotes. DinB and its orthologs possess a specialized lesion bypass function but also display potentially deleterious -1 frameshift mutagenic phenotypes when overproduced. We show that the DNA damage-inducible proteins UmuD(2) and RecA act in concert to modulate this mutagenic activity. Structural modeling suggests that the relatively open active site of DinB is enclosed by interaction with these proteins, thereby preventing the template bulging responsible for -1 frameshift mutagenesis. Intriguingly, residues that define the UmuD(2)-interacting surface on DinB statistically covary throughout evolution, suggesting a driving force for the maintenance of a regulatory protein-protein interaction at this site. Together, these observations indicate that proteins like RecA and UmuD(2) may be responsible for managing the mutagenic potential of DinB orthologs throughout evolution.     

Model-based strategy for cell culture seed train layout verified at lab scale

Cell culture seed trains—the generation of a sufficient viable cell number for the inoculation of the production scale bioreactor, starting from incubator scale—are time- and cost-intensive. Accordingly, a seed train offers potential for optimization regarding its layout and the corresponding proceedings. A tool has been developed to determine the optimal points in time for cell passaging from one scale into the next and it has been applied to two different cell lines at lab scale, AGE1.HN_AAT and CHO-K1. For evaluation, experimental seed train realization has been evaluated in comparison to its layout. In case of the AGE1.HN_AAT cell line, the results have also been compared to the formerly manually designed seed train. The tool provides the same seed train layout based on the data of only two batches.     

Hypothalamus–Pituitary–Thyroid Feedback Control: Implications of Mathematical Modeling and Consequences for Thyrotropin (TSH) and Free Thyroxine (FT4) Reference Ranges

The components of thyrotropic feedback control are well established in mainstream physiology and endocrinology, but their relation to the whole system’s integrated behavior remains only partly understood. Most modeling research seeks to derive a generalized model for universal application across all individuals. We show how parameterizable models, based on the principles of control theory, tailored to the individual, can fill these gaps. We develop a system network describing the closed-loop behavior of the hypothalamus–pituitary (HP)–thyroid interaction and the set point targeted by the control system at equilibrium. The stability of this system is defined by using loop gain conditions. Defined points of homeostasis of the hypothalamus–pituitary–thyroid (HPT) feedback loop found at the intersections of the HP and thyroid transfer functions at the boundaries of normal reference ranges were evaluated by loop gain calculations. At equilibrium, the feedback control approaches a point defined in both dimensions by a [TSH]–[FT4] coordinate for which the loop gain is greater than unity. This model describes the emergence of homeostasis of the HPT axis from characteristic curves of HP and thyroid, thus supporting the validity of the translation between physiological knowledge and clinical reference ranges.     

Electrophoretic mobilities and diffusion coefficients of hemoglobin at high pH.

Diffusion studies by photon correlation of scattered laser light confirm the dissociation of the tetrameric form of human carboxyhemoglobin to dimers above pH 10 and provide new estimates of the subunit dissociation equilibrium constants in this pH range. Electrophoretic light-scattering experiments under the same conditions reveal that the electrophoretic mobilities of tetramers and dimers are indistinguishable to within instrumental resolution (ca. 7% in these experiments). The data imply an increase of the electrical charge on the dimer of at least 2.8 to 4.4 net negative charges upon dissociation. Mechanisms for the accumulation of negative charge by the dimer upon dissociation of the tetramer are proposed.     

Impact of 2,4-diacetylphloroglucinol-producing biocontrol strain Pseudomonas fluorescens F113 on intraspecific diversity of resident culturable fluorescent pseudomonads associated with the roots of field-grown sugar beet seedlings

The impact of the 2,4-diacetylphloroglucinol-producing biocontrol agent Pseudomonas fluorescens F113Rif on the diversity of the resident community of culturable fluorescent pseudomonads associated with the roots of field-grown sugar beet seedlings was evaluated. At 19 days after sowing, the seed inoculant F113Rif had replaced some of the resident culturable fluorescent pseudomonads at the rhizoplane but had no effect on the number of these bacteria in the rhizosphere. A total of 498 isolates of resident fluorescent pseudomonads were obtained and characterized by molecular means at the level of broad phylogenetic groups (by amplified ribosomal DNA restriction analysis) and at the strain level (with random amplified polymorphic DNA markers) as well as phenotypically (55 physiological tests). The introduced pseudomonad induced a major shift in the composition of the resident culturable fluorescent Pseudomonas community, as the percentage of rhizoplane isolates capable of growing on three carbon substrates (erythritol, adonitol, and L-tryptophan) not assimilated by the inoculant was increased from less than 10% to more than 40%. However, the pseudomonads selected did not display enhanced resistance to 2,4-diacetylphloroglucinol. The shift in the resident populations, which was spatially limited to the surface of the root (i.e., the rhizoplane), took place without affecting the relative proportions of phylogenetic groups or the high level of strain diversity of the resident culturable fluorescent Pseudomonas community. These results suggest that the root-associated Pseudomonas community of sugar beet seedlings is resilient to the perturbation that may be caused by a taxonomically related inoculant.     

The "typical" immunophenotype of acute promyelocytic leukemia (APL-M3): does it prove true for the M3-variant?

The immunophenotypes of 12 acute promyelocytic leukemias (APL-M3; eight hypergranular, four microgranular) with documented PML-RAR-alpha fusion gene are presented. Bone marrow mononuclear cells were immunophenotyped using a panel of 20 monoclonal antibodies. The hypergranular APLs exhibited a mature myeloid phenotype as it has been described to be typical for M3. No lineage infidelity was detectable in classic M3 cases. In contrast, among the four cases of M3 variant, all leukemias showed marked expression of CD34 and two of four cases expressed the HLA-DR antigen. The CD2 antigen was expressed in three of four cases. Furthermore, one case showed expression of the CD56 antigen, and one case was positive for the blood group H antigen. The data suggest that microgranular APL is a heterogeneous entity with regard to the immunologic phenotype.