Absorbance signals from resting frog skeletal muscle fibers injected with the pH indicator dye, phenol red

Singly dissected twitch fibers from frog muscle were studied on an optical bench apparatus after micro-injection with the pH indicator dye, phenol red. Dye-related absorbances in myoplasm, denoted by A0(lambda) and A90(lambda), were estimated as a function of wavelength lambda (450 nm less than or equal to lambda less than or equal to 640 nm) with light polarized parallel (0 degrees) and perpendicular (90 degrees) to the fiber axis respectively. At all lambda, A0(lambda) was slightly greater than A90(lambda), indicating that some of the phenol red molecules were bound to oriented structures accessible to myoplasm. The phenol red "isotropic" signal, [A0(lambda) + 2A90(lambda)]/3, a quantity equal to the average absorbance of all the dye molecules independent of their orientation, had a spectral shape that was red-shifted by approximately 10 nm in comparison with in vitro dye calibration curves measured in 140 mM KCl. The red-shifted spectrum also indicates that some phenol red molecules were bound in myoplasm. A quantitative estimate of indicator binding was obtained from measurements of the dye's apparent diffusion constant in myoplasm, denoted by Dapp. The small value of Dapp, 0.37 x 10(-6) cm2 s-1 (at 16 degrees C), can be explained if approximately 80% of the dye was bound to myoplasmic sites of low mobility. To estimate the apparent myoplasmic pH, denoted by pHapp, the isotropic absorbance of phenol red was fitted by in vitro calibration spectra. pHapp was found to be independent of dye concentration (0.2-2 mM), but varied widely (range, 6.8-7.5; mean value, 7.17) among fibers judged from functional characteristics to be normal. When fibers were subjected to acid or alkaline loads by exposure to Ringer's solution containing, respectively, dissolved CO2 or NH3, the changes in pHapp were in agreement with those expected from pH micro-electrode studies. It is concluded that in spite of the several indications for the presence of bound phenol red inside muscle cells, the pHapp signal from the indicator is useful for monitoring changes in myoplasmic pH in response to physiological and pharmacological manipulations.     

Host/vector interactions which affect the viability of recombinant phage lambda clones

A class of recombinant phage lambda clones are recovered from human genomic libraries on Escherichia coli recB21 recC22 sbcB15 cells, which fail to form plaques on wild-type cells. We report experiments which address the mechanism of this inhibition. The introduction of the recombination-stimulating sequence chi into one such clone allows growth of this phage on Rec+ cells. In addition, the insertion of lambda gam+ gene into a rec+-inhibited clone results in the ability of the phage to form plaques on wild-type cells. Since lambda Gam protein is an inhibitor of host RecBC enzyme, we tested a collection of such phage for growth on a variety of hosts altered in RecBC function. Host permissiveness correlated with the inactivation of the RecBC nucleolytic activities and not with the recombinational activities. These observations suggest that the inserted DNA sequences of these phage limit the production of packageable chromosomes. This conclusion is easily reconciled with our current knowledge of the interaction of the host recombination systems with lambda replication and encapsidation. Based on these experiments we have constructed strains, both recombination-proficient and recombination-deficient, which serve as improved hosts for the recovery of genomic sequences which are otherwise inhibitory to the growth of phage lambda.     

Electrodiffusion of ions approaching the mouth of a conducting membrane channel.

The movement of ions in the aqueous medium as they approach the mouth (radius a) of a conducting membrane channel is analyzed. Starting with the Nernst-Planck and Poisson equations, we derive a nonlinear integrodifferential equation for the electric potential, phi(r), a less than or equal to r less than infinity. The formulation allows deviations from charge neutrality and dependence of phi(r) on ion flux. A numerical solution is obtained by converting the equation to an integral equation that is solved by an iterative method for an assumed mouth potential, combined with a shooting method to adjust the mouth potential until the numerical solution agrees with an asymptotic expansion of the potential at r-a much greater than lambda (lambda = Debye length). Approximate analytic solutions are obtained by assuming charge neutrality (Läuger, 1976) and by linearizing. The linear approximation agrees with the exact solution under most physiological conditions, but the charge-neutrality solution is only valid for r much greater than lambda and thus cannot be used unless a much greater than lambda. Families of curves of ion flux vs. potential drop across the electrolyte, phi(infinity)-phi (a), and of permeant ion density at the channel mouth, n1(a), vs. flux are obtained for different values of a/lambda and S = a d phi/dr(a). If a much greater than lambda and S = O, the maximum flux (which is approached when n1(a)----0) is reduced by 50% compared to the value predicted by the charge-neutrality solution. Access resistance is shown to be a factor a/[2 (a + lambda)] times the published formula (Hille, 1968), which was derived without including deviations from charge neutrality and ion density gradients and hence does not apply when there is no counter-ion current. The results are applied to an idealized diffusion-limited channel with symmetric electrolytes. For S = O, the current/voltage curves saturate at a value dependent on a/lambda; for S greater than O, they increase linearly for large voltage.     

Access permeability of ionic channels. Dependence on aqueous jump distance

The access diffusion permeability of pores with diameters comparable to the aqueous jump distance is characterized using a rate theory analysis for the aqueous diffusion process. It is found that this process gives rise to two permeability terms, one associated with bulk diffusion and the other a jump from the aqueous solution into a position where it has access to the channel. The latter term dominates for small channel diameters and vice versa for large channel diameters. The properties of access diffusion with respect to concentration polarization is shown to be different in the two limits of large and small values of the channel radius. A necessary criterion for bulk access diffusion to be rate limiting is given in terms of measured channel conductance G, aqueous jump distance lambda and aqueous resistivity rho, G greater than pi lambda/rho, which does not require a knowledge of channel geometry.     

Effects of divalent cations on the ultrasonic absorption coefficient of negatively charged liposomes (LUV) near their phase transition temperature

The ultrasonic absorption coefficient per wavelength (alpha lambda), as a function of temperature and frequency, was determined for large unilamellar vesicles (LUV) in the vicinity of their phospholipid phase transition temperature, using a double crystal acoustic interferometer. (The vesicles were composed of a 4:1 (w/w) mixture of dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylglycerol (DPPG). It has been found that alpha lambda reaches a maximum (alpha lambda)max at the phase transition temperature (tm) of the phospholipids in the bilayer, at an ultrasonic relaxation frequency of 2.1 MHz. Divalent cations (Ca2+ and Mg2+), added to LUV suspensions, shifted (alpha lambda)max to higher temperatures, dependent upon the concentration of divalent cation. It was also found that the shape of the alpha lambda versus t curve was significantly changed, representing changes in the Van't Hoff enthalpy of the transition, and therefore, the cooperative unit of the transition. This suggests that divalent cations interact individually with the negatively charged phospholipid headgroups of DPPG and with DPPC headgroups, thus decreasing the cooperative unit of the transition. The observed upward shift in tm suggests an interaction that increases the activation energy and, therefore, the temperature of the phase transition. However, alpha lambda as a function of frequency did not change with the addition of divalent cations and, thus, the relaxation time of the event responsible for the absorption of ultrasound is not changed by the addition of divalent cations.     

The elastic behavior of the urinary bladder for large deformations

The purpose of this paper is to investigate the theoretical basis for the pressure-distension behavior of the urinary bladder. A finite strain theory is developed for hollow spherical structures and it is shown that the Treloar model is a good prototype only for rubber balloons. The pressure-extension ratio relationship is inverted to lead a general form of strain energy function, and fitted by an empirical relation involving one exponential. The following form of strain energy function is derived: W(lambda, lambda, lambda -2) = C1 (P(1), a) + P(1)C2 (a, lambda)ea(lambda -1). Where C1(P(1), a) is a constant (N m-2), P(1) is the initial pressure, a is the rate of pressure increase and C2 (a, lambda) a third degree polynomial relation. P(1) and a are experimentally determined through volumetric pressure-distension data. It is verified that this type of energy function is also valid for uniaxial loading experiments by testing strips coming from the same bladder for which P(1) and a were computed. There is a good agreement between the experimental points and the theoretical stress-strain relation. Finally, the strain energy function is plotted as a function of the first strain invariant and appears to be of an exponential nature.     

Red system of bacteriophage lambda complements the growth of a bacteriophage T1 gene 4 mutant.

The ability of phage lambda to complement the growth of T1am23, a T1 gene 4 mutant with a DNA arrest phenotype, has been shown to require both lambda Red functions, redX and redB. lambdagam function, however, is not required. Therefore, the lambda Red function can substitute for T1 gene 4 function. However, T1+ does not substitute for lambda Red in allowing lambda to grow in a polA host.     

Isolation of different bacteriophages using the LamB protein for adsorption on Escherichia coli K-12.

Ten phages which use the LamB protein for adsorption have been isolated from sewage waters. Nine have a shape similar to lambda and require only the LamB protein for adsorption. One has a shape similar to T phages and can use either the LamB or the OmpC protein. Preliminary characterization by a number of criteria showed that at least nine of these phages were different and also differed from other known phages which use the LamB protein, such as lambda, 21, and K10.     

Two conserved essential motifs of the murine immunoglobulin lambda enhancers bind B-cell-specific factors.

Two highly homologous enhancers associated with the two murine immunoglobulin lambda constant-region clusters were recently identified. In order to better understand the molecular basis for the developmental stage- and cell-type-restricted expression of lambda genes, we have undertaken an analysis of the putative regulatory domains of these enhancers. By using a combination of DNase I footprinting, electrophoretic mobility shift assay, and site-specific mutations, four candidate protein binding sites have been identified at analogous positions in both enhancers. A mutation of any of these sites decreases enhancer activity. Two of the sites, lambda A and lambda B, are essential for enhancer function, and both of these sites appear to bind both B-cell-specific and general factors. Nevertheless, isolated lambda A and lambda B sites show no evidence of inherent transactivating potential, alone or together, even when present in up to three copies. We suggest that the generation of transactivating signals from these enhancers may require the complex interaction of multiple B-cell-specific and nonspecific DNA-binding factors.     

Circular dichroism studies on alpha- and beta-ketosides of 5-acetamido-3,5-dideoxy-D-glycero-D-galacto- nonulopyranosonic acid (N-acetylneuraminic acid) and of some of its derivatives.

The circular dichroism spectra of a number of N-acetylneuraminic acid derivatives in aqueous solution were studied. For all compounds, the Cotton effects were found to be in the spectral range of the acetamido and carboxyl chromophores. The c.d. curves of the methy, ethyl, and allyl alpha-D-ketosides are characterized by a broad, positive band centered at lambda similar to 195 nm with a slight skew towards the higher wavelengths and weak bands between lambda 225 and 255 nm, whereas the methyl beta-D-ketoside and the corresponding methyl ester show only an intense positive band with a broad shoulder in the same spectral range. 5-Acetamido-3,5-dideoxy-D-glycero-beta-D-galacto-nonulopyranose, its methyl beta-D-ketoside, and 5-acetamido-3,5-dideoxy-D-glycero-D-galacto-nonulopyranosonamide containing only the acetamido chromophore showed one single positive Cotton effect centered at lambda similar to 192 nm. The c.d. spectrum of 5-acetamido-3,5-dideoxy-D-glycero-D-galacto-nonulopyranosonic acid confirms the beta-D configuration of the free acid in aqueous solution, whereas the shape of the c.d. curve of O-(N-acetyl-alpha-D-neuraminopyranosyl)-(2yields3)-O-beta-D-galactopyranosyl-(1 yields 4)-D-glucopyranose resembles that of the methyl, ethyl, and allyl alpha-D-ketosides 2-4.     

Sequence organization of the origins of DNA replication in lambdoid coliphages

We have determined the sequences of the ori region DNA of several phage lambda mutants and hybrids, which shed light on the mechanism of DNA replication in the lambdoid phages. These include the heterologous substitution hybrids lambda rep82:lambda and lambda rep80:lambda, a pseudorevertant of the ori-r93 mutant lambda r93hot5, and the insertion mutant lambda pk35. The ori regions of the three lambdoid phages, lambda, phi 80 and 82, all have repeated sequences, termed iterons, and A . T-rich zones. We note that a similar arrangement of DNA is also found in several other prokaryotic origins of replication. lambda and phi 80 have four iterons, and 82 has five. The origin of lambda r93hot5 is unusual in that contains only three iterons, yet the phage grows normally. Analysis of this mutant indicates that the spacing of iterons is crucial to ori function, whereas their number is not. This argues against the cloverleaf model for lambda ori structure (Hobom et al., 1979). In lambda pk35 the drug resistance element Tn903 is inserted into the "inceptor" (ice) site, proposed to be crucial for lambda replication initiation (Hobom et al., 1979); yet this phage grows normally.     

Fibrin aggregation before sol-gel transition.

Fibrinogen solutions (concentrations 2 mg/ml, 0.15-M Tris-NaCl buffer, pH 7.4) were incubated at 20 degrees C with quantities of reptilase or thrombin that were so small that the polymerization process could be followed for several hours by means of static and dynamic light scattering. The scattered intensity and its correlation function were recorded at scattering angles between 30 degrees and 150 degrees. The measured data were compared with model calculations based on the Flory-Stockmayer distribution, which predicts a sol-gel phase transition. This distribution is characterized by a parameter, lambda, that indicates the extent of aggregation. lambda = 0 corresponds to the monomeric solution, and lambda = 1 indicates the sol-gel transition. Good agreement was found for monomeric units of 75-nm length aggregating (a) end-to-end in the early stage (0 less than or equal to lambda less than or equal to 0.3), and (b) in a staggered overlap pattern for the progressing polymerization (0.3 less than or equal to lambda less than 1). Before the gel point was reached, no systemic difference was observed between the data obtained after activation with thrombin which releases both fibrinopeptides A and B from fibrinogen, and reptilase, which exclusively releases the fibrinopeptides A. This confirms that the release of the fibrinopeptides A is the essential prerequisite for the aggregation process.     

Induction of prophage lambda during the irradiation of E. coli cells exposed to radiations of various LET

Induction of lambda prophage in lysogenic E. coli cells exposed to ionizing radiation of different LET was studied as a function of dose I(D). Activities of pleiotropic RecA protein were shown to contribute to the shape of the I(D) curve. The experimental data were fitted by the function I(D) = alpha D(1-exp(-D0-1.D]exp(-beta D). Inducibility alpha increased with increasing LET which was related to the increased incidence of DNA lesions being a SOS-system call.     

Defective and plaque-forming lambda transducing bacteriophage carrying penicillin-binding protein-cell shape genes: genetic and physical mapping and identification of gene products from the lip-dacA-rodA-pbpA-leuS region of the Escherichia coli chromosome

A series of defective lambda transducing phage carrying genes from the lip-leuS region of the Escherichia coli chromosome (min 14 on the current linkage map) has been isolated. The phage defined the gene order as lac---lip-dacA-rodA-pbpA-leuS---gal. These included the structural genes for penicillin-binding protein 2 (pbpA) and penicillin-binding protein 5 (dacA) as well as a previously unidentified cell shape gene that we have called rodA. rodA mutants were spherical and very similar to pbpA mutants but were distinguishable from them in that they had no defects in the activity of penicillin-binding protein 2. The separation into two groups of spherical mutants with mutations that mapped close to lip was confirmed by complementation analysis. The genes dacA, rodA, and pbpA lie within a 12-kilobase region, and represent a cluster of genes involved in cell shape determination and peptidoglycan synthesis. A restriction map of the lip-leuS region was established, and restriction fragments were cloned from defective transducing phage into appropriate lambda vectors to generate plaque-forming phage that carried genes from this region. Analysis of the proteins synthesized from lambda transducing phage in ultraviolet light-irradiated cells of E. coli resulted in the identification of the leuS, pbpA, dacA, and lip gene products, but the product of the rodA gene was not identified. The nine proteins that were synthesized from the lip-leuS region accounted for 57% of its coding capacity. Phage derivatives were constructed that allowed about 50-fold amplification of the levels of penicillin-binding proteins 2 and 5 in the cytoplasmic membrane.     

A comparison of BRCT domains involved in nonhomologous end-joining: introducing the solution structure of the BRCT domain of polymerase lambda

Three of the four family X polymerases, DNA polymerase lambda, DNA polymerase mu, and TdT, have been associated with repair of double-strand DNA breaks by nonhomologous end-joining. Their involvement in this DNA repair process requires an N-terminal BRCT domain that mediates interaction with other protein factors required for recognition and binding of broken DNA ends. Here we present the NMR solution structure of the BRCT domain of DNA polymerase lambda, completing the structural portrait for this family of enzymes. Analysis of the overall fold of the polymerase lambda BRCT domain reveals structural similarity to the BRCT domains of polymerase mu and TdT, yet highlights some key sequence and structural differences that may account for important differences in the biological activities of these enzymes and their roles in nonhomologous end-joining. Mutagenesis studies indicate that the conserved Arg57 residue of Pol lambda plays a more critical role for binding to the XRCC4-Ligase IV complex than its structural homolog in Pol mu, Arg43. In contrast, the hydrophobic Leu60 residue of Pol lambda contributes less significantly to binding than the structurally homologous Phe46 residue of Pol mu. A third leucine residue involved in the binding and activity of Pol mu, is nonconservatively replaced by a glutamine in Pol lambda (Gln64) and, based on binding and activity data, is apparently unimportant for Pol lambda interactions with the NHEJ complex. In conclusion, both the structure of the Pol lambda BRCT domain and its mode of interaction with the other components of the NHEJ complex significantly differ from the two previously studied homologs, Pol mu and TdT.