Stages of bacteriophage lambda head morphogenesis: physical analysis of particles in solution

Intermediates in the morphogenesis of bacteriophage lambda are characterized in solution by classical light-scattering, using a modified version of the Zimm plot procedure, by quasi-elastic light-scattering and analytical ultracentrifugation. Partial specific volumes are determined simultaneously with molecular weights by a variant of the conventional combination of sedimentation and diffusion constants. Our measurements were performed within a short time and allowed the characterisation of metastable intermediates.Comparison of hydration of DNA-containing and empty heads shows that dehydration plays a minor role in the stabilisation of the DNA within the heads. The molecular weight of the scaffolding protein is 4 × 10⁶, about twice the value estimated so far. Enlargement of preheads (21% and 13% increase in dry and hydrodynamic radius, respectively) leaves the molecular weight unchanged, whereas the volume of hydration water increases from 70% to 90% of the total hydrodynamic volume. Addition of protein pD to the enlarged preheads leads to a further increase in the radius, indicating that pD is attached to the outside of the protein shell.In order to determine simultaneously the molecular weight and the partial specific volume of large and sometimes labile structures, such as a virus, the conventional sedimentation-diffusion method is modified by measuring sedimentation and diffusion coefficients in buffers containing different amounts of ²H₂O. If diffusion coefficients are determined by quasi-elastic light-scattering, experiments can be performed in a few hours. In addition, the method allows a check on the sample for changes in the frictional coefficient due, for instance, to DNA abortively ejected from a virus preparation. This method is described in the Appendix.     

Aggregation and dispersity of isolated chromaffin granules studied by intensity fluctuation spectroscopy

The aggregation and dispersity of isolated bovine adrenal secretory vesicles (chromaffin granules) were studied by intensity fluctuation spectroscopy. The degree of dispersity and the Z-average translational diffusion coefficients were calculated from the autocorrelation functions of the intensity fluctuations in lase light scattered from the granules in solution. Granules purified by sedimentation through 0.3 M sucrose/Ficoll/²H₂O showed greater dispersity than granules purified by sedimentation through 1.6 M sucrose. By monitoring the scattered light intensity and the diffusion coefficients of the granules, many of the difficulties encountered in the interpretation of absorbance measurements were avoided. Measurements over a range of granule concentrations in sucrose solutions (10 mM HEPES, pH 7.0), indicated that aggregation of the granules occurred at concentrations above 150 μg protein/ml. At low granule concentrations (15–30 μg protein/ml) Ca²⁺-induced aggregation was detected at a threshold of 2–10 mM calcium.     

Studies on Transformations of Hemophilus influenzae : II. The molecular weight of transforming DNA by sedimentation and diffusion measurements

The sedimentation and diffusion coefficients have been determined for Hemophilus influenzae transforming activity and DNA using P³²-labeled DNA. The methods employed the Spinco fixed boundary separation cell for measurements of the sedimentation coefficient and the Northrop-Anson diffusion cell to determine the diffusion coefficient. There was a very close correlation between the amount of DNA and transforming activity sedimented or diffused. The sedimentation coefficient (s_20°), for both biological activity and DNA was 27 and the diffusion coefficient (D_20°) 1 x 10^-8 cm²/sec. The molecular weight calculated from these coefficients gave a value of 16 million. There was no difference in the sedimentation coefficients for the two unlinked markers, streptomycin and erythromycin resistance, and the diffusion coefficients for single markers or the linked markers, streptomycin and cathomycin, were the same.     

Dynamic light scattering of the bacteriophage T4B: determination of translational diffusion coefficients and centres of rotation

Using dynamic light scattering, the translational diffusion coefficient (D_T) and the distance between the hydrodynamic centre and the centre of the head (r₀) of the bacteriophage T4B have been determined. For a particle with retracted tail fibres we found D_T20.w =2.88 (2.88 ± 0.02) × 10^?8cm²s^?1 and r₀ = 52 ± 1 nm. For a phage with fully extended tail fibres D_T20w = (.210 ± 0.02) × 10^?8cm²s^?1 and r₀ = 112 ± nm. These data were obtained by interpreting the correlation function using a theory which takes into account the influence of the lollipop shape of the phage. In the literature this influence has not been taken into account, which has led to erroneous values of diffusion coefficients for T4B and other phages. The sedimentation coefficient of T4B phage is 1040 ± 5 S (fibres retracted) or 829 ± 4 S (fibres extended). With the above mentioned diffusion coefficients, these values correspond to a molecular weight of 236 × 10⁶ ± 3 × 10⁶. Finally, the theory used in this study is applied to other bacterial viruses, to correct reported values of the translational diffusion coefficients and of the corresponding molecular weights of these viruses.     

Partitioning Average and Heterotic Components of Direct and Maternal Genetic Effects on Growth in Mice Using Crossfostering Techniques

Crossfostering was performed using lines selected for increased 6-week body weight (H₆) and increased 3-to 6-week postweaning gain (M16) and their reciprocal F₁ crosses as nurse dams in the selected crossfostering group, and base population controls (C₂, ICR) and their reciprocal F₁ crosses in the control group. The offspring suckled were H₆, M16 and F₂ crosses in the selected group, and C₂, ICR and their F₂ crosses in the control group. Measurements taken on the individual offspring were body weights at birth (WB) and at 12, 21, 31, 42, and 63 days (W12, W21, W31, W42 and W63, respectively) and weight gains between adjacent ages (GB-12, G12–21, G21–31, G31–42 and G42–63, respectively). Least squares constants fitted to populations of genetic and nurse dams were used to calculate specific linear contrasts. Correlated responses to selection in average direct genetic effects were significant and positive for all traits examined in both H₆ and M16, while the correlated responses in average maternal genetic effects were negative in M16 and negligible in H₆. Selection response was primarily due to average direct genetic effects while the contribution of average maternal genetic effects was of secondary importance. The response in average direct genetic effects was smaller in M16 than in H₆ through weaning (WB, W12 and W21), but was larger in M16 for postweaning weights (W31, W42 and W63). The correlated responses in average maternal genetic effects were consistently smaller in M16 than in H₆. Direct heterosis was significant for all traits except for G12–21 and G42–63 in the control group, whereas maternal heterosis was significant for weight gains at early ages and for body weights. Direct heterosis tended to be larger than maternal heterosis in both selected and control crosses. Percent direct heterosis for body weight was larger in the selected crosses relative to the control crosses through 31 days of age, but the trend was reversed by 63 days. Percent maternal heterosis was consistently larger in the selected crosses.     

Intensity fluctuation spectroscopy and transfer RNA conformation. III. Influence of NaCl concentration on the size and shape of the initially salt-free tRNA in solution.

The influence of sodium ion concentration in solution on the initially salt-free conformation of bulk tRNA from baker's yeast has been investigated by means of photon correlation spectroscopy. From the measured values of translational (D^T) and rotational (D^R) diffusion coefficients, the semiaxes of an ellipsoid of revolution, which are hydrodynamically equivalent to the tRNA molecule, were calculated for tRNA solutions in pure H₂O as well as in 0.005, 0.1, 0.5M NaCl and 0.01M MgCl₂ solutions at pH 4.2 and 7.5. These data, combined with our previous studies, suggested a model which describes the formation of an ordered tRNA structure due to increasing NaCl concentrations. Furthermore, we have obtained information concerning intermolecular interactions between tRNA molecules in solution. In low-salt or salt-free tRNA solutions, we detected in the linewidth distribution function an extra-fast component which can be attributed as possibly due to charge fluctuations related to the reaction of ionization of organic bases. In our light-scattering linewidth measurements, we do not see fluctuations of charged and uncharged states directly as concentration fluctuations. Rather, we postulate a modulation of long-range intermolecular electrostatic interactions between the tRNA molecules due to such charge fluctuations. It is this modulation which is related to the fast component of the time correlation function at finite concentrations. A quantitative theory is needed to provide a more definitive explanation of the dynamical behavior of tRNA in salt-free or low-salt solutions.     

Fluctuations, exchange processes, and water diffusion in aqueous protein systems: A study of bovine serum albumin by diverse NMR techniques

Experimental frequency, concentration, and temperature dependences of the deuteron relaxation times T₁ and T₂ of D₂O solutions of bovine serum albumin are reported and theoretically described in a closed form without formal parameters. Crucial processes of the theoretical concept are material exchange, translational diffusion of water molecules on the rugged surfaces of proteins, and tumbling of the macromolecules. It is also concluded that, apart from averaging of the relaxation rates in the diverse deuteron phases, material exchange contributes to transverse relaxation by exchange modulation of the Larmor frequency. The rate limiting factor of macromolecular tumbling is determined by the free water content. In a certain analogy to the classical free-volume theory, a “free-water-volume theory” is presented. There are two characteristic water mass fractions indicating the saturation of the hydration shells (C_s ≈ 0.3) and the onset of protein tumbling (C₀ ≈ 0.6). The existence of the translational degrees of freedom of water molecules in the hydration shells has been verified by direct measurement of the diffusion coefficient using an NMR field-gradient technique. The concentration and temperature dependences show phenomena indicating a percolation transition of clusters of free water. The threshold water content was found to be C_c^w ≈ 0.43.     

Motion of myosin fragments during actin-activated ATPase: fluorescence correlation spectroscopy study.

The rates of the translational motion of myosin fragments, heavy meromyosin (HMM), and heavy meromyosin subfragment-1 (HMM S-1) were measured during actin-activated ATPase reaction by the method of fluorescence correlation spectroscopy. This technique monitors the random fluctuations in the concentration of fluorescent molecules in an open volume which result from the translational diffusion of the molecular species under observation. The statistical behavior of the fluctuations is represented in the form of the autocorrelation function, which is related to the translational diffusion coefficient of the fluorescent molecules. The translational motion of fluorescently labeled myosin fragments was progressively slowed down after additions of increasing amounts of actin in the presence of excess MgATP. When these results are interpreted according to a simple binding scheme, the extent of the retardation can be used to obtain the apparent association constant for binding of S-1 and HMM to actin in the presence of MgATP. In 0.1M KCl and at 23°C, the apparent association constants were determined as K_app^HMM = 2.2 × 10⁴M^?1 and K_app^S-1 = 8.8 × 10³ for HMM and S-1, respectively.     

Dynamic light scattering by kappa- and lambda-carrageenan solutions

The intensity correlation functions of kappa- and lambda-carrageenan in various salt solutions and at different concentrations have been determined with the help of dynamic light scattering. From the first cumulant of these correlation functions the values of the translational diffusion coefficients D have been derived. They increase with macromolecular concentration. The extrapolated values to infinite dilution of the diffusion coefficients increase with increasing salt concentration as expected from the salt concentration dependence of the r.m.s. radii of gyration determined previously by static light scattering. The translational diffusion coefficient of lambda-carrageenan in 0.1 M NaCl is smaller than the corresponding value for the kappa species. This is consistent with the difference in contour length and linear charge density of the two samples used. No satisfactory interpretation for the concentration dependence of the diffusion coefficient seems to be possible at present. Although current theories for the macromolecular and salt concentration dependence of D, taking into account charge effects, seem to be applicable, they do not allow for a consistent interpretation of the data. No specific difference between the solution behaviour of kappa- and lambda-carrageenan has been detected.     

Photon correlation spectroscopy of hemoglobin: Diffusion of Oxy-HbA and Oxy-HbS

Translation diffusion coefficients have been measured for oxyhemoglobin A and oxyhemoglobin S over the concentration range 0.1–37 g/dl by means of photon correlation spectroscopy. The solutions were 0.1 M in KCl and in each case the pH was adjusted to the isoelectric point of the hemoglobin species present. No significant differences were found between the HbA and HbS results; and after correction to water at 20°C, the diffusion coefficients could be described by the equation where D_o = (6.93 ± 0.06) × 10^?7 cm²/sec and c ids the concentration in units of g/dl. No evidence was found for the aggregation of oxy-HbS at high concentrations which was reported by Lindstrom et. al. [(1976) Biophys. J. 16 , 679–689].     

A rotational diffusion coefficient of the 70S ribosome determined by depolarized laser light scattering

We have obtained a rotational diffusion coefficient of the 70S ribosome isolated from Escherichia-coli (MRE-600), from the depolarized light scattering spectrum measured by photon correlation spectroscopy. The intensity correlation function of depolarized scattered light contains contributions due to multiple scattered and anisotropy scattered light from the ribosomal particle. We discuss extensively the subtraction procedure used to obtain the rotational correlation time from the experimental correlation function. We have also obtained the translational diffusion coefficient from the same sample by determining the polarized correlation function. The hydrodynamic radius determined from the rotational diffusion coefficient is only slightly larger than the radius obtained from the translational diffusion coefficient. Therefore the ribosomal particle has a non-spherical shape. This conclusion, however, could be impaired by the effect of free draining of the ribosome.     

Distribution and diffusion of alamethicin in a lecithin/water model membrane system

Summary Attenuated total reflection infrared spectroscopy has been used to determine the equilibrium distribution of the peptide antibiotic alamethicinR _F30 between dipalmitoyl phosphatidylcholine bilayers and the aqueous environment. The distribution coefficientK=c _eq ^W /c _eq ^M turned out to be concentration dependent, pointing to alamethicin association in the membrane with increasing concentration in the aqueous phase (c _eq ^W ). This concentration was varied within 28 and 310nm, i.e., in a range typical for black film experiments. Furthermore, diffusion coefficients of alamethicin in the hydrophobic phase of the membrane (D _M) and across the membrane/water interface (D _I) have been estimated from the time course of the equilibration process. It was found that the diffusion rate of the uncharged analogueR _F50 is about 10 times higher than that of theR _F30 component, exhibiting one negative charge at theC-terminus. The time constants for transmembrane diffusion of alamethicinR _F30 varied between 2.2 hr at low concentration and 3.2 hr at higher concentration. The corresponding low concentration value of theR _F50 component was found to be 0.25 hr.     

Ivermectin and curcumin cause plasma membrane rigidity in Leishmania amazonensis due to oxidative stress

Spin label electron paramagnetic resonance (EPR) spectroscopy was used to study the mechanisms of action of ivermectin and curcumin against Leishmania (L.) amazonensis promastigotes. EPR spectra showed that treatment of the parasites with both compounds results in plasma membrane rigidity due to oxidative processes. With the IC₅₀ and EPR measurements for assays using different parasite concentrations, estimations could be made for the membrane-water partition coefficient (K_M/W), and the concentration of the compound in the membrane (c_m50) and in the aqueous phase (c_w50), which inhibits cell growth by 50%. The K_M/W values indicated that ivermectin has a greater affinity than curcumin for the parasite membrane. Therefore, the activity of ivermectin was higher for experiments with low cell concentrations, but for concentrations greater than 1.5 × 10⁸ parasites/mL the compounds did not show significantly different results. The c_m50 values indicated that the concentration of compound in the membrane leading to growth inhibition or membrane alteration is approximately 1 M for both ivermectin and curcumin. This high membrane concentration suggests that many ivermectin molecules per chlorine channel are needed to cause an increase in chlorine ion influx.     

The molecular weight of xanthan polysaccharide produced under oxygen limitation

Summary The molecular weight distributions of xanthan polysaccharide were determined by size-exclusion chromatography during batch fermentations in a bubble column. Xanthan with lower weight-mean molecular weight M _W ^* was formed after growth had ceased. Under oxygen limitation, M _W ^* decreased linearly with the specific oxygen uptake rate resulting in lower viscosity yield of the product.     

Effects of exercise-stress on ventilation,cardiac output and blood respiratory parameters in the carp,Cyprinus carpio

• 1.1. Carp (Cyprinus carpio) were stressed to exercise by rolling in a respiration chamber. Ventilatory water flow rate, cardiac output and blood respiratory parameters were determined.
• 2.2. During exercise, oxygen uptake increased about 3.5-fold and returned to pre-exercise level within 15 min.
• 3.3. This exercise-stress resulted in no plasma acidosis and in no swelling of the erythrocytes.
• 4.4. Ventilatory water flow rate increased 6-fold, whereas cardiac output increased 2-fold. Hence the ventilation-perfusion ratio increased during exercise.
• 5.5. During exercise, arterial O₂ content (CaO₂) increased due to increases in O₂ tension (PaO₂), O₂ saturation of hemoglobin (SaO₂) and hemoglobin concentration (Hb). On the other hand, Pv̄O₂ and Sv̄O₂ remained at the resting levels but Cv̄O₂ slightly increased due to an increase in Hb.
• 6.6. Arterial-venous O₂ difference (CaO₂-Cv̄O₂) increased by 38%, which was met by a much greater increase in CaO₂ than Cv̄O₂.

     

Modeling population dynamics of yeast-like symbionts (Ascomycota: Pyrenomycetes: Clavicipitaceae) of the planthopper <Emphasis Type="Italic">Delphacodes kuscheli</Emphasis> (Hemiptera: Delphacidae)

Delphacodes kuscheli establish mutualistic relationship with yeast-like symbionts (YLS) that live in the fat body and are necessary for host survival and reproduction. We estimated for a host of age t, its body weight, W_(t), and the number of YLS per host, YLS_(t). The host body weight was calculated as: W_(t)?=?Lm/[1+ e ^(d–kt)], (Lm?=?the maximum observed weight, and d and k are constants), and the fat body was considered a fixed proportion of W_(t). We calculated the number of YLS per unit host body mass: α_(t)?=?YLS_(t)/W_(t). We also calculated the number of YLS per host, cYLS_(t), and analyzed the pattern of variation in both sexes adapting the expression of the logistic model: cYLS_(t)?=?KN_oe^rt/K+(e^rt -1)N_o, (N_o?=?initial number of YLS, r?=?intrinsic per capita rate of natural increase, and K?=?variable carrying capacity). In females the carrying capacity varied according to a constant proportion of the host’s weight: K_(t)?=?αW_(t). In males α_(t) was considered a decreasing function of the host age: K_(t)?=?α_(t)W_(t). The coefficients No, α, and r were subjected to parameterization. We found that the patterns of W_(t) and YLS_(t) of D. kuscheli were similar to other planthoppers. In females YLS increased up to the adult stage and then remained almost constant, varying similarly to individual weight. In males YLS increased up to the 5th instar nymph as the individual weight did, but the number of YLS decreased in the adult stage and the correlation was not so good. The calculated number of YLS per host matches reasonably well with the number estimated experimentally both in females and males. This is the first study that quantified and modeled the dynamics of YLS endosymbionts in a Neotropical planthopper pest. The models will be used in future studies for better understand the experimental reduction of YLS in young nymphal stages.     

Self‐assembly of lipid rafts revealed by fluorescence correlation spectroscopy in living breast cancer cells

Lipids and proteins in the plasma membrane are laterally heterogeneous and formalised as lipid rafts featuring unique biophysical properties. However, the self‐assembly mechanism of lipid raft cannot be revealed even its physical properties and components were determined in specific physiological processes. In this study, two‐photon generalised polarisation imaging and fluorescence correlation spectroscopy were used to study the fusion of lipid rafts through the membrane phase and the lateral diffusion of lipids in living breast cancer cells. A self‐assembly model of lipid rafts associated with lipid diffusion and membrane phase was proposed to demonstrate the lipid sorting ability of lipid rafts in the plasma membrane. The results showed that the increased proportion of slow subdiffusion of G_M1‐binding cholera toxin B‐subunit (CT‐B) was accompanied with an increased liquid‐ordered domain during the β‐estradiol‐induced fusion of lipid rafts. And slow subdiffusion of CT‐B was vanished with the depletion of lipid rafts. Whereas the dialkylindocarbocyanine (DiIC₁₈) diffusion was not specifically regulated by lipid rafts. This study will open up a new insight for uncovering the self‐assembly of lipid rafts in specific pathophysiological processes.     

A rotational diffusion coefficient of the 70S ribosome determined by depolarized laser light scattering.

We have obtained a rotational diffusion coefficient of the 70S ribosome isolated from Escherichia-coli (MRE-600), from the depolarized light scattering spectrum measured by photon correlation spectroscopy. The intensity correlation function of depolarized scattered light contains contributions due to multiple scattered and anisotropy scattered light from the ribosomal particle. We discuss extensively the subtraction procedure used to obtain the rotational correlation from the time from the experimental correlation function. We have also obtained the translational diffusion coefficient from the same sample by determining the polarized correlation function. The hydrodynamic radius determined from the rotational diffusion coefficient is only slightly larger than the radius obtained from the translational diffusion coefficient. Therefore the ribosomal particle has a non-spherical shape. This conclusion, however, could be impaired by the effect of free draining of the ribosome.     

Hydrodynamic properties and structure of fd virus

A length of 8950 ± 200 Å and a diameter of 90 ± 10 Å have been obtained for fd virus from a simultaneous solution of the Broersma equations relating the length and diameter of a rod-like particle to its rotational, D_R, and translational, D_T, diffusion coefficients. Measurements of D_R were by transient electric birefringence, and of D_T by low-angle intensity fluctuation spectroscopy. A mass of (16.4 ± 0.6) × 10⁶ daltons was calculated from the Svedberg equation using our measured values of D_T, the sedimentation coefficient and the density increment. These results, together with the molecular weight of fd DNA, give a total number of major coat protein subunits of 2710 ± 110 and a ratio of nucleotides to protein subunits which is definitely non-integral, 2.30 ± 0.11. These measurements help delineate significant structural differences between fd and other filamentous viruses. Also included in this paper is an Appendix (by L. A. Day & S. A. Berkowitz) concerning the number of nucleotides, 6370 ± 140, and the density and refractive index increments of fd DNA.