Collagen I Self-Assembly: Revealing the Developing Structures that Generate Turbidity

Type I collagen gels are routinely used in biophysical studies and bioengineering applications. The structural and mechanical properties of these fibrillar matrices depend on the conditions under which collagen fibrillogenesis proceeds, and developing a fuller understanding of this process will enhance control over gel properties. Turbidity measurements have long been the method of choice for monitoring developing gels, whereas imaging methods are regularly used to visualize fully developed gels. In this study, turbidity and confocal reflectance microscopy (CRM) were simultaneously employed to track collagen fibrillogenesis and reconcile the information reported by the two techniques, with confocal fluorescence microscopy (CFM) used to supplement information about early events in fibrillogenesis. Time-lapse images of 0.5 mg/ml, 1.0 mg/ml, and 2.0 mg/ml acid-solubilized collagen I gels forming at 27°C, 32°C, and 37°C were collected. It was found that in situ turbidity measured in a scanning transmittance configuration was interchangeable with traditional turbidity measurements using a spectrophotometer. CRM and CFM were employed to reveal the structures responsible for the turbidity that develops during collagen self-assembly. Information from CRM and transmittance images was collapsed into straightforward single variables; total intensity in CRM images tracked turbidity development closely for all collagen gels investigated, and the two techniques were similarly sensitive to fibril number and dimension. Complementary CRM, CFM, and in situ turbidity measurements revealed that fibril and network formation occurred before substantial turbidity was present, and the majority of increasing turbidity during collagen self-assembly was due to increasing fibril thickness.     

Elastic Moduli of Collagen Gels Can Be Predicted from Two-Dimensional Confocal Microscopy

We quantitatively compare data obtained from imaging two-dimensional slices of three-dimensional unlabeled and fluorescently labeled collagen gels with confocal reflectance microscopy (CRM) and/or confocal fluorescence microscopy (CFM). Different network structures are obtained by assembling the gels over a range of concentrations at various temperatures. Comparison between CRM and CFM shows that the techniques are not equally sensitive to details of network structure, with CFM displaying higher fidelity in imaging fibers parallel to the optical axis. Comparison of CRM of plain and labeled collagen gels shows that labeling itself induces changes in gel structure, chiefly through inhibition of fibril bundling. Despite these differences, image analyses carried out on two-dimensional CFM and CRM slices of collagen gels reveal identical trends in structural parameters as a function of collagen concentration and gelation temperature. Fibril diameter approximated from either CRM or CFM is in good accord with that determined via electron microscopy. Two-dimensional CRM images are used to show that semiflexible polymer theory can relate network structural properties to elastic modulus successfully. For networks containing bundled fibrils, it is shown that average structural diameter, rather than fibril diameter, is the length scale that sets the magnitude of the gel elastic modulus.     

The gelation of deoxyhemoglobin S in erythrocytes as detected by transverse water proton relazation measurements

At 37 °C, when samples of blood, washed erythrocytes, or isolated hemoglobin from individuals with sickle cell disease are deoxygenated, the transverse water proton relaxation time is sharply decreased. In similar samples from normal adults homozygous for hemoglobin A, only a slight decrease in t₂ is observed upon deoxygenation at 37 °C. In samples containing deoxyhemoglobin S the value of t₂ increases as the temperature is decreased from 37 °C to 4 °C, in contrast to samples containing oxyhemoglobin S, oxyhemoglobin A, or deoxyhemoglobin A where t₂ decreases as the temperature decreases. It is suggested that this decrease in t₂ observed in samples of deoxyhemoglobin S at 37 °C is the result of an increase in the amount of preferentially oriented water at macromolecular interfaces which occurs under conditions known to produce deoxyhemoglobin S gelation. Conditions which reverse deoxyhemoglobin S gelation such as lowering the temperature to 4 °C decrease the amount of preferentially oriented water which results in an increase in the value of t₂. Thus, measurement of the transverse water proton relaxation time can be used to monitor the gelation of deoxyhemoglobin S inside the erythrocyte.     

Life tables ofProctolaelaps deleoni Nawar,Childers and Abou-Setta (Gamasida: Ascidae) at different temperatures

Life tables were constructed for the predatory mite,Proctolaelaps deleoni, fed on the free-living nematodeRhabditis scanica at 20, 25, 28, 30 and 32°C. The intrinsic rate of increase (r _m ) and the net reproduction rate (R _o ) reach maximum values at 28°C. Mean generation time (t _g ) decreases with increasing temperature reaching its lowest value at 32°C.     

Kinetic Hysteresis in Collagen Folding

The triple helix of collagen shows a steep unfolding transition upon heating, whereas less steep and more gradual refolding is observed upon cooling. The shape of the hysteresis loop depends on the rate of temperature change as well as the peptide concentration. Experimental heating and cooling rates are usually much faster than rates of unfolding and refolding. In this work, collagen model peptides were used to study hysteresis quantitatively. Their unfolding and refolding profiles were recorded at different heating and cooling rates, and at different peptide concentrations. Data were fitted assuming kinetic mechanisms in which three chains combine to a helix with or without an intermediate that acts as a nucleus. A quantitative fit was achieved with the same kinetic model for the forward and backward reactions. Transitions of exogenously trimerized collagen models were also analyzed with a simplified kinetic mechanism. It follows that true equilibrium transitions can only be measured at high concentrations of polypeptide chains with slow scanning rates, for example, 0.1°C/h at 0.25 mM peptide concentration of (Gly-Pro-Pro)₁₀. (Gly-Pro-4(R)Hyp)₁₀ folds ∼2000 times faster than (Gly-Pro-Pro)₁₀. This was explained by a more stable nucleus, whereas the rate of propagation was almost equal. The analysis presented here can be used to derive kinetic and thermodynamic data for collagenous and other systems with kinetically controlled hysteresis.     

Thermotropic gelation of ovalbumin: 2. Boundary conditions on the formation and the concentration dependence of equilibrium elastic modulus for thermotropic ovalbumin gels

Studies were made on the boundary conditions for thermotropic ovalbumin gelation at pH within the range 2.5 to 10.0. The pH dependence of the gelation threshold, C₀, and denaturation temperature, T_d, were obtained. The dependence C₀(pH) has a sharp minimum close to the isoelectric point (pl). Over pH range 2.5 to 4.0 the dependence T_d(pH) is linear; although above pI it shows unusual behaviour. T_d increases smoothly, becoming a constant value (T_d=80°C) at pH 7. Analysis of the temperature dependence of Leu's line integral intensity in the p.m.r. spectrum of ovalbumin shows that the temperature threshold of thermotropic gelation closely approximates to T_d. A diagram for the state of an ovalbumin -water system was constructed in temperature-concentration-pH coordinates. The dependences of the initial shear modulus for thermotropic ovalbumin gels on the concentration (0.06≤C≤0.25g/cm³ were obtained at pH 4.0, 7.0, 8.5, 10.0. They are equivalent to the concentration dependence of the equilibrium elastic modulus E_e(C). The dependences obtained may be reduced to the theoretical master dependence of Hermans, $\text{E}{}_{\mathrm{e}}\text{(rm}\text{C}\text{?}\text{)}$, where $\text{C}\text{?}\text{=}\text{C}\text{/}\text{C}{}_0$ is the reduced concentration. Hermans' theory, based o the model for random cross-linking of linear identical macromolecules without cyclization, adequately describes the equilibrium elastic properties of thermotropic ovalbumin gels.     

Planktonic rotifers and temperature

The influence of temperature (t) upon rotifer embryonic development rate (D_e) has been analysed using data from the literature, and the author's own results from experimental and natural populations. For Keratella cochlearis (Gosse), within the temperature range of 1–28°C, this relationship is best expressed by the equation: 1/D_e = 0.002 + 0.00025t + 0.000065t².For Brachionus calyciflorus Pallas, between 8°C and 35°C, the best relationship is given by the equation: 1/D_e = 0.005 + 0.00013t + 0.00013t².Increasing the incubation temperature to 37–40°C resulted in a decrease in development rate and a sharp reduction in life length.Analysis of the relationship between respiration rate and temperature in experimental and natural populations of Brachionus calyciflorus and Hexarthra mira (Hudson) showed that the maximum rate of oxygen consumption occurred at 32–33°C.The effects of temperature upon the ingestion rates of rotifers is greatly influenced by food concentration. Consequently, this factor also influences the secondary production of experimental populations at different temperatures.     

A [P]NAD-based method to identify and quantitate long residence time enoyl-acyl carrier protein reductase inhibitors

The classical methods for quantifying drug–target residence time (t_R) use loss or regain of enzyme activity in progress curve kinetic assays. However, such methods become imprecise at very long residence times, mitigating the use of alternative strategies. Using the NAD(P)H-dependent FabI enoyl-acyl carrier protein (enoyl-ACP) reductase as a model system, we developed a Penefsky column-based method for direct measurement of t_R, where the off-rate of the drug was determined with radiolabeled [adenylate-³²P]NAD(P⁺) cofactor. In total, 23 FabI inhibitors were analyzed, and a mathematical model was used to estimate limits to the t_R values of each inhibitor based on percentage drug–target complex recovery following gel filtration. In general, this method showed good agreement with the classical steady-state kinetic methods for compounds with t_R values of 10 to 100 min. In addition, we were able to identify seven long t_R inhibitors (100–1500 min) and to accurately determine their t_R values. The method was then used to measure t_R as a function of temperature, an analysis not previously possible using the standard kinetic approach due to decreased NAD(P)H stability at elevated temperatures. In general, a 4-fold difference in t_R was observed when the temperature was increased from 25 to 37 °C.     

Defective assembly of a hybrid vacuolar H-ATPase containing the mouse testis-specific E1 isoform and yeast subunits

Mammalian vacuolar-type proton pumping ATPases (V-ATPases) are diverse multi-subunit proton pumps. They are formed from membrane V_o and catalytic V₁ sectors, whose subunits have cell-specific or ubiquitous isoforms. Biochemical study of a unique V-ATPase is difficult because ones with different isoforms are present in the same cell. However, the properties of mouse isoforms can be studied using hybrid V-ATPases formed from the isoforms and other yeast subunits. As shown previously, mouse subunit E isoform E1 (testis-specific) or E2 (ubiquitous) can form active V-ATPases with other subunits of yeast, but E1/yeast hybrid V-ATPase is defective in proton transport at 37 °C (Sun-Wada, G.-H., Imai-Senga, Y., Yamamoto, A., Murata, Y., Hirata, T., Wada, Y., and Futai, M., 2002, J. Biol. Chem. 277, 18098-18105). In this study, we have analyzed the properties of E1/yeast hybrid V-ATPase to understand the role of the E subunit. The proton transport by the defective hybrid ATPase was reversibly recovered when incubation temperature of vacuoles or cells was shifted to 30 °C. Corresponding to the reversible defect of the hybrid V-ATPase, the V_o subunit a epitope was exposed to the corresponding antibody at 37 °C, but became inaccessible at 30 °C. However, the V₁ sector was still associated with V_o at 37 °C, as shown immunochemically. The control yeast V-ATPase was active at 37 °C, and its epitope was not accessible to the antibody. Glucose depletion, known to dissociate V₁ from V_o in yeast, had only a slight effect on the hybrid at acidic pH. The domain between Lys26 and Val83 of E1, which contains eight residues not conserved between E1 and E2, was responsible for the unique properties of the hybrid. These results suggest that subunit E, especially its amino-terminal domain, plays a pertinent role in the assembly of V-ATPase subunits in vacuolar membranes.     

The naturally split Npu DnaE intein exhibits an extraordinarily high rate in the protein trans-splicing reaction

We have studied the naturally split α subunit of the DNA polymerase III (DnaE) intein from Nostoc punctiforme PCC73102 (Npu) using purified proteins and determined an apparent first-order rate constant of (1.1±0.2)×10^-2 s⁻¹ at 37 °C. This represents the highest rate reported for the protein trans-splicing reaction so far (t_1/2 of ∼ 60 s). Furthermore, the reaction was very robust and high-yielding with respect to different extein sequences, temperatures from 6 to 37 °C, and the presence of up to 6 M urea. Given these outstanding properties, the Npu DnaE intein appears to be the intein of choice for many applications in protein and cellular chemistry.     

A study on the nature of intermolecular links in the cryotropic weak gels of hyaluronan

In this study, the influence of acidification and salting effect on the properties of hyaluronan (HA) aqueous solutions and cryotropic weak gels were investigated by dynamic rheometry, polarizing and optical microscopy, scanning electron microscopy (SEM), X-ray diffraction (XRD), differential scanning calorimetry (DSC) and FTIR spectroscopy. The forming mechanism of HA cryotropic weak gels was also discussed. Experimental results indicated that the HA weak gel showing a thermoreversible property was constructed by entangled bundle-like structures that could be melted at elevated temperature above 70 °C, and that the junction knots of three-dimensional polymeric network were not the ordinary microcrystalline zones that are generally of detectable crystallinity and thermal effect. The intermolecular hydrogen bonding induced from -COOH and -NHCOCH₃ in HA chains played a predominant role in respect to the network formation and stabilization of HA weak gel.     

Germanium tetra(tertiary butoxide): Synthesis, structure and its utility as a precursor for the preparation of europium doped germanium oxide nanoparticles

Germanium tetra(tertiary butoxide), [Ge(O^tBu)₄], has been prepared by the reaction of GeCl₄ with KOBu^t in benzene. It is a monomeric crystalline solid having a distorted tetrahedral configuration, defined by the coordination of four OBu^t groups around germanium atom. The TG analysis showed that the compound is thermally stable and volatilizes at around 130 °C. Europium doped and un-doped germanium oxide nanoparticles were prepared based on the urea hydrolysis of Ge(O^tBu)₄/Eu(OOCCH₃)₃ in ethylene glycol medium at 150 °C followed by heating the resulting product at 750 °C. The nanoparticles were characterized by XRD, TEM and PL measurements. The europium doped nanoparticles, which were nearly monodispersed (∼30 nm), showed luminescence and the Eu³⁺ ions were occupying the surface of the GeO₂ nanoparticles.     

Nodulation and N2 fixation of guar at high root temperature

Guar (Cyamopsis tetregonoloba (L.) may be grown when soil temperatures are potentially high enough at the time of planting to inhibit nodulation and N₂ fixation. An experiment was conducted using controlled conditions to determine the influence of high root temperature on growth and N₂ fixation of guar. The experiment included two strains of rhizobia, two varieties of guar, two mineral N treatments, and root temperatures of 34, 37, and 40°C. Plants were grown for 44 days. The root temperature of 40°C reduced N fixation by at least 80% and nodule weight by more than 50%. Significant interactions occurred between most factors in influencing nodulation, N₂ fixation and dry matter production. Guar, nodulated by rhizobial strain GAR022-1 and fully dependent on N₂ fixation or provided with starter mineral N (25 mg pot^–1), was not influenced by the root temperature of 37°C as compared to 34°C. Nodulation and N₂ fixation by strain 32H1 was reduced by at least 40% when no starter mineral N was provided and the root temperature was 37°C. Providing starter mineral N to one variety of guar doubled the quantity of N₂ fixed by strain 32H1 at both 34 and 37°C but N₂ fixation was lower at the higher root temperature. It appears that root temperatures between 37° and 40°C bracketed the critical root temperature for N₂ fixation by nodulated guar and that the critical root temperature for guar dependent on mineral N was above 40°C.     

Body heat responsive gelation of methylcellulose formulation containing betaine

We examined a methylcellulose (MC) formulation that gels at body temperature for enteral alimentation. Betaine was found to have a lowering effect on the gelation temperature of the MC solution. The thermal gelation temperature of a body heat-responsive (BHR) gelling MC formulation, consisting of 2% MC, 15% glucose, 1.2% sodium citrate, and 3.5% betaine mixture, was approximately 32 °C, indicating that it could gel in response to body heat. Glucose release from the BHR gels was delayed at 37 °C in an in vitro study. In rats, oral administration of BHR gelling MC formulation delayed an increase in blood glucose and appearance of ¹³CO₂ in expired air in a ¹³C-acetate breath test in comparison with the control. These results suggested that the BHR gelling MC formulation was gelled in the stomach and delayed gastric emptying after oral administration and glucose in the gels was absorbed slowly.     

Collagen/cellulose hydrogel beads reconstituted from ionic liquid solution for Cu(II) adsorption

A novel adsorbent, biodegradable collagen/cellulose hydrogel beads (CCHBs), was prepared by reconstitution from a 1-butyl, 3-methylimidazolium chloride ([C₄mim]Cl) solution. The adsorption properties of the CCHBs for Cu(II) ion removal from aqueous solutions were investigated and compared with those of cellulose hydrogel beads (CHBs). The CCHBs have a three-dimensional macroporous structure whose amino groups are believed to be the main active binding sites of Cu(II) ions. The equilibrium adsorption capacity (q_e) of the CCHBs is greatly influenced by the collagen/cellulose mass ratio, and steeply increases until the collagen/cellulose mass ratio exceeds 2/1. The maximum adsorption is obtained at pH 6. The q_e of Cu(II) ions increases with increased initial concentration of the solution. Based on Langmuir isotherms, the maximum adsorption capacity (q_m) of CCHB3 (collagen/cellulose mass ratio of 3/1) is 1.06 mmol/g. The CCHBs maintain good adsorption properties after the fourth cycle of adsorption–desorption.     

Pericellular Conditions Regulate Extent of Cell-Mediated Compaction of Collagen Gels

Cell-mediated compaction of the extracellular matrix (ECM) plays a critical role in tissue engineering, wound healing, embryonic development, and many disease states. The ECM is compacted as a result of cellular traction forces. We hypothesize that a cell mechanically remodels the nearby ECM until some target conditions are obtained, and then the cell stops compacting. A key feature of this hypothesis is that ECM compaction primarily occurs in the pericellular region and the properties of the ECM in the pericellular region govern cellular force generation. We developed a mathematical model to describe the amount of macroscopic compaction of cell-populated collagen gels in terms of the initial cell and collagen densities, as well as the final conditions of the pericellular environment (defined as the pericellular volume where the collagen is compacted (V^∗) and the mass of collagen within this volume (m^∗)). This model qualitatively predicts the effects of varying initial cell and collagen concentrations on the extent of gel compaction, and by fitting V^∗ and m^∗, provides reasonable quantitative agreement with the extent of gel compaction observed in experiments with endothelial cells and fibroblasts. Microscopic analysis of compacted gels supports the assumption that collagen compaction occurs primarily in the pericellular environment.     

Effect of Soy Protein Subunit Composition on the Rheological Properties of Soymilk during Acidification

The effect of soy protein subunit composition on the acid-induced aggregation of soymilk was investigated by preparing soymilk from different soybean lines lacking specific glycinin and β-conglycinin subunits. Acid gelation was induced by glucono-δ-lactone (GDL) and analysis was done using diffusing wave spectroscopy and rheology. Aggregation occurred near pH 5.8 and the increase in radius corresponded to an increase in the elastic modulus measured by small deformation rheology. Diffusing wave spectroscopy was also employed to follow acid gelation, and data indicated that particle interactions start to occur at a higher pH than the pH of onset of gelation (corresponding to the start of the rapid increase in elastic modulus). The protein subunit composition significantly affected the development of structure during acidification. The onset of aggregation occurred at a higher pH for soymilk samples containing group IIb (the acidic subunit A₃) of glycinin, than for samples prepared from Harovinton (a commercial variety containing all subunits) or from genotypes null in glycinin. The gels made from lines containing group I (A₁, A₂) and group IIb (A₃) of glycinin resulted in stiffer acid gels compared to the lines containing only β-conglycinin. These results confirmed that the ratio of glycinin/β-conglycinin has a significant effect on gel structure, with an increase in glycinin causing an increase in gel stiffness. The type of glycinin subunits also affected the aggregation behavior of soymilk.     

Preparation, properties, and uses of two fluorogenic substrates for the detection of 5'-(venom) and 3'-(spleen) nucleotide phosphodiesterases

A new method for ³H-labeling of native collagen and a specific microassay for collagenase activity are presented. Acid-soluble type I collagen derived from rat tail tendons was reacted with pyridoxal phosphate and then reduced with NaB³H₄ to yield [³H]collagen with a specific activity of more than 10 μCi/mg. With respect to rate of hydrolysis, trypsin susceptibility, and gelling properties this collagen compares favorably with biosynthetically labeled preparations. It was shown that chemical labeling procedures such as this, or N-acetylation with acetic anhydride, do not adversely affect properties of collagen which are important for its use as substrate in specific assays. The microassay employs 50-μl [³H]collagen gels (1 mg/ml) dispensed in microtest plates. At 36°C this assay combines rapid rate of hydrolysis with low trypsin susceptibility. As little as 1 ng of clostridial collagenase activity can be measured reproducibly. The high specific activity of the [³H]collagen allowed us to explore microassay conditions employing minute quantities of substrate in solution. These studies indicated that native type I collagen whether labeled or not, is cleaved in the helical region by trypsin at subdenaturation temperatures. It was concluded that, in order to remain specific, collagenase assays with collagen in solution as with collagen in fibrils must be performed at 10–12°C below the denaturation temperature, i.e., at 35–37°C with collagen gels and 27–29°C with collagen in solution.     

Effect of temperature on production of aflatoxin on rice by Aspergillus flavus

Summary The effect of temperature on formation of aflatoxin on solid substrate (rice) byAspergillus flavus NRRL 2999 has been studied in some detail. The optimum temperature for production of both aflatoxin B₁ and G₁ under the conditions employed is 28° C. Comparable yields of B₁ were obtained at 32° C, but considerably less G₁ was produced at this temperature. Both B₁ and G₁ were found in lesser amounts at temperatures above 32° C, and the aflatoxin content of rice incubated at 37° C was low (300–700 ppb) even though growth was good.Reducing the temperature from 28° to 15° C resulted in progressively less aflatoxin, but 100 ppb of B₁ was detected in cultures incubated 3 weeks at 11° C. No aflatoxin was produced at 8° C.The ratio of the four aflatoxins is affected by temperature. At the lower temperatures, essentially equal amounts of aflatoxin B₁ and G₁ were produced, whereas at 28° C, approximately four times as much B₁ was detected as G₁. At the higher temperatures, relatively less G was formed, until at 37° C, less than 10 ppb was detected.This is a laboratory of the Northern Utilization Research and Development Division, Agricultural Research Service, U.S. Department of Agriculture.     

Effect of co-solute and gelation temperature on milk protein and gum tragacanth interaction in acidified gels

The aim of this study was to investigate the role of process conditions and system composition on the acid-induced gelation of a mixture of milk protein and gum tragacanth. This was studied by determining the effects of co-solute (lactose) addition (3, 5 and 7%) and gelation temperature (25, 37 and 45°C) on the mixture's rheological properties and microstructure using a combination of techniques including small-deformation rheology and scanning electron microscopy. The presence of lactose played an important role in the microstructure formation of gels but did not change most rheological properties. The microstructure of gels formed in the presence of lactose was coarser and more particulate, but less interconnected; this can be explained by lactose's role in improving protein aggregation. Gels prepared at a lower temperature had a high structure strength, as indicated by their high storage modulus, τ(f) and G(f) values. Low gelation temperature also caused a more branched and homogenous microstructure.