A new method for the preparation of a calcium activated neutral protease highly sensitive to calcium ions

A Ca²⁺--activated neutral protease has been purified from chicken skeletal muscle to homogeneity by a new method which employs affinity chromatography on casein CH-Sepharose 4B. SDS polyacrylamide gel electrophoresis shows that the purified enzyme consists of a single polypeptide chain with a molecular weight of 76,000. For half-maximum activity this protease requires 50 μM Ca²⁺ ions and its optimum pH is 7.6. The protease is inhibited by leupeptin, antipain, E-64 and endogenous inhibitor. The purified protease is very labile upon storage; after 3 days at 4°C no detectable activity remained.     

A systematic mutagenesis-driven strategy for site-resolved NMR studies of supramolecular assemblies

Obtaining sequence-specific assignments remains a major bottleneck in solution NMR investigations of supramolecular structure, dynamics and interactions. Here we demonstrate that resonance assignment of methyl probes in high molecular weight protein assemblies can be efficiently achieved by combining fast NMR experiments, residue-type-specific isotope-labeling and automated site-directed mutagenesis. The utility of this general and straightforward strategy is demonstrated through the characterization of intermolecular interactions involving a 468-kDa multimeric aminopeptidase, PhTET2.     

A new sampling formula for neutral biodiversity

The neutral model of biodiversity, proposed by Hubbell (The Unified Neutral Theory of Biodiversity and Biogeography, Princeton University Press, Princeton, NJ, 2001) to explain the diversity of functionally equivalent species, has been subject of hot debate in community ecology. Whereas Hubbell studied the model mostly by simulations, recently analytical treatments have yielded expressions of the expected number of species of a particular abundance in a local community with dispersal limitation. Moreover, a formula has been offered for the joint likelihood of observing a given species‐abundance dataset in a local community with dispersal limitation, but this formula is too complicated to allow practical applications. Here, I present a much simplified expression that can be regarded as an enhanced version of the famous Ewens sampling formula. It can be used in maximum likelihood methods for quick estimation of the model parameters, using all information in the data, and for model comparison. I also show how to rapidly generate examples of species‐abundance distributions for a given set of model parameters and how to calculate Simpson's diversity index.     

A new ditopic GdIII complex functionalized with an adamantyl moiety as a versatile building block for the preparation of supramolecular assemblies

A dimeric GdAAZTA-like complex (AAZTA is 6-amino-6-methylperhydro-1,4-diazepinetetraacetic acid) bearing an adamantyl group (Gd₂ L1) able to form strong supramolecular adducts with specific hosts such as β-cyclodextrin (β-CD), poly-β-CD, and human serum albumin (HSA) is reported. The relaxometric properties of Gd₂ L1 were investigated in aqueous solution by measuring the ¹H relaxivity as a function of pH, temperature, and magnetic field strength. The relaxivity of Gd₂ L1 (per Gd atom) at 40 MHz and 298 K is 17.6 mM^?1 s^?1, a value that remains almost constant at higher fields owing to the great compactness and rigidity of the bimetallic chelate, resulting in an ideal value for the rotational correlation time for high-field MRI applications (1.5–3.0 T). The noncovalent interaction of Gd₂ L1 with β-CD, poly-β-CD, and HSA and the relaxometric properties of the resulting host–guest adducts were investigated using ¹H relaxometric methods. Relaxivity enhancements of 29 and 108 % were found for Gd₂ L1–β-CD and Gd₂ L1–poly-β-CD, respectively. Binding of Gd₂ L1 to HSA (K _A = 1.2 × 10⁴ M^?1) results in a remarkable relaxivity of 41.4 mM^?1 s^?1 for the bound form (+248 %). The relaxivity is only limited by the local rotation of the complex within the binding site, which decreases on passing from Gd₂ L1–β-CD to Gd₂ L1–HSA. Finally, the applicability of Gd₂ L1 as tumor-targeting agent through passive accumulation of the HSA-bound adduct was evaluated via acquisition of magnetic resonance images at 1 T of B16-tumor-bearing mice. These experiments indicate a considerable signal enhancement (+160 %) in tumor after 60 min from the injection and a very low hepatic accumulation.     

A new solvent system for the separation of neutral glycosphingolipids

A solvent system and a column for high performance liquid chromatography for the separation of glycosphingolipids without derivatization is described. A column pakced with porous silica gel (latrobeads) and eluted with a mixture of isopropanol-hexane-water with increasing water content and decreasing hexane content was used. Glycosphingolipids with mono- to dodeca- or tetrakaidecasaccharides were separated within 60 min and the separation pattern was highly reproducible. The method was applied for preparative separation of highly complex glycolipids with blood group activity.     

A new reagent for the preparation of glycoconjugates

The thioglycoside derivative 2-carbazoylethyl 1-thio-beta-D-galactopyranoside hydrochloride was synthesized. Conversion of the carbazoyl group into the reactive azidocarbonyl function leads to a well suited reagent for the preparation of glycoconjugates via amidation of proteins or synthetic carriers in aqueous media.     

A short oxazolidine-2-one containing peptide forms supramolecular hydrogels under controlled conditions

Low-molecular-weight hydrogels are made of a small percentage of small organic molecules dispersed in an aqueous medium, which may aggregate in several manners using different methods. However, often the organic gelator in water has poor solubility, so the addition of a solubilising agent is required. In the case of acidic gelators, this mainly consists of the addition of a strong base, that is sodium hydroxide, that deprotonates the acidic moiety, so the gelator molecules become more soluble and tend to assemble into micelles, forming a dispersion. Some gelators, however, are sensitive to the harsh pH and get hydrolysed. This is the case of some molecules presenting carbamates in their features, like Fmoc-protected or oxazolidinone-containing peptides. In this paper, we present a valid alternative to sodium hydroxide, by dissolving a tripeptide containing an oxazolidinone moiety in a phosphate buffer (PB) medium at pH 7.4. The results obtained with the NaOH dissolution are compared with the ones with PB, as both methods present advantages and drawbacks. The use of NaOH produces transparent but weak hydrogels, as it exposes the gelator to harsh conditions that end up in its partial hydrolysis, which is more pronounced at high concentrations (≥10 mM). Using PB to dissolve the gelator, this problem is completely avoided as no hydrolysis product has been detected in the hydrogels, which are very stiff although more opaque. By tuning the preparation conditions, we can obtain a wide variety of hydrogels, with the properties required by the final application.     

A new strategy for recycling and preparation of poly(L-lactic acid): hydrolysis in the melt

Poly(L-lactide) [i.e., poly(L-lactic acid) (PLLA)] was hydrolyzed in the melt in high-temperature and high-pressure water at the temperature range of 180-350 degrees C for a period of 30 min, and formation, racemization, and decomposition of lactic acids and molecular weight change of PLLA were investigated. The highest maximum yield of l-lactic acid, ca. 90%, was attained at 250 degrees C in the hydrolysis periods of 10-20 min. Too-high hydrolysis temperatures such as 350 degrees C induce the dramatic racemization and decomposition of formed lactic acids, resulting in decreased maximum yield of L-lactic acid. The hydrolysis of PLLA proceeds homogeneously and randomly via a bulk erosion mechanism. The molecular weight of PLLA decreased exponentially without formation of low-molecular-weight specific peaks originating from crystalline residues. The activation energy for the hydrolysis (deltaE(h)) of PLLA in the melt (180-250 degrees C) was 12.2 kcal x mol(-1), which is lower than 20.0 kcal x mol(-1) for PLLA and 19.9 kcal x mol(-1) for poly(dl-lactide) [i.e., poly(DL-lactic acid)] as a solid in the temperature range below the glass-transition temperature (21-45 degrees C). This study reveals that hydrolysis of PLLA in the melt is an effective and simple method to obtain l-lactic acid and to prepare PLLA having different molecular weights without containing the specific low-molecular-weight chains, because of the removal of the effect caused by crystalline residues.     

A new method for the preparation of solid-phase immunoadsorbents

A solid-phase immunoadsorbent was prepared by insolubilization of antibody during precipitation with Na₂SO₄. The polyaldehyde macromolecule created by periodate oxidation of dextran (macrofixative) served as the insolubilizing, crosslinking agent. After appropriate fixation, the precipitate was stable to removal of the precipitating salt, to washing, and, to a large extent, to heating in the denaturing detergent, sodium dodecyl sulfate. Under proper conditions, the precipitate retained satisfactory antibody activity, although very high molecular weight antigens were apparently excluded from the internal active sites of the solid-phase matrix. This method provides the advantage of insolubilization of the primary antibody in a small volume; for analytical work, the entire precipitate, with bound antigen, may be quantitatively applied to a polyacrylamide gel (tube or slab) and electrophoresed without overloading by the binding antibody. This method might also be extended for use in immunoisolation procedures employing standard eluting agents, as well as insolubilization of proteins other than immunoglobulin.     

A new strategy for the detection of subtelomeric rearrangements

We present a new strategy for the detection of subtelomeric rearrangements. This approach is based on two hybridizations with different probe sets. The first set consists of microdissected subtelomeric probes (each 5-10 megabases in size) labeled combinatorially employing 7 different fluorochromes. With this set, subtelomeric interchromosomal exchanges can be detected in a 24-color experiment. The second set comprises a second generation of subtelomeric PAC-, P1- and BAC-clones. Probes for p- and q-arms are labeled with two different colors. This second set detects small deletions; in addition it provides regional information, so that translocated material identified by the first probe set can be assigned to the p- or q-arm of a chromosome. The test has been evaluated in a blind study on a series of subtle translocations and deletions.     

A novel strategy for the preparation of liposomes: rapid solvent exchange

During the preparation of multi-component model membranes, a primary consideration is that compositional homogeneity should prevail throughout the suspension. Some conventional sample preparation methods pass the lipid mixture through an intermediary, solvent-free state. This is an ordered, solid state and may favor the demixing of membrane components. A new preparative method has been developed which is specifically designed to avoid this intermediary state. This novel strategy is called rapid solvent exchange (RSE) and entails the direct transfer of lipid mixtures between organic solvent and aqueous buffer. RSE liposomes require no more than a minute to prepare and manifest considerable entrapment volumes with a high fraction of external surface area. In phospholipid/cholesterol mixtures of high cholesterol content, suspensions prepared by more conventional methods reveal evidence of artifactual demixing, whereas samples prepared by rapid solvent exchange do not. The principles which may lead to artifactual demixing during conventional sample preparation are discussed.     

A new method for fish chromosome preparation

A new method for the preparation of fish chromosomes from abdominal cavity fluid has been developed. Cells were collected from fish abdominal cavity fluid after an in vivo PHA treatment, and cultured for a short time in medium with colchicine. After 30 min hypotonic treatment for marine fish and 35 min for freshwater fish, slides were prepared by the conventional air-drying method. The advantages ofthe method are: (1) it is technically simple; (2) it produces a reasonably high mitotic index; (3) chromosome spreading is good (4) there is very little cell breakage. Using this method, the chromosomes ofrainbow trout (2n=62); cod (2n=4546) and plaice (2n=46,47 and 48) were investigated.     

微生物降解纤维素的新机制

自然界中能够降解纤维素的微生物分布广泛,纤维素降解的方式也呈现很高的多样性。Cytophaga hutchinsonii属于拟杆菌门,具有很强的结晶纤维素降解能力,但是其降解机制既异于已经发现的游离纤维素酶降解体系,也不同于纤维小体的降解模式,推测其存在第3种细胞结合型纤维素降解模式。本文简要阐述以C. hutchinsonii为代表的一种新的纤维素降解策略。