Reactions of papain and of low-molecular-weight thiols with some aromatic disulphides. 2,2′-Dipyridyl disulphide as a convenient active-site titrant for papain even in the presence of other thiols

1. The u.v.-spectral characteristics of 5,5'-dithiobis-(2-nitrobenzoic acid) (Nbs(2)), 2,2'-dipyridyl disulphide (2-Py-S-S-2-Py), 4,4'-dipyridyl disulphide (4-Py-S-S-4-Py), 5-mercapto-2-nitrobenzoic acid (Nbs), 2-thiopyridone (Py-2-SH) and 4-thiopyridone (Py-4-SH) were determined over a wide range of pH and used to calculate their acid dissociation constants. 2. The reactions of l-cysteine, 2-mercaptoethanol and papain with the above-mentioned disulphides were investigated spectrophotometrically in the pH range 2.5-8.5. 3. Under the conditions of concentration used in this study the reactions of both low-molecular-weight thiols with all three disulphides resulted in the stoicheiometric release of the thiol or thione fragments Nbs, Py-2-SH and Py-4-SH at all pH values. The rates of these reactions are considerably faster at pH8 than at pH4, which suggests that the predominant reaction pathway in approximately neutral media is nucleophilic attack of the thiolate ion on the unprotonated disulphide. 4. The reaction of papain with Nbs(2) is markedly reversible in the acid region, and the pH-dependence of the equilibrium constant for this system in the pH range 5-8 at 25 degrees C and I=0.1 is described by: [Formula: see text] 5. Papain reacts with both 2-Py-S-S-2-Py and 4-Py-S-S-4-Py in the pH range 2.5-8.5 to provide release of the thione fragments, stoicheiometric with the thiol content of the enzyme. 6. Whereas the ratios of the second-order rate constant for the reaction at pH4 to that at pH8 for the cysteine-2-Py-S-S-2-Py reaction (k(pH4)/k(pH8)=0.015) and for the papain-4-Py-S-S-4-Py reaction (k(pH4)/k(pH8)=0.06) are less than 1, that for the papain-2-Py-S-S-2-Py reaction is greater than 1 (k(pH4)/k(pH8)=15). 7. This high reactivity of papain has been shown to involve reaction of the thiol group of cysteine-25, the enzyme's only cysteine residue, which is part of its catalytic site. 8. That this rapid and stoicheiometric reaction of the thiol group of native papain is not shown either by low-molecular-weight thiols or by the thiol group of papain after its active conformation has been destroyed by acid or heat denaturation, strongly commends 2-Py-S-S-2-Py as one of the most useful papain active-site titrants discovered to date. This reagent has been shown to allow accurate titration of papain active sites in the presence of up to 10-fold molar excess of l-cysteine and up to 100-fold molar excess of 2-mercaptoethanol.     

Recovery of the motor function of the arm with the aid of a hand exoskeleton controlled by a brain–computer interface in a patient with an extensive brain lesion

The dynamics of motor function recovery in a patient with an extensive brain lesion has been investigated during a course of neurorehabilitation assisted by a hand exoskeleton controlled by a brain–computer interface. Biomechanical analysis of the movements of the paretic arm recorded during the rehabilitation course was used for an unbiased assessment of motor function. Fifteen procedures involving hand exoskeleton control (one procedure per week) yielded the following results: (a) the velocity profile for targeted movements of the paretic hand became nearly bell-shaped; (b) the patient began to extend and abduct the hand, which was flexed and adducted at the beginning of the course; and (c) the patient started supinating the forearm, which was pronated at the beginning of the rehabilitation course. The first result is interpreted as improvement of the general level of control over the paretic hand, and the two other results are interpreted as a decrease in spasticity of the paretic hand.     

Electrooptical properties of the microbial suspensions during a cell’s interaction with the antibodies of a different specificity

The electrooptical abilities of the microbial suspensions during a cells interaction with antibodies (ABs) of a different specificity have been studied on the example of the Azospirillum brasilense Sp245 cells and their interaction with the polyclonal monospecific and polyspecific antibodies. Measuring of the orientational spectra of the cells has been performed using the ELUS electrooptical analyzer. A discrete frequency set of an orienting electric field (740, 1000, 1450, 2000, and 2800 kHz) was used. It has been shown that an interaction of the polyspecific AB with the investigated cells redoubles the value of an electrooptical signal of the cells’ suspension as compared with the monospecific antibodies. These findings can be used for a development a new method of microorganism detection.     

Computational analysis of the interactions of a novel cephalosporin derivative with β-lactamases

Background

One of the main concerns of the modern medicine is the frightening spread of antimicrobial resistance caused mainly by the misuse of antibiotics. The researchers worldwide are actively involved in the search for new classes of antibiotics, and for the modification of known molecules in order to face this threatening problem. We have applied a computational approach to predict the interactions between a new cephalosporin derivative containing an additional β-lactam ring with different substituents, and several serine β-lactamases representative of the different classes of this family of enzymes.

Results

The results of the simulations, performed by using a covalent docking approach, has shown that this compound, although able to bind the selected β-lactamases, has a different predicted binding score for the two β-lactam rings, suggesting that one of them could be more resistant to the attack of these enzymes and stay available to perform its bactericidal activity.

Conclusions

The detailed analysis of the complexes obtained by these simulations suggests possible hints to modulate the affinity of this compound towards these enzymes, in order to develop new derivatives with improved features to escape to degradation.

     

β-N-Acetylhexosaminidases in the spleen of a patient with hairy-cell leukaemia

The spleen from a patient with hairy-cell leukaemia had β-N-acetylhexosaminidase activity that could be resolved into three isoenzymes by chromatography on phenyl boronate agarose. Two of these were the major forms, A and B, found in normal tissues but, in addition, there was an ‘extra’ form that accounted for 15% of total activity. The ‘extra’ form hydrolysed the synthetic substrate 4-methylumbelliferyl-β-N-acetylglucosamine 6-sulphate, indicating the presence of α-subunits. It was more acidic than A, was less heat-stable and showed no generation of B on denaturation under a variety of conditions. These findings and the immunoblot (Western blotting) analysis demonstrate that the ‘extra’ form is entirely composed of α-subunits, and most closely resembles S, the residual activity in Sandhoff's disease.     

Crystal structure of tarocystatin–papain complex: implications for the inhibition property of group-2 phytocystatins

Tarocystatin (CeCPI) from taro (Colocasia esculenta cv. Kaohsiung no. 1), a group-2 phytocystatin, shares a conserved N-terminal cystatin domain (NtD) with other phytocystatins but contains a C-terminal cystatin-like extension (CtE). The structure of the tarocystatin–papain complex and the domain interaction between NtD and CtE in tarocystatin have not been determined. We resolved the crystal structure of the phytocystatin–papain complex at resolution 2.03 Å. Surprisingly, the structure of the NtD–papain complex in a stoichiometry of 1:1 could be built, with no CtE observed. Only two remnant residues of CtE could be built in the structure of the CtE–papain complex. Therefore, CtE is easily digested by papain. To further characterize the interaction between NtD and CtE, three segments of tarocystatin, including the full-length (FL), NtD and CtE, were used to analyze the domain–domain interaction and the inhibition ability. The results from glutaraldehyde cross-linking and yeast two-hybrid assay indicated the existence of an intrinsic flexibility in the region linking NtD and CtE for most tarocystatin molecules. In the inhibition activity assay, the glutathione-S-transferase (GST)-fused FL showed the highest inhibition ability without residual peptidase activity, and GST-NtD and FL showed almost the same inhibition ability, which was higher than with NtD alone. On the basis of the structures, the linker flexibility and inhibition activity of tarocystatins, we propose that the overhangs from the cystatin domain may enhance the inhibition ability of the cystatin domain against papain.     

Separation of two functional domains of the family F/10 β-xylanase from Streptomyces olivaceoviridis E-86 limited proteolysis with papain and some of their properties

Limited proteolysis of papain on the intact family F/10 -xylanase (45 kDa) led to the isolation of catalytic domain (32 kDa) and xylan-binding domain (15 kDa). The two truncated protein fragments were purified by gel filtration to homogeneity. The purified catalytic domain was fully active against 4-O-methyl-d-glucurono-d-xylan, and the action mode on xylan was the same as the intact xylanase. However, the removal of xylan-binding domain reduces dramatically the capability of adsorption for xylan.     

Molecular basis for subtypic differences of the “a” subunit of coagulation factor XIII with description of the genesis of the subtypes

The “a” subunit of human coagulation factor XIII (F13A) exhibits genetic polymorphism defined by four common alleles, F13A*1A, *1B, *2A, and *2B. We have previously suggested on the basis of the isoelectric focusing patterns of the four allele products that point mutations at two separate sites and one intragenic crossing over might be involved in the genes of F13A polymorphism. Here, we report nucleotide substitutions associated with F13A polymorphism. A C/T transition of the second nucelotide of codon 564 in exon 12 is responsible for the difference between F13A*1A and *1B and that between F13A*2A and *2B, and a set of two base changes in codons 650 and 651 in exon 14 leads to the differences between F13A*1A and *2A and those between F13A*1B and *2B. The four combinations of the point mutations at the two exons thus correspond to the four alleles, two of which were generated by the point mutations from ancestral monomorphic gene. The results suggest strongly that intragenic crossing over must be involved in the genesis of the fourth allele. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) methods discriminating these base changes in exons 12 and 14 are also presented.     

Transformation of a Leu− Mutant of Rhizopus niveus with the leuA Gene of Mucor circinelloides

The 2287-bp cryptic plasmid, pMA1, from Microcystis aeruginosa f. aeruginosa Kützing, a unicellular cyanobacterium originally derived from Kasumigaura lake, was completely sequenced and analyzed. The predicted amino acid sequence (253 residues) of an open reading frame had identities of 47%, 48%, and 53% with replication-associated proteins of Bacillus amyloliquefaciens’s plasmid, pFTB14, B. subtilis BAA1’s pBAA1, and B. coagulans’s pBC1, respectively, when conservative amino acid substitutions were included. Such high-level identities were also shown with rep proteins and ori regions in a group of Gram-positive bacterial plasmids such as Lactococci and Staphylococci that are known to replicate via single-stranded intermediates. The pMA1 does hybridize with a plasmid, pUS1-3, derived from another unicellular cyanobacterium, Synechocystis sp. PCC 6803. Novel features of pMA1 are discussed.     

Is the shoot a root with a view?

Recently, it has been shown that the same sets of genes act in both root and shoot to regulate cell fate and patterning. One gene cassette regulates epidermal cell fate, another cassette regulates ground tissue derived cell fate and organization. Ectopic expression and laser ablation have been used to probe the mechanisms by which these genes perform their tissue and organ-specific functions.     

Quantification of the secretory glycoproteins of the subcommissural organ by a sensitive sandwich ELISA with a polyclonal antibody and a set of monoclonal antibodies against the bovine Reissner’s fiber

The subcommissural organ (SCO) is an ependymal brain gland that releases glycoproteins into the ventricular cerebrospinal fluid where they condense to form the Reissner’s fiber (RF). We have developed a highly sensitive and specific two-antibody sandwich enzyme-linked immunosorbent assay (ELISA) for the quantification of the bovine SCO secretory material. The assay was based on the use of the IgG fraction of a polyclonal antiserum against the bovine RF as capture antibody and a pool of three peroxidase-labeled monoclonal antibodies that recognize non-overlapping epitopes of the RF glycoproteins as detection antibody. The detection limit was 1 ng/ml and the working range extended from 1 to 4000 ng/ml. The calibration curve, generated with RF glycoproteins, showed two linear segments: one of low sensitivity, ranging from 1 to 125 ng/ml, and the other of high sensitivity between 125 and 4000 ng/ml. This assay was highly reproducible (mean intra- and interassay coefficient of variation 2.2% and 5.3%, respectively) and its detectability and sensitivity were higher than those of ELISAs using exclusively either polyclonal or monoclonal antibodies against RF glycoproteins. The assay succeeded in detecting and measuring secretory material in crude extracts of bovine SCO, culture medium supernatant of SCO explants and incubation medium of bovine RF; however, soluble secretory material was not detected in bovine cerebrospinal fluid.     

The association of the human ε-globin gene with the nuclear matrix: a reconsideration

The association of the human -globin gene with the nuclear matrix was studied in erythroid and non-erythroid cell lines. Using a high salt method to prepare histone depleted nuclei we studied the association of variety of fragments covering a 7.8 kb region which contains the human -globin gene. We furthermore studied the association of a set of DNA fragments covering the 13 kb human G/A-globin gene domain, the 16 kb /-globin gene domain and the 10 kb -globin gene domain with the nuclear matrix of K562 and Raji cells. The results show that all fragments studied are easily released from the nuclear matrix, indicating no specific association.Summarizing our results we could say that a region starting 5.7 kb 5 to the human -globin gene and ending 4 kb 3 to the human -globin gene seems to contain no attachment sites with the nuclear matrix of both erythroid and non-erythroid cells.     

Methylation of methyl α-d-hexopyranosides with diazo-methane in the presence of a small amount of water

The long-time reduction of methyl α-d-gluco-, α-d-manno-, and α-d-galactopyranosides with excess diazomethane-diethyl ether at 25° in the presence of water gave all partially methylated methyl α-d-hexopyranosides which differ in number and position of methyl substitution. The presence of electrolytes, such as potassium or sodium phosphate, in the reaction medium enhanced the degree of methylation, resulting in preferential formation of tri-O-methyl derivatives of methyl α-d-hexopyranosides.     

Methylene blue as a suppessor of the genotoxic effect of ultraviolet radiation with a wavelength of 300–400 nm

Ultraviolet radiation with a wavelength of 300–400 nm is characteristic of sunlight at the earth surface and causes DNA damage mediated by energy transfer to O₂ with the transformation of the latter in the singlet state. In connection with this, scavengers of reactive oxygen species (ROSs) are potential protectors against the genotoxic effect of this kind of radiation. It was found that the methylene blue dye at doses differing by several orders of magnitude from those that are toxic for humans is able to suppress completely the SOS response induced by UV with a wavelength of 300–400 nm in Escherichia coli.     

Q- and C-band polymorphism of the chromosomes in a group of patients with the Shereshevski?-Turner syndrome

Q- and C-band polymorphism of heterochromatic regions of chromosomes were studied in a group of patients with Turner's syndrome (30 girls with the karyotype 45, X) and in 105 normal individuals. No significant differences in the frequencies of Q-polymorphic variants for the most part of chromosomes studied (with the exception of chromosome 13 satellites) were obtained between patients with Turner's syndrome and the control. There were no differences in the mean number of Q-variants per individual in both groups investigated. An increase in the frequency of large C-segments of chromosome 9 was detected in patients with Turner's syndrome. An increase in the frequency of individuals carrying a combination of several extreme variants in the individual karyotype was found for patients with Turner's syndrome. The differences revealed are of non-specific character for a given form of developmental pathology.     

Association of calpastatin with inactive calpain: a novel mechanism to control the activation of the protease?

It is generally accepted that the Ca(2+)-dependent interaction of calpain with calpastatin is the most relevant mechanism involved in the regulation of Ca(2+)-induced proteolysis. We now report that a calpain-calpastatin association can occur also in the absence of Ca(2+) or at very low Ca(2+) concentrations, reflecting the physiological conditions under which calpain retains its inactive conformational state. The calpastatin binding region is localized in the non-inhibitory L-domain containing the amino acid sequences encoded by exons 4-7. This calpastatin region recognizes a calpain sequence located near the end of the DII-domain. Interaction of calpain with calpastatins lacking these sequences becomes strictly Ca(2+)-dependent because, under these conditions, the transition to an active state of the protease is an obligatory requirement. The occurrence of the molecular association between Ca(2+)-free calpain and various recombinant calpastatin forms has been demonstrated by the following experimental results. Addition of calpastatin protected calpain from trypsin digestion. Calpain was coprecipitated when calpastatin was immunoprecipitated. The calpastatin molecular size increased following exposure to calpain. The two proteins comigrated in zymogram analysis. Furthermore, calpain-calpastatin interaction was perturbed by protein kinase C phosphorylation occurring at sites located at the exons involved in the association. At a functional level, calpain-calpastatin interaction at a physiological concentration of Ca(2+) represents a novel mechanism for the control of the amount of the active form of the protease potentially generated in response to an intracellular Ca(2+) influx.     

Measurement of THz Spot Sizes with a λ/200 Diameter in the Near-Field of a Metal Tip

We report on a method to obtain asub-wavelength resolution in terahertz time-domain imaging. In our method,a sharp copper tip is used to locallydistort and concentrate the THz electricfield. The distorted electric field, presentmainly in the near field of the tip, iselectro-optically measured in an (100)oriented GaP crystal. By raster scanning the tipalong the surface of the crystal we find asmallest THz spot size of 10 m forfrequencies from 0.1 to 2.5 THz. For ourpeak frequency of 0.15 THz this corresponds to aresolution of /200. Our setup has thepotential to reach a resolution down to afew m, and is a promising candidate tostudy single, living cells in the THzfrequency range.