Neutron star mass estimates from gamma-ray eclipses in spider millisecond pulsar binaries.

Reliable neutron star mass measurements are key to determining the equation of state of cold nuclear matter, but such measurements are rare. Black widows and redbacks are compact binaries consisting of millisecond pulsars and semi-degenerate companion stars. Spectroscopy of the optically bright companions can determine their radial velocities, providing inclination-dependent pulsar mass estimates. Although inclinations can be inferred from subtle features in optical light curves, such estimates may be systematically biased due to incomplete heating models and poorly understood variability. Using data from the Fermi Large Area Telescope, we have searched for gamma-ray eclipses from 49 spider systems, discovering significant eclipses in 7 systems, including the prototypical black widow PSR B1957+20. Gamma-ray eclipses require direct occultation of the pulsar by the companion, and so the detection, or significant exclusion, of a gamma-ray eclipse strictly limits the binary inclination angle, providing new robust, model-independent pulsar mass constraints. For PSR B1957+20, the eclipse implies a much lighter pulsar (1.81 ± 0.07 solar masses) than inferred from optical light curve modelling.

Differential colchicine-binding across eukaryotic families: the role of highly conserved Pro268beta and Ala248beta residues in animal tubulin.

Colchicine-tubulin interaction, responsible for the disruption of microtubule formation, has immense pharmacological importance but is poorly understood in terms of its biological significance. The interaction is characterized by a marked higher affinity of colchicine for animal tubulins compared to tubulins from plants, fungi and protists. From an analysis of tubulin sequences and colchicine-tubulin crystal structure, we propose that Pro268beta and Ala248beta (270beta and 250beta in the crystal structure 1SA0) in animal tubulin are crucial for the observed differential binding. We also suggest that mediated by the binding of endogenous molecules to the colchicine-binding site, microtubule assembly in eukaryotes may be modulated in a family specific manner.

Comparative profiles of alpha-amylase production in conventional tray reactor and GROWTEK bioreactor.

GROWTEK bioreactor was used as modified solid-state fermentor to circumvent many of the problems associated with the conventional tray reactors for solid-state fermentation (SSF). Aspergillus oryzae IFO-30103 produced very high levels of alpha-amylase by modified solid-state fermentation (mSSF) compared to SSF carried out in enamel coated metallic trays utilizing wheat bran as substrate. High alpha-amylase yield of 15,833 U g(-1) dry solid in mSSF were obtained when the fungus were cultivated at an initial pH of 6.0 at 32 degrees C for 54 h whereas alpha-amylase production in SSF reached its maxima (12,899 U g(-1) dry solid ) at 30 degrees C after 66 h of incubation. With the supplementation of 1% NaNO(3), the maximum activity obtained was 19,665 U g(-1) dry solid (24% higher than control) in mSSF, whereas, in SSF maximum activity was 15,480 U g(-1) dry solid in presence of 0.1% Triton X-100 (20% higher than the control).

Ester antibiotic accumulation by Streptomyces hygroscopicus.

In an attempt to maximize the ester antibiotic production by Streptomyces hygroscopicus D1.5, its efficacy was found to be enhanced by manipulation of the nutrient and physical environment. The two stage fermentation using seed inoculum (10% v/v) resulted in better production while fermentation continued for 5 days in pH 7.0 at 30 degrees C. Enhanced yield was also observed in whole cell immobilization. Under entrapment, maximum yield was achieved at 7th and 9th day of fermentation for mycelia and spore. In addition, the beads could be reused up to the 3rd cycle.

Conformational properties of alpha-tubulin tail peptide: implications for tail-body interaction.

The carboxy-terminal segments of the alpha/beta-tubulins are flexible regions rich in acidic amino acid residues. It is generally believed that these regions play crucial roles in tubulin polymerization and interaction with many ligands, including colchicine. Exactly how these effects are exerted are not known at present. One such interesting aspect is the pH dependence of colchicine-tubulin interaction and the influence of the alpha-tail on the binding interaction. We have investigated the location of the colchicine-binding site on tubulin by docking. It has been located on the alpha/beta interface on the N-terminal side, which is also supported by much of the solution data. This location is too far from the tail regions, suggesting that influence of the tail region is transmitted by a pH-dependent conformational change. Two-dimensional NMR studies indicate that at pH 7 a 13-residue peptide corresponding to alpha-tubulin tail shows little NOE constraints, suggesting extended conformation. On the contrary, at pH 5, a relatively compact structure was deduced from the interproton NOE constraints. Pulsed field gradient measurement of diffusion constant indicates that the peptide at pH 5 is substantially faster diffusing than at pH 7. The Perrin factors calculated from diffusion data indicates that the peptide structure at pH is significantly more compact than at pH 7. Temperature coefficients of several amide protons at pH 5 fall below 5 ppb/(o)K, indicating a degree of protection. A difference is also seen in the CD spectra obtained at different pHs, consistent with the NMR data. We have investigated the probable spatial organization of the tail of the alpha-subunit of tubulin, in the high pH extended form and the low pH compact form. On the basis of correlation of pH dependence of many properties of tubulin and the conformation of the alpha-tail peptide, we propose that the intrinsic conformational preference of the tail-region modulate the tail-body interaction, which in turn has important bearing on colchicine binding properties.

Chaperone-like activity of tubulin. binding and reactivation of unfolded substrate enzymes.

The eukaryotic cytoskeletal protein tubulin is a heterodimer of two subunits, alpha and beta, and is a building block unit of microtubules. In a previous communication we demonstrated that tubulin possesses chaperone-like activities by preventing the stress-induced aggregation of various proteins (Guha, S., Manna, T. K., Das, K. P., and Bhattacharyya, B. (1998) J. Biol. Chem. 273, 30077-30080). As an extension of this observation, we explored whether tubulin, like other known chaperones, also protected biological activity of proteins against thermal stress or increased the yields of active proteins during refolding from a denatured state. We show here that tubulin not only prevents the thermal aggregation of alcohol dehydrogenase and malic dehydrogenase but also protects them from loss of activity. We also show that tubulin prevents the aggregation of substrates during their refolding from a denatured state and forms a stable complex with denatured substrate. The activity of malic dehydrogenase, alpha-glucosidase, and lactate dehydrogenase during their refolding from urea or guanidium hydrochloride denatured states increased significantly in presence of tubulin compared with that without tubulin. These results suggest that tubulin, in addition to its role in mitosis, cell motility, and other cellular events, might be implicated in protein folding and protection from stress.

Role of the carboxy-termini of tubulin on its chaperone-like activity.

Mutational analysis and the enzymatic digestion of many chaperones indicate the importance of both hydrophobic and hydrophilic residues for their unique property. Thus, the chaperone activity of alpha-crystallin is lost due to the substitution of hydrophobic residues or upon enzymatic digestion of the negatively charged residues. Tubulin, an eukaryotic cytoskeletal protein, exhibits chaperone-like activity as demonstrated by prevention of DTT-induced aggregation of insulin, thermal aggregation of alcohol dehydrogenase, betagamma-crystallin, and other proteins. We have shown that the tubulin lost its chaperone-like activity upon digestion of its negatively charged C-termini. In this article, the role of the C-terminus of individual subunits has been investigated. We observe that the digestion of C-terminus of beta-subunit with subtilisin causes loss of chaperone-like activity of tubulin. The contribution of C-terminus of alpha-subunit is difficult to establish directly as subtilisin cleaves C-terminus of beta-subunit first. This has been ascertained indirectly using a 14-residue peptide P2 having the sequence corresponding to a conserved region of MHC class I molecules and that binds tightly to the C-terminus of alpha-subunit. We have shown that the binding of P2 peptide to alphabeta-tubulin causes complete loss of its chaperone-like activity. NMR and gel-electrophoresis studies indicate that the P2 peptide has a significant higher binding affinity for the C-terminus of alpha-subunit compared to that of beta-subunit. Thus, we conclude that both the C-termini are necessary for the chaperone-like activity of tubulin. Implications for the chaperone functions in vivo have been discussed.

Unipolar brush cells form a glutamatergic projection system within the mouse cerebellar cortex.

Unipolar brush cells (UBCs) of the mammalian vestibulocerebellum receive mossy fiber projections primarily from the vestibular ganglion and vestibular nuclei. Recently, the axons of UBCs have been shown to generate an extensive system of cortex-intrinsic mossy fibers, which resemble traditional cerebellar mossy fiber afferents and synapse with granule cell dendrites and other UBCs. However, the neurotransmitter used by the UBC axon is still unknown. In this study, we used long-term organotypic slice cultures of the isolated nodulus (lobule X) from postnatal day 8 mouse cerebella to identify the neurotransmitter and receptors at synapses of the UBC axon terminals, relying on the notion that, in these cultures, all of the cortex-extrinsic fibers had degenerated during the first few days in vitro. Quantification of glutamate immunogold labeling showed that the UBC axon terminals have the same high gold-particle density as the glutamatergic parallel fiber varicosities. Furthermore, UBCs identified by calretinin immunoreactivity expressed the glutamate receptor subunits GluR2/3, NMDAR1, and mGluR2/3, like they do in the mature mouse cerebellum in situ. Evoked excitatory postsynaptic currents (EPSCs), spontaneous EPSCs, and burst discharges were demonstrated in UBCs and granule cells by patch-clamp recording. Both the evoked and spontaneous EPSCs were blocked by ionotropic glutamate receptor antagonists CNQX and D-AP5. We conclude that neurotransmission at the UBC axon terminals is glutamatergic. Thus, UBCs provide a powerful network of feedforward excitation within the granular layer, which may amplify vestibular signals and synchronize activity in clusters of functionally related granule cells which project vertically to patches of Purkinje cells.

Model for the generation of toroidal and poloidal magnetic fields in a laser-produced plasma.

A mechanism of simultaneous generation of toroidal and poloidal magnetic fields in an underdense region of a laser-produced plasma is discussed. The mechanism relies on the fact that at least a part of the incident transverse mode of the laser field undergoes a linear conversion into a longitudinal mode in the thermal plasma. It involves the conversion of ordered kinetic motion of the charged particles in the presence of the field into the energy of the induced magnetic fields both in poloidal and toroidal directions. The analysis is based on obtaining perturbative solutions of the two-fluid model of a hot nondissipative plasma. Our numerical results show that both the toroidal and poloidal fields increase with the laser intensity, and that the former dominates over the latter. Further, the toroidal fields decrease with increasing pulse lengths and increase rather slowly with an increase in laser wavelengths. However, the poloidal fields seem to be insensitive to the laser pulse lengths but they increase exponentially with the laser wavelengths. Finally, toroidal fields have a tendency to decrease as the critical surface is approached. The poloidal fields show a contrary behavior.

Optimization of some additives to improve protease production under SSF.

In a locally isolated Rhizopus oryzae strain highest-production of protease (388.54/g wheat bran) was observed in presence of Tween-80 and dioctyl sodium sulfosuccinate individually at 40mg/g wheat bran concentration. Under solid state fermentation biotin (0.0025mg/g wheat bran); Ca2+ (0.05mg/g wheat bran) and 1-Naphthyl acetic acid (0.01mg/g wheat bran) also showed some inducing effect on the synthesis of the enzyme protease by solid state fermentation.

A study of energetics of cooperative interaction using a mutant lambda-repressor.

A lambda-repressor mutant, S228N, which is defective in tetramer formation in the free state but retains full cooperativity, was studied in detail. Isolated single operator-bound S228N repressor shows association properties similar to those of the wild-type repressor. Fluorescence anisotropy studies with dansyl chloride-labeled repressor show a dimer-monomer dissociation constant of around 10(-5) M. The structure of the mutant repressor was studied by circular dichroism, acrylamide quenching and sulfhydryl reactivity at protein concentrations of < or =10(-6) M, where it is predominantly monomeric. The results suggest no significant perturbations in the structure of the S228N mutant repressor from that of the wild-type repressor. Urea denaturation studies also indicate no significant change in the stability of the repressor. The results were used to calculate energetics of loop formation in the cooperative binding process.

Sulfhydryls of tubulin. A probe to detect conformational changes of tubulin.

The 20 cysteine residues of tubulin are heterogeneously distributed throughout its three-dimensional structure. In the present work, we have used the reactivity of these cysteine residues with 5, 5'-dithiobis(2-nitrobenzoic acid) (DTNB) as a probe to detect the global conformational changes of tubulin under different experimental conditions. The 20 sulfhydryl groups can be classified into two categories: fast and slow reacting. Colchicine binding causes a dramatic decrease in the reactivity of the cysteine residues and causes complete protection of 1.4 cysteine residues. Similarly, other colchicine analogs that bind reversibly initially decrease the rate of reaction; but unlike colchicine they do not cause complete protection of any sulfhydryl groups. Interestingly, in all cases we find that all the slow reacting sulfhydryl groups are affected to the same extent, that is, have a single rate constant. Glycerol has a major inhibitory effect on all these slow reacting sulfhydryls, suggesting that the reaction of slow reacting cysteines takes place from an open state at equilibrium with the native. Ageing of tubulin at 37 degrees C leads to loss of self-assembly and colchicine binding activity. Using DTNB kinetics, we have shown that ageing leads to complete protection of some of the sulfhydryl groups and increased reaction rate for other slow reacting sulfhydryl groups. Ageing at 37 degrees C also causes aggregation of tubulin as indicated by HPLC analysis. The protection of some sulfhydryl groups may be a consequence of aggregation, whereas the increased rate of reaction of other slow reacting sulfhydryls may be a result of changes in global dynamics. CD spectra and acrylamide quenching support such a notion. Binding of 8-anilino-1-naphthalenesulfonate (ANS) and bis-ANS by tubulin cause complete protection of some cysteine residues as indicated by the DTNB reaction, but has little effect on the other slow reacting cysteines, suggesting local effects.

NBD-isocolcemid-tubulin interaction: a novel one-step reaction involving no conformational adjustment of reactants.

Isocolcemid, a colcemid analogue in which the positions of the C-ring methoxy and carbonyl are exchanged, is virtually inactive in binding to tubulin and inhibiting the formation of microtubule assembly. We have found that the substitution of a NBD group in the side chain of the B-ring of isocolcemid can reverse the effect of these structural alterations (at the C-ring) and the newly synthesized NBD-isocolcemid restores the lost biological activity. It inhibits microtubule assembly with an IC(50) of 12 microM and competes efficiently with [(3)H]colchicine, for binding to tubulin. NBD-isocolcemid has two binding sites on tubulin; one is characterized by fast binding, whereas the binding to the other site is slow. These two sites are independent and unrelated to each other. Colchicine and its analogues compete with NBD-isocolcemid for the slow site. Association and dissociation rate constants for the fast site, obtained from the stopped-flow measurements, are (7.37 +/- 0. 70) x 10(5) M(-1) s(-1) and 7.82 +/- 2.74 s(-1), respectively. While the interaction of colchicine and its analogues with tubulin involves two steps, NBD-isocolcemid binding to tubulin at the slow site has been found to be a one-step reaction. This is evident from the linear dependence of the observed rate constant (k(obs)) with both NBD-isocolcemid and tubulin concentrations. The interaction of NBD-isocolcemid with tubulin does not involve the conformational change of NBD-isocolcemid, as is evident from the unchanged CD spectra of the drug. The absence of enhanced GTPase activity of tubulin and the native-like protease cleavage pattern of the NBD-isocolcemid-tubulin complex suggest an unaltered conformation of tubulin upon NBD-isocolcemid binding to it as well. Implications of this on the mechanism of polymerization inhibition have been discussed.

Purification and characterization of a thermostable alpha-amylase from Bacillus stearothermophilus.

A soil isolate of Bacillus stearothermophilus was found to synthesize thermostable alpha-amylase. The enzyme was purified to homogeneity by ammonium sulfate fractionation and IECC on DEAE-cellulose column. The purified enzyme was considered to be a monomeric protein with a molar mass of 64 kDa, as determined by SDS-PAGE. The enzyme showed a wide range of pH tolerance and maximum activity at pH 7.0. The temperature tolerance was up to 100 degrees C with more than 90% catalytic activity; the maximum activity was observed at 50 degrees C. Divalent metal ions exhibited inhibitory effect on the enzyme activity. However, proteinase inhibitor did not react positively.

Nucleotide-dependent bisANS binding to tubulin.

Non-covalent hydrophobic probes such as 5, 5'-bis(8-anilino-1-naphthalenesulfonate) (bisANS) have become increasingly popular to gain information about protein structure and conformation. However, there are limitations as bisANS binds non-specifically at multiple sites of many proteins. Successful use of this probe depends upon the development of binding conditions where only specific dye-protein interaction will occur. In this report, we have shown that the binding of bisANS to tubulin occurs instantaneously, specifically at one high affinity site when 1 mM guanosine 5'-triphosphate (GTP) is included in the reaction medium. Substantial portions of protein secondary structure and colchicine binding activity of tubulin are lost upon bisANS binding in absence of GTP. BisANS binding increases with time and occurs at multiple sites in the absence of GTP. Like GTP, other analogs, guanosine 5'-diphosphate, guanosine 5'-monophosphate and adenosine 5'-triphosphate, also displace bisANS from the lower affinity sites of tubulin. We believe that these multiple binding sites are generated due to the bisANS-induced structural changes on tubulin and the presence of GTP and other nucleotides protect those structural changes.

Amino acid changes in the repressor of bacteriophage lambda due to temperature-sensitive mutations in its cI gene and the structure of a highly temperature-sensitive mutant repressor.

The mutant cIts genes from seven different lambdacIts phages carrying tsU50, tsU9, tsU46, ts1, tsU51, tsI-22 and ts2 mutations were cloned in plasmid. The positions of these mutations and the resulting changes of amino acids in the repressor were determined by DNA sequencing. The first four mutations mapping in the N-terminal domain show the following changes: I21S, G53S, A62T and V73A, respectively. Of the three remaining mutations mapping in the C-terminal domain, cItsI-22 and cIts2 show N207T and K224E substitutions respectively, while the mutant cItsU51 gene carries F141I and P153L substitutions. Among these ts repressors, CIts2 having the charge-reversal change K224E was overexpressed from tac promoter in a plasmid and purified, and its structure and function were studied. Operator-binding studies suggest that the ts2 repressor is somewhat defective in monomer-dimer equilibrium and/or cooperativity even at permissive temperatures and loses its operator-binding ability very rapidly above 25 degrees C. Comparative studies of fluorescence and CD spectra, sulfhydryl group reactivity and elution behaviour in size-exclusion HPLC of both wild-type and ts2-mutant repressors at permissive and non-permissive temperatures suggest that the C-terminal domain of the ts2 repressor carrying a K224E substitution has a structure that does not favor tetramer formation at non-permissive temperatures.

Antibiotic resistance--a close look.

The discovery of antibiotic greatly improved the human health care system. But today we are facing the problem of antibiotic resistance due to uncontrolled use of the compound. This can be by passed by controlled use of antibiotic and formulation of newer ones.

Optimization of n variable biological experiments by evolutionary operation-factorial design technique.

Since the traditional approach to optimization of biological systems based on the single variable search technique is incapable of detecting the true optimum when a number of chemicals are used together as inducers, the Evolutionary Operation (EVOP)-factorial design technique was applied successfully to optimize the concentrations of vitamin (biotin), metal ion (CaCl(2)) and plant hormone (NAA) for maximizing enzyme (protease) production by Rhizopus oryzae (RO IIT RB-13, NRRL-21498) in solid state fermentation. Addition of inducers to an optimal level (biotin-2.5 ppm, CaCl(2)-20 ppm and NAA-12.5 ppm) resulted in a approximately 1.9-fold increase in protease production.

Chaperone-like activity of tubulin.

Tubulin, a ubiquitous protein of eukaryotic cytoskeleton, is a building block unit of microtubule. Although several cellular processes are known to be mediated through the tubulin-microtubule system, the participation of tubulin or microtubule in protein folding pathway has not yet been reported. Here we show that goat brain tubulin has some functions and features similar to many known molecular chaperones. Substoichiometric amounts of tubulin can suppress the non-thermal and thermal aggregation of a number of unrelated proteins such as insulin, equine liver alcohol dehydrogenase, and soluble eye lens proteins containing beta- and gamma-crystallins. This chaperone-like activity of tubulin becomes more pronounced as temperature increases. Aging of tubulin solution at 37 degreesC also enhances its chaperone-like activity. Tubulin loses its chaperone-like activity upon removal of its flexible hydrophilic C-terminal tail. These results suggest that both electrostatic and hydrophobic interactions are important in substrate binding by tubulin and that the negatively charged C-terminal tails play a crucial role for its chaperone-like activity.