Independency of anti-HIV-1 activity from ribosome-inactivating activity of trichosanthin

Trichosanthin (TCS) is a type I ribosome-inactivating (RI) protein possessing multiple biological and pharmacological activities. Its major action is inhibition of human immunodeficiency virus (HIV) replication but the mechanism is still elusive. All evidences showed that this action is related to its RI activity. Previous studies found that TCS mutants with reduced RI activity simultaneously lost some anti-HIV activity. In this study, an exception was demonstrated by two TCS mutants retaining almost all RI activity but were devoid of anti-HIV-1 activity. Five mutants were constructed by using site-directed mutagenesis with either deletion or addition of amino acids to the C-terminal sequence. Results showed that the RI activity of mutants with C-terminal deletion mutants (TCS(C2), TCS(C4), and TCS(C14)) decreased by 1.2-3.3-fold with parallel downshifting of its anti-HIV-1 activity (1.4-4.8-fold). Another two mutants, TCS(C19aa) and TCS(KDEL) having 19 amino acid extension and a KDEL signal sequence added to the C-terminal sequence, retained all RI activity but subsequently lost most of the anti-HIV-1 activity. These findings suggested that ribosome inactivation alone might not be adequate to explain the anti-HIV action of TCS.     

Evoking spontaneous activity

In this issue of Neuron, MacLean et al. report that thalamic stimulation can evoke spatiotemporal sequences of neocortical neuronal activity similar to spontaneous UP states. This could suggest that the patterns of activity associated with the processing of sensory input might be replayed during spontaneous activity.     

Influence of memory demand and contra lateral activity on muscle activity.

Computer mouse work often includes memory demands and contra lateral activity. This study simulated video display unit (VDU) mouse-work and the focus was on forearm muscle activity as a result of standardised postural loads, memory demands and contra lateral activity. Surface and intramuscular electromyography (EMG) were recorded from the right forearm muscles during finger elevation and rest with and without memory demands and with and without contra lateral activity i.e. activity of the left hand. In most situations, memory demand increased activity in the m. extensor carpi radialis brevis and m. flexor digitalis superficialis. Also contra lateral activity increased activity in situations with and without memory demands. While surface EMG level of the m. extensor digitorum communis did not increase during memory demands, intramuscular EMG level increased when memory demands and contra lateral activity was combined. Influence of memory demands and contra lateral activity were most pronounced, in situations where activity levels were small.We presume that it is not only prolonged time of active computer mouse use that is a risk for development of musculoskeletal disorders, but also the time when people interact with the computer mentally or with the 'non-mouse hand', while resting their 'mouse-hand' on the mouse.     

A special deoxyribonuclease activity accompanying competence-inducing activity

A deoxyribonuclease activity accompanies the competence substance isolated from transformableDiplococcus pneumoniae even in well purified fractions. The deoxyribonuclease seems to exhibit a rather different kind of activity from the one found as a major nuclease in a partially purified competence substance. The products of interaction between the enzyme and double-stranded DNA would indicate that the enzyme might act as an “unwindase” on the double-stranded DNA.     

Contact-dependent hemolytic activity distinct from deforming activity of Bartonella bacilliformis

Although Bartonella bacilliformis causes a severe anemia in humans, this study presents the first report of hemolytic activity by B. bacilliformis. The activity was not apparent in culture supernatants but was reliably detected when B. bacilliformis cells were centrifuged onto erythrocytes prior to incubation. Abrogation of hemolytic activity by proteinase K treatment suggested the hemolysin was a Bartonella protein. Even though hemolysis required relatively long incubation times, de novo protein synthesis was not required to produce the protein. A preparation containing factors released by B. bacilliformis, including deformin, a B. bacilliformis protein able to induce pits and invaginations in erythrocyte membranes, had some ability to lyse erythrocytes. However, pre-deformed erythrocytes did not lyse faster or to a greater extent than control erythrocytes after the addition of B. bacilliformis cells. Inhibition of deformation caused by B. bacilliformis cells with the erythrocyte ATPase inhibitor, vanadate, did not affect hemolytic activity. This study suggests hemolytic activity and deforming activity are attributable to different B. bacilliformis proteins.     

CRAMP analog having potent antibiotic activity without hemolytic activity

CRAMP-18 is an 18-residue functional region, corresponding to residues 16-33 of a mouse-derived antibiotic peptide CRAMP. To develop novel antibiotic peptides possessing strong antibiotic activity against bacterial, fungal and tumor cells without hemolytic activity, three analogs of CRAMP-18 were synthesized containing either Leu- or Lys-substitution. Leu-substitution ([L(1, 8)]-CRAMP-18) in the hydrophobic helix face of CRAMP-18 induced a dramatic increase in antibiotic activity without a significant increase in hemolytic activity. Lys-substitution ([K(2, 13)]-CRAMP-18 or [K(9, 16)]-CRAMP-18) in the hydrophilic helix face produced a smaller response. Therefore, [L(1, 8)]-CRAMP-18 may be an attractive candidate for developing novel peptide antibiotics.     

Separation of toxic activity and ADP-ribosylation activity of botulinum neurotoxin D

Neurotoxin from Clostridium botulinum type D strain South African (neurotoxin D) has shown ADP-ribosylation activity as well as toxic activity (Matsuoka, I., Sakuma, H., Syuto, B., Moriishi, K., Kubo, S., and Kurihara, K. (1989) J. Biol. Chem. 264, 706-712). Separation of these activities from each other was attempted by means of gel filtration, hydroxylapatite column chromatography, or immunoaffinity chromatography. Approximately 90% of toxic activity was recovered in each chromatography. Although ADP-ribosylation activity was incompletely separated from neurotoxin D by gel filtration, it was separated by hydroxylapatite column chromatography. In immunoaffinity chromatography with a column of Sepharose 4B coupling antibodies against botulinum ADP-ribosyltransferase, no ADP-ribosylation activity was detected by autoradiography in the unabsorbed toxic fraction. These results indicate that neurotoxin D does not have ADP-ribosylation activity.     

Units of enzyme activity

Units of enzymes activity, recommended by the Nomenclature Committee on enzymes of the International Union of Biochemistry are described the unit E (U), introduced in 1961 and its derivatives: specific activity, molecular (molar) activity, enzyme catalytic centre activity, enzyme solution concentration; the unit catal, introduced in 1972 and its derivatives. Information presented is essential to ensure correct expression of enzyme activity.     

Proteolytic activity of largomycin

Largomycin, an antibiotic and antitumor protein, purified from the culture broth of Streptomyces pluricolorescens, displayed specific proteolytic activity. Pure largomycin did not degrade a number of substrates commonly used for detection of aminopeptidase, endopeptidase and carboxypeptidase activity. Pure largomycin degraded angiotensin II, bradykinin, a few dipeptides and a number of proteins of KB cell plasma membranes. The biological activity and the proteolytic activity of largomycin showed similar temperature-dependent patterns, suggesting that one protein is responsible for both activities. The apoprotein of largomycin, which did not show antibiotic activity, contained the proteolytic activity.     

Synthesis and neuroprotective activity of bergenin derivatives with antioxidant activity

Norbergenin, which is the O-demethyl derivative of bergenin, the main component of Mallotus japonicus, has been found to show moderate antioxidant activity (IC(50) 13 microM in DPPH radical scavenging; 32 microM in superoxide anion scavenging). Modification of sugar part on norbergenin by coupling with a variety of fatty acids was employed for increasing its antioxidant activity. Selective esterification of hydroxyl groups on the sugar part enhanced greatly antioxidant activity. The most potent one is norbergenin 11-caproate, which not only exhibits stronger antioxidant activity than that of catechin but also prevents neuronal death at 10 microM on the primary culture of rat cortical neurons in DMEM supplemented with N2.     

Determination of ITPase activity in erythrocyte lysates obtained for determination of TPMT activity

The indication for the determination of both thiopurine methyltransferase (TPMT) and inosine triphosphate pyrophosphohydrolase is identical (i.e., adverse drug reactions toward mercaptopurines). Therefore, we tested whether or not our standard procedure to prepare erythrocyte lysates for measurement of TPMT activity, which includes treatment with Chelex 100 (a chelating resin), was suitable for the measurement of ITPase activity. It also was tested to see if ITPase activity differs in EDTA and Heparin anti-coagulated blood samples. We found that there was no difference between the ITPase activity in erythrocyte lysates prepared from EDTA or Heparin anti-coagulated blood. Treatment with a chelating resin or omission of magnesium from the assay procedure resulted in decreased and nearly absent ITPase activity, respectively. We conclude that untreated erythrocyte lysates obtained for determination of TPMT activity are suitable for determination of ITPase activity. However, after treatment with Chelex 100 the erythrocyte lysates become unsuitable for determination of ITPase activity.     

Serotonin and motor activity

The activity of brain serotonergic neurons in both the pontine-mesencephalic and medullary groups is positively correlated with the level of behavioral arousal and/or the behavioral state. This, in turn, appears to be related to the level of tonic motor activity, especially as manifested in antigravity muscles and other muscle groups associated with gross motor activity. In addition, a subset of serotonergic neurons displaysa further increase in activity in association with repetitive, central pattern generator mediated responses. Accumulating evidence indicates that this relation to motor activity is related both to the co-activation of the sympathetic nervous system and to the modulation of afferent inputs.     

Water activity and substrate concentration effects on lipase activity

Catalytic activity of lipases (from Rhizopus arrhizus, Canadida rugosa, and Pseudomonas sp. was studied in organic media, mainly diisopropyl ether. The effect of water activity (a(w)) on V(max) showed that the enzyme activity in general increased with increasing amounts of water for the three enzymes. This was shown both for esterification and hydrolysis reactions catalyzed by R. arrhizus lipase. In the esterification reaction the K(m) for the acid substrate showed a slight increase with increasing water activities. On the other hand, the K(m) for the alcohol substrate increased 10-20-fold with increasing water activity. The relative changes in K(m) were shown to be independent of the enzyme studied and solvent used. The effect was attributed to the increasing competition of water as a nucleophile for the acyl-enzyme at higher water activities. In a hydrolysis reaction the K(m) for the ester was also shown to increase as the water activity increased. The effect of water in this case was due to the fact that increased concentration of one substrate (water), and thereby increased saturation of the enzyme, will increase the apparent K(m) of the substrate (ester) to be determined. This explained why the hydrolysis rate decreased with increasing water activity at a fixed, low ester concentration. The apparent V(max) for R. arrhizus lipase was similar in four of six different solvents that were tested; exceptions were toulene and trichloroethylene, which showed lower values. The apparent K(m) for the alcohol in the solvents correlated with the hydrophobicity of the solvent, hydrophobic solvents giving lower apparent K(m). (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 798-806, 1997.     

Protective activity of 28-kDa 1-Cys peroxiredoxin determines its peroxidase activity]

The 28 kDa peroxiredoxin from rat exhibited peroxidase activity only in the presence of dithiothreitol. Both organic and nonorganic peroxidases were found to be substrates for the 28-kDa peroxiredoxin activity. Analysis of the protective antioxidant activity of the 28-kDa peroxiredoxin revealed that it is accounted for by its peroxidase activity.     

Amylase activity inPichia polymorpha

The amylase activity ofPichia polymorpha was studied during growth. The localization of activity in the cell and the general properties of the enzyme are described. Two types of activity were observed but they could not be ascribed to two distinct enzymes.     

Vision drives correlated activity without patterned spontaneous activity in developing Xenopus retina

Developing amphibians need vision to avoid predators and locate food before visual system circuits fully mature. Xenopus tadpoles can respond to visual stimuli as soon as retinal ganglion cells (RGCs) innervate the brain, however, in mammals, chicks and turtles, RGCs reach their central targets many days, or even weeks, before their retinas are capable of vision. In the absence of vision, activity-dependent refinement in these amniote species is mediated by waves of spontaneous activity that periodically spread across the retina, correlating the firing of action potentials in neighboring RGCs. Theory suggests that retinorecipient neurons in the brain use patterned RGC activity to sharpen the retinotopy first established by genetic cues. We find that in both wild type and albino Xenopus tadpoles, RGCs are spontaneously active at all stages of tadpole development studied, but their population activity never coalesces into waves. Even at the earliest stages recorded, visual stimulation dominates over spontaneous activity and can generate patterns of RGC activity similar to the locally correlated spontaneous activity observed in amniotes. In addition, we show that blocking AMPA and NMDA type glutamate receptors significantly decreases spontaneous activity in young Xenopus retina, but that blocking GABA(A) receptor blockers does not. Our findings indicate that vision drives correlated activity required for topographic map formation. They further suggest that developing retinal circuits in the two major subdivisions of tetrapods, amphibians and amniotes, evolved different strategies to supply appropriately patterned RGC activity to drive visual circuit refinement.     

"Matrigenin" activity from bovine bone--I. Partial purification of activity

1. Bovine bone contains an extractable activity which stimulated the synthesis of glycosaminoglycans by bovine synovial, human synovial and mouse 3T3 fibroblastic cells in culture. Human cells were used to develop an assay for purification of the stimulatory activity ("matrigenin" activity) from bovine bone. 2. Partial purification of "matrigenin" activity was achieved by precipitation of the EDTA extract at pH 3.5 and Sepharose CL-6B chromatography in 4 M guanidinium HCl. Dissociative conditions were necessary to prevent aggregation. 3. On SDS-polyacrylamide gel electrophoresis the activity ran with a mobility equivalent to a Mr = 27,500 and could be recovered from the SDS gels.     

The kinase activity of PKR represses inflammasome activity

The protein kinase R (PKR) functions in the antiviral response by controlling protein translation and inflammatory cell signaling pathways. We generated a transgenic, knock-in mouse in which the endogenous PKR is expressed with a point mutation that ablates its kinase activity. This novel animal allows us to probe the kinase-dependent and -independent functions of PKR. We used this animal together with a previously generated transgenic mouse that is ablated for PKR expression to determine the role of PKR in regulating the activity of the cryopyrin inflammasome. Our data demonstrate that, in contradiction to earlier reports, PKR represses cryopyrin inflammasome activity. We demonstrate that this control is mediated through the established function of PKR to inhibit protein translation of constituents of the inflammasome to prevent initial priming during innate immune signaling. These findings identify an important role for PKR to dampen inflammation during the innate immune response and caution against the previously proposed therapeutic strategy to inhibit PKR to treat inflammation.     

Lactase activity of microorganisms

Sixty-two strains of yeasts, molds and bacteria were screened for lactase (β-D-galactosidase) activity. Strains exhibiting the enzyme activity were evaluated for cell yield as well as enzyme units available per litre of the medium, per g cell dry weight and per mg protein of their cell-free extracts. The molds exhibited lowest enzyme activity but highest cell yields, bacteria produced lowest cell yield and maximum enzyme activity. Cultures exhibiting very high activity among yeasts wereSaccharomyces fragilis (strain 3217) and among bacteriaStreptococcus cremoris (strain H),Lactobacillus bulgaricus (strain RTS and 1373) andLeuconostoc citrovorum (strain 8081).