Voltage-dependent capacitance as a probe for albumin adsorption onto a solid surface

The process of adsorption of bovine serum albumin onto a platinum electrode was monitored through the measurement of a nonlinear electrochemical property. The principle of the new method is that a sinusoidal voltage source is applied to a test solution and the waveform of the output current is analyzed by Fourier transformation. It was found that the intensities of the higher harmonics in the Fourier transformation change depending on the concentration of albumin and with time. From the higher harmonics, voltage dependence of the capacitance was quantitatively evaluated. The change of the state of albumin adsorbed onto the platinum plate was also monitored from the pattern of 'crack' of adsorbed albumin by using scanning electron microscopy. These results were discussed in relation to the mechanism of bimodal adsorption of albumin.     

Developmental change of an enzyme activity oxidizing γ-aminobutyraldehyde to γ-aminobutyric acid in the chick embryonic brain

An enzyme activity oxidizing -aminobutyraldehyde (ABAL) to GABA reflecting an alternative pathway for GABA synthesis was assayed in the developing chick embryonic brain and was compared with glutamate decarboxylase (GAD) activity. An enzyme activity oxidizing ABAL to GABA showed almost constant level during development in the chick embryonic brain, and was present at low levels compared with GAD activity. The results indicate that GABA synthesis via an alternative pathway is always much less than synthesis via the GAD-dependent pathway in the developing chick embryonic brain.     

Analysis of chromosome-sized DNA from the bacterial genome of thermophilic Campylobacter laridis by pulsed-field gel electrophoresis and physical mapping.

Three restriction enzymes ApaI, SalI and SmaI, among nine enzymes tested, were found to produce distributions of DNA fragments which were useful for analysis of chromosome-sized DNA from thermophilic Campylobacter laridis by pulsed-field gel electrophoresis. From experiments with C. laridis JCM2530T and four isolates of C. laridis, the size of the genome of C. laridis was calculated to range from 1,590 to 1,700 kb, with a mean of 1,640 kb. An SmaI restriction map was derived by the partial digestion of the DNA from C. laridis JCM2530T.     

Molecular cloning of feline interferon cDNA by direct expression.

A cDNA sequence coding for feline interferon has been cloned for the first time by screening a cDNA library constructed using Okayama-Berg vector and mRNA derived from the feline cells (LSA-I) induced by TPA (12-o-tetradecanoylphorbor 13-acetate) for the ability of transient expression to produce feline interferon in COS1 monkey cells. The amino acid sequence, deduced from the nucleotide sequence by comparing it with the sequences of other mammalian IFNs, consists of 171 amino acids with 6 cysteins and an N-glycosylation site at the amino acid position 79, and has about 60% homology to human IFN alpha 1. The interferon was partially purified through Blue Sepharose, and its molecular weight was estimated to be 2.4 x 10(4). The antiviral activity was acid stable, and glycosylation was suggested.     

Metabolic response to treatment with cold, paraquat, or 3-amino-1,2,4-triazole in leaves of winter wheat.

We treated leaves of winter wheat (Triticum aestivum L.) with cold, paraquat, or 3-amino-1,2,4-triazole and compared the responses. We assayed the activities of glucose-6-phosphate dehydrogenase, catalase, dehydroascorbate reductase and ascorbate free radical reductase and levels of hydrogen peroxide, glucose-6-phosphate, fructose-6-phosphate, ascorbate, dehydroascorbate, reduced and oxidized glutathione. With any of the three treatments, contents of cellular peroxides and hexose phosphates were raised. The content of ascorbate was lowered markedly by paraquat treatment, which produces active oxygen species, whereas such a decrease did not occur in other two treatments. When the plants were treated with 3-amino-1,2,4-triazole, which is a specific inhibitor of catalase, the content of oxidized glutathione increased severalfold. The glucose-6-phosphate dehydrogenase activity increased with all three treatments, but it decreased after glyphosate treatment, which does not stimulate the formation of peroxides. The activities of catalase and dehydroascorbate reductase were increased by the treatment of cold and paraquat, while 3-amino-1,2,4-triazole did not affect the dehydroascorbate reductase activity. The activity of ascorbate free radical reductase increased after treatment by paraquat only.     

Human liver manganese superoxide dismutase. Purification and crystallization, subunit association and sulfhydryl reactivity

Manganese superoxide dismutase (Mn-SOD) has been purified with a high yield (320 mg) from human liver (2 kg) and crystallized. Low-angle laser light scattering of the enzyme has shown that native enzyme is a tetrametic form. Four of the eight cysteine residues in the tetramer reacted with 5,5'-dithiobis(2-nitrobenzoic acid) or with iodoacetamide. The others were only reactive in protein heated with SDS or urea after reduction with dithiothreitol or 2-mercaptoethanol. The reactive sulfhydryl group was found to be located at Cys196 by amino acid sequence analysis of Nbs2-reactive peptides isolated by activated thiol-Sepharose covalent chromatography. Incubation of Mn-SOD in 1% SDS for 2 or 3 days at 25 degrees C or 5 min at 100 degrees C gave material showing two prominent components on polyacrylamide gel electrophoresis in the presence of 0.1% SDS. The major component had a molecular mass of 23 kDa; the other, 25 kDa. Reduction of the protein by dithiothreitol or 2-mercaptoethanol heated in SDS produced only the 25-kDa monomer species. Essentially, no thiol groups were detected in the 23-kDa form, in which two cysteine residues appear to have been oxidized to form an intrasubunit disulfide. This indicates that Cys196 has a reactive sulfhydryl and appears to be a likely candidate for a mixed disulfide formation in vivo.     

Neuromuscular blocking in acutely tetanus intoxicated mice

The effects of tetanus toxin on neuromuscular transmission of mice in acute intoxication produced by intravenous injection of a large amount of the toxin were examined by (1) recording the phrenic nerve impulses, the electromyograms (EMGs) of the diaphragm and the electrocardiograms, and (2) the evoked EMGs of the gastrocnemius muscle in response to electrical stimulation of the sciatic nerve. The evoked EMGs of the gastrocnemius muscle were analyzed in terms of kinetic and tonic components by their different latencies. Just before death of animals, the EMGs of the diaphragm appeared with some delay relative to the corresponding phrenic discharges. Finally, the EMG of the diaphragm disappeared even in the presence of phrenic discharge, but cardiac electrical activities continued. The amplitudes of the evoked EMGs of the gastrocnemius muscle invariably became low before death, but the muscle action potential could be recorded by direct muscle stimulation for several minutes after death. The latencies of the evoked EMGs were constant until about the middle of the survival time when the latencies suddenly became prolonged. The longer latency was the same as that of the tonic action potentials. Thus, in acutely tetanus-intoxicated mice, neuromuscular transmission was blocked rapidly and the kinetic component of the muscle was blocked earlier than the tonic component.     

TRF2-mediated ORC recruitment underlies telomere stability upon DNA replication stress

Telomeres are intrinsically difficult-to-replicate region of eukaryotic chromosomes. Telomeric repeat binding factor 2 (TRF2) binds to origin recognition complex (ORC) to facilitate the loading of ORC and the replicative helicase MCM complex onto DNA at telomeres. However, the biological significance of the TRF2–ORC interaction for telomere maintenance remains largely elusive. Here, we employed a TRF2 mutant with mutations in two acidic acid residues (E111A and E112A) that inhibited the TRF2–ORC interaction in human cells. The TRF2 mutant was impaired in ORC recruitment to telomeres and showed increased replication stress-associated telomeric DNA damage and telomere instability. Furthermore, overexpression of an ORC1 fragment (amino acids 244–511), which competitively inhibited the TRF2–ORC interaction, increased telomeric DNA damage under replication stress conditions. Taken together, these findings suggest that TRF2-mediated ORC recruitment contributes to the suppression of telomere instability.     

Promoting effects of 105K glycoprotein on cell proliferation in chicken lymphoblastoid cell lines

Abstract The promoting effects on cell proliferation of the 105K glycoprotein (105Kgp), purified from sera of chickens to which a Marek's disease (MD) lymphoblastoid cell line, MDCC-MSB1-41C (MSB1-41C), had been transplanted, were examined using culture cells from various sources. The MSB1-41C line as well as the other chicken lymphoblastoid cell lines examined were sensitive to the 105Kgp. The growth-promoting effects of 105Kgp showed a biphasic pattern depending upon the amount of 105Kgp added into the culture medium.
These findings indicate that the 105Kgp may be a promoting factor for chicken growing cells, especially lymphoblastoid cell lines.