Purification and properties of a lambda operator-binding protein which is expected to be autorepressor (tof protein) from E. coli carrying lambdadv plasmid.

Summary In order to study the mode of action of the tof gene product, which is an autorepressor of the bacteriophage and plasmid dv, we have purified a DNA-binding protein which is specifically produced in bacteria carrying dv. This protein possesses characteristics expected for the product of the tof gene, since it is produced under conditions where cI-repressor is not made, and since it binds to oL and oR operators on the phage genome. The molecular weight of the native protein is 16,000–17,000 daltons, and the monomeric molecular weight as measured by gel electrophoresis in the presence of sodium dodecyl sulfate is about 10,000 daltons. Denaturation and renaturation experiments demonstrated that the native protein is a dimer of 10,000-dalton monomers. The DNA-specific binding protein is not produced in cells carrying i ²¹dv or 80dv.     

Random replication and random assortment model for plasmid incompatibility in bacteria

To understand the incompatibility between two related plasmids, both of which replicate in an autonomous state under a common control mechanism, we have developed a model that assumes a random choice mechanism for replication of plasmid copies and their random assortment into daughter cells upon cell division. Segregation kinetics by this model is analyzed mathematically and the number of generations required for segregation is calculated as a function of plasmid copy number per cell. The results obtained offer enough quantitative data to make our model reasonably realistic.     

Dynamic sweating response of man to infrared irradiation in various spectral regions

In an attempt to detect differences in the thermal effect of infrared irradiation of different wavelengths, transient sweating response to infrared irradiation in various spectral regions was examined. In Series 1, the ventral or dorsal surface of the nude subject was irradiated repetitively for a period of 4 min (2 min on, 2 min off) by each of three kinds of infrared heaters with main emissivity in near-infrared (NIR; 0.7–2.8 m), intermediate-infrared (MIR; 1.5–5.8 m), and far-infrared (FIR; 2.8–25 m) regions. The sweating response on a non-irradiated area tended to be the greatest with MIR, while the magnitude of the sweating response on the irradiated area showed no consistent differences among various wavelengths. The results infer that MIR stimulated cutaneous thomoreceptors most effectively, while its direct effect on local sweat gland activity was minimal. In Series 2, the effects of 9–12 min irradiations in more restricted ranges of wavelength were compared by the combination of the three kinds of heaters with filters (translucent to wavelength ranges of 1.3–2.7, 2.7–3.5, 3.6–8.0 m, respectively). The sweating response on a remote area was predominantly greater with the range of 2.7–3.5 m than with the other wavelength ranges, while the local effect on sweating was minimal with this range. The results of Series 2 reinforce those of Series 1, indicating that the degree of stimulation of cutaneous thermoreceptors and of direct thermal effect on sweat gland activity differ with spectral regions incident on the skin, thus affecting local and remote effects on the sweating response.     

Potato and rabbit muscle phosphorylases: comparative studies on the structure,function and regulation of regulatory and nonregulatory enzymes

Summary Phosphorylases (EC 2.4.1.1) from potato and rabbit muscle are similar in many of their structural and kinetic properties, despite differences in regulation of their enzyme activity. Rabbit muscle phosphorylase is subject to both allosteric and covalent controls, while potato phosphorylase is an active species without any regulatory mechanism. Both phosphorylases are composed of subunits of approximately 100 000 molecular weight, and contain a firmly bound pyridoxal 5-phosphate. Their actions follow a rapid equilibrium random Bi Bi mechanism. From the sequence comparison between the two phosphorylases, high homologies of widely distributed regions have been found, suggesting that they may have evolved from the same ancestral protein. By contrast, the sequences of the N-terminal region are remarkably different from each other. Since this region of the muscle enzyme forms the phosphorylatable and AMP-binding sites as well as the subunit-subunit contact region, these results provide the structural basis for the difference in the regulatory properties between potato and rabbit muscle phosphorylases. Judged from CD spectra, the surface structures of the potato enzyme might be significantly different from that of the muscle enzyme. Indeed, the subunit-subunit interaction in the potato enzyme is tighter than that in the muscle enzyme, and the susceptibility of the two enzymes toward modification reagents and proteolytic enzymes are different. Despite these differences, the structural and functional features of the cofactor, pyridoxal phosphate, site are surprisingly well conserved in these phosphorylases. X-ray crystallographic studies on rabbit muscle phosphorylase have shown that glucose-1-phosphate and orthophosphate bind to a common region close to the 5-phosphate of the cofactor. The muscle enzyme has a glycogen storage site for binding of the enzyme to saccharide substrate, which is located away from the cofactor site. We have obtained, in our reconstitution studies, evidence for binding of saccharide directly to the cofactor site of potato phosphorylase. This difference in the topography of the functional sites explains the previously known different specificities for saccharide substrates in the two phosphorylases. Based on a combination of these and other studies, it is now clear that the 5-phosphate group of pyridoxal phosphate plays a direct role in the catalysis of this enzyme. Information now available on the reaction mechanism of phosphorylase is briefly described.     

Stimulative effect of serum on the growth of HeLa cells suppressed in a K+-deficient chemically defined medium

Growth of HeLa cells cultured with a chemically defined medium was slightly stimulated in the presence of 5% dialyzed calf serum. The growth-promoting action of serum was more conspicuous when cell growth was suppressed in the same medium, in which K⁺ was replaced by Rb⁺ to various ratios. The growth-promoting factors(s) of serum was heat-labile. Upon addition of dialyzed serum, passive K⁺ or Rb⁺ influx was increased, whereas the active cation uptake was unaffected and cell K⁺ was rather decreased. The serum did not alter uptake of [³H] amino acids. Also, protein synthesis inhibited in the Rb⁺-substituted medium was not stimulated significantly, except that observed only when the external K⁺/Rb⁺ ratio was $\text{1}\text{5}$. From the distinct effects of serum on cell growth and protein synthesis, we conclude that (i) the serum-induced stimulation of cell growth, which is suppressed in the Rb⁺-substituted medium, is not a result of the direct effect of serum on synthesis of bulk protein, but a reflection of the effect on another mechanism(s) required for cell growth; and that (ii) this action is basically identical with the growth-promoting action on cells cultured in the normal medium.     

Asymmetrical radical formation in D- and L-alanines irradiated with yttrium-90β-rays

Radical formation in^{9 0}Y--irradiated D- and L-alanines was studied using ESR. It was observed that the relative radical concentration by -irradiation was distinguishably (13.9–21.5%) more in D-alanine than in L-alanine. Discussion was made on the possible mechanisms for the observed results.     

The variable refractivity of the protein or polypeptide hormone-producing cells showing a unique luminescence in the dark-field microscope

Synopsis When cryostat sections of endocrine tissue were examined in a dark-field microscope, a brilliant granular luminescence was revealed in the endocrine cells thought to be concerned with protein or polypeptide hormone production. The sections were prepared from fresh materials either frozen in a cryostat chamber at –25°C, in dry ice-acetone, or fixed in formalin-calcium for 24 hr. The neurosecretory substance in the hypothalamus and the posterior lobe of the pituitary showed a blue luminescence; the acidophil cells of the anterior lobe of the pituitary, orange; basophil cells, green or blue; intermediate lobe cells, no luminescence; thyroid C cells, white-blue; pancreatic A cells, blue; B cells, orange; adrenomedullary cells, greenish blue; enterochromatin cells, green; and other endocrine cells in the gastrointestinal tract, blue or orange. After tearing and spreading the pituitary and hypothalamus with a pair of needles on a glass slide, and examining the teased specimen by dark-field microscopy, various cells of different luminescent colours became apparent in the anterior lobe of the pituitary, a blue fluorescent substance in the posterior lobe, and neurosecretory cell bodies in the hypothalamus. The different colours appear to be inherent in the granules of living tissues.     

Electrochemical detection of nucleic base mismatches with ferrocenyl naphthalene diimide

Electrochemical detection of nucleic base mismatches was attempted successfully with ferrocenyl naphthalene diimide (FND) in a model system with 20-meric double-stranded oligonucleotides with or without a mismatch(es). Thus, dA(20) or a 20-meric sequence of the lac Z gene was immobilized on a gold electrode and complementary oligonucleotides with different numbers of mismatches were allowed to hybridize in the presence of FND to give rise to an electrochemical signal. The signal intensity varied depending on the number of unpaired bases on the DNA duplex. From experiments with a quartz crystal microbalance, eight molecules of FND were found to bind to the 20-meric double-stranded oligos and this number decreased as the number of mismatches increased. These findings were further supported by matrix-assisted laser desorption ionization time-of-flight mass spectroscopy. This novel method will be useful for the analysis of single-nucleotide polymorphisms present on human genes.     

Effect of recent changes in atomic bomb survivor dosimetry on cancer mortality risk estimates

The Radiation Effects Research Foundation has recently implemented a new dosimetry system, DS02, to replace the previous system, DS86. This paper assesses the effect of the change on risk estimates for radiation-related solid cancer and leukemia mortality. The changes in dose estimates were smaller than many had anticipated, with the primary systematic change being an increase of about 10% in gamma-ray estimates for both cities. In particular, an anticipated large increase of the neutron component in Hiroshima for low-dose survivors did not materialize. However, DS02 improves on DS86 in many details, including the specifics of the radiation released by the bombs and the effects of shielding by structures and terrain. The data used here extend the last reported follow-up for solid cancers by 3 years, with a total of 10,085 deaths, and extends the follow-up for leukemia by 10 years, with a total of 296 deaths. For both solid cancer and leukemia, estimated age-time patterns and sex difference are virtually unchanged by the dosimetry revision. The estimates of solid-cancer radiation risk per sievert and the curvilinear dose response for leukemia are both decreased by about 8% by the dosimetry revision, due to the increase in the gamma-ray dose estimates. The apparent shape of the dose response is virtually unchanged by the dosimetry revision, but for solid cancers, the additional 3 years of follow-up has some effect. In particular, there is for the first time a statistically significant upward curvature for solid cancer on the restricted dose range 0-2 Sv. However, the low-dose slope of a linear-quadratic fit to that dose range should probably not be relied on for risk estimation, since that is substantially smaller than the linear slopes on ranges 0-1 Sv, 0-0.5 Sv, and 0- 0.25 Sv. Although it was anticipated that the new dosimetry system might reduce some apparent dose overestimates for Nagasaki factory workers, this did not materialize, and factory workers have significantly lower risk estimates. Whether or not one makes allowance for this, there is no statistically significant city difference in the estimated cancer risk.