A near-infrared method for studying hydration changes in aqueous solution: Illustration with protease reactions and protein denaturation

Proteins undergoing protease reactions, heat denaturation, or interactions with sodium dodecyl sulfate (SDS) were used to demonstrate the effectiveness of a near-infrared method for the quantitative study of changes in hydration or water binding during such processes. The spectra of different proteins showed that the liberation of $\text{COO}{}^{\mathrm{?}}\text{and NH}{}_3{}^{\mathrm{+}}$ groups during a protease reaction is associated with a large increase in hydration and excluded volume. On the basis of experiments with model compounds, other spectral changes, including development of continuum absorbance between 1.55 and 1.85 μm and a band with a peak near 2.1 μm, were also attributed to the liberation of these groups. After heat denaturation or in the presence of SDS, the rate of proteolytic hydrolysis was markedly increased, consistent with the view that some preliminary denaturation is necessary for protease activity. The validity of the hydration changes calculated for protease reactions was supported by model studies with l-lysine, and with poly-l-lysine before and after hydrolysis. The near-infrared spectrum of the protein substrate with no added protease was largely unaffected by heat treatment alone, indicating that the hydration as such was not changed to a large extent by the structural modifications of denaturation. In contrast to the protease reaction, the interactions between SDS and the proteins resulted in a decrease in hydration. Results of this paper are compared with those obtained from other methods. Some unique advantages of the near-infrared method for the study of hydration changes during reactions in aqueous solution are described.     

Uncoupling of mitochondrial oxidative phosphorylation by thallium.

The mechanism of integration of λ$\text{bio}$ll, which is deleted of all the known λ recombination genes, was studied using $\text{bio}$ deleted hosts as recipients. The presence of $\text{rec}$BC DNase and $\text{exo}$I in the recipient cells affected the fate of λ$\text{bio}$ll DNA. In nine of ten $\text{imm}\text{λ}{}^{\mathrm{+}}$ transductants, insertion of the λ$\text{bio}$ll genome took place somewhere between J and N and the remaining one had abnormally permuted prophage λ. In this lysogen (#42), the sequence of prophage genes was similar to that of vegetative phage λ. The properties of lysogen #42 were compared with those of other lysogens.     

Artificial Brassica napus flowering in Bangladesh

Summary Natural rapeseed (Brassica napus L.; AACC 2n=38), originated in the temperate climate of the Southwest European Mediterranean region, fails to complete its generative phase in the subtropics and is thus not cultivated in countries like Bangladesh. Adapted agroecotypes are available from the diploid representatives of its genome A (B. campestris/pekinensis, 2n=20) and C (B. oleracea/alboglabra, 2n=18). An artificial resynthesis based on carefully selected progenitor lines was expected to give a photoperiodically better adapted rapeseed. B. pekinensis x B. oleracea/alboglabra gave 2 hybrids and 87 matromorphous plants from 1,448 crossed flowers and the reciprocal combination gave no hybrid but 11 matromorphous plants from 2,228 pollinated flowers. The two true hybrids were vegetatively propagated and chromosome doubled. Part of the F₂ was grown in Sweden (all plants flowered and the most early ones were selected), part in Bangladesh (13 out of 706 plants flowered). The selected F₃ material flowered in Bangladesh and transgressions in earliness could be recorded, some lines were of definite agronomic potential. A correlation in earliness between reaction in Sweden (long day) and Bangladesh (short day) was observed.     

Fertile revertants from S-type male-sterile maize grown in vitro

Summary Plants were regenerated from callus cultures of maize inbred W182BN with the S(USDA) type of cytoplasmic male sterility (cms). Some regenerates from 16 of 18 separate cultures had fertile tassels. Many other regenerates, whose fertility could not be scored accurately because of abnormal plant morphology, produced fertile progeny after pollination with N cytoplasm W182BN. Revertant plants and/or progeny were obtained from all 18 cultures, which included the CA, D, LBN, and S sources of cmsS. More revertants were recovered from cultures maintained as callus for 12 months than from 3–4 month old cultures. Several types of evidence (absence of segregation for fertility after selfing or pollination of revertants with standard W182BN, pollen viability counts, failure of revertants to restore sterile cmsS lines to fertility, mitochondrial DNA analyses) indicated that the reversion to fertility involved cytoplasmic rather than nuclear alterations. All revertants examined lacked the S1 and S2 plasmid-like DNAs characteristic of the mitochondrial genome of sterile cmsS lines. Most callus cultures lost S1 and S2 after 13–20 months in vitro. No revertants were seen among thousands of W182BN cmsS plants grown from seed in the field or among plants from tissue cultures of W182BN with the C or T types of cms. The cytoplasmic revertants recovered from culture may be useful for the molecular analysis of cmsS.     

Glutathione status and sensitivity to GSH-reacting compounds of Escherichia coli strains deficient in glutathione metabolism and/or catalase activity

The intracellular concentrations of total glutathione, GSSG and protein · S-SG, the total excreted glutathione concentration, and the susceptibility towards GSH-reacting compounds were assayed in strains of Escherichia coli deficient in biosynthesis and/or reduction of glutathione. A deficiency in glutathione reductase displaced the glutathione status towards the oxidized forms. This displacement was more clearly appreciated in strains additionally deficient in glutathione biosynthesis. A deficiency in catalase activity also produced an increase in the oxidation of glutathione. The most severe changes were observed in the concentrations of protein-glutathione mixed disulfides and in the amount of glutathione excreted to the medium. Increased sensitivities towards compounds known to interact with cellular GSH were observed in glutathione reductase deficient strains, although these effects were enhanced in strains additionally deficient in GSH biosynthesis     

A comparison of anther and microspore culture as a breeding tool in Brassica napus

Summary A direct comparison of microspore culture and anther culture was made in Brassica napus using F₁ crosses of Regent (canola) by Golden (rapeseed), and their reciprocals, as well as a hybrid between Reston and a highly embryogenic, canola-quality breeding line (G231) as donor plants. The study confirmed that microspore culture can be ten times more efficient than anther culture for embryo production. Embryo yields from cultures initiated from the Reston x G231 were four-fold greater than those initiated from the Regent x Golden crosses, and significant differences were also detected among cultures initiated from the different Regent x Golden crosses. These results illustrate the influence that donor plant genotype has on embryo production. However, superior embryogenic potential among donor material was not always coincident with superior plant production. The average haploid-todiploid ratio in microspore-derived regenerates was 21 for the population obtained from the Regent x Golden crosses but 11 for the Reston x G231 cross. For both types of material, the frequency of diploids increased upon repeated cycles of explanting. A field study showed that there were no differences between the populations of anther-derived and microspore-derived spontaneous diploid and doubled haploid lines, with respect to the days required for them to flower or to mature. The information is valuable for canola breeding programs considering the use of haploidy.     

Binding of the transition metal ion [(H20)(NH3)5Ru(II)]2+ to nucleosomal core and internucleosomal DNA

The goal of this study is to establish the nature of pentammineruthenium(III) binding to DNA in intact mouse liver nuclei. Also, we wish to determine whether the nucleosomal organization of mouse chromatin has a substantial effect on the relative Ru(III) binding levels of internucleosomal and nucleosomal core DNA. These questions are important because ammineruthenium compounds share chemical and biological properties with the cis-dichlorodiammineplatinum(II) or cisplatin chemotherapeutic agent. Therefore, they represent a potential class of new chemotherapeutic agents. We find that in intact nuclei the predominant DNA binding site for pentammineruthenium(II), followed by air oxidation to pentammineruthenium(III), is N-7 guanine, as is the case with cisplatin. Also, the Ru(III) distribution between internucleosomal and nucleosomal core DNA was found to be nearly identical as probed with three non-specific deoxyribonucleases.     

Pathogenicity of some chrysosporium species isolated in France

In order to appreciate the pathogenicity of several geophilic Chrysosporium species (including Anixiopsis stercoraria, Chrysosporium keratinophilum, C. tropicum, C. pannorum, C. state of Arthroderma curreyi, C. state of A. multifidum, and C. state of A. tuberculatum), the authors have realized two series of experimental infestations. Inoculation of these fungi on the back of guinea pigs produced rare erythematous scaling lesions which spontaneously disappeared 3–5 weeks later. No real hair invasion was observed. In white mice, eight weeks after intraperitoneal inoculation, granulomas with necrotic center were observed in the peritoneal tissue with C. keratinophilum, C. tropicum, C. state of A. curreyi and C. state of A. tuberculatum. Conidia were often intact in necrotic centers and retrocultures were positive. With C. state of A. curreyi, spherical spores associated with rare budding cells were noted. The pathogenic role of these keratinophilic fungi is uncertain. However, their ability to remain viable for several weeks in skin and peritoneal tissue indicates that they could become pathogen in certain circumstances.This paper was presented at the X^th congress of the International Society for Human and Animal Mycology at Barcelona, Spain from June 27 to July 1, 1988.     

Effect of Moniliformis moniliformis density on distribution within the definitive host population (Rattus norvegicus)

The population dynamics of Moniliformis moniliformis was studied in ‘free-ranging’ laboratory rats, Rattus norvegicus, presented with different relative density levels of M. moniliformis in cockroaches, Periplaneta americana. Changes in selected population parameters of the negative binomial distribution were evaluated as indicators of changes in aggregation. A significant increase in the degree of aggregation of parasites occurred as a result of the increase in relative density of infective stages available to the rats. This increase in aggregation was due to the increase in over-dispersion that occurred in female rats only. The degree of aggregation in females was found to be significantly higher than that in males at both treatment levels. The best indicators of the degree of aggregation were found to be the ratio of the variance to the relative density and the ratio of the log-variance to log-relative density. Changes in k were not correlated with changes in over-dispersion or the relative density.