Modified spectrophotometer for multi-dimensional circular dichroism/fluorescence data acquisition in titration experiments: application to the pH and guanidine-HCI induced unfolding of apomyoglobin.

In a previous paper (Ramsay and Eftink, Biophys. J. 66:516-523) we reported the development of a modified spectrophotometer that can make nearly simultaneous circular dichroism (CD) and fluorescence measurements. This arrangement allows multiple data sets to be collected during a single experiment, resulting in a saving of time and material, and improved correlation between the different types of measurements. The usefulness of the instrument was shown by thermal melting experiments on several different protein systems. This CD/fluorometer spectrophotometer has been further modified by interfacing with a syringe pump and a pH meter. This arrangement allows ligand, pH, and chemical denaturation titration experiments to be performed while monitoring changes in the sample's CD, absorbance, fluorescence, and light scattering properties. Our data acquisition program also has an ability to check whether the signals have approached equilibrium before the data is recorded. For performing pH titrations we have developed a procedure which uses the signal from a pH meter in a feedback circuit in order to collect data at evenly spaced pH intervals. We demonstrate the use of this instrument with studies of the unfolding of sperm whale apomyoglobin, as induced by acid pH and by the addition of guanidine-HCI.     

A histological and immunohistochemical study of the effects of N-acetyl cysteine on retinopathy of prematurity by modifying insulin-like growth factor-1

Retinopathy of prematurity (ROP) is a vasoproliferative disorder that occurs in premature infants and may lead to permanent visual impairment. We investigated both the possible protective role of N-acetyl cysteine (NAC) for preventing ROP and the role of IGF-1 in the disorder. Forty-five newborn rats were divided into three groups. Group 1 was raised in room air as controls. Group 2 was exposed to 60% oxygen for 14 days after birth, then transferred to room air. Group 3 was exposed to the same conditions as group 2, but received intraperitoneal injections of NAC on postnatal days 7–17. After 35 days, both eyes of all rats were processed for histology. Some sections were stained with hematoxylin and eosin to assess structural changes and other sections were immunostained to determine the location of IGF-1. Frozen sections also were prepared and stained for adenosine triphosphatase to detect retinal blood vessels. Compared to the controls, more blood vessels, many of which were abnormal, and increased IGF-1 expression were observed in group 2. In group 3, abnormal blood vessels and IGF-1 expression were less evident. NAC appeared to be an effective vascular-protective agent for ROP by decreasing IGF-1 expression.     

Water exchange through erythrocyte membranes: Nuclear magnetic resonance studies on the effects of inhibitors and of chemical modification of human membranes

The changes in water diffusion across human erythrocyte membranes following exposure to various inhibitors and proteolytic enzymes have been studied on isolated erythrocytes suspended in isotonic buffered solutions. An important issue was to investigate whether the sulfhydryl reacting reagents that have been applied in osmotic experiments showed similar effects on diffusional permeability. It was found that mercurials, including mersalyl, were the only sulfhydryl reacting reagents that were efficient inhibitors. Under optimal conditions a similar degree of inhibition (around 45%) was found with all mercury-containing sulfhydryl reagents. Other reagents, including the sulfhydryl reagent DTNB, phloretin, or H2DIDS, the specific inhibitor of the anion transport system in erythrocyte membrane, did not appear to inhibit significantly the diffusional permeability. No changes in water diffusion were noticed after exposure to erythrocytes to trypsin and chymotrypsin. A new kind of experiments was that in which the effects of exposure of erythrocytes to two or more agents were studied. It was found that none of the chemical manipulations of membranes that did not affect water diffusion hampered the inhibitory action of mercurials. These findings show that the SH groups involved in water diffusion across erythrocyte membrane do not react with any of the other SH reagents aside from mercurials and that the molecular mechanism of water transport is not affected by chymotryptic cleavage of band 3 protein into the 60 and 35 kD fragments. The NMR method appears as a useful tool for studying changes in water diffusion in erythrocyte membranes following various chemical manipulations of the membranes with the aim of locating the water channel.     

Antityphoid and antitetanic intradermal vaccination of chronic neuropsychic patients in a typhoid fever outbreak.

Intradermal immunization of 229 chronic neuropsychic patients in Gura Ocnitzei Sanatorium, D?mbovitza County, where a typhoid fever outbreak burst, was performed with a freeze-dried typhoid vaccine, suspended in purified and concentrated tetanic anatoxin. Adverse reactions were minimal and immunologic response, evaluated by laboratory tests, was good, reaching the level of that obtained as a result of classic vaccination methods (subcutaneous antityphoid and intramuscular anti-tetanic). Both after vaccination and also booster O and H agglutinating titers increased 4-7 times and anti-S. typhi. seroprotecting effect reached and maintained a high level. Moreover, antityphoid vaccination permitted epidemic focus limiting and its complete extinguishing after booster. Antitetanic vaccination resulted in protecting titers settlement (greater than or equal to 0.01 IU/ml) in all previously non-protected persons (1/3 of all subjects) and in the increase of protecting titers, existing before vaccination in the other persons.     

Electron microscopic studies on protoplast release from I.C.A.-1.65 line (I.F.C.-1.65 line) of Bacillus subtilis cells.

Under lysozyme action a minicell-forming line (I.C.A.-1.65) of B. subtilis releases protoplasts. The main cytologic events which proceed protoplast releasing are described. Different areas of the cell wall prove a remarkable difference in their sensitivity to enzymatic lysozyme action. Central areas of the cell wall are most sensitive and the polar areas are most resistant. Mesosomal vesicles and tubules are extruded and released together with other cytoplasmic extrusion during the protoplasting process. The cell wall of minicells does not prove resistance particularities to lysozyme action. The minicells release protoplasts.