Solubilization and reconstitution of rat liver mitochondrial carnitine acylcarnitine translocase

Carnitine acylcarnitine translocase has been solubilized from inverted inner membrane vesicles of rat liver mitochondria with octyl glucoside and reconstituted into asolectin liposomes. For both processes, optimization of the detergent to phospholipid ratio was found crucial for obtaining reconstitutively active liposomes. Reassembly of the solubilized carrier into asolectin liposomes was achieved either by the octyl glucoside dilution method or by Extracti-Gel D column chromatography. The reconstituted system catalyzed exchange diffusion of carnitine, exhibited the expected inhibitor and temperature sensitivity, and discriminated between stereoisomers of octanoylcarnitine. The activity of unidirectional import of carnitine was low compared to exchange diffusion. It showed high-temperature sensitivity and a loss of activity on prolonged sonication that was regained by an appropriate freeze-thaw step subsequently.     

Extensive heterogeneity of the protein composition of Chlamydia trachomatis following serial passage in two different cell lines

To determine if the host-modulated adherence characteristics of the intracellular bacterial pathogen Chlamydia trachomatis were due to the acquisition of altered surface-exposed proteins, highly purified chlamydiae grown in two different host cells were analysed. Two serovars, L1 and E, were grown for multiple passages in both HeLa and McCoy host cells. Numerous protein differences in the chlamydial elementary bodies (EB) of each serovar grown in the two different hosts were detected by two-dimensional (2-D) gel electrophoresis and fluorography of radioactively labelled proteins. At least four to six serial passages in the alternative host were necessary before the changes were apparent. Iodination of suspensions of purified chlamydiae and 2-D electrophoresis revealed several surface proteins that were determined by the host cells in which the bacteria had replicated. These iodinated chlamydial proteins were removed by treatment of the iodinated EB with trypsin, indicating their location at the bacterial surface. Two of the major constituents of the outer-membrane complex, the cysteine- and methionine-rich 60 kDa and 40 kDa proteins, remained unchanged in both molecular mass and charge during the host adaptation. Several chlamydial proteins capable of binding iodinated host membrane preparations also exhibited host-dependent alterations. Immunoblotting experiments with a rabbit and a human polyclonal sera indicated that distinct host-specified chlamydial proteins were reactive with the two sera.     

Regulation of tubulin by triiodothyronine in hypothyroid rat brain

The effect of T₃ (triiodothyronine) on the induction of tubulin in hypothyroid developing rat brain has been examined using organ cultures of brains from late fetal, neonatal and postnatalrats. The neonatal brain displayed maximum sensitivity to T₃. Hypothyroidism resulted in a 26% decline in the level of tubulin in the neonatal brain as opposed to a 5–15% decline in the fetal or postnatal brain. Exposure of the hypothyroi d neonatal brain to T₃ for 2 h in culture led to a 61% rise in the level of tubulin in contrast to a 41% increase seen in the case of normal brain. Total protein synthesis was not significantly affected . The preferential decline of tubulin in the neonatal hypothyroid brain, its enhanced sensitivity to T₃ compared to normal brain, and the coincidence of the period of sensitivity to that of brain maturation indicate that the regulation of the level of tubulin by T₃ in the developing brain is a natural ontogenic phenomenon.     

Paper from Thysanolaena maxima

Thysanolaena maxima (Roxb) (tiger or broom grass), is a tall reed-like perennial grass, grown on shady slopes in forests in India and the Nicobar Islands. Culms are solid, smooth and rounded and up to a height of 4 m. The leaves and the tips are used as fodder; the bushy, fox-tail-like panicles (30–90 cm) are used for making brooms. When the panicles are cut, the stem portion (3–4 m) is left out in the field and is burnt. Fibres, of average 1.25 mm in length at 45% yield (unbleached), could be obtained from this grass. The laboratory handmade paper sheets exhibited good properties, with a burst factor of 30, a breaking length of 3555 m and a tear factor of 106. Hence, it can be suggested that T. maxima may become a potential source of raw material for pulp- and paper-making either alone or in combination with the conventional pulp- and paper-making raw materials. This material could help to meet the future demand for pulp- and paper-making raw material, if properly exploited.     

Influence of Pulsed Electric Fields and Mitochondria-Cytoskeleton Interactions on Cell Respiration

Pulsed electric fields with microsecond pulse width (μsPEFs) are used clinically; namely, irreversible electroporation/Nanoknife is used for soft tissue tumor ablation. The μsPEF pulse parameters used in irreversible electroporation (0.5–1 kV/cm, 80–100 pulses, ～100 μs each, 1 Hz frequency) may cause an internal field to develop within the cell because of the disruption of the outer cell membrane and subsequent penetration of the electric field. An internal field may disrupt voltage-sensitive mitochondria, although the research literature has been relatively unclear regarding whether such disruptions occur with μsPEFs. This investigation reports the influence of clinically used μsPEF parameters on mitochondrial respiration in live cells. Using a high-throughput Agilent Seahorse machine, it was observed that μsPEF exposure comprising 80 pulses with amplitudes of 600 or 700 V/cm did not alter mitochondrial respiration in 4T1 cells measured after overnight postexposure recovery. To record alterations in mitochondrial function immediately after μsPEF exposure, high-resolution respirometry was used to measure the electron transport chain state via responses to glutamate-malate and ADP and mitochondrial membrane potential via response to carbonyl cyanide-p-trifluoromethoxyphenylhydrazone. In addition to measuring immediate mitochondrial responses to μsPEF exposure, measurements were also made on cells permeabilized using digitonin and those with compromised cytoskeleton due to actin depolymerization via treatment with the drug latrunculin B. The former treatment was used as a control to tease out the effects of plasma membrane permeabilization, whereas the latter was used to investigate indirect effects on the mitochondria that may occur if μsPEFs impact the cytoskeleton on which the mitochondria are anchored. Based on the results, it was concluded that within the pulse parameters tested, μsPEFs alone do not hinder mitochondrial physiology but can be used to impact the mitochondria upon compromising the actin. Mitochondrial susceptibility to μsPEF after actin depolymerization provides, to our knowledge, a novel avenue for cancer therapeutics.