Assessing post-induction cellular response in a recombinant E. coli lactose-inducible system by monitoring β-galactosidase levels

Summary A synthetic lactose-inducible promoter was chosen to study host cell responses to the over-expression of heterologous genes. Fermentations were conducted to compare the effect of induction strategies on the synthesis of -galactosidase versus the production of recombinant protein. The levels of lactose, IPTG and glucose during induction were manipulated to adjust the utilization of lactose as the inducer and/or the carbon source. In addition, the involvement of the gal operon in lactose metabolism was also explored in order to optimize lactose transport and utilization during induction.     

Post-translational modification of proteins and the discovery of new medicine

Post-translational modifications are fundamental to processes controlling behaviour, including cellular signaling, growth and transformation. As the molecular basis of protein modifications in normal and disease processes are becoming better defined, so new strategies for designing therapeutic entities to control complex disease processes are emerging.     

T cell receptor-mediated Ca2+ signaling: Release and influx are independent events linked to different Ca2+ entry pathways in the plasma membrane

In this study, we showed that cross-linking CD3 molecules on the T cell surface resulted in Ca²⁺ release from the intracellular stores followed by a sustained Ca²⁺ influx. Inhibition of release with TMB-8 did not block the influx. However, inhibition of phospholipase C activity suppressed both Ca²⁺ release and influx. Once activated, the influx pathway remained open in the absence of further hydrolysis of PIP2. Thapsigargin, a microsomal Ca²⁺ -ATPase inhibitor, stimulated Ca²⁺ entry into the cells by a mechanism other than emptying Ca²⁺ stores. In addition, Ca²⁺ entry into the Ca²⁺ -depleted cells was stimulated by low basal level of cytosolic Ca²⁺, not by the emptying of intracellular Ca²⁺ stores. Both the Ca²⁺ release and influx were dependent on high and low concentrations of extracellular Ca²⁺. At low concentrations, Mn²⁺ entered the cell through the Ca²⁺ influx pathway and quenched the sustained phase of fluorescence; whereas, at higher Mn²⁺ concentration both the transient and the sustained phases of fluorescence were quenched. Moreover, Ca²⁺ release was inhibited by low concentrations of Ni²⁺, La³⁺, and EGTA, while Ca²⁺ influx was inhibited by high concentrations. Thus, in T cells Ca²⁺ influx occurs independently of IP3-dependent Ca²⁺ release. However, some other PIP2 hydrolysis-dependent event was involved in prolonged activation of Ca²⁺ influx. Extracellular Ca²⁺ influenced Ca²⁺ release and influx through the action of two plasma membrane Ca²⁺ entry pathways with different pharmacological and biochemical properties.     

Cytochromes inAzospirillum brasilense

Difference spectra of the crude cell-free extract ofAzospirillum brasilense sp. 7 indicate the presence of cytochrome b, cytochrome c, and one CO-binding pigment that exhibits the spectral characteristics of cytochrome o. All the pigments are present in varying concentrations at all stages of growth. With progress of the bacterial growth, there is a linear increase in the level of cytochrome b with a disproportionate increase in the level of cytochrome c. At the stationary phase, the amount of cytochrome b and c is increased by about sevenfold compared with that in the early log phase. The increase in the concentration of total cytochrome is not accompanied by an increase in the respiration rate of the cells. Both cytochrome b and cytochrome c are located in the particulate fraction of the cells and are not fully reducible by succinate alone.     

Optical characteristics of carboxyl group in relation to the circular dichroic properties and dissociation constants of glycosaminoglycans

Circular dichroism studies of glycosaminoglycan including chemically transformed heparins at various pH values reveal that carboxyl chromophore plays an important role in the dichroic behavior of the polymers. With decreasing pH, iduronic acid-containing glycosaminoglycans show increased negative ellipticity near 220 nm whereas the polymers containing glucuronic acid display enhanced negative dichroism near 230 nm and decreased negative dichroism around 210 nm. The pH-dependent optical properties have been utilized to determine the pK_a values of uronic acid moieties. The acid strengths of the iduronic acid-containing glycosaminoglycans are inherently smaller than those of corresponding glucuronic acid-containing polymers. Glycosaminoglycans in which the amino sugars are linked with iduronic acid display a very weak n → π^* amide transition, or none. The rotational strength at 210 nm of these polymers is largely due to iduronic acid moieties. The CD variations above 200 nm with change in pH do not indicate any major conformational transition of the molecules but the difference between dermatan sulfate and heparin can be attributed to difference either in iduronic acid conformation or in intersaccharide linkages.     

Vasoactive intestinal peptide causes peristaltic contractions in the esophageal body

Vasoactive intestinal peptide (VIP) caused a dose-dependent fall in lower esophageal sphincter (LES) pressure and dose-dependent contractions in the body of the esophagus. The response to VIP in the esophagus or LES was not modified by atropine, phentolamine, haloperidol, pyrilamine, methysergide, indomethacin and tetrodotoxin, showing that it exerts direct action at the esophageal smooth muscle. These studies suggest that VIP causes contraction in the esophageal body and relaxation of the LES by a direct action on the smooth muscle. It is possible that VIP may be the common mediator of noncholinergic, nonadrenergic neurons that cause relaxation of the lower esophageal sphincter and contraction in the esophageal body.     

The late stage following continuous amphetamine administration to rats is correlated with altered dopamine but not serotonin metabolism

In previous experiments rats pretreated with slow-release d-amphetamine (d-Amp) pellets for $\text{4}\text{1}\text{2}$ days, given a 12-hr drug-free period, and then injected with d-Amp have been found to show a behavioral syndrome which has similarities to that induced by acute injections of the hallucinogens LSD and mescaline. The present results indicate that rats administered this same drug regimen have large decreases in Dopamine (DA), dihydroxyphenyl acetic acid (Dopac), and homovanillic acid (HVA) in caudate nucleus, smaller decreases in DA with no changes in Dopac and HVA levels in nucleus accumbens, but no alterations in 5-hydroxytryptamine (5HT) and 5-hydroxyindole acetic acid (5HIAA) levels in caudate, accumbens, brainstem and hippocampus. Increased 5HIAA levels are found in rats sacrificed with pellets intact following 3 days of continuous d-Amp administration, while sleep deprived and in motor stereotypies. The late and hallucinatory stage following continuous d-amp is correlated more closely with alterations in dopamine than of 5HT.     

The cytosol receptors for progesterone in the different parts of rabbit fallopian tube and uterus during ovum transport

Progesterone receptors were determined in the cytosol from the ampulla, ampullaryisthmic junction and isthmus of rabbit fallopian tube and uterus of estrus and pregnant rabbits. The receptor levels when compared among its various anatomical segments, were the same in ampulla, isthums and uterus but maximum in ampullary-isthmic junction. Significant differences were observed in mated animals at 14, 24, 34, 48, 70 and 144 h after coitus. The receptor concentrations in portions of the fallopian tube showed no significant change between 14 and 24 h after coitus, except for a decrease in ampullary-isthmic junction at 24 h. At 34 h the concentration of receptor further decreased in all parts of the tube. At 48 and 70 h after coitus, receptor concentrations decreased gradually in ampulla and ampullary-isthmic junction, while isthmus showed a gradual increase. At 144 h, the receptor concentration showed no further change in ampulla and ampullary-isthmic junction; however, isthmus showed a decline. The uterine receptor concentration declined steadily from estrus till 70 h after coitus, however, it was increased at 144 h. The dissociation constant (K_d) of cytosol receptor in all the tissues at estrus and during early pregnancy was found similar. The implications of these changes in relation to the normal ovum transport have been correlated in this paper.     

Influence of free fatty acid anion on the binding of warfarin to cytoplasmic proteins from rat liver

In vitro binding studies have shown that warfarin binds strongly to both ligandins (Y) and Z protein obtained from rat liver cytosol with dissociation constants of 11.7 and 10.1 μM respectively. Increasing concentrations of oleate ion significantly increased the dissociation constant of warfarin with either protein, whereas laurate ion showed the same behavior only with Z protein. On the other hand, the binding of warfarin to liver cytoplasmic proteins in vivo was decreased in 72-h-pre-fasted rats, although such fasting failed to produce any increase in the in vivo levels of the cytoplasmic free fatty acids (FFA). However, based on the results of the in vitro binding study, it is suggested that changes in the composition of hepatic cytoplasmic free fatty acids as a result of fasting could reduce the in vivo binding of warfarin to Y and Z proteins and hence could lead to an increase of unbound warfarin in liver cytosol.