Reversible covalent immobilization of ligands and proteins on polyacrylamide gels

Ligands and proteins were covalently but reversibly immobilized on polyacrylamide gels using novel acrylic monomers whose syntheses are reported here. These reagents have an acrylyl group at one end for copolymerization into gels, an N-succinimidyl ester at the other allowing rapid immobilization of molecules having an available primary amino group, and a cleavable disulfide bond in the middle. Two immobilization methods were developed using these reagents. In the first method, a ligand with a primary amino group was treated with the immobilization reagent in anhydrous ethanol and the resulting amide derivative was purified and copolymerized with acrylamide and bisacrylamide resulting in the desired reversible immobilization. In the second method, the immobilization reagents (at densities up to 50 mumol/ml) were directly copolymerized with acrylamide and bisacrylamide to form activated gels of the desired shape and porosity. Proteins or other ligands in aqueous buffers were then added to the activated gels resulting in their covalent immobilization. Ligands or proteins immobilized using the methods reported here remained stably bound even when gels were subjected to boiling in detergents or high-ionic-strength buffers. Immobilized ligands were readily released (greater than 97%) from gels by treatment with quantitative amounts of aqueous dithiothreitol (DTT) under mild conditions. Immobilized proteins were also released (up to 87%) from the gels by DTT treatment. Small ligands (e.g., aminohexyl glycosides), active enzymes, and glycoproteins were immobilized, and then recovered, using these reagents.     

Reverse transformation of Chinese hamster ovary cells by methyl xanthines : Structure-function relationships

Using a number of drugs that increase cellular cAMP levels, alterations in the amount of cell surface fibronectin and other transformation parameters were studied in Chinese hamster ovary (CHO) cells. The drugs include db-cAMP, different methylxanthines (theophylline, aminophylline, methyl isobutyl xanthine (MIX), caffeine and theobromine), papaverine and cholera toxin. Methylxanthines that have a methyl group at the seventh position lack reverse transforming potential; those that lack a methyl group at the seventh position induced reverse transformation in CHO cells, causing an increase in surface fibronectin, cell substratum adhesive strength and anchorage dependence for growth. Further, as methyl xanthines are substituted in other positions different from the seventh position, the more efficient they become in restoring normal phenotypic properties; the later agents also induced low saturation density via a cytostatic state causing accumulation of cells in the S and G2 phases of the cycle in contrast to the G1 arrest of normal cells at low saturation density. db-cAMP and cholera toxin induced cell elongation but like caffeine and theobromine, did not induce surface fibronectin. The non-methylxanthine phosphodiesterase inhibitor papaverine induced neither cell elongation nor surface fibronectin but produced a cytostatic effect similar to aminophylline and MIX. These studies suggest that the reverse transformation properties fall into two groups: (a) Differentiation-related properties including cell morphology, parallel alignment and surface matrix fibronectin, etc.; (b) cell cycle-related properties-low saturation density, cell arrest at G1 phase and anchorage-dependent growth. Phosphodiesterase inhibitors reversibly eliminate indefinite division potential of CHO cells by inducing a cytostatic situation and not by inducing a G1-specific arrest.     

Adrenergic Regulation of the Reduction in Acetyl Coenzyme A: Arylamine 7V-Acetyltransferase Activity in the Rat Pineal

Abstract: Pharmacologically active agents were employed to study the mechanisms that control the reduction in levels of acetyl-coA: arylamine N-acetyltransferase activity (NAT) (EC 2.3.1.5) in the rat pineal. Pretreatment of rats with phenoxybenzamine or phentolamine prevented the rapid light-mediated decrease in NAT activity, although pretreatment with yohimbine or atropine did not alter this effect of light. Administration of mecamylamine resulted in a rapid reduction in enzyme activity prior to light exposure. When clonidine was administered intraperitoneally to animals with elevated NAT levels, there was a rapid decrease in enzyme activity, mimicking the effects of light. However, intraperitoneal injections of norepinephrine, methoxamine and phenylephrine into similar groups of animals had no significant effect on enzyme acitivity. When clonidine and norepinephrine were administered intraventricularly, there was a rapid reduction in enzyme activity. On the other hand, intraventricular administration of phenylephrine did not result in reduced enzyme activity. Pretreatment of animals with phenoxybenzamine failed to block the reduction in NAT activity precipitated by low doses of clonidine. This clonidine-mediated reduction in enzyme activity was, however, blocked by yohimbine. When animals were simultaneously exposed to light and administered clonidine, the rapid reduction in NAT activity was affected only when animals were pretreated with both yohimbine and phenoxybenzamine. In contrast to the decrease in pineal NAT activity observed in in vivo preparations, incubation of pineals with clonidine in an organ culture system produced a moderate, but consistent, rise in enzyme activity. These results suggest that stimulation of a receptor with α-adrenergic characteristics mediates the reduction in NAT activity produced by light. Stimulation of yet a second adrenergic-like receptor appears to mediate a reduction in pineal NAT activity precipitated by clonidine. Our evidence suggests that one or both of these receptors are located within the central nervous system.     

Developmental delays in exploration and locomotor activity in male rats exposed to low level lead

Delays in the development of exploratory and locomotor behavior in neonatal male rats (up to 21 days of age) are shown to accrue as a consequence of low level lead exposure. Cross fostering experiments indicate that these delays are primarily due to prenatal exposure. These Pb induced behavioral modifications appear to be associated with the delays in synaptogenesis and biochemical development of the cerebral cortex reported previously (4, 18). A new behavioral bioassay for detecting delays in brain development is described.     

Deletion of the penicillin-binding protein 5 gene of Escherichia coli.

A strain of Escherichia coli that has a deletion of the entire dacA gene has been constructed. The complete lack of penicillin-binding protein 5 in this strain establishes that the activity of this protein is not essential for the growth of E. coli.     

Rat lens beta-crystallins are internally duplicated and homologous to gamma-crystallins

The nucleotide sequence of two cloned rat lens beta-crystallin cDNAs pRL beta B3-2 and pRL beta B1-3 has been determined. pRL beta B3-2 contains the complete coding information for a beta-crystallin, designated beta B3, of 210 amino acid residues. pRL beta B1-3 is incomplete at its 5' end; the 5' codogenic information which is not present in this cDNA clone was deduced from the cloned gene. pRL beta B1-3 codes for a beta-crystallin polypeptide, designated beta B1, whose full length is 247 amino acid residues. Considerable sequence homology is noted between the amino- and carboxy-terminal halves of each protein. The two rat beta-crystallins show a substantial sequence homology with each other (60%) as well as with the published sequences of rat gamma-crystallin (37%) and bovine and murine beta-crystallins (55 and 45%). All these proteins have a two-domain structure which, like the bovine gamma II-crystallin, might be folded into four remarkably similar protein motifs. Our data further indicate that the beta-crystallins can be subdivided into two groups which are evolutionarily related. Both groups are, although more distantly, also related to the gamma-crystallins.