Functional equivalence of the uridine turn and the hairpin as building blocks of tertiary structure in the Neurospora VS ribozyme.

Mutational, kinetic, and chemical modification experiments show that one of the three-way helical junctions in the Neurospora VS ribozyme contains a uridine turn that is important for organizing the functional three-dimensional structure of this junction. Disruption of the uridine turn disrupts the structure of the junction and decreases the self-cleavage activity of the ribozyme; however, substitution of the uridine turn with a variety of different hairpins, thereby transforming the three-way junction into a four-way junction, maintains catalytic activity. Chemical modification structure probing reveals that both the native junction and the hairpin-containing junction support the same tertiary interactions required elsewhere in the ribozyme for catalysis. These observations show that functionally equivalent three-dimensional RNA structures can be built from different secondary structure elements.     

An automated technique for monitoring carbon dioxide respired by insects*

ABSTRACT. A non-dispersive infrared gas analyser equipped with a Luft-type sonic detector and flow-through reference cell was automated to monitor the total volume of carbon dioxide (CO₂) respired by single insects or groups of insects. The infrared analyser was interfaced with an integrator for quantification, a microprocessor to control intermittent air flow through the insect respiration chambers, and a microcomputer for data storage and reduction. This technique has been used to monitor the CO₂ Output of diapausing and non-diapausing mature fifth instar larvae and of developing pupae of the codling moth, Cydia pomonella (L.) (Lepidoptera: Tortricidae). The resulting data were accurate, quantitative and reproducible.     

Inhibition of DNA synthesis in relation to enhanced survival of UV-damaged herpes virus in monkey cells treated by a variety of 2-nitronaphthofurans

Various 2-nitronaphthofuran derivatives (related to each other by simple structural modifications) were tested for 2 different effects in CV-1 monkey kidney cell cultures: the immediate inhibition of normal DNA synthesis and the capacity of pretreated cultures (40 h of contact) to support the replication of UV-damaged Herpes simplex virus (HSV). For all compounds tested, a fair correlation was found between their efficiencies to inhibit cellular DNA synthesis and to provoke an increase in UV-HSV production (virus reactivation). Virus reactivation was due to an increase in both the number of virus-producing cells and the amount of infectious particles produced per cell. The most efficient 2-nitronaphthofurans (particularly 2-nitro-7-methoxy-naphtho[2,1-b]furan-R 7000) were at least as potent as aflatoxin B1 in inducing virus reactivation.     

Amyloid fibrils derived from V-region together with C-region fragments from a lambda II-immunoglobulin light chain (HAR)

Amyloid fibril proteins were isolated from the spleen of a patient with IgD(lambda)-plasmocytoma by extraction and gel filtration in 5M guanidine hydrochloride. The molecular mass of the predominant polypeptide chain was approximately 5000 Da. Its complete amino-acid sequence was elucidated by stepwise automated degradation of the carboxymethylated polypeptide chain and by structural studies of tryptic and thermolysinolytic cleavage products. The length of the polypeptide chain was 58 to 59 residues and it was homologous to the amino acids in positions 8 through 65 of the variable part of an lambda-type immunoglobulin light chain, which was most closely related to the lambda II subgroup. The N-terminal sequence of this amyloid fibril protein proved to be heterogeneous, indicating cleavage after the amino acids in positions 7 and 8. Peptides from the constant part of the lambda-chain were unexpectedly found in the tryptic digest of the denatured amyloid protein HAR. One polypeptide derived from the constant region was separated from the main component by high performance liquid chromatography. Its amino-acid sequence commenced at position 111 and could be traced in 41 steps. In this case, at least two constant region fragments were shown to be constituents of the amyloid fibril protein. The association of fragments from the variable as well as the constant region is discussed with respect to amyloid formation.     

Hormonal regulation of skeletal muscle glycogen synthase through covalent phosphorylation

Studies have been initiated to determine the hormonal regulation of glycogen synthase in rabbit skeletal muscle. It was found that glycogen synthase purified from control animals was quite highly phosphorylated (2.35 mol phosphate/mol synthase subunit) with 40% of the phosphate in the trypsin-sensitive or COOH-terminal domain, and 60% in the trypsin-insensitive or NH2-terminal domain. The phosphorylation state of synthase was elevated (3.9 mol/mol) by epinephrine injection and in the diabetic condition. With epinephrine, about 76% of the additional phosphate was incorporated in the trypsin-sensitive domain, which strongly supports the contention that this hormone acts through the cyclic AMP (cAMP)-dependent protein kinase. In the synthase purified from diabetic rabbits, 90% of the additional phosphate was in the trypsin-insensitive domain. Insulin treatment of the diabetics resulted in specific dephosphorylation of the trypsin-insensitive domain. These results indicate that in this system insulin is not acting by inhibition of the cAMP-dependent protein kinase.     

Demonstration of a COOH-terminal extension on equine lutropin by means of a common acid-labile bond in equine lutropin and equine chorionic gonadotropin

The beta subunits of equine lutropin and equine chorionic gonadotropin were incubated in 0.013 N HCl for 30 min at 110 degrees C and separated into two fragments by reverse-phase high performance liquid chromatography. The amino acid and carbohydrate compositions of both fragments from each subunit were analyzed. The results demonstrated that equine lutropin-beta has a glycosylated COOH-terminal extension that differs only in carbohydrate composition from the COOH-terminal portion of equine chorionic gonadotropin-beta. This is the first demonstration of a glycosylated COOH-terminal extension in a pituitary glycoprotein hormone.