Methods for study of mutations and mutagenesis in human lymphocytes

Detailed methods are presented for measurement and study of in vivo mutations and in vitro mutagenesis in human lymphocytes. The methods described include preparation of conditioned medium containing interleukin-2, enumeration of mutant clones, in vitro mutagenesis, and expansion of mutant clones for further study.     

Presence of sex steroid receptors in normal breast of human females.

Receptors for estradiol, progesterone and dihydrotestosterone have been demonstrated in normal growing breasts of young females below 25 years.     

Mean geometry of the thiopeptide unit and conformational features of dithiopeptides and polythiopeptides

The mean geometry of the thiopeptide [Ca-N-C(=S)-Ca] unit has been derived from an analysis of X-ray crystal structure data, as well as MM2 and Gaussian 80/82 calculations. The conformational flexibilities of dithiopeptides with glycl- and alanyl-side chains have been investigated by molecular mechanics. Minimum energy conformations were examined using interactive computer graphics molecular modeling techniques. Alanyl-dithiopeptide substitution within an oligopeptide results in considerable restriction of conformational freedom whereas the effect is minimal for glycyl-dithiopeptide substitution. Polyglycyl-thiopeptide adopts a left-handed three or fourfold or right-handed threefold helical structure with favorable interchain C = S...H-N hydrogen bond interactions. A poly-L-alanyl-thiopeptide prefers a left-handed threefold poly-L-proline-like helical structure.     

Increase in hepatic microsomal lipid peroxidation mediated by α-adrenergic system under cold stress and noradrenaline treatment

A membrane protein recognized by monoclonal antibody SQM1 was identified in human squamous carcinomas, including those originating in the head and neck (SqCHN), lung and cervix. Cell lines derived from SqCHN of previously untreated patients expressed high amounts of this protein. In contrast, many cell lines established from SqCHN of patients previously treated with chemotherapy and/or radiation showed diminished amounts of this SQM1 protein. The expression of SQM1 antigen was determined in several SgCHN cell lines made resistant by exposure to methotrexate (MTX) in vitro. The parent cell lines all exhibited strong binding to SQM1 antibody. The MTX-resistant sublines showed much lower membrane binding of SQM1. The lowest SQM1 reactivity was found in cell lines with high resistance to MTX and with diminished rate of MTX transport. Some highly MTX-resistant cell lines which had high levels of dihydrofolate reductase, but which retained a high rate of MTX transport, also retained high levels of SQM1 binding. Reduced SQM1 protein was also found in SgCHN cells which developed resistance to the alkylating drug cis-platinum (CDDP) and which showed reduced membrane transport of CDDP. Cell growth kinetics and non-specific antigenic shifts were not responsible for the differences in SQM1 binding between the parent cell lines and their drug-resistant sublines. The finding of a novel protein which is reduced in cells resistant to MTX and CDDP could contribute to our understanding of the basic mechanisms of drug resistance. By detecting SQM1 protein in clinical specimens, it may be possible to monitor the development of drug resistance in tumors.Abbreviations SqCHN Squamous Carcinoma of the Head and Neck - MTX Methotrexate - CDDP Cis-Platinum - DHFR Dihydrofolate Reductase     

Modes of binding of 2'-AMP to RNase T1. A computer modeling study.

The modes of binding of adenosine 2'-monophosphate (2'-AMP) to the enzyme ribonuclease (RNase) T1 were determined by computer modelling studies. The phosphate moiety of 2'-AMP binds at the primary phosphate binding site. However, adenine can occupy two distinct sites--(1) The primary base binding site where the guanine of 2'-GMP binds and (2) The subsite close to the N1 subsite for the base on the 3'-side of guanine in a guanyl dinucleotide. The minimum energy conformers corresponding to the two modes of binding of 2'-AMP to RNase T1 were found to be of nearly the same energy implying that in solution 2'-AMP binds to the enzyme in both modes. The conformation of the inhibitor and the predicted hydrogen bonding scheme for the RNase T1-2'-AMP complex in the second binding mode (S) agrees well with the reported x-ray crystallographic study. The existence of the first mode of binding explains the experimental observations that RNase T1 catalyses the hydrolysis of phosphodiester bonds adjacent to adenosine at high enzyme concentrations. A comparison of the interactions of 2'-AMP and 2'-GMP with RNase T1 reveals that Glu58 and Asn98 at the phosphate binding site and Glu46 at the base binding site preferentially stabilise the enzyme-2'-GMP complex.     

Molecular dynamics studies on nucleoside 2',3'-cyclic phosphates.

2',3'-cyclic nucleotides are intermediates and substrates of Ribonuclease (RNase)-catalysed reactions. The characterization of the equilibrium conformation as well as the flexibility inherent in these molecules helps in understanding the enzymatic action of RNases. The present study explores parameters like phase angle, glycosydic torsion angle and hydrogen bond to find possible interrelationship between them through Molecular Dynamics (MD) simulations on 3'-GMP,3'-UMP, A greater than p, G greater than p, U greater than p, C greater than p, GpA greater than p and UpA greater than p. Interesting results of the effect of cyclisation and other constraints such as hydrogen bond between certain groups on the equilibrium ribose conformation have emerged from this study.     

Molecular dynamics simulations of oligosaccharides and their conformation in the crystal structure of lectin-carbohydrate complex: importance of the torsion angle {psi} for the orientation of {alpha}1,6-arm

The conformation of the heptasaccharide Man-1,6-(Man-1,3)(Xyl-ß1,2)-Man-ß,4-GlcNAc₂-ß1,4-(L-Fuc-1,3)-GlcNAc₁,the carbohydrate moiety of Erythrina corallodendron lectin (EcorL),the hexasaccharide Man-1,6-(Man-1,3) (GlcNAc-ß1,4)-Man-ß1,4-GlcNAc-ß1,4-GlcNAcand their disaccharide fragments have been studied by moleculardynamics (MD) simulations for 1000 ps with different initialconformations. In the isolated heptasaccharide, the most frequentlyaccessed conformation during MD has a value of 180° aroundMan-1,6-Man linkage. This conformation is stabilized by theformation of a hydrogen bond between the carbonyl oxygen ofGlcNAc₂ with the O3/O4 hydroxyls of the 1,6-linked mannose residue.The conformation of the heptasaccharide found in the crystalstructure of the EcorL-lactose complex (Shaanan et al., Science,254, 862, 1991), that has a value of 76° around Man-1,6-Manlinkage, is accessed, although less frequently, during MD ofthe isolated oligosaccharide. The ,, = 58°,–134°,–60°conformation around Man-₁,6-Man fragment observed in the crystalstructure of the Lathyrus ochnrs lectin complexed with a biantennaryoctasaccharide (Table I in Homans,S.W., Glycobiology, 3, 551,1993) has also been accessed in the present MD simulations.These values for the 1,6-linkage, which are observed in theprotein-carbohydrate crystal structures and are accessed inthe MD simulations, though occasionally, have not been predictedfrom NMR studies. Furthermore, these different values of leadto significantly different orientations of the 1,6-arm for thesame value of . This contrasts with the earlier predictionsthat only different values of can bring about significant changesin the orientation of the ₁,6-arm. The MD simulations also showthat the effects of bisecting GlcNAc or ß1,2-xyloseare very similar on the ₁,3-arm and slightly different on the₁,6-arm. bisecting GlcNAc carbohydrates glycoprotein lectinsaccharide complex     

Molecular dynamics simulations of high-mannose oligosaccharides

Conformations of several high-mannose-type oligosaccharidesthat are generated during the biosynthetic degradation of Man₉GlcNAc₂to Man₅GlcNAc₂ have been studied by molecular dynamics (MD).Simulations were performed on NCI-FCRDC's Cray Y-MP 8D/8128supercomputer using Biosym's CVFF force field for 1000 Ps withdifferent initial conformations. The conformations of the two1,3- and the two 1,6-linkages in each oligomannose were different,suggesting that deriving oligosaccharide conformations basedon the conformational preferences of the constituent disaccharidefragments will not always yield correct results. Unlike otheroligomannoses, Man₉GlcNAc₂ appears to take more than one distinctconformation around the core 1,6-linkage. These various conformationsmay play an important role in determining the processing pathways.Using the data on the preferred conformations of these oligomannosesand the available experimental results, possible pathways forprocessing Man₉GlcNAc₂ to Man₅GlcNAc₂ by 1,2-linkage-specificmannosidases have been proposed. Conformational analysis ofMan₅GlcNAc₂ indicates that the addition of ß1,2-GlcNActo the 1,3-linked core mannose, besides serving as a prerequisitefor mannosidase II action as suggested earlier, may also preventthe removal of 1,3-mannose. The MD simulations also suggestthat the processing of the precursor oligosaccharide duringAsn-linked complex and hybrid glycan biosynthesis proceeds ina well-defined pathway involving more than one 1,2-linkage-specificmannosidase. Knowledge of the conformation of the processingintermediates obtained from the present study can be used todesign highly specific substrate analogues to inhibit a particularmannosidase, thereby blocking one processing pathway withoutinterfering with the others. carbohydrates conformation glycosidase inhibitors mannosidase oligosaccharide processing