Size of the directing moiety at carbon 5 of cytosine and the activity of human DNA(cytosine-5) methyltransferase

M13 DNAs in which carbon 5 of each deoxycytidine residue in one strand is replaced with a bulky group are very good substrates for human DNA (cytosine-5) methyltransferase. Rate enhancements of up to 35 fold are obtained depending on the size of the moiety at C-5. The enzyme appears optimally suited to sense a methyl group in one strand at this position. Alkaline density gradient analyses of the distribution of methyl groups applied to 5-BrdCyd or 5-IdCyd substituted DNA reveal that these groups serve to direct the enzyme to methylate the unsubstituted strand.     

Human DNA (cytosine-5)methyltransferase selectively methylates duplex DNA containing mispairs.

The presence of the C.C mispair in a defined duplex oligodeoxynucleotide enhanced its capacity to serve as a substrate for highly purified human DNA methyltransferase. Analysis of tritiated reaction products showed that the C.C mispair acted as a "methylation acceptor" in that it was itself rapidly methylated. The m5C.G base pair also enhanced the capacity of the oligodeoxynucleotide to serve as a substrate for the enzyme. However, this complementary base pair was found to act as a "methylation director". That is, the presence of the m5C in one strand induced the enzyme to rapidly methylate at the cytosine residue on the opposite strand in an adjacent C.G base pair.     

Rescue of developmental lens abnormalities in chimaeras of noncataractous and congenital cataractous mice

In the study of the lens of a congenital cataractous mouse mutant (CAT), it has been shown that a loss of growth regulation at the cellular level causes gross lens abnormalities. The phenotypic characteristics of the cataractous mouse lens are similar to those seen in human congenital cataract and thus serves as a model system for medical research. In this present investigation, we have demonstrated that the abnormalities of the congenital cataractous lens can be rescued by forming chimaeras between DBA/2 (a noncataractous strain of mouse) and the CAT mutant. This report describes the histological, cellular and biochemical analysis of the resultant chimaeric eyes, and discusses possible mechanisms by which these results were achieved.     

Viability of an ectomycorrhizal fungus during cross-flow filtration

Factors affecting the viability and infectivity of an ectomycorrhizal fungus during moderate concentration by cross-flow filtration were determined. Mycelial suspensions were concentrated with three commercial membrane filters (Prostak Millipore Co., M14 Tech-Sep Co. and Ceraflo Norton Co.) under aseptic conditions. Medium components may reduce the filtration rate due to their low solubility. An antifoam agent did not reduce the average flux rates as much as did the malt extract. Clear unobstructed channels (I.D. 6mm) of the tubular modules (Tech-Sep) gave the best results both in terms of performance (filtration rate) and cell viability. Shear stresses caused by pumping and flow through narrow retentate channels were probably responsible for lowering viability and infectivity. There was no linear relationship between permeate fluxes and cell concentration. There is an optimum pore size both in terms of performance (filtration rate) and cell viability. Physical blockage of large pores by hyphae could explain lower permeate flux rates than those obtained with lower pore sizes membranes.     

Caprine acrosin. Purification, characterization and proteolysis of the porcine zona pellucida.

Acrosin purified from an acidic extract of ejaculated goat spermatozoa migrated as a single 42,000-Mr band in SDS/polyacrylamide-gel electrophoresis. Reduction and alkylation of caprine acrosin produced two polypeptides, one of Mr 40,000 (heavy chain) and the other of Mr 3700 (light chain). The light chain purified by reversed-phase h.p.l.c. was a glycosylated octadecapeptide with an amino acid sequence similar to that of the N-terminal 18 residues of porcine acrosin light chain (78% positional identity). The sequence of the N-terminal 37 amino acids of purified caprine acrosin heavy chain is similar to that of porcine acrosin heavy chain (70% positional identity through 37 residues). Studies with synthetic substrates and synthetic and natural proteinase inhibitors confirmed both the specificity of the purified proteinase for Arg-Xaa and Lys-Xaa bonds and a serine-proteinase mechanism. Purified caprine acrosin hydrolysed the 90 kDa and 65 kDa components, but did not hydrolyse the 55 kDa component of the porcine zona pellucida. The action of the enzyme on the porcine zona pellucida was indistinguishable from that previously reported for porcine acrosin.     

Bronchoconstrictor and antibronchoconstrictor properties of inhaled prostacyclin in asthma

Prostacyclin (PGI2) is generated in appreciable amounts during allergic reactions in human lung tissue. To define its activity on human airways we have studied the effects of doubling concentrations of inhaled PGI2 and its hydrolysis product 6-oxoprostaglandin F1 alpha (6-oxo-PGF1 alpha) on specific airway conductance (sGaw), maximum expiratory flow at 30% vital capacity (Vmax30), forced expiratory volume in 1 s (FEV1), and static lung volumes in subjects with mild allergic asthma. In a second study the effect of inhaled PGI2 on bronchoconstriction provoked by increasing concentrations of inhaled prostaglandin (PG) D2 and methacholine was observed. Inhalation of PGI2 up to a concentration of 500 micrograms/ml had no significant effect on sGaw but produced a concentration-related decrease in FEV1 and Vmax30 in all subjects. In two of four subjects inhalation of PGI2 also increased residual volume and decreased vital capacity but had no effect on total lung capacity. PGI2, but not 6-oxo-PGF1 alpha, protected against bronchoconstriction provoked by either PGD2 or methacholine whether airway caliber was measured as sGaw, FEV1, or Vmax30. The apparent disparity between the bronchoconstrictor and antibronchoconstrictor effects of PGI2 might be explained by its potent vasodilator effect in causing airway narrowing through mucosal engorgement and reducing the spasmogenic effects of other inhaled mediators by increasing their clearance from the airways.     

High level of divergence of male-reproductive-tract proteins, between Drosophila melanogaster and its sibling species, D. simulans

We compared male-reproductive-tract polypeptides of Drosophila melanogaster and D. simulans by using two-dimensional gel electrophoresis. Approximately 64% of male-reproductive-tract polypeptides were identical between two randomly chosen isofemale lines from these two species, compared with 83% identity for third-instar imaginal wing-disc polypeptides. Qualitatively similar differences were found between reproductive tracts and imaginal discs when D. sechellia was compared with D. melanogaster and with D. simulans. When genic polymorphism was taken into account, approximately 10% of male- reproductive-tract polypeptides were apparently fixed for different alleles between D. melanogaster and D. simulans; this proportion is the same as that found for soluble enzymes by one-dimensional gel electrophoresis. Strikingly, approximately 20% of male-reproductive- tract polypeptides of either D. melanogaster or D. simulans had no detectable homologue in the other species. We propose that proteins of the Drosophila male reproductive tract may have diverged more extensively between species than have other types of proteins and that much of this divergence may involve large changes in levels of polypeptide expression.      

Profiles of hypoxanthine guanine phosphoribosyl transferase and adenine phosphoribosyl transferase activities measured in single preimplantation human embryos by high-performance liquid chromatography

The profiles of hypoxanthine guanine phosphoribosyl transferase (HGPRT) and adenine phosphoribosyl transferase (APRT) activities were examined in normally fertilized human embryos developing at the normal rate in vitro between the 2-4-cell stage on Day 2 and the blastocyst stage on Day 6 after insemination. The activities of both enzymes were assayed simultaneously in extracts of single embryos by measuring the rate of production of the reaction products, inosine monophosphate (IMP) and adenine monophosphate (AMP), separated by high-performance liquid chromatography (HPLC). The activity profiles of the two enzymes over this period showed marked differences. The activity of HGPRT, coded by the X chromosome, increased between Days 2 and 4 (P less than 0.01) but declined sharply by Day 6 (P less than 0.001), whereas autosome-coded APRT activity remained low between Days 2 and 5, but increased on Day 6 (P less than 0.05). The profile of HGPRT activity may reflect a combination of decreasing levels of maternal enzyme inherited from the oocyte and the initiation of embryonic gene expression followed by X inactivation at the blastocyst stage on Day 6.     

A review of the USAF/NASA proton bioeffects project: rationale and acute effects.

Ionizing radiation represented one of the important hazards facing the first manned lunar mission. A combined USAF/NASA project was conducted from 1963 through 1969 to estimate the relative biological effectiveness (RBE) of the radiations of space. Approximately 2000 primates (Macaca mulatta) and 5000 mice were irradiated with protons and electromagnetic radiations. The proton energies studied were selected to be representative of the proton spectrum in space. Much of the project was concerned with the use of cyclotrons for proton irradiations and with dosimetry. Biological measurements included clinical findings, physiological changes, hematological changes, histopathology, and mortality. When allowance was made for variation of response as a consequence of depth-dose distribution, the RBE for protons was approximately 1. This was anticipated from earlier theoretical studies and radiation therapy in humans with high-energy charged-particle beams.