Stereospecific binding of diastereomeric peptides to salmon sperm deoxyribonucleic acid. Further evidence for partial intercalation

A series of diastereomeric dipeptide amides, containing an N-terminal L-lysyl residue and a C-terminal L- or D-amino acid with a derivatized aromatic ring on the side chain, was synthesized to determine the dependence of (1) the chirality of the N-terminal amino acid alpha-carbon and (2) the length of the N-terminal amino acid side chain for intercalation of the aromatic ring. The nature of the complex between the peptide and DNA (i.e., electrostatic, intercalative, or a combination of these) was determined by UV and CD studies, viscometric titrations, and 1H NMR studies. The results of these studies reveal distinct differences in the binding site of the aromatic rings of the various peptides. In particular, the results suggest that the alpha- and epsilon-amino groups of the lysyl residue bind electrostatically to adjacent phosphates on the DNA backbone in a stereospecific manner. As a result of this stereospecificity, the aromatic rings of the peptides with the L-L designation point toward the DNA helix, while those of the peptides of the L-D designation point away from the helix. This is completely consistent with previously reported work [Gabbay, E.J., Adawadkar, P. D., & Wilson, W. D. (1976) Biochemistry 15, 146; Gabbay, E. J., Adawadkar, P. D., Kapicak, L., Pearce, S., & Wilson, W. D. (1976) Biochemistry 15, 152]. The results also indicate a great dependence on the length of the side chain for intercalation of the aromatic ring. Specifically, if the side chain is long enough, and flexible enough, the aromatic ring can fully or partially intercalate, regardless of the chirality of the N-terminal amino acid alpha-carbon. However, if the side chain is too short, only partial intercalation is observed for peptides of the L-D designation, and no intercalation is observed for peptides of the L-D designation.     

Phylogenetic distribution in the genus Mus of t-complex-specific DNA and protein markers: inferences on the origin of t-haplotypes

We have examined the phylogenetic distribution of two t-specific markers among representatives of various taxa belonging to the genus Mus. The centromeric TCP-1a marker (a testicular protein variant specific for all t-haplotypes so far studied) has also been apparently detected in several non-t representatives of the Mus IVA, Mus IVB, and probably M. cervicolor species. By contrast, a t-specific restriction- fragment-length polymorphism allele (RFLP) of the telomeric alpha- globin pseudogene DNA marker alpha-psi-4 was found only in animals belonging to the M. musculus-complex species either bearing genuine t- haplotypes or, like the M. m. bactrianus specimen studied here, likely to do so. This t-specific alpha-psi-4 RFLP allele was found to be as divergent from the RFLP alleles of the latter, non-t, taxonomical groups as it is from Mus 4A, Mus 4B, or M. spretus ones. These results suggest the presence of t-haplotypes and of t-specific markers in populations other than those belonging to the M. m. domesticus and M. m. musculus subspecies, implying a possible origin for t-haplotypes prior to the radiation of the most recent offshoot of the Mus genus (i.e., the spretus/domesticus divergence), some 1-3 Myr ago.      

Growth hormone regulates the abundance of insulin-like growth factor I RNA in adult rat liver

Insulin-like growth factor I (IGF-I) is a mitogenic polypeptide present in the plasma of man and rat that is thought to mediate the actions of pituitary growth hormone on cartilage to promote skeletal elongation. In the rat, plasma levels of IGF-I show both developmental and hormonal regulation: levels are low at birth, increase with age, and are decreased in growth hormone-deficient adult animals. The present study demonstrates that these changes in plasma IGF-I reflect the abundance of IGF-I RNA in rat liver. A human IGF-I cDNA probe hybridized to multiple RNA species in adult rat liver with sizes 8.6, 4.6, 3.2, 2.1, and 1.0-1.4 kilobases. These RNA species were decreased by greater than 80% in neonatal (2- and 12-day-old) rat liver and by greater than 90% in liver from adult rats made growth hormone-deficient by hypophysectomy. Treatment of hypophysectomized rats with growth hormone increased the abundance of all species of IGF-I RNA. These results suggest that growth hormone regulates the expression of its physiological mediator by altering the synthesis, stability, or both of IGF-I RNA in rat liver.     

Linkage of the VNTR/insulin-gene and type I diabetes mellitus: increased gene sharing in affected sibling pairs.

Ninety-six multiplex type I diabetic families were typed at the 5' flanking region of the insulin gene by using a PCR assay that better resolves the VNTR into multiple alleles. Affected sibling pairs shared 2, 1, and 0 VNTR alleles--identical by descent--at a frequency of .47, .45, and .08, respectively, a ratio that deviated from the expected 1:2:1 ratio (P < .001). These results confirm linkage of the chromosome 11p15.5 region with type I diabetes mellitus susceptibility.     

Relationship between immunoglobulin levels and extremes of solar activity

The possible relationship between epidemics and extremes of solar activity has been discussed previously. The purpose of the present study was to verify whether differences in the levels of immunoglobulins (IgA, IgG, IgM) could be noted at the highest (July 1989) and lowest (September 1986) points of the last (21st) and present (22nd) 11-year solar cycle. The work was divided into a 1-month study (covering the month of minimal or maximal solar activity), a 3-month study (1 month before and after the month of minimal or maximal solar activity) and a 5-month study (2 months before and after the month of minimal or maximal solar activity). A trend of a drop-off for all three immunoglobulins was seen on the far side of the maximal point of the solar cycle. Statistical significance was achieved in the 5-month study for IgM (P=0.04), and a strong trend was shown for IgG (P=0.07). Differences between the sexes were also noted.     

Visualization of drug-nucleic acid interactions at atomic resolution. IX. Structures of two N,N-dimethylproflavine: 5-iodocytidylyl (3'-5') guanosine crystalline complexes

This paper describes two complexes containing N,N-dimethylproflavine and the dinucleoside monophosphate, 5-iodocytidylyl (3'-5') guanosine (iodoCpG). The first complex is triclinic, space group P1, with unit cell dimensions a = 11.78 A, b = 14.55 A, c = 15.50 A, alpha = 89.2 degrees, beta = 86.2 degrees, gamma = 96.4 degrees. The second complex is monoclinic, space group P21, with a = 14.20 A. b = 19.00 A, c = 20.73 A, beta = 103.6 degrees. Both structures have been solved to atomic resolution and refined by Fourier and least squares methods. The first structure has been refined anisotropically to a residual of 0.09 on 5,025 observed reflections using block diagonal least squares, while the second structure has been refined anisotropically to a residual of 0.13 on 2,888 reflections with full matrix least squares. The asymmetric unit in both structures contains two dimethylproflavine molecules and two iodoCpG molecules; the first structure has 16 water molecules (a total of 134 non-hydrogen atoms), while the second structure has 18 water molecules (a total of 136 non-hydrogen atoms). Both structures demonstrate intercalation of dimethylproflavine between base-paired iodoCpG dimers. In addition, dimethylproflavine molecules stack on either side of the intercalated duplex, being related by a unit cell translation along b and a axes, respectively. The basic structural feature of the sugar-phosphate chains accompanying dimethylproflavine intercalation in both structures is the mixed sugar puckering pattern: C3' endo (3'-5') C2' endo. This same structural information is again demonstrated in the accompanying paper, which describes a complex containing dimethylproflavine with deoxyribo-CpG. Similar information has already appeared for other "simple" intercalators such as ethidium, acridine orange, ellipticine, 9-aminoacridine, N-methyl-tetramethylphenanthrolinium and terpyridine platinum. "Complex" intercalators, however, such as proflavine and daunomycin, have given different structural information in model studies. We discuss the possible reasons for these differences in this paper and in the accompanying paper.     

The contractile basis of amoeboid movement: V. The control of gelation, solation, and contraction in extracts from dictyostelium discoideum

Motile extracts have been prepared from Dictyostelium discoideum by homogenization and differential centrifugation at 4 degrees C in a stabilization solution (60). These extracts gelled on warming to 25 degrees Celsius and contracted in response to micromolar Ca++ or a pH in excess of 7.0. Optimal gelation occurred in a solution containing 2.5 mM ethylene glycol-bis (β-aminoethyl ether)N,N,N',N'-tetraacetate (EGTA), 2.5 mM piperazine-N-N'-bis [2-ethane sulfonic acid] (PIPES), 1 mM MgC1(2), 1 mM ATP, and 20 mM KCI at ph 7.0 (relaxation solution), while micromolar levels of Ca++ inhibited gelation. Conditions that solated the gel elicited contraction of extracts containing myosin. This was true regardless of whether chemical (micromolar Ca++, pH >7.0, cytochalasin B, elevated concentrations of KCI, MgC1(2), and sucrose) or physical (pressure, mechanical stress, and cold) means were used to induce solation. Myosin was definitely required for contraction. During Ca++-or pH-elicited contraction: (a) actin, myosin, and a 95,000-dalton polypeptide were concentrated in the contracted extract; (b) the gelation activity was recovered in the material sqeezed out the contracting extract;(c) electron microscopy demonstrated that the number of free, recognizable F-actin filaments increased; (d) the actomyosin MgATPase activity was stimulated by 4- to 10-fold. In the absense of myosin the Dictyostelium extract did not contract, while gelation proceeded normally. During solation of the gel in the absense of myosin: (a) electron microscopy demonstrated that the number of free, recognizable F- actin filaments increased; (b) solation-dependent contraction of the extract and the Ca++-stimulated MgATPase activity were reconstituted by adding puried Dictyostelium myosin. Actin purified from the Dictyostelium extract did not gel (at 2 mg/ml), while low concentrations of actin (0.7-2 mg/ml) that contained several contaminating components underwent rapid Ca++ regulated gelation. These results indicated : (a) gelation in Dictyostelium extracts involves a specific Ca++-sensitive interaction between actin and several other components; (b) myosin is an absolute requirement for contraction of the extract; (c) actin-myosin interactions capable of producing force for movement are prevented in the gel, while solation of the gel by either physical or chemical means results in the release of F-actin capable of interaction with myosin and subsequent contraction. The effectiveness of physical agents in producting contraction suggests that the regulation of contraction by the gel is structural in nature.     

Chemical quantitation of hemoglobin glycosylation: fluorometric detection of formaldehyde released upon periodate oxidation of glycoglobin

A sensitive fluorometric method for the quantitation of hemoglobin glycosylation, based upon periodate oxidation of the carbohydrate moieties present on both the α- and ?-amino groups of globin is described. The formaldehyde product is measured as the fluorescent 3,5-diacetyl-1,4-dihydrolutidine formed from the condensation of formaldehyde with acetylacetone and ammonia.This method is rigorously designed to assay glycosylated hemoglobin levels and to give a direct measure of the number of glycogroups per milligram of hemoglobin. It requires only 1 mg of protein and may also be used to determine the extent of the nonenzmatic glycosylation of other proteins.