Methylated poly(L-lysine): conformational effects and interactions with polynucleotides

Methylated lysine, arginine and histidine residues are found in a number of proteins (for example, histones, non-histone chromosomal proteins, ribosomal proteins, calmodulin, cytochrome C, etc.). We are studying the effects of methylation on the conformations of poly(lysine) and of the effects of methylation of poly(lysine) and poly(arginine) on interactions with polynucleotides. The conformational properties of epsilon-amino-methylated poly(lysine) differ from those of unmodified poly(lysine). Methylation increases resistance to thermally-induced and NaCl-induced changes in the CD spectrum. Guanidinium chloride increases (proportional to the degree of methylation) the extent of approach to the conformation in dispute as to its being a random coil or an extended helix. Methylation enhances aggregation in the helix-inducing solvent 0.5 M Ca(ClO4)2. With increasing methylation of poly(lysine), the conformation in dodecyl sulfate changes from beta, to 50% alpha, to random coil at the maximum methylation. Increasing methylation of poly(lysine) weakens the interaction with polynucleotides in respect to dissociation by salt, linearly with methyl content. Complexes of (dAdT)n.(dAdT)n with the polypeptides are increasingly stabilized to heat denaturation by progressive methylation. However, with a series of synthetic double-stranded RNA's and DNA's a more complex situation exists, Tm increasing or decreasing, depending on the base composition, sequence and type of sugar. Methylation of poly(lysine) and poly(arginine) can have opposite effects on Tm based on results with complexes with (dI)n.(dC)n. Methylated poly(lysine) affects the CD spectrum of polynucleotides, in a manner dependent on base composition and sequence. In some cases large positive or negative psi-spectra are induced, which, in the case of (dGdC)n.(dGdC)n, can be positive or negative depending on the degree of methylation of the polypeptide and the salt concentration. It is suggested that the biological effects of methylation proteins may be evoke by salt changes in the cell cycle, and that methylation can affect local interactions with nucleic acids and larger scale structure, and interactions with lipids.     

Genetic polymorphism of the sixth component (C6) of rat complement

The complement protein C6 has been shown to be genetically polymorphic in the rat. Isoelectric focusing of plasma samples from 19 inbred strains demonstrated two electrophoretically distinguishable migration patterns, each consisting of three bands. Breeding studies with the use of the BN and DA strains showed that the C6 patterns were inherited in a manner consistent with the co-dominant autosomal expression of two alleles (C6 A and C6 B). The distribution of the C6 alleles in a backcross mating was compared with eight independently segregating marker genes: RT1.A, RT2, Gdc -1, Igk-1, Hbb, Svp-1, Fh-1, and Es-6. There was no detectable linkage between C6 and any of these eight loci.     

Genetic polymorphism of the human sex hormone-binding globulin: evidence of an isoelectric focusing variant with normal androgen-binding affinities

Human sex hormone-binding globulin (hSHBG) is a plasma glycoprotein composed of two identical subunits. The protein, which has high affinity for testosterone and estradiol has been purified to homogeneity. In this study we have investigated, on neuraminidase-treated serum samples, the presence of genetic variations of hSHBG by polyacrylamide gel isoelectric focusing (IEF). Based on IEF analyses of 110 serum samples from adult Mexican individuals we have identified two distinct IEF-patterns. The most frequent phenotype (95.45%) was characterized by two IEF-bands with pIs of 6.50 and 6.63, respectively. In five serum samples, a different 4-band pattern with pIs of 6.50, 6.63, 6.70 and 6.76 was identified. Family studies showed that this pattern was genetically determined. The frequency of this variant was 4.55%, and the observed phenotypes were consistent with the expression of an autosomal genetic system. The estimated gene frequencies for both alleles were shown to be in genetic equilibrium. Affinity constants, binding kinetics and serum concentrations of hSHBG from individuals having a 4-band pattern were similar to those obtained in individuals with a 2-band pattern, thus suggesting that the mechanism responsible for the generation of polymorphic variants of hSHBG reported herein did not involve the steroid binding site of the molecule. These findings may be of broad interest, as other serum binding proteins express genetic variants, which may permit their further structural and functional subclassification.     

Human C4 haplotypes with duplicated C4A or C4B

In the course of study of families for the sixth chromosome markers HLA-A, C, B, D/DR, BF, and C2, the two loci for C4, C4A, and C4B, and glyoxalase I, we encountered five examples of probable duplication of one or the other of the two loci for C4. In one of these, both parents and one sib expressed two different structural genes for C4B, one sib expressed one, and one sib expressed none, suggesting that two C4B alleles were carried on a single haplotype: HLA-A2, B7, DR3, BFS1, C2C, C4A2, C4B1, C4B2, GLO1. In a second case, two siblings inherited C4B*1 and C4B*2 from one parent and C4B*Q0 from the other. This duplication appeared on the chromosome as HLA-AW33, B14, DR1, BFS, C2C, C4A2, C4B1, C4B2, GLO2. In a third, very large family with 3 generations, a duplication of the C4B locus occurred which was followed in 2 generations. In one individual, there were three C4B alleles and two C4A alleles. One of the C4B alleles had a hemolytically active product with electrophoretic mobility near C4B2 and was designated C4B*22. It segregated with C4B1 in the family studied. The complete haplotype was HLA-A11, CW1, BW56, DR5, BFS, C2C, C4A3, C4B22, C4B1, GLO2. In another family with 12 siblings, one parent and eight children expressed two C4A alleles on the haplotype HLA-AW30, BW38, DR1, BFF, C2C, C4A3, C4A2, C4BQ0, GLO1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Screening of insect cell lines for the production of recombinant proteins and infectious virus in the baculovirus expression system.

Eight cell lines derived from the insects Spodoptera frugiperda, Trichoplusia ni, Mamestra brassicae, and Estigmene acrea were evaluated for recombinant beta-galactosidase and infectious virus production following infection with the baculovirus Autographa californica multiple nuclear polyhedrosis virus (AcMNPV). Production was assessed on a specific (per cell and per microgram of uninfected cellular protein) and on a volumetric (per milliliter) basis. Cell density was found to be an important factor in comparing the cell lines due to a density-dependent inhibition of specific protein and virus production that appeared to result from cell-cell contact. After infection of cells at low-density specific beta-galactosidase production per cell would drop between 3- and 6-fold in five of the eight cell lines when plated on tissue culture plates at near-confluent and confluent cell densities. The cell lines Sf 21 and Sf 9 were least sensitive to cell density. After accounting for cell density effects and differences in cell size, two cell lines, BTI Tn 5B1-4 and BTI TnM, were identified that were superior to the other cell lines, including Sf 21 and Sf 9, in beta-galactosidase production. Optimal volumetric and specific beta-galactosidase production from Tn 5B1-4 and TnM cells was 2-fold and 5-fold higher, respectively, in both cell lines than the optimal production from Sf 9 or Sf 21 cells. The Tn 5B1-4 cell line also had the highest viability of all the cell lines at 3 days postinfection and could be adapted to serum-free media.(ABSTRACT TRUNCATED AT 250 WORDS)     

Detection of Autographa californica and Heliothis zea baculovirus proteins by enzyme-linked immunosorbent assay (ELISA)

The structural proteins of Autographa californica (AcMNPV) and Heliothis zea (HzSNPV) nuclear polyhedrosis viruses were detected by indirect enzyme-linked immunosorbent assay (ELISA). The immunoassay detected less than 1 ng of AcMNPV protein. The extent of immunological relatedness between AcMNPV-occluded virus and AcMNPV polyhedral protein, AcMNPV-nonoccluded virus, Estigmene acrea granulosis virus, Amsacta moorei entomopoxvirus Heliothis zea NPV, and Lymantria dispar NPV was determined. No immunological relatedless was detected between HzSNPV, AcMNPV, and a persistent rod-shaped virus isolated from the Heliothis zea cell line (IMC-Hz-1). The polyhedral proteins of HzSNPV and AcMNPV were found to be immunologically identical.     

Infectivity and mode of action of Baculoviruses

The genus Baculovirus contains three subgroups of viral types: (1) nuclear polyhedrosis viruses (NPVs), (2) granulosis viruses (GVs), and (3) nonoccluded baculoviruses. While little information is available for viruses from the third subgroup, several aspects of the infectivity and mode of action of NPVs and GVs have been studied. The most common route of entry of a virus into an insect host is per os, and both virus types enter midgut cells (primary site of infection) by membrane fusion. However, two distinct mechanisms of virus uncoating occur among the baculoviruses: NPVs uncoat within the nucleus, whereas GVs uncoat at the nuclear pore complex. Baculoviruses of subgroup 3 appear to uncoat by either mechanism. In addition to replicating within the nucleus, NPV inoculum virus may pass through the intestinal epithelium immediately after ingestion, thereby establishing a systemic infection of the hemocoel prior to virus replication in midgut cells. The GVs do not appear to pass through midgut cells as rapidly as NPVs and in general, the developmental cycle of GVs is longer than that of NPVs. NPVs have been grown in cell culture while GVs have not.     

Absolute configuration of phomactatriene diterpenoids obtained by Wagner‐Meerwein rearrangement of epimeric verticillols

The epimeric diterpenes (+)‐(1S,3E,7E,11S,12S)‐verticilla‐3,7‐dien‐12‐ol ( 1 ), isolated from Bursera suntui, and (+)‐(1S,3E,7E,11S,12R)‐verticilla‐3,7‐dien‐12‐ol ( 2 ), isolated from Bursera kerberi, gave the same Wagner‐Meerwein rearrangement product (?)‐(1E,4Z,8Z,11S,12R)‐phomacta‐1,(15)4,8‐triene ( 3 ). The Et₂O:BF₃‐induced transformations evidence that verticillenes and phomactanes, both containing the bicyclo[9.3.1]pentadecane skeleton, are biogenetically related through the verticillen‐12‐yl cation ( A ⁺ ), which also is a key intermediate in the biosynthetic pathways to generate antitumor taxanes. Molecular modeling using the Monte Carlo protocol, followed by density functional theory (DFT) geometry optimization employing the hybrid functionals B3LYP and B3PW91, both with the DGDZVP basis set, secured the configuration of 3 as followed from the good agreement between the calculated and experimental vibrational circular dichroism spectra. Similar DFT calculations allowed determining the absolute configuration of (+)‐(1R,4R,5R,8S,9S,11S,12R,15R)‐1,15:4,5:8,9‐triepoxyphomactane ( 9 ), which surprisingly derives from epoxidation of the second minimum energy conformer of 3 .     

In vitro antimicrobial effect of metallic nanoparticles on phytopathogenic strains of crop plants

In this research, the in vitro antimicrobial effect of zinc oxide (ZnO), copper oxide (CuO) and iron oxide (Fe₂O₃) nanoparticles (NPs)—with average sizes of 20, 46 and 30 nm, respectively—on the root rot disease caused by the fungus Fusarium oxysporum and on blight disease caused by the fungus Alternaria solani were studied. Also, bacterial diseases caused by Clavibacter michiganensis and Pseudomonas syringae that infects a wide range of plant species were assessed. Different concentrations of NPs (0, 100, 250, 500, 700 and 1,000 mg/L) were prepared on PDA agar or King's B medium in a complete randomized design with four replicates. According to the results, ZnO NPs exhibited an outstanding inhibitory effect against fungi and bacteria strains. The above results were associated with the smaller particle size. Fungi strains showed a differential sensitivity depending on the kind of NPs used. A. solani showed the highest sensitivity to ZnO NPs at 1,000 mg/L (99%), followed by CuO NPs at the same dose (95%). Fe₂O₃ NPs at all evaluated doses had no inhibitory effects on the mycelia growth of this strain, although F. oxysporum revealed greater effectiveness of the CuO NPs (96%) compared with ZnO NPs since it only inhibited 91% of the mycelial growth. The antibacterial activity was studied through optical density. C. michiganensis was found to be more sensitive to ZnO NPs because a lesser dose (700 mg/L) was required to reduce the bacterial growth (90%); in comparison, P. syringae required a dose of 1,000 mg/L to inhibit its growth (67%). CuO NPs displayed the smallest growth inhibition against the bacteria strains analysed. The antimicrobial effect of the metallic NPs that were assayed increased with higher doses.