Lysine Dendrimers and Their Starburst Polymer Derivatives: Possible Application for DNA Compaction and in vitro Delivery of Genetic Constructs

We attempted to find some compounds for the effective delivery of gene constructs into cells and obtained two trispherical dendrimers on the basis of lysine, (Lys)₈-(,-Lys)₄-(,-Lys)₂-(,-Lys)-Ala-NH₂ (D1) and ((Lys)₈-(,-Lys)₄-(,-Lys)₂-,-Lys)-Ala-[Lys(Plm)]₂-Ala-NH₂ (D2), as well as the starburst polymeric derivatives of D1, (pVIm) ₈ -D1 and (pLys) _n -D1, containing poly(N-vinylimidazole) and polylysine chains single-point bound to the dendrimer amino groups. The conditions of dendrimer–plasmid DNA complex formation were studied. The intracellular localization of these complexes and the expression of gene constructs delivered with their help were analyzed in transfection experiments on the HeLa cell cultures of human epithelial carcinoma and on mouse C2C12 myoblasts. It was found that the chemical structure of dendrimer D1 and its derivatives significantly affected the structure and properties of complex.     

Gender- and age-specific contributions of additional DNA sequence variation in the 5′ regulatory region of the <Emphasis Type="Italic">APOE</Emphasis> gene to prediction of measures of lipid metabolism

In the present study of 9,000 individuals representative of the general population, we have considered whether the addition of common single nucleotide polymorphisms (SNPs) in the promoter region of Apolipoprotein E (APOE) improve the statistical explanation of variation in lipid traits and test the hypothesis that the estimated genotype effects are independent of factors indexed by gender and age. To address these questions, we have asked, for each gender and for each 20-year age strata (young: 20–39 years; middle-aged: 40–59 years; old: 60–79 years; very old: 80–100 years), how much trait variation is associated with the traditional 2, 3, and 4 allelic variations defined by the g.2059TC and g.2197CT SNPs in the fourth exon of the APOE gene, and how much additional trait variation is associated with genotypes defined by combining the g.2059TC and g.2197CT SNPs with one, two, or three promoter SNPs. Our study demonstrates that the pleiotropic effects of genotype variation defined by the traditional 2, 3, and 4 alleles on five plasma measures of lipid metabolism manifest differently in women and men and change significantly during the life cycle for high-density lipoprotein cholesterol in women. Multi-site genotypes defined by adding SNPs located in the 5 promoter region to the traditional g.2059TC and g.2197CT SNPs doubled the estimate of genetic variance of high-density lipoprotein and apolipoprotein Al in middle-aged females.     

ε-caprolactam,a new powerful inducer for the formation of Rhodococcus rhodochrous J1 nitrilase

We found that -caprolactam is a new powerful inducer for the formation of Rhodococcus rhodochrous J1 nitrilase. When Rhodococcus rhodochrous J1 cells were cultivated at 28°C for 120 h in a nutrient medium supplemented with 0.5% (w/v) -caprolactam, an enormous amount of nitrilase was formed in the cells which corresponded to approximately 30% of all soluble protein. The level of -caprolactam in the culture broth barely decreased in the course of cultivation. -Butyrolactam and -valerolactam also caused effective induction. The induction of nitrilase formation by -caprolactam was also observed in some other Rhodococcus strains.     

Pyridoxal-5′-phosphate derivatives of substance P: Preparation,spasmogenic activity,and degradation by human plasma

Two fluorescent derivatives of substance P (SP) (Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH₂) were prepared by chemical modification of the native peptide by pyridoxal-5-phosphate (pyridoxal-P). The formation of both pyridoxal-P-derivatives of SP is the result of one modification procedure. The determination of the amino acid composition showed that in one of the derivatives the -amino group of the Lys residue [-(P-pxy)-SP] and in the other the -amino group of the Lys residue and also the N-terminal amino group [,-di-(P-pxy)-SP] of SP had been substituted by pyridoxal-P. -(P-pxy)-SP and ,-di-(P-pxy)-SP have spasmogenic activity with ED₅₀ of 1.8×10^–9 and 4×10^–9 M, respectively, tested on isolated guinea pig ileum. The fluorescence of P-pxy residues permits detection of as little as 1 pmol/ml of -(P-pxy)-SP and 0.5 pmol/ml of ,-di-(P-pxy)-SP. Both analogues of SP obtained are degraded by human plasma more slowly than the native peptide.Abbreviations SP substance P - pyridoxal-P pyridoxal-5-phosphate - P-pxy phospho-pyridoxyl residue - -(P-pxy)-SP substance P modified by pyridoxal-P at the -amino group of the Lys residue - ,-di-(P-pxy)-SP substance P modified by pyridoxal-P at the -amino group of the Lys residue and the N-terminal amino group of SP - (P-pxy)-Lys Lys modified by pyridoxal-P at the -amino group     

Substitutions of the Conserved Thr42 Increased the Roles of the ε-Subunit of Maize CF1 as CF1 Inhibitor and Proton Gate

The conserved residue Thr42 of -subunit of the chloroplast ATP synthase of maize (Zea mays L.) was substituted with Cys, Arg, and Ile, respectively, through site-directed mutagenesis. The over-expressed and refolded -proteins were purified by chromatography on DEAE-cellulose and FPLC on mono-Q column, which were as biologically active (inhibiting Ca²⁺-ATPase activity and blocking proton gate) as the native subunit isolated from chloroplasts. The T42C and T42R showed higher inhibitory activities on the soluble CF₁(–) Ca²⁺-ATPase than the WT. The T42I inhibited the Ca²⁺-ATPase activity of soluble CF₁ and restored photophosphorylation activity of membrane-bound CF₁ deficient in the most efficiently. Far-ultraviolet CD spectra showed that the portions of -helix and -sheet structures of the three mutants were somewhat different from WT. Thus the conserved residue Thr42 may be important for maintaining the structure and function of the -subunit and the basic functions of the -subunit as far as an inhibitor of Ca²⁺-ATPase and the proton gate are related.     

IgE heavy chain constant region genes in atopic dermatitis and senile erythroderma patients

By Southern hybridization using a genomic DNA fragment carrying a human IgE heavy chain constant region gene (C ) as a probe, we analyzed the organization of human C genes and their flanking regions in 23 atopic dermatitis and 6 senile erythroderma patients with elevated serum IgE levels, and 6 atopic dermatitis patients with normal IgE levels. On Barn HI, Hind III, and Eco RI digestions, we detected three hybridizable fragments containing three human C genes, C 1, C 2, and C 3, respectively, in all leukocyte DNAs. These fragments were almost identical in size among patients and healthy donors. Pst I digestion generated a genetic polymorphism. We, however, could find no correlation between this polymorphism and the disorders. It was concluded that among the patients and healthy donors, there was no marked difference in the organization of the functional C gene and its flanking region containing a class switch region. Our conclusion cannot rule out the presence of genetic abnormalities of this region in some atopic dermatitis patients which are not resolvable by our method. In the course of this study, we found a novel C -like gene in placenta DNA which differs from the three C genes commonly present in normal human DNA.     

Differences in the number of embryonic and pseudo-β-globin genes between HbA and HbB sheep

DNA samples obtained from 8 goats, 1 moufflon, and 84 sheep with HbA, HbAB, and HbB belonging to different breeds were digested withBamHI,EcoRI,HindIII andPstI and probed with the 5 end of the goat ^IV- and ^Z-globin genes. Sheep homozygous for HbA show a different restriction pattern than sheep homozygous for HbB with each of these endonucleases. The main differences is that HbB sheep lack the ^H and ^X genes. These results, in addition to those previously obtained using a probe specific for -globin genes, suggest that HbB sheep probably lack the preadult four-gene set. The DNAs from moufflon and sheep homozygous for HbA show indistinguishable restriction patterns. Furthermore, a number of restriction fragment length polymorphisms (RFLPs) are detected in the ^IV and ^Z DNA regions, and oneHindIII RFLP in the ^VI DNA region.This work was supported by the Ministero della Pubblica Istruzione and, in part, by the Consiglio Nazionale delle Ricerche.     

Phylogeny of immunoglobulin heavy chain isotypes: structure of the constant region of Ambystoma mexicanum υ chain deduced from cDNA sequence

An RNA polymerase chain reaction strategy was used to amplify and clone a cDNA segment encoding for the complete constant part of the axolotl IgY heavy (C) chain. C is 433 amino acids long and organized into four domains (C1–C4); each has the typical internal disulfide bond and invariant tryptophane residues. Axolotl C is most closely related to Xenopus C (40% identical amino acid residues) and C1 shares 46.4% amino acid residues among these species. The presence of additional cysteines in C1 and C2 domains is consistent with an additional intra-domain S-S bond similar to that suggested for Xenopus C and C, and for the avian C and the human C. C4 ends with the Gly-Lys dipeptide characteristic of secreted mammalian C3, human C4, and avian and anuran C4, and contains the consensus [G/GT(AA)] nucleotide splice signal sequence for joining C4 to the transmembrane region. These results are consistent with the hypothesis of an ancestral structural relationship between amphibian, avian chains, and mammalian chains. However, these molecules have different biological properties: axolotl IgY is secretory Ig, anuran and avian IgY behave like mammalian IgG, and mammalian IgE is implicated in anaphylactic reactions.The nucleotide sequence data reported in this paper have been submitted to the EMBL/GenBank nucleotide sequence database and have been assigned the accession number X69492. Correspondence to: J. S. Fellah.     

Genetic Association between the Apolipoprotein E (APOE) Gene and Different Forms of Alzheimer's Disease

Allele 4of the apolipoprotein E (APOE) gene is associated with higher risk for family or sporadic Alzheimer's disease (AD) in many, though not all, ethnic groups. The APOEallele and genotype frequency distributions were evaluated in 207 AD patients without vascular disorders, 62 AD patients with vascular disorders (combined AD), and 206 control individuals (ethnic Russians from the Russian population). The frequency of allele 4in patients with early-onset and late-onset AD was three times higher than in controls (P< 0.000001). The increase in the frequency of 4in mixed dementia cases over controls was somewhat less but still significant (P= 0.0019). Relative risk of AD in carriers of allele 4was five times higher than in carriers of alleles 2and 3(P< 0.000001). Allele 2showed evidence of a protective effect in the early-onset AD group (P= 0.015). These results suggest that APOEallele 4is a universal factor of early-onset, late-onset, and combined AD in ethnic Russians from Russia.     

Protein kinase C isoenzymes in mouse Harderian gland. Differential expression of the α- and ε-isoforms during pregnancy

Protein kinase C (PKC) is known to be involved in the regulation of exocytosis in different cell lines and tissues. Experiments were designed to determine whether the Harderian gland of CD-1 mouse produces PKC isoenzymes and whether the expression of the isoforms changes during pregnancy. The presence of the isoenzymes was assessed by immunoblotting experiments using extract of total Harderian gland and polyclonal antisera specific for nine different PKC isoforms. Antisera giving a positive staining on Western blots were subsequently used for immunohistochemical investigation using a secondary antibody conjugated to alkaline phosphatase. Immunoblotting experiments revealed that the Harderian gland from female mouse expresses PKC isoforms-,-,- and-. These isoforms were also detected in the Harderian gland from 13-day pregnant mouse; however, striking quantitative changes were seen concerning the - and -isoforms. The 80-kDa native form of PKC- almost doubled in the pregnant mouse in comparison with normal female mouse whereas the amount of 50-kDa catalytic domain did not change. Protein kinase C- appeared as a 92- to 93-kDa form and a 67-kDa form. While the 92- to 93-kDa protein was expressed to a similar extent in both types of mouse, the 67-kDa form was more abundant inthe Harderian gland from normal female mouse. These data were corroborated by immunohistochemical experiments and showing a diffuse and granular staining of the adenomeres. These observations demonstrate for the first time (to our knowledge) that the mouse Harderian gland produces several PKC isoenzymes that could be involved in the regulation of exocytosis and/or other functions. Moreover, the expression of the - and -isoforms could be regulated by sexual hormones, as suggested by the differential abundance of these two proteins in the gland of pregnant mouse compared with normal female mouse.     

PKCε inhibits the hyperglycemia-induced apoptosis signal in adult rat ventricular myocytes

Recruitment of the protein kinase C (PKC) family of isozymes is an integral component of the signaling events that direct cardiac phenotype expressed during postnatal development and in response to pathologic stimuli. Hyperglycemia is a potent activating signal for cardiac PKC isozymes and induces the apoptosis program in cardiac muscle cells. To determine whether cardiac PKC isozymes modulate transmission of the hyperglycemia apoptosis signal, we have employed isozyme-specific peptide modulators to selectively inhibit (PKC _I/_II, and ) or activate (PKC). PKC peptides were delivered to primary cultures of serum starved adult rat ventricular myocytes (ARVM), by conjugation to the homeodomain of drosophila antennapedia. As expected, hyperglycemia induced a 35% increase in ARVM apoptosis. Peptide inhibitors of PKC _I/_II and blocked transmission of the hyperglycemia apoptosis signal, whereas the isozyme specific inhibitor of PKC (V1-2) did not alter the magnitude of glucose-induced ARVM apoptosis. Alternatively, the PKC translocation activator (RACK) abolished hyperglycemia-induced apoptosis, strongly suggesting a cardioprotective role for PKC in this system. Therefore, we conclude that cardiac PKC isozymes modulate hyperglycemia-induced apoptosis and activation of cardiac PKC protects ARVM from the hyperglycemia-induced death signal. (Mol Cell Biochem 268: 169–173, 2005)     

Effect of dexamethasone on T-cell receptor/CD3 expression

Glucocorticoid hormones (GCH) are anti-inflammatory and immunosuppressive agents that inhibit T-cell growth and activation. Since the T-cell receptor (TCR)/CD3 complex mediates T-lymphocyte activation, we studied the effect of in vitro dexamethasone (DEX), a synthetic GCH, on TCR/CD3 expression.DEX-treatment of a hybridoma T-cell line and normal un-transformed T-cell clones induced a decrease of the TCR/CD3 membrane expression after 4 days. After 4 weeks, TCR/CD3 was undetectable. However, the amount of mRNAs coding TCR/CD3 chains, including TCR, TCR, CD3, CD3 and CD3, as well as the amount of CD3 protein, a major component of the complex, were unaltered. By contrast, a decrease of the mRNAs deriving from the TCR gene locus, as well as of the TCR protein which is responsible for the membrane expression of the TCR/CD3 complex, was induced.These data suggest that the down-modulation of TCR expression is due to the diminution of TCR gene products in DEX-treated cells. (Mol Cell Biochem 167: 135-144, 1997)     

Kinetic comparison of caiman ε-crystallin and authentic lactate dehydrogenases of vertebrates

Kinetic comparison of -crystallins isolated from the avian and reptilian species and the authentic lactate dehydrogenases (LDHs) was undertaken in order to clarify the identities of these structural lens proteins in relation to their enzymatic activity. Caiman -crystallin similar to the previously characterized duck -crystallin appeared to possess a genuine and stable LDH activity as detected by nitro blue tetrazolium staining on polyacrylamide gels and conventional kinetic assays. Kinetic parameters for pyruvate,l-lactate, NAD⁺, and three structural analogues of the coenzyme in this -crystallin catalyzed reaction were also determined and compared. Despite the structural similarities between -crystallins and chicken heart LDH, differences in charge and kinetic properties have been revealed by native isozyme electrophoresis and kinetic analysis as examined by initial velocity and substrate inhibition studies. It is found that the kinetic data analyzed for caiman -crystallin were more fitted with a compulsory ordered Bi-Bi sequential mechanism similar to those for the authentic LDHs and duck -crystallin. Caiman -crystallin has for the first time been established as a heart-type LDH based on the kinetic analysis and comparison with the authentic heart- and muscle-type LDHs from pig and chicken.     

Interaction of Apolipoprotein E ε 4 With Other Genetic and Non-Genetic Risk Factors in Late Onset Alzheimer Disease: Problems Facing the Investigator

The Apolipoprotein E4 allele (Apo-4) is the major susceptibility gene for late onset Alzheimer Disease (AD) but epidemiological data suggest that the effect of this allele is modified in different individuals by genetic or environmental factors. Age and head injury are major non-genetic factors modifying the Apo-4 risk. There is conflicting data as to whether alleles of other chaperon proteins (such as alpha-1-antichymotrypsin (ACT)) or Apo-4 receptors (such as the VDRL receptor) modify the Apo-4 risk for AD. We analyze problems posed by genetic association studies including those of multiple comparisons and selection of controls, the latter problem exacerbated by the wide variations in Apolipoprotein E allele frequencies observed in different groups and localities.     

Decreases in Plasma Aβ1-40 Levels with Aging in Non-Demented Elderly with ApoE-ε4 Allele

This report examines plasma amyloid proteins A40 and A42 and apolipoprotein E (apoE) levels and their relationships with age in non-demented older adults with (N = 32) or without the apoE-4 allele (N = 94). A levels did not differ between the groups whereas the 4 allele was associated with a significant reduction in plasma apoE. In subjects with the 4 allele, increasing age was associated with significant reduction in plasma A40. Subjects without the 4 allele showed a significant positive correlation between A40 and A42 levels. There was also a significant correlation between plasma A40 and apoE levels in all subjects.     

Detection of ε(γ-glutamyl) lysine

Summary Detection of (-glutamyl) lysine crosslinks is not only necessary for establishing the importance of the dipeptide as a post-translational modification of proteins, but provides information as to the importance of the transglutaminase enzyme in a biological system. The crosslink may be detected using both indirect and direct methodology. Indirect methods for its detection include measurement of masked lysines within a protein, detection of polymer formation by gel-electrophoresis and the inhibition of crosslinking by the incorporation of small molecular weight amines into the substrate protein. Direct methods for the detection of (-glutamyl) lysine require the actual isolation of the dipeptide following its release from the sample protein by exhaustive proteolytic digestion. Separation of the dipeptide from other components of the digest may be achieved by either ion-exchange chromatography or gel filtration and its qualitative identification achieved by techniques such as paper-electrophoresis or thin layer chromatography. Quantitative estimation of (-glutamyl) lysine normally involves its further separation by ion-exchange chromatography and its post-column detection following derivatisation with ninhydrin. More recent techniques include pre-column derivatisation of the dipeptide with fluorogenic reagents such as -pthalaldehyde and separation by reverse phase HPLC. With the recent advances in liquid chromatography resulting in the improved resolution of amino acids, increased sensitivity, rapid analysis times, and small sample sizes, it appears likely that direct quantitation of (-glutamyl) lysine will be the preferred method for the future.     

Intracellular sites of the synthesis of sweet potato mitochondrial F1ATPase subunits

Summary Five subunits (-, -, -, - and -subunits) of the six -and -subunits) in the F₁ portion (F₁ATPase) of sweet potato (Ipomoea batatas) mitochondrial adenosine triphosphatase were isolated by an electrophoretic method. The - and -subunits were not distinguishable immunologically but showed completely different tryptic peptide maps, indicating that they were different molecular species. In vitro protein synthesis with isolated sweet potato root mitochondria produced only the -subunit when analyzed with anti-sweet potato F₁ATPase antibody reacting with all the subunits except the -subunit. Sweet potato root poly(A)⁺RNA directed the synthesis of six polypeptides which were immunoprecipitated by the antibody: two of them immunologically related to the -subunit and the others to the - and -subunits. We conclude that the -subunit of the F₁ATPase is synthesized only in the mitochondria and the -, - and -subunits are in the cytoplasm.