Different sensitivities of mutants and chimeric forms of human muscle and liver fructose-1,6-bisphosphatases towards AMP

AMP is an allosteric inhibitor of human muscle and liver fructose-1,6-bisphosphatase (FBPase). Despite strong similarity of the nucleotide binding domains, the muscle enzyme is inhibited by AMP approximately 35 times stronger than liver FBPase: I0.5 for muscle and for liver FBPase are 0.14 microM and 4.8 microM, respectively. Chimeric human muscle (L50M288) and chimeric human liver enzymes (M50L288), in which the N-terminal residues (1-50) were derived from the human liver and human muscle FBPases, respectively, were inhibited by AMP 2-3 times stronger than the wild-type liver enzyme. An amino acid exchange within the N-terminal region of the muscle enzyme towards liver FBPase (Lys20-->Glu) resulted in 13-fold increased I0.5 values compared to the wild-type muscle enzyme. However, the opposite exchanges in the liver enzyme (Glu20-->Lys and double mutation Glu19-->Asp/Glu20-->Lys) did not change the sensitivity for AMP inhibition of the liver mutant (I0.5 value of 4.9 microM). The decrease of sensitivity for AMP of the muscle mutant Lys20-->Glu, as well as the lack of changes in the inhibition by AMP of liver mutants Glu20-->Lys and Glu19-->Asp/Glu20-->Lys, suggest a different mechanism of AMP binding to the muscle and liver enzyme.     

Maternal-fetal distribution and prenatal toxicity of 2,2,4-trimethyl-1,2-dihydroquinoline in the rat

BACKGROUND: Polnoks R (poly‐2,2,4‐trimethyl‐1,2‐dihydroquinoline) is used as an antioxidant in elastomer processing. It is an embryotoxic and fetotoxic agent. This chemical given per os to female rats induces also teratogenic effect but only at doses toxic to the mother. The aim of the study was to evaluate prenatal development and tissue distribution in rats exposed to 2,2,4‐trimethyl‐1,2‐dihydroquinoline (TMDHQ), a monomer of Polnoks R. METHODS: Females were exposed orally to unlabeled TMDHQ during organogenesis at doses 50–400 mg/kg to asses prenatal toxicity and to radiolabeled ¹⁴C monomer at a dose 210 mg/kg to evaluate tissues distribution. RESULTS: TMDHQ administered to pregnant females per os at doses 100 mg/kg and higher produced teratogenic effect (cleft palate, wavy ribs, kyphoscoliosis, exencephaly, external hydrocephalus, hydronephrosis, and renal hypoplasia). Peak ¹⁴C‐radioactivity was found in mothers' plasma about 10 hr after administration of this compound at dose 210 mg/kg. The accretion of ¹⁴C proceeded with a kinetic constant of 0.35 hr ^?1 and a half‐life of 53.3 hr. Kidneys are the main organs of monomer excretion. The highest concentration of ¹⁴C in maternal tissues 24 hr after oral dosing was found in adipose tissue, sciatic nerve, muscles, kidneys, and liver. Radiocarbon retention in fetuses was the highest in kidneys at all time points after dosing. CONCLUSIONS: This study has demonstrated that transplacental exposure to Polnoks R monomer is teratogenic in rats. ¹⁴C retention in placenta, amniotic fluid, and fetal tissues indicates that this compound or its metabolites penetrate into placenta to the fetus. Birth Defects Res B 68:375–382, 2003. © 2003 Wiley‐Liss, Inc.     

Water-soluble polycationic dendrimers with a phosphoramidothioate backbone: preliminary studies of cytotoxicity and oligonucleotide/plasmid delivery in human cell culture

A series of water-soluble polycationic dendrimers with a phosphoramidothioate backbone (P-dendrimers) was studied in human cell culture. Preliminary studies have shown that P-dendrimers of series 1 and 2, possessing N,N-diethyl-ethylenediamine hydrochloride functions at the surface, show rather moderate cytotoxicity toward HeLa, HEK 293, and HUVEC cells in a standard MTT assay in serum-containing medium, generally lower than lipofectin. The experiments of cellular uptake have shown the necessity for the presence of serum for transfection with P-dendrimers of series 1 and 2. These compounds efficiently delivered fluorescein-labeled oligodeoxyribonucleotide into HeLa cells in serum-containing medium, but they failed to do so in HUVEC cell culture. The dendrimers were found to be successful mediators of transfection of the HeLa cells with a DNA plasmid containing the functional gene of enhanced green fluorescent protein (EGFP).     

Accumulation of CD45RO<Superscript>+</Superscript> cells in peritoneal carcinomatous fluid favours survival of ovarian carcinoma patients

In 44 patients with advanced ovarian carcinoma (OC) a fraction of CD45RO(+) lymphocytes in the blood and peritoneal carcinomatous fluid (PCF) was investigated. Thirty-one patients received cisplatinum with cyclophosphamide +/- doxorubicin. This group was followed from 2.2 to 9 years (mean: 45 months). In 23 out of 31 patients, the percentage of CD45RO(+) lymphocytes was higher in the PCF than in the blood samples. Patients with these higher lymphocyte levels experienced longer survival than those who did not show any excess of CD45RO(+) lymphocytes in PCF ( P=0.02). This was further verified by the use multivariate Cox analysis which included an assessment of the percentage of CD45RO(+) lymphocytes in PCF, age, FIGO status, histology, treatment (CAP or CP) and residual disease (RD) post-surgery. This analysis revealed that two factors had an independent power of prediction: RD ( P=0.02) and the percentage of CD45RO(+) cells in PCF ( P=0.04). Therefore, CD45RO(+) lymphocytes were studied in further detail in a group of 20 patients. This study revealed that PCF CD45RO(+) lymphocytes were characterized by: (1) a higher proportion of cells co-expressing activation markers (HLA-DR, CD28) and higher levels of mRNA for CXC chemokines (IP-10, IL-8) and for IL-10, but with lower levels for IL-2; (2) higher levels of Ki67, bcl-2 and p53 mRNA as compared to those in blood. In conclusion, in the present study it was found that an accumulation of activated CD45RO(+) cells in PCF had a beneficial effect on the survival of patients receiving platinum-based chemotherapy.     

Effect of exogenous pulmonary surfactant preparations on the structure of pulmonary alveoli in newborn rats

Treatment of pre-term newborns with exogenous surfactant preparation is a well established part of the therapy for respiratory distress syndrome of the newborns (RDS). Since the introduction of surfactant into clinical practice in 1980, hundreds of studies have been published describing beneficial effects of such treatment. There is only limited number of morphological publications reporting adverse effects of surfactant administration. The aim of the present study is to describe morphological changes in the lung after surfactant administration to healthy newborn rats. Two types of surfactant were used: Exosurf (Glaxo Wellcome, England) and Survanta (Abbott Laboratories, USA). Surfactant preparation were given intratracheally in single dose (bolus) (100 mg of lipids per kg b.w.). Animals from control group received 0.9% saline in equivalent volume. Lung specimens were taken 15, 20, 25 and 30 minutes after drug administration and evaluated by light and electron microscopy. There was no damage in lungs from the control group. Tissue specimens from the Exosurf group revealed severe pathological changes: foci of atelectasis, frank edema in the parenchyma, focal disruption of air-blood barrier, hemorrhages in many alveoli, surfactant particles in many alveolar capillaries, and strongly activated alveolar macrophages. In this group changes appeared as early as 15 min after surfactant administration and intensity of lung injury increased with time. Also, Survanta administration caused damage to the lung tissue. However, the changes were less intense and appeared later (20-25 minutes after Survanta treatment). In conclusion, the presented morphological findings proved that exogenous surfactant administration to healthy rat newborns caused lung damage. Comparing two different surfactant preparation, Exosurf and Survanta, it was shown that the former one produced stronger and faster damage to lung alveoli than the latter one.     

Induction of tumor-reactive CTL by C-side chain variants of the CTL epitope HER-2/neu protooncogene (369-377) selected by molecular modeling of the peptide: HLA-A2 complex

To design side chain variants for modulation of immunogenicity, we modeled the complex of the HLA-A2 molecule with an immunodominant peptide, E75, from the HER-2/neu protooncogene protein recognized by CTL. We identified the side chain orientation of E75. We modified E75 at the central Ser(5) (E75 wild-type), which points upward, by removing successively the HO (variant S5A) and the CH2-OH (variant S5G). Replacement of the OH with an aminopropyl (CH2)3-NH3 (variant S5K) maintained a similar upward orientation of the side chain. S5A and S5G were stronger stimulators while S5K was a weaker stimulator than E75 for induction of lytic function, indicating that the OH group and its extension hindered TCR activation. S5K-CTL survived longer than did CTL induced by E75 and the variants S5A and S5G, which became apoptotic after restimulation with the inducer. S5K-CTL also recognized E75 endogenously presented by the tumor by IFN-gamma production and specific cytolysis. S5K-CTL expanded at stimulation with E75 or with E75 plus agonistic anti-Fas mAb. Compared with S5K-CTL that had been restimulated with the inducer S5K, S5K-CTL stimulated with wild-type E75 expressed higher levels of E75(+) TCR and BCL-2. Activation of human tumor-reactive CTL by weaker agonists than the nominal Ag, followed by expansion with the nominal Ag, is a novel approach to antitumor CTL development. Fine tuning of activation of tumor-reactive CTL by weak agonists, designed by molecular modeling, may circumvent cell death or tolerization induced by tumor Ag, and thus, may provide a novel approach to the rational design of human cancer vaccines.     

New carbocyclic analogues of netropsin: synthesis and inhibition of topoisomerases

A series of carbocyclic analogues of netropsin were synthesized and evaluated for their capacity to inhibit human topoisomerases I and II in vitro. The compounds are oligopeptides containing 1,4-di- and 1,2,5-trisubstituted benzene rings and unsubstituted N-terminal NH2 groups. Compounds 4-7 consist of two netropsin-like units linked by aliphatic (tetra- and hexamethylene) chains. In the topoisomerase I and II assay, the relaxation of pBR322 plasmid was inhibited by compounds 4-7 at 100 microM concentration.     

Directionality of kinesin motors

Kinesins are molecular motors that transport various cargoes along microtubule tracks using energy derived from ATP hydrolysis. Although the motor domains of kinesins are structurally similar, the family contains members that move on microtubules in opposite directions. Recent biochemical and biophysical studies of several kinesins make it possible to identify structural elements responsible for the different directionality, suggesting that reversal of the motor movement can be achieved through small, local changes in the protein structure.     

Direct sequencing of PCR products for mutation detection in osteogenesis imperfecta

This work present a short and simple method for mutation detection in type I collagen genes, based on the direct sequencing of single-stranded DNA. The sequencing of type I collagen genes is complicated and difficult because of their large size and highly repetitive and GC-rich coding regions. Although many techniques have been developed for mutation screening in osteogenesis imperfecta (OI), they represent different degrees of sensitivity and are difficult to reproduce and too expensive for application in each laboratory. The method described here is short, easy and especially useful for sequencing of collagen genes in OI cases, in which the region with a suspected structural defect is localized by collagen analysis.