New directions in liposome gene delivery

The history of liposomes, progress in liposome gene delivery, and future directions are discussed. Specific characteristics of liposomes and DNA:liposome complexes have been identified that are essential for optimal delivery and gene expression. Of particular interest are the requirements for increased delivery and high levels of gene expression in vivo. At present, significant efforts are focused towards achieving specific delivery and gene expression in target organs and tissues.     

Expression vectors for the construction of hybrid Ty-VLPs

Purification of expressed proteins can be facilitated by expressing the rccombinant protein as a fusion with a carrier protein that assembles into paniculate structures. This article describes the use of expression vectors in producing a hybrid of the yeast retrotransposon Ty, which self-assembles into virus-like particles (VLPs). Hybrid VLPs can be used in such laboratory applications as the production of polyclonal and monoclonal antibodies, structure/function analyses, the detection of important antigenic determinants, and cpitope mapping of monoclonal antibodies.     

A new technique for the study of erythrocyte survival by double labeling and use of a well-type Ge detector

A double label procedure with⁵⁷Co and⁵⁸Co has been developed for detailed in vivo studies of erythrocyte survival. A well-type Ge detector is used in the measurements. The activities necessary for these experiments are very low, and the associated dose received by the test persons can be neglected.     

Interference of Manganese on neuroendocrinal system in exposed workers

The neurotoxic effects of Manganese (Mn) are well known, and are usually caused by high occupational exposure over long periods of time. The effects caused by low doses of this metal have been poorly evaluated. For this reason, we decided to evaluate neuroendocrinal tests in a group of 14 male workers employed in a ferrousmanganese foundry (exposed to Mn air concentrations within the TLV-TWA) and in 14 male control subjects, by analyzing FSH, LH, prolactin, and cortisol. The urinary Mn, prolactin, and cortisol levels were significantly higher in the worker’s group. The preliminary results of this research seem to show that for exposure below the TLV, Mn can somehow interfere with the neuroendocrine system. In order to confirm the existence of these effects and to verify their possible correlation with the dopaminergic control system, further studies are necessary.     

Milk transfer of inorganic mercury to suckling rats

The transport of mercury into rat milk, and uptake in the suckling offspring was studied after peroral administration of inorganic mercury to lactating control rats, and to rats fed selenite in the diet. On day 8, 9, 10, or 11 of lactation, dams were administered a single oral dose of 0.1, 0.4, 0.7, 1.3, or 5.8 mg Hg/kg bw labeled with 203mercuric acetate. There was a linear relationship between mercury concentrations in dam's plasma and milk. The level of mercury in milk was approximately 25% of the level in plasma. After 3 d, milk levels were reduced to half the levels at 24 h. In the suckling offspring, exposed to mercury via milk during 3 d, the mercury level in blood was approximately 1% of the level in maternal blood. Mercury concentration in milk was linearly correlated to the levels in kidney, liver, and brain in the suckling offspring after 3 d exposure to mercury via milk. Selenite treatment of rats, 1.3 micrograms Se/g diet for 5 mo, resulted in increased transport of mercury to milk, probably because of increased plasma levels of mercury. However, selenite treatment of the dams did not cause any increased tissue levels of mercury in the suckling offspring.     

Enhancement of the kidney Cd burden by SH-containing chelating agents

The accumulation of Cd in the kidneys is enhanced markedly if chelating agents that contain SH-groups like 2,3 dimercaptopropanol (BAL) are injected immediately after the metal. This is not a transient effect but persists for more than 3 d. It is less pronounced at higher chelate doses or when the pH of urine is increased. Our experiments indicate that chelating agents, which form unstable complexes at acid pH and are able to pass through the cell membrane, will cause metal accumulation in the kidneys.     

Characterization of a nickel resistant mouse cell line

Nickel is a potent carcinogen and, at high concentrations, is toxic to mammalian cells. The effects associated with nickel exposure are well-documented but its mechanism of action in the cell has not yet been fully described. In order to understand the metabolic fate of nickel in mammalian cells, a variant cell population has been selected that continues to grow and divide in the presence of nickel chloride concentrations that are toxic to the parental cell line (Balb/c-3T3 mouse fibroblasts). Nickel resistance is not caused by altered uptake of nickel from the medium or increased clearance from the cells and is not associated with changes in metallothionein expression. Compared to the normal cells, the nickel resistant cells have a decreased number of chromosomes and numerous centromeric fusions. The expression of some proteins and the distribution of nickel bound by various proteins are altered in the nickel resistant cells. Preliminary results indicate that the nickel resistant phenotype may be transferred by genomic DNA-mediated transfection into a recipient NIH-3T3 cell line. Current investigations are directed at identifying a gene responsible for nickel resistance.     

DNA double staining for a fluorescence energy transfer study of chromatin in liver cells

Methodological aspects related to the application of techniques based on fluorescence energy transfer in the study of chromatin structure, were first examined. Fluorochromes specific for DNA with different interaction mechanisms were employed, both in single and double stainings. The following dye pairs were considered as donor/acceptor couples: Hoechst 33342 or DAPI/Mithramycin A or Chromomycin A₃, Hoechst 33342 or DAPI/Propidium Iodide, and Mithramycin A or Chromomycin A₃/Propidium Iodide. Spectrofluorometric analysis showed that the spectral distribution of the dye pair Ho/PI is more suitable for the evaluation of energy transfer efficiency. This dye pair was used in the study of the chromatin microstructure in rat hepatocytes isolated from livers at two different growth stages. In particular, diploid mono- and binucleated cells from young and adult rats were considered. The results indicated the existence of a more homogeneous situation in young than in adult rats. In the latter case, the statistical analysis indicates the presence of two groups of energy transfer values. The different efficiency values in energy transfer can be considered a consequence of chromatin structure rearrangements and are tentatively interpreted according to the functional role of the diploid cells in the two stages of liver growth.     

Characterization of protein transacetylase from human placenta as a signaling molecule calreticulin using polyphenolic peracetates as the acetyl group donors

We have earlier shown that a unique membrane-bound enzyme mediates the transfer of acetyl group(s) from polyphenolic peracetates (PA) to functional proteins, which was termed acetoxy drug: protein transacetylase (TAase) because it acted upon several classes of PA. Here, we report the purification of TAase from human placental microsomes to homogeneity with molecular mass of 60 kDa, exhibiting varying degrees of specificity to several classes of PA confirming the structure-activity relationship for the microsome-bound TAase. The TAase catalyzed protein acetylation by a model acetoxy drug, 7,8-diacetoxy-4-methyl coumarin (DAMC) was established by the demonstration of immunoreactivity of the acetylated target protein with anti-acetyl lysine antibody. TAase activity was severely inhibited in calcium-aggregated microsomes as well as when Ca2+ was added to purified TAase, suggesting that TAase could be a calcium binding protein. Furthermore, the N-terminal sequence analysis of purified TAase (EPAVYFKEQFLD) using Swiss Prot Database perfectly matched with calreticulin (CRT), a major microsomal calcium binding protein of the endoplasmic reticulum (ER). The identity of TAase with CRT was substantiated by the observation that the purified TAase avidly reacted with commercially available antibody raised against the C-terminus of human CRT (13 residues peptide, DEEDATGQAKDEL). Purified TAase also showed Ca2+ binding and acted as a substrate for phosphorylation catalyzed by protein kinase C (PKC), which are hallmark characteristics of CRT. Further, purified placental CRT as well as the commercially procured pure CRT yielded significant TAase catalytic activity and were also found effective in mediating the acetylation of the target protein NADPH cytochrome P-450 reductase by DAMC as detected by Western blot using anti-acetyl lysine antibody. These observations for the first time convincingly attribute the transacetylase function to CRT. Hence, this transacetylase function of CRT is designated calreticulin transacetylase (CRTAase). We envisage that CRTAase plays an important role in protein modification by way of acetylation independent of Acetyl CoA.     

Accumulation of cesium and radiocesium in forest litter in selected regions of poland and its influence on litter-to-mushroom transfer factor

The aim of this work was to check whether the stable cesium content in forest litter affects the value of radiocesium from litter-to-mushroom transfer factorTf or not. Total cesium in litter, measured by AAS, varied from 0.1–2.7 μg/g. These data, combined with earlier results for mushrooms, showed no simple correlation forTf. More complex relationships provided very high correlation coefficients, but their validity needs further investigation.     

A review of protein engineering for the food industry

In this review I briefly describe the technique of protein engineering and indicate how the present state of knowledge allows proteins to be mutated to increase or decrease stability. I discuss experiments on both model proteins and those of relevance to the food industry and show how hydrophobic forces are a major driving force for folding as well as having a major role in thermostability, I also indicate the large contribution that hydrogen bonding, electrostatic interactions and, in a less well predicted way, disulfide bridges make to thermostability.     

Cell orientation induced by extracellular signals

Cells like fibroblasts and osteoblasts are oriented by different extracellular guiding signals like an electric field, a bent surface, and a periodically stretched surface. An automatic controller is responsible for the cell alignment. The controller contains both a deterministic and a stochastic signal. The following machine properties were determined: (1) The angle dependence of the cellular signal transformer is cos 2(psi 0 - psi). (2) The set point of the automatic controller is psi 0 = +/- 90 degrees. The cells like to orient their long axis perpendicular to the direction of the applied guiding signal. (3) The signal transformer measures the extracellular signal in a quadratic fashion. The cells cannot register the sign of the guiding field. (4) The stochastic signal in the automatic controller can be quantified by a characteristic time (approximately 130 min for fibroblasts). (5) The extracellular signal is registered in cell-made standards (ratio of the deterministic and stochastic signal equals one): 0.3 +/- 0.05 V/mm for human fibroblasts (electric field) and 85 +/- 3 microns for human fibroblasts and osteoblasts (cyclindrically bent surface). (6) The lag-time in the signal transduction system of fibroblasts is approximately 4 min.     

Effects of doxorubicin on the sensitivity of L1210 leukemia cells to deformation-associated trauma

Most of the cancer cells arrested in the microcirculation during hematogenous metastasis are rapidly killed; one major mechanism is surface-membrane rupture, associated with the mechanical deformation of cancer cells in capillaries. The feasibility of increasing the susceptibility of cancer cells to lethal, deformation-associated trauma by doxorubicin, was tested in an in vitro mechanical model system, by filtering suspensions of L1210 leukemia cells through 8-μm pore-size Nuclepore® membranes, with or without prior incubation with 10^-7M doxorubicin. The results showed that mechanically-induced loss of cancer cells immediately after filtration was increased from 18 to 55% in cells previously exposed to doxorubicin for 48 h. The results indicate the feasibility of chemotherapeutic enhancement of the mechanical killing-action of the microvasculature as a potential rate-regulator of hematogenous metastasis.     

Copper deficiency and hyperlipoproteinemia induced by a tetramine cupruretic agent in rabbits

The effectiveness of a cupruretic agent, N,N'-bis-(2 amino ethyl)-1,3-propanediamine HCl or 2,3,2-tetramine HCl (TETA), in the induction of copper (Cu) deficiency and the ability of a Cu-deficient diet in the maintenance of the depressed Cu status 10 wk after TETA treatment were examined in this study. In the first experiment, 42 male New Zealand White rabbits, 35 d of age, were randomly divided into three dietary treatments: a copper (Cu)-deficient (2.3 mg Cu/kg diet), a Cu-adequate (13.5 mg Cu/kg diet), and a commercial ration (21.6 mg Cu/kg diet) group. A single oral dose of 100 mg of 2,3,2-tetramine HCl TETA/kg body wt/d were administered to half of the rabbits from each treatment group for 10 d while the remaining rabbits were untreated. In the second experiment, 10 similar rabbits were assigned to three treatments: Cu-deficient plus TETA (n = 4); Cu-adequate plus TETA (n = 3); and Cu-adequate alone (n = 3). The rabbits were fed a TETA dose of 100 mg/d for three 4-d periods over 3 wk, and thereafter maintained on the diets for another 10 wk. Rabbits from the first experiment fed Cu-deficient diet and treated with TETA demonstrated cardiac hypertrophy and markedly reduced plasma and liver Cu concentrations that indicated that the animals were Cu-deficient. Significant elevations (twofold) in low density lipoprotein (LDL) protein, cholesterol, triglyceride, and apolipoprotein B (apo B) concentrations were observed in TETA treated rabbits fed Cu-deficient diet. In the second experiment, the plasma LDL protein level remained elevated, the plasma Cu level was reduced 45%, and the Cu level of the heart when expressed as microgram/g dry tissue was reduced, 10 wk post TETA treatment in rabbits maintained on Cu-deficient diet. Thus, Cu deficiency and hyperlipoproteinemia was rapidly induced by TETA and was still evident 10 wk posttreatment in rabbits maintained on a Cu-deficient diet.     

Transformation of human kidney epithelial cells to tumorigenicity by nickel(II) and V-HA-RAS oncogene

Nickel is a toxic metal of environmental concern that has been found to be carcinogenic in man and animals. Primary human kidney (NHKE) cells were immortalized or rescued from senescence after exposure to NiSO₄. The cell lines (IHKE) displayed abnormal karyotype and anchorage independent growth was observed. However, none of the IHKE cells produced tumor upon injection into athymic nude mice. Transfer of the v-Ha-ras oncogene into IHKE cells induced conversion of the immortalized cells into cell lines (THKE) that were tumorigenic when transplanted into athymic nude mice. Ha-ras DNA was present in the transformed cell lines and expressed at high level.     

Global amplification of cDNA from limiting amounts of tissue. An improved method for gene cloning and analysis

In this study we present an improved polymerase chain reaction (PCR)-based methodology to generate large amounts of high-quality complementary DNA (cDNA) from small amounts of initial total RNA. Global amplification of cDNA makes it possible to simultaneously clone many cDNAs and to construct directional cDNA libraries from a sequence-abundance-normalized cDNA population, and also permits rapid amplification of cDNA ends (RACE), from a limited amount of starting material. The priming of cDNAs with an adapter oligo-deoxythymidine (oligo-dT) primer and the ligation of a modified oligonucleotide to the 3′ end of single-stranded cDNAs, through the use of T4 RNA ligase, generates known sequences on either end of the cDNA population. This helps in the global amplification of cDNAs and in the sequence-abundance normalization of the cDNA population through the use of PCR. Utilization of a long-range PCR enzyme mix to amplify the cDNA population helps to reduce bias toward the preferential amplification of shorter molecules. Incorporation of restriction sites in the PCR primers allows the amplified cDNAs to be directionally cloned into appropriate cloning vectors to generate cDNA libraries. RACE-PCR done with biotinylated primers and streptavidin-coated para-magnetic particles are used for the efficient isolation of either full-length coding or noncoding strands.