Gene delivery mediated by polyplexes such as DNA complexed with polylysine conjugates is limited by the low efficiency of escape of DNA from the endosomes. One of the strategies which favors the transmembrane passage of polyplexes consists of adding anionic amphipathic peptides capable of destabilizing membranes in an acidic medium. Although less efficient than replication-defective adenoviruses, fusogenic peptides increase the expression of the reporter gene by a factor between 100 and 1000 depending on the cell line. However, the activity of a given peptide depends on the composition of the lipid bilayer. We were interested in developing a polyplex (glycoplex) formulation comprising a glycosylated polylysine, a fusogenic peptide and a plasmid which would be useful for efficient transfection (glycofection) of a large panel of cells, even in the presence of serum. We synthesized several peptides and tested their efficiency in combination with different glycoplex formulations. We found that glycofection with a quaternary complex (called one pot formulation) made of lactosylated-polylysine, polylysine, DNA, and the dimeric peptide (E5-WYGG)2-KA was less cell-type dependent than other peptide-based formulations. In addition, its efficiency was not affected by the presence of serum (up to 20%).

Amino Acid Sequence↗

PubMedDate not supplied

Factors influencing survival in patients with hepatic metastases from adenocarcinoma of the colon or rectum.

The median survival of all patients with hepatic metastases from colorectal cancer referred to the Sidney Farber Cancer Institute during a five-year period was 12.5 months. Two major factors influenced survival. The first was extent of disease at presentation. The second was the histologic grade of the cancer. The median survival of patients presenting with the least disease, characterized by less than four liver nodules visible on liver scan (n = 38), normal liver size on physical examination (n = 60), normal liver function test results (n = 30), and normal performance status (n = 91), was between 18 and 24 months, regardless of treatment. The median survival of those few patients (n = 13) who had objective responses to a variety of treatments, most of whom also had minimal disease at presentation, was also 24 months. Patients whose tumors were poorly differentiated or who had abnormal performance status or weight loss of greater than 10 per cent at presentation survived only six months (median). Those with four or more liver nodules, hepatomegaly (greater than 16-cm vertical span on physical examination), or abnormal liver function test results, survived ten, eight, and 12 months (median), respectively. It is concluded that a significant group of patients survived longer than would have been predicted by earlier literature surveys after the diagnosis of colorectal cancer metastatic to the liver. It is suggested that future therapeutic trials, using survival as a measure of response of patients with liver metastases from colorectal cancer, must be prospectively controlled before selection factors can be differentiated from significant therapy effect.

Adenocarcinoma↗

PubMedDate not supplied

Endocytosis mediated by monocyte and macrophage membrane lectins--application to antiviral drug targeting.

Sugar receptors, or membrane lectins, have been evidenced at the surface of various normal and tumour cells using fluoresceinylated neoglycoproteins (glycosylated bovine serum albumin (BSA]. By flow cytometry we have shown that macrophages bind and internalize mannosylated and 6-phosphomannosylated ligands in acidic compartments. Freshly isolated monocytes and U937, a promonocytic cell line, lack a mannose-specific receptor, but express mannose-6-phosphate (Man-6P) membrane lectin. Neoglycoproteins are potent drug carriers: muramyl dipeptide (MDP), an immunoactivator, when bound to Man-BSA or Man-6P-BSA, is 100 times more efficient than free MDP in activating macrophages; in vivo, it enables eradication of lung metastases in mice. Recently, neutral glycosylated biodegradable and nonimmunogenic polymers, were synthesized and found to be as efficient as neoglycoproteins. Antiviral drug conjugates were more active than the free drug, inhibiting the multiplication of virus (herpes) in human macrophages in vitro.

Animals↗

PubMedDate not supplied

PCDD/F levels in food and canteen meals from southern Germany.

Analysis of food samples collected in 1997 and 1998 in Bavaria, Germany showed that PCDD/F levels in food of animal origin, with the exception of fish, have decreased to low levels way below 1 pg I-TEQ/g fat. Lowest PCDD/F contents were found in pork (mean: 0.27 pg I-TEQ/g fat) followed by poultry and cow's milk (mean: approx. 0.5 pg I-TEQ/g fat). Average contamination levels determined in beef and eggs lay in the range from 0.7 to 0.8 pg I-TEQ/g fat. PCDD/F levels in fish were clearly higher than levels in meat, milk and egg samples (mean: 5.6 pg I-TEQ/g fat). Comparison of topical levels with former results from Bavaria show that the recently detected decline of contamination of food has continued during the past few years. In addition to investigations of unprepared food, we analysed prepared canteen meals over a period of 1 year. The average PCDD/F level found in canteen food (0.37 pg I-TEQ/g fat) was in good conformity with determined PCDD/F levels in unprepared food regarding the estimated fat composition of prepared meals. The average consumption of fat per meal via canteen food was 21.1 g and the average PCDD/F intake per meal was 7.6 pg I-TEQ. On the supposition that determined PCDD/F levels in canteen food on fat basis are representative of total fat consumed by humans (102 g fat/day), the daily PCDD/F intake would amount to a mean value of 36.7 pg I-TEQ corresponding to 0.52 pg I-TEQ/kg body weight.

Animals↗

PubMedDate not supplied

Results of an "emergency quality control study" as confirmation of a PCDD/PCDF-contamination of milk and butter samples.

Elevated PCDD/F levels in four butter samples and one milk sample had to be confirmed under considerable time pressure. These samples should demonstrate a raising dioxin contamination in different regions of Germany caused by the use of Brazilian citrus pulp as feedstuff. Above all, the increase of 2,3,7,8-TCDD and 1,2,3,7,8-PeCDD had to be confirmed. Thus, an "emergency quality control study" was performed including five laboratories from the official food control in Germany. The results had to be reported about two to three weeks after the first announcement of the samples (including time for shipment). The five laboratories applied different extraction and clean-up methods, used different GC-columns and GC/MS-equipment and different standard solutions. The results are in a good agreement. Slightly different results for most individual congeners were of minor importance. A difference was observed only in the 2,3,7,8-TCDF content of one laboratory which was not relevant for the evaluation of the increase of the dioxin contamination in milk and dairy products. The laboratory's individual methods proved to give correct results even under a very tight schedule.

Animal Feed↗

PubMedDate not supplied

Production of macrophage-derived cytotoxic factor by N-[3-[(carbamoylmethyl)thio]propionylated] neoglycoproteins.

6-Phosphomannosylated bovine serum albumin (Man6P-BSA), a neoglycoprotein endocytosed by macrophages, bearing either 3-(2-pyridyldithio)propionyl or 3-[(carbamoylmethyl)thio]propionyl residues coming from alkylation of thiol residues by iodoacetamide were prepared and tested for their immunomodulator properties. The supernatants of mouse peritoneal macrophages incubated with Man6P-BSA bearing 3-[(carbamoylmethyl)thio]propionyl groups, and by a lesser extent 3-(2-pyridyldithio)propionyl groups, were cytotoxic to L929 cells, suggesting the presence of a tumor necrosis factor like compound. This macrophage-activation process is linked to the capacity of Man6P-BSA to be endocytosed via membrane lectins of macrophages, because the supernatants of macrophages incubated with unglycosylated conjugates were not cytotoxic. The cytotoxic activity induced by 3-[(carbamoylmethyl)thio]propionyl groups bound onto Man6P-BSA was similar to that induced by Man6P-BSA bearing muramyl dipeptide, indicating that endocytosed neoglycoproteins bearing 3-[(carbamoylmethyl)thio]propionyl residues are potent macrophage activators.

Acetylmuramyl-Alanyl-Isoglutamine↗

PubMedDate not supplied

Synthesis and antiviral activity of peptide-oligonucleotide conjugates prepared by using N alpha-(bromoacetyl)peptides.

Antisense oligonucleotides represent an interesting tool for selective inhibition of gene expression. In order to direct oligonucleotides to specific compartments within the cell, we have investigated the possibility of coupling them to a signal peptide Lys-Asp-Glu-Leu (KDEL). This sequence should be able to convey oligonucleotides to the endoplasmic reticulum and from there to the cytosol and the nucleus where their targets are located. On this basis we prepared peptide-oligonucleotide conjugates by coupling, in a single step, a Nalpha-bromoacetyl peptide with an oligonucleotide bearing a thiol group, through a thioether bond. This paper deals with the definition of the optimal pH and temperature conditions leading to an efficient synthesis of peptide-oligonucleotide conjugates: the reaction was quantitative at pH 7.5 within few hours. This method was first set up using a 5',3'-modified dodecanucleotide and a (bromoacetyl)pentapeptide as a conjugation model. Then a 5',3'-modified pentacosanucleotide, complementary to the translation initiation region of the gag mRNA of HIV, was coupled to a (bromoacetyl)dodecapeptide containing a KDEL signal sequence. The anti-HIV activity of the pentacosanucleotide was compared with that of pentacosanucleotide-dodecapeptide conjugates linked through either a thioether bond or a disulfide bridge. The conjugate with a thioether bond has a higher antiviral activity than the peptide-free oligonucleotide and the conjugate linked via a disulfide bond.

Amino Acid Sequence↗