Pronounced Cytotoxic Effect Research Articles

Hemoglobin induces cytotoxic damage of glycine-preserved renal tubules

In isolated tubular segments (ITS) of rat kidney cortex, we studied the effect of hemoglobin (Hb) on reoxygenation damage. All tubules were suspended in Ringer's solution containing 5-mm glycine and oxygenated for 30 min with 95% O(2):5% CO(2), followed by a 30-min period with 95% N(2):5% CO(2), and final reoxygenation for 60 min. Untreated tubules served as controls. Different concentrations of free Hb and equivalent amounts of intact erythrocytes were added to the incubation medium. Secondly, we added deferoxamine (DFO) to Hb and erythrocytes. Membrane leakage and lipid peroxidation were measured by lactate dehydrogenase and glutamate dehydrogenase and the development of thiobarbituric acid reactive substances. Cell function was quantified by gluconeogenesis and intracellular potassium accumulation. Hb exerted concentration-dependent cytotoxic effects indicated by significantly increased enzyme leakage rates, lipid peroxidation and a significantly decreased cell function (P < 0.05), in ITS during hypoxia, and subsequent reoxygenation. Moreover, we found that toxicity of both Fe(2+) and Fe(3+) ions increased with rising concentration. However, Fe(2+) showed a higher tissue toxicity than Fe(3+). DFO reduced significantly the reoxygenation damage of free Hb and iron ions. Our data clearly demonstrate a pronounced cytotoxic effect of free Hb in ITS, which critically depended on the reduction state of the iron ions.

Antibacterial and cytotoxic activities of extracts from (Tunisian)Rhamnus alaternus (Rhamnaceae)

The petroleum ether, chloroformic, ethyl acetate, methanolic, Total Oligomers Flavonoids (TOF) enriched extracts, water extract as well as its fractions A1, A2, A3 obtained from aerial parts ofRhamnus alaternus, a Tunisian-Mediterranean medicinal species, were investigated for the contents of phenolic compounds, cytotoxic activity against the K562 human chronic myelogenous leukaemia cell line and L1210 leukaemia murine cells and for antibacterial activity against Gram positive and Gram negative bacterial reference strains. A pronounced cytotoxic effect on both the cell lines was shown in the TOF, ethyl acetate, methanolic, aqueous extracts and A2 fraction, with respectively IC50 values 75, 232, 298, 606 and 571 μg/ml on K562 cells and 198, 176, 767, 560 and 614 μg/ml on L1210 cell line. Significant activity against bacterial reference strains:Staphylococcus aureus, Enterococcus faecalis, Escherichia coli, Salmonella enteritidis andSalmonella typhimurium was shown with ethyl acetate, TOF extracts and A2 fraction. The antimicrobial and cytotoxic activities showed byR. alatemus depended on the chemical composition of the tested extracts.

Biologically Active Indole Alkaloids from Kopsia arborea

Nine new indole alkaloids, rhazinoline (1), 19(S)-methoxytubotaiwine (2), 19(R)-methoxytubotaiwine (3), kopsamidine A (4), kopsamidine B (5), kopsinidine A (6), kopsinidine B (7), paucidactine C (8), and pericine N-oxide (9), in addition to several recently reported novel indoles and 34 other known ones, were obtained from the stem-bark extract of the Malayan Kopsia arborea. The structures were determined using NMR and MS analysis. Valparicine (12) showed pronounced cytotoxic effects against KB and Jurkat cells (IC(50) 13.0 and 0.91 microM, respectively).

Phospholipid Transfer Protein Augments Apoptosis in THP-1–Derived Macrophages Induced by Lipolyzed Hypertriglyceridemic Plasma

Lipolysis of triglyceride-rich lipoproteins (TGRLPs) generates phospholipid-rich surface remnants and induces cytotoxic effects in adjacent vascular cells. We hypothesized that by integrating surface remnants into HDL, phospholipid transfer protein (PLTP) alleviates cytotoxicity. To test this hypothesis and gain insight into cytotoxicity during the postprandial phase in vivo, we injected normo-TG and hyper-TG human volunteers after a standardized fat meal (postprandial sample) with heparin, thereby stimulating lipolysis (postprandial heparinized sample). Incubation of (primary) human macrophages and primary human endothelial cells with postprandial heparinized hyper-TG plasma induced pronounced cytotoxic effects that were dose dependent on the TG content of the sample. No such effects were seen with normo-TG and postprandial hyper-TG plasma. In vitro lipolysis of VLDL and chylomicrons indicated that both lipoprotein fractions can cause cytotoxicity. Interestingly, in experiments with THP-1-derived macrophages stably transfected with PLTP, PLTP substantially augmented both net phospholipid uptake and apoptotic cell death due to postprandial heparinized hyper-TG plasma. We observed that activation of caspase-3/7, poly-ADP-ribose polymerase, and enhanced bioactivity of acid sphingomyelinase may all contribute to this augmented apoptosis. Our data show that lipolysis of TGRLPs and their remodelling by PLTP interact to disturb cellular phospholipid flux and intracellular signaling processes, ultimately leading to apoptosis in human macrophages and endothelial cells.

The degree and kind of agglomeration affect carbon nanotube cytotoxicity

The urgent need for toxicological studies on carbon nanotubes (CNTs) has arisen from the rapidly emerging applications of CNTs well beyond material science and engineering. In order to provide a basis for comparison to existing epidemiological data, we have investigated CNTs at various degrees of agglomeration using an in vitro cytotoxicity study with human MSTO-211H cells. Non-cytotoxic polyoxyethylene sorbitan monooleate was found to well-disperse CNT. In the present study, the cytotoxic effects of well-dispersed CNT were compared with that of conventionally purified rope-like agglomerated CNTs and asbestos as a reference. While suspended CNT-bundles were less cytotoxic than asbestos, rope-like agglomerates induced more pronounced cytotoxic effects than asbestos fibres at the same concentrations. The study underlines the need for thorough materials characterization prior to toxicological studies and corroborates the role of agglomeration in the cytotoxic effect of nanomaterials.

Construction and anti-tumor effects of recombinant fowlpox virus expressing Newcastle disease virus hemagglutinin-neuramidinase gene

Hemagglutinin-neuramidinase (HN), a Newcastle disease virus-derived protein, not only mediates receptor recognition but also possesses neuraminidase (NA) activity, the ability to cleave a component of those receptors, N-acetylneuraminic acid (NAcneu, sialic acid). It is known that this protein in mammalian species, including human beings, has interesting anti-neoplastic as well as immune stimulating properties. To explore the use of the HN gene in cancer gene therapy, we constructed a recombinant fowlpox virus expressing the HN protein (vFV-HN) and compared the anti-tumor activity of the recombinant virus with that of wild-type fowlpox virus (FPV) in vivo and in vitro. Here we found that although B16 cells were somewhat resistant to the basal cytotoxic effect of wild-type fowlpox virus, infection with vFV-HN caused a pronounced cytotoxic effect and, the survival of tumor-bearing mice immunized with vFV-HN was significantly increased compared with the survival of mice immunized with the FPV alone. Furthermore, the immunization of mice with vFV-HN elicited a B16 tumor-specific cytotoxic T lymphocyte (CTL) response and clonal expansion of both CD4+ and CD8+ T cell populations in vivo. In addition, T cells from lymph nodes of mice vaccinated with vFV-HN secreted high levels of the Th1 cytokine IL-2 and IFN-γ, indicating that the regression of tumor cells is related to a Th1-type dominant immune response. These results demonstrate that vaccination with vFV-HN may be a potential strategy for cancer gene therapy.

Small-Molecule MDM2 Antagonists as a New Therapy Concept for Neuroblastoma

Circumvention of the p53 tumor suppressor barrier in neuroblastoma is rarely caused by TP53 mutation but might arise from inappropriately increased activity of its principal negative regulator MDM2. We show here that targeted disruption of the p53-MDM2 interaction by the small-molecule MDM2 antagonist nutlin-3 stabilizes p53 and selectively activates the p53 pathway in neuroblastoma cells with wild-type p53, resulting in a pronounced antiproliferative and cytotoxic effect through induction of G(1) cell cycle arrest and apoptosis. A nutlin-3 response was observed regardless of MYCN amplification status. Remarkably, surviving SK-N-SH cells adopted a senescence-like phenotype, whereas CLB-GA and NGP cells underwent neuronal differentiation. p53 dependence of these alternative outcomes of nutlin-3 treatment was evidenced by abrogation of the effects when p53 was knocked down by lentiviral-mediated short hairpin RNA interference. The diversity of cellular responses reveals pleiotropic mechanisms of nutlins to disable neuroblastoma cells and exemplifies the feasibility of exploiting, by a single targeted intervention, the multiplicity of anticancer activities exerted by a key tumor suppressor as p53. The observed treatment effects without the need of imposing a genotoxic burden suggest that selective MDM2 antagonists might be beneficial for treatment of neuroblastoma patients with and without MYCN amplification.

Cytotoxicity of six South African medicinal plant extracts used in the treatment of cancer

Aqueous extracts prepared from six South African medicinal plants, with cancer-related ethnobotanical uses, were tested for their cytotoxic ability in vitro against three human cancer cell lines: DU-145 prostate cancer cells, MDA-MB-231 and MCF-7 breast cancer cells and a non-malignant breast cell line, MCF-12A. The plants studied were: Bidens pilosa, Centella asiatica, Cnicus benedictus, Dicoma capensis, Hypoxis hemerocallidea and Sutherlandia frutescens. Of these plants, only D. capensis exhibited pronounced cytotoxic effects in two of the cell lines tested: MCF-7 and MCF-12A.

Preliminary antiproliferative effects of some species of Terminalia, Combretum and Pteleopsis collected in Tanzania on some human cancer cell lines

Methanolic extracts (25 μg/ml) of species belonging to the genera of Combretum, Terminalia and Pteleopsis, collected during a field expedition in Tanzania in 1999, were screened for their antiproliferative and cytotoxic effects against three human cancer cell lines (HeLa, cervical carcinoma; T 24, bladder carcinoma; and MCF 7, breast carcinoma). A leaf extract of Combretum fragrans and a fruit extract of C. zeyheri gave the strongest antiproliferative and cytotoxic effects of all the twenty-four extracts screened in this investigation. In contrast to the highly powerful leaf extract of C. fragrans, the root extract of this species gave no cytotoxic effects against the investigated cancer cell lines at a concentration of 25 μg/ml. The other investigated species of Combretum and Terminalia differed greatly in their cytotoxic potential. Root extracts of Terminalia sambesiaca and T. sericea gave the strongest cytotoxic effects of the five species of Terminalia used in this study. Eight of the twenty-four investigated plant extracts showed pronounced cytotoxic effects (< 30% proliferation compared to the control) against the T 24 bladder cancer cells, seven against the HeLa cells and four against the MCF 7 cells.

Cytotoxic, antioxidant and antibacterial activities of Varthemia iphionoides Boiss. extracts

The hexane, ethyl acetate, chloroform, ethanol and water extracts of aerial parts of Varthemia, Varthemia iphionoides, were investigated for cytotoxic activity against human myelocytic leukemia (HL-60) cells; DPPH radical-scavenging activity; antioxidative activity in the linoleic acid system; reducing power; antibacterial activity; the contents of phenolic compounds. A pronounced cytotoxic effect on human leukemia (HL-60) cells was shown in the hexane, chloroform and ethanol extracts, with inhibition of 89.0, 68.4 and 62.3%, respectively, at a concentration of 200 μg extract/ml. High DPPH radical-scavenging activity, antioxidative activity in the linoleic acid system and reducing power were found in the water and ethanol extracts, and were correlated to the contents of phenolic compounds. Antibacterial activity against Staphylococcus aureus, Bacillus subtilis, Micrococcus luteus, Escherichia coli, Bacillus cereus and Salmonella enteritides was shown in the ethyl acetate and chloroform extracts. A compound responsible for the antibacterial activity was isolated from the ethyl acetate extract, and identified as 3-oxocostusic acid.

Novel Thermosensitive 5-Fluorouracil−Cyclotriphosphazene Conjugates: Synthesis, Thermosensitivity, Degradability, and in Vitro Antitumor Activity

A novel thermosensitive macromolecular prodrug of 5-fluorouracil (5-FU) was synthesized using cyclotriphosphazene, and its thermosensitivity, degradability, and in vitro antitumor activity were studied. A series of alpha-substituted glycine derivatives of 5-FU containing carboxylic groups were prepared, and cyclotriphosphazenes with amino groups were synthesized via the stepwise substitution of hexachlorocyclotriphosphazene (NPCl(2))(3) with methoxy-poly(ethylene glycol) (MPEG) or alkoxy ethylene oxide and lysine ethyl ester (LysOEt). The coupling reaction of the two derivatives, and their subsequent deprotection, yielded a thermosenstive 5-FU-cyclotriphosphazene conjugate, which exhibited a unique octopus-shaped molecular structure, in which the three hydrophilic PEG groups (or alkoxy ethylene oxides) were oriented in one direction, opposing the other three hydrophobic groups containing 5-FU, with respect to the trimer ring plane. This conjugate exhibited a reversible and thermosensitive phase transition in an aqueous medium, from soluble to insoluble states. The lower critical solution temperature (LCST) of the conjugate was controlled by substitution with different hydrophilic/hydrophobic side groups, and a few of the conjugates displayed LCSTs which were just below body temperature. This, of course, implies possible applications for local drug delivery by direct intratumoral injection. The conjugate exhibited gradual degradation at 37 degrees C in both neutral and acidic buffer solutions, and high temperature significantly facilitated its hydrolytic degradation. All of the conjugates displayed dose-dependent cytotoxicity against the leukemia L1210 cell line and exhibited more pronounced cytotoxic effects than did 5-FU.

Cytosolic RNase Inhibitor Only Affects RNases with Intrinsic Cytotoxicity

Cytosolic RNase inhibitor binds to and neutralizes most members of the pancreatic type RNase superfamily. However, there are a few exceptions, e.g. amphibian onconase and bovine seminal RNase, and these are endowed with cytotoxic activity. Also, RNase variants created by mutagenesis to partially evade the RNase inhibitor acquire cytotoxic activity. These findings have led to the proposal that the cytosolic inhibitor acts as a sentry to protect mammalian cells from foreign RNases. We silenced the expression of the gene encoding the cytosolic inhibitor in HeLa cells and found that the cells become more sensitive to foreign cytotoxic RNases. However foreign, non-cytotoxic RNases remain non-cytotoxic. These results indicate that the cytosolic inhibitor neutralizes those foreign RNases that are intrinsically cytotoxic and have access to the cytosol. However, its normal physiological role may not be to guard against foreign RNases in general.

In vitro cell death induced by irradiation and chemicals relevant for dental applications; dose-response and potentiation effects.

Resin-based dental materials polymerized using blue light are frequently used in dental practice and may come in contact with the oral mucosa. Remnants from oral hygiene product ingredients, such as sodium lauryl sulfate (SLS), add to the chemical exposure of the mucosa. The aim of the present in vitro study was to elucidate the cytotoxic effects in terms of apoptosis and necrosis after exposures to combinations of an adhesive (0.5% and 0.6%), SLS (concentration range 0.0025%-0.0075%), and irradiation from a dental curing lamp (radiant exposure of 8 J cm(-2)). The test system chosen was rat submandibular salivary gland acinar cells, and the cytotoxic effects were measured by fluorescence microscopy and flow cytometry methods. Cytotoxicity was observed as a result of irradiation. The most pronounced cytotoxic effects were seen in cells exposed to a combination of adhesive and SLS compared with those exposed to either agent alone. Necrosis was the dominating form of cell death for all exposures, except for the highest concentration of SLS. Apoptosis was dose-dependent on SLS in the rat submandibular acinar cells. Cytotoxic considerations of dental materials should include contributions from irradiation and other chemicals that might be present in the oral cavity.

A Rapid Colorimetric Assay for Sulfur Mustard Cytotoxicity Using Isolated Human Peripheral Blood Lymphocytes and Keratinocytes

Sulfur mustard (SM) is a potent vesicating agent that has pronounced cytotoxic effects as well as mutagenic, carcinogenic, and radiomimetic properties. Isolated human peripheral blood lymphocytes (PBLs) and human epidermal keratinocytes (HEKs) have been used as in vitro models for determining SM-induced cytotoxicity. A recently developed colorimetric assay (the CellTiter 96 AQ ueous Non-radioactive Cell Proliferation Assay) was assessed using both of the in vitro models described above. Using 24- or 96-well microplates, reproducible (± 10%) SM dose/response curves for both types of human cells were obtained using a spectrophotometric microplate reader set at 490 nm. After a 4-h incubation time, as many as 96 sample wells could be measured within 45 s using this commonly available equipment. Multiple plates of samples can be run immediately. This technique may facilitate cytotoxicity investigations of new candidate compounds for both prophylaxis of and therapy for SM intoxication.

Long-Term Cytocompatibility of Various Endodontic Sealers Using a New Root Canal Model

It was the purpose of our study to determine the cytotoxicity of several types of root canal sealers in vitro over the period of 1 yr by using a new test model. Roots of extracted human teeth were filled with N2, Apexit, Roekoseal, AH Plus, Ketac Endo, Endomethasone, and one gutta-percha point. In addition, roots filled with laterally condensed gutta-percha/N2. Teeth filled with one gutta-percha point only were controls. All specimens were consecutively extracted with distilled water for a total period of 1 yr. Extracts were investigated for cytotoxicity by using immortalized 3T3 fibroblasts and primary human periodontal ligament fibroblasts. Results were statistically analyzed with Dunnett's t tests (p < 0.05). Pronounced cytotoxic effects were only caused by N2-extracts in both cell cultures (p < 0.05). Furthermore, statistically significant cytotoxic alterations were also induced by 10-week eluates of Endomethasone (p < 0.05). All other investigated materials did not significantly alter cell metabolism.

Cytotoxicity of some medicinal plant extracts used in Tanzanian traditional medicine

Using the ethnomedical data approach, some Tanzanian plants that are used in Tanzanian traditional medicine for cancer or non-cancer diseases were collected and evaluated for cytotoxic activity. The antiproliferative effect of the methanolic extracts (10 and 100 μg/ml) of 47 plants was evaluated in vitro on three human cell lines (HeLa, cervical carcinoma; HT29, colon adenocarcinoma; and A431, skin carcinoma). From the nine plants that are used to treat cancer, two plants (22%) exhibited pronounced cytotoxic effect (<25% cell proliferation) at least in one of the tested cell lines. For the 38 plants that are used to treat non-cancer diseases, 14 plants (37%) exhibited pronounced cytotoxic effect (<25% cell proliferation). Cell type cytotoxic specificity was observed in some extracts. Overall, the A431 cells were much more sensitive to most of the extracts than the other cell lines. For the plants that are used as anticancer herbal drugs, our results indicate that there is no correlation between the reported use of these plants and their cytotoxic activity obtained in this study. However, plants that have shown pronounced cytotoxic activity will be evaluated further for the possible isolation of active antitumor compounds.

Toxicity of cytostatic drugs to normal bone marrow cells in vitro.

In this study we compared how different concentrations and periods of incubation of anthracyclines, amsacrine, and cytosine arabinoside would affect normal hematopoietic bone marrow cells in terms of interindividual differences in toxicity, the age of the donor, and the proliferative capacity of the bone marrow. Bone marrow was obtained from 36 donors in connection with bone marrow transplantation. After separation the mononuclear cell fraction was incubated with doxorubicin, 4-epidoxorubicin, daunorubicin, idarubicin, aclarubicin, mitroxantrone, amsacrine, and cytosine arabinoside for 1 h, for 3 h, or continuously. The cells were thereafter cultured in soft agar and CFU-GM were counted after 10-12 days. The results showed a large interindividual variation in toxicity for all drugs tested. Daunorubicin, idarubicin, aclarubicin, and mitoxantrone had a pronounced cytotoxic effect after 1 h of incubation. Doxorubicin and 4-epi-doxorubicin showed the greatest cytotoxic effect after 3 h and were also more toxic to normal bone marrow cells from donors over 40 years of age. Ara-C had a low cytotoxic effect after 1 and 3 h of incubation, even at high concentrations, but exerted a pronounced degree of toxicity during continuous incubation. Daunorubicin, idarubicin, and ara-C also showed increased toxicity to cell samples with a low proliferating capacity in the control. The conclusions drawn from these results are that interindividual variation, proliferation capacity, incubation conditions, and the age of the donors are factors of importance in the toxicity of drugs to normal hematopoietic bone marrow cells.

DNA binding-independent anti-proliferative action of benzazolo[3,2-alpha]quinolinium DNA intercalators.

The proposed mechanism of action of the antineoplastic drug 3-nitrobenzothiazolo[3,2-alpha]quinolinium chloride (NBQ-2) involves its interaction with DNA by intercalation and inhibition of topoisomerase II activity by arresting the enzyme in a covalent cleavage complex. In an attempt to identify some structural determinants for activity and develop a molecular structure/cytotoxicity correlation, four new structural analogs of the antitumor NBQ-2 were prepared and their cytotoxic activity and DNA binding properties were investigated. The cytotoxic activity was evaluated against six different human tumor cell lines: U937, K-562, HL-60, HT-29, HeLa, and A431. The results showed that these new drugs elicit pronounced cytotoxic effects against U937, K-562, HL-60 and A431 while HeLa and HT-29 were less sensitive to the new drugs. This apparent selectivity was different to that of m-AMSA, a drug currently used for cancer treatment. Since the interaction of NBQ-2 to DNA by intercalation has been proposed as the initial step leading to its antineoplastic activity, DNA binding and changes in DNA contour length induced by the new NBQ-2 structural analogs were also investigated using calf thymus and human DNA. The drug, 7-(1-propenyl)-3-nitrobenzimidazolo[3,2-alpha]quinolinium chloride (NBQ-59) was the most cytotoxic agent of the analog series (IC50 = 16 microM for HL-60 cells), however, it demonstrated the weakest binding to DNA (Kint = 0.9 x 10[5] M-1 for calf thymus DNA). NBQ-59 was also found to be a poor intercalator into the DNA double helix. Therefore, our results suggest that DNA binding is not the primary mechanism of drug action for this family of compounds. In addition structural determinants important for cytotoxicity of the benzazolo quinolinium chlorides were suggested by our results. In particular, the nitro group in the 3 position does not seem to be necessary for bioactivity, while substitutions in the benzazolo moiety have striking effects on the biological activity of the drugs.

Estramustine-binding protein (EMBP) in renal cell carcinoma immunohistochemistry, immunoscintigraphy and in vitro estramustine effects.

The present report shows that the human renal cell carcinoma (RCC) cell lines, A498 and CAKI-2, express the estramustine-binding protein (EMBP). The RCC cell lines investigated were highly sensitive for estramustine, with cell arrest in atypical metaphase. In vitro experiments using a fluorimetric cytotoxicity assay (FMCA) showed a pronounced cytotoxic effect mediated by estramustine. Immunohistochemical analysis of tumour specimens from patients with RCC showed positive staining for EMBP in 12/16 cases. Immunoscintigraphy was performed in an experimental system in nude mice, heterotransplanted with the CAKI-2 cell line. A radiolabelled monoclonal anti-EMBP antibody was used. The results show a specific uptake of the antibody in the RCC tumour, expressed as a percentage of the injected dose per gram tissue, which ranged from 4.03 to 6.9. The results obtained form the basis for clinical studies on the feasibility of utilizing estramustine in the management of RCC. Immunoscintigraphy using the monoclonal anti-EMBP antibody is of potential use for in vivo characterization of the malignancy and in the selection patients suitable for treatment with estramustine.

An active-site mutation in the human immunodeficiency virus type 1 proteinase (PR) causes reduced PR activity and loss of PR-mediated cytotoxicity without apparent effect on virus maturation and infectivity.

Infectious retrovirus particles are derived from structural polyproteins which are cleaved by the viral proteinase (PR) during virion morphogenesis. Besides cleaving viral polyproteins, which is essential for infectivity, PR of human immunodeficiency virus (HIV) also cleaves cellular proteins and PR expression causes a pronounced cytotoxic effect. Retroviral PRs are aspartic proteases and contain two copies of the triplet Asp-Thr-Gly in the active center with the threonine adjacent to the catalytic aspartic acid presumed to have an important structural role. We have changed this threonine in HIV type 1 PR to a serine. The purified mutant enzyme had an approximately 5- to 10-fold lower activity against HIV type 1 polyprotein and peptide substrates compared with the wild-type enzyme. It did not induce toxicity on bacterial expression and yielded significantly reduced cleavage of cytoskeletal proteins in vitro. Cleavage of vimentin in mutant-infected T-cell lines was also markedly reduced. Mutant virus did, however, elicit productive infection of several T-cell lines and of primary human lymphocytes with no significant difference in polyprotein cleavage and with similar infection kinetics and titer compared with wild-type virus. The discrepancy between reduced processing in vitro and normal virion maturation can be explained by the observation that reduced activity was due to an increase in Km which may not be relevant at the high substrate concentration in the virus particle. This mutation enables us therefore to dissociate the essential function of PR in viral maturation from its cytotoxic effect.