Formation of trans-verbenol and verbenone from alpha-pinene catalysed by immobilised Picea abies cells

Both enantiomers and the racemate of alpha-pinene were transformed by Picea abies cells immobilised on alginate. The main products were cis- and trans-verbenol, the later being further transformed to verbenone. The enantiomeric purity of each product more or less corresponded to that of the substrate. Transformation by free cells was faster than that by the immobilised cells. The ratio of products differed to some extent between the transformation by free and immobilised cells.     

The effect of new non-cross resistant antitumour agents on the energy state of human erythrocytes

Multidrug resistance (MDR) of tumour cells is related to the overexpression of ATP-dependent pumps responsible for the active efflux of antitumour agents out of resistant cells. Benzoperimidine and anthrapyridone compounds exhibit comparable cytotoxic activity against sensitive and MDR tumour cells. They diffuse extremely rapidly across the plasma membrane and render the ATP-dependent efflux inefficient. Such uptake could disturb an energy metabolism of normal cells possessing an elevated level of ATP-dependent proteins, especially erythrocytes having a high level of the MRP1, MRP4 and MRP5 proteins. In this study the effect of five antitumour agents: benzoperimidine (BP1), anthrapyridones (CO1, CO7) and reference drugs used in the clinic: doxorubicin (DOX) and pirarubicin (PIRA), on the energetic state in human erythrocytes has been examined. These compounds have various types of structure and kinetics of cellular uptake (slow--DOX, CO7, moderate--PIRA, fast--BP1, CO1) resulting in their different ability to saturate ATP-dependent transporters. The energetic state of erythrocytes was examined by determination of purine nucleotide contents (ATP, ADP, AMP), NAD(+) and values of adenylate energy charge (AEC) using an HPLC method. It was found that the level of nucleotides as well as the AEC value of erythrocytes were not changed during 24 h of incubation with these agents independently of their structure and ability to saturate ATP-dependent pumps. This is a very promising result in view of their potential use in the clinic as antitumour drugs against multidrug resistant cancers.     

Porphyrin depth in lipid bilayers as determined by iodide and parallax fluorescence quenching methods and its effect on photosensitizing efficiency

Photosensitization by porphyrins and other tetrapyrrole chromophores is used in biology and medicine to kill cells. This light-triggered generation of singlet oxygen is used to eradicate cancer cells in a process dubbed "photodynamic therapy," or PDT. Most photosensitizers are of amphiphilic character and they partition into cellular lipid membranes. The photodamage that they inflict to the host cell is mainly localized in membrane proteins. This photosensitized damage must occur in competition with the rapid diffusion of singlet oxygen through the lipid phase and its escape into the aqueous phase. In this article we show that the extent of damage can be modulated by employing modified hemato- and protoporphyrins, which have alkyl spacers of varying lengths between the tetrapyrrole ring and the carboxylate groups that are anchored at the lipid/water interface. The chromophore part of the molecule, and the point of generation of singlet oxygen, is thus located at a deeper position in the bilayer. The photosensitization efficiency was measured with 9,10-dimethylanthracene, a fluorescent chemical target for singlet oxygen. The vertical insertion of the sensitizers was assessed by two fluorescence-quenching techniques: by iodide ions that come from the aqueous phase; and by spin-probe-labeled phospholipids, that are incorporated into the bilayer, using the parallax method. These methods also show that temperature has a small effect on the depth when the membrane is in the liquid phase. However, when the bilayer undergoes a phase transition to the solid gel phase, the porphyrins are extruded toward the water interface as the temperature is lowered. These results, together with a previous publication in this journal, represent a unique and precedental case where the vertical location of a small molecule in a membrane has an effect on its membranal activity.     

Sulforaphane-mediated induction of a phase 2 detoxifying enzyme NAD(P)H:quinone reductase and apoptosis in human lymphoblastoid cells

The effect of sulforaphane on human lymphoblastoid cells originating from a patient of a high cancer risk was studied. Sulforaphane (SFN) is a naturally occurring substance of chemopreventive activity. In our study, changes in cell growth, induction of apoptosis and phase 2 enzymes as well as glutathione level were examined. Apoptosis was tested by confocal microscopy at three stages: change in mitochondrial membrane potential, caspase activation and phosphatidylserine externalization. We show that SFN increases the activity of the detoxification system: it increases quinone reductase activity at low concentration (0.5-1 microM) and raises glutathione level in a dose-dependent manner. At higher doses (2.5-10 microM) sulforaphane is a cell growth modulator, as it caused cell growth cessation (IC50 = 3.875 microM), and apoptosis inducer. The results obtained suggest that sulforaphane acts as a chemopreventive agent in human lymphoblastoid cells.     

Human herpesvirus 8 in primary effusion lymphoma in an HIV-seronegative male. A case report

BACKGROUND: AIDS-related body cavity-based lymphoma, or primary effusion lymphoma (PEL), is a distinct clinicopathologic entity that occurs predominantly in immunosuppressed patients infected with human herpesvirus 8 (HHV-8), also known as Kaposi's sarcoma-associated herpesvirus. Although it rarely occurs in human immunodeficiency virus (HIV)-negative patients, we report such a case here. CASE: A 74-year-old male, who was HIV and Epstein-Barr virus (EBV) negative, was admitted to the hospital with dyspnea and chest pain. Chest radiography and computed tomography showed right pleural effusion. Cytologic analysis of the pleural effusion revealed a high grade lymphoma with round nuclei, prominent nucleoli and abundant cytoplasm. Polymerase chain reaction performed on the pleural effusion was positive for HHV-8 and negative for EBV. On molecular studies, the immunoglobulin heavy and kappa light chains were rearranged. Flow cytometry revealed a hyperploid fraction with DNA index of 1.29 expressing CD30. Immunostaining for HHV-8 from a cell block was positive. Electron microscopy revealed lymphomalike cells, many in various stages of apoptosis, with large nucleoli and clusters of viruslike particles in the nucleoplasm. CONCLUSION: A firm diagnosis of PEL can be established by the examination of cells from the lymphomatous effusion by a combination of cytology, molecular genetics, phenotypic features, immunostaining and electron microscopy. To our knowledge, this is the first case in which immunostaining for anti-HHV-8 monoclonal antibodies was used to support the diagnosis.     

The effects of leflunomide on clinical parameters and serum levels of IL-6, IL-10, MMP-1 and MMP-3 in patients with resistant rheumatoid arthritis

OBJECTIVE: The purpose of this open pilot study was to assess possible mechanisms of the effects of leflunomide by studying the influence of the drug on the serum levels of MMP-1, MMP-3, IL-10, IL-6 and their possible correlation with clinical disease parameters. PATIENTS AND METHODS: Thirty patients with long standing active rheumatoid arthritis were enrolled in this study. All patients failed at least 5 DMARDs in the past and were on stable treatment for at least 3 months before starting the protocol. The patients received a loading dose of 100 mg for 3 days followed by 20 mg/day thereafter and followed up monthly for 6 months. Disease activity was assessed at baseline, 2 weeks, and every month of therapy thereafter using the following variables: tender joint count, swollen joint count, morning stiffness duration, pain, tiredness, physician's and patient's global assessment, using VAS, ESR and CRP. Clinical effects of the treatment regimen were calculated using the American College of Rheumatology (ACR) criteria for clinical response. Adverse events were recorded. Serum levels of MMP-1, MMP-3, IL-10 and IL-6 were measured before and 3 months after starting the protocol. RESULTS: Except for tiredness, a statistically significant improvement in all clinical and laboratory parameters of disease activity was reached after 3 months. At this time point the ACR-20 response rate was 46.2%. The levels of MMP-1, MMP-3, IL-6 and IL-10 decreased significantly after 3 months. A statistically significant correlation between serum levels of MMP-1, IL-10 and IL-6 and clinical and laboratory parameters was also shown. After 6 months, 16 out of 30 patients withdrew from the study [adverse events (35.4%), lack of efficacy (9.7%), and low compliance (6.4%)]. CONCLUSIONS: Leflunomide was clinically efficacious in this group of long standing resistant RA in an open study "real life" design. These results comply with those reported in previous clinical trials. Serum MMP-1, MMP-3, IL-10 and IL-6 levels decreased significantly. Despite high withdrawal rate, no serious adverse effects were recorded.     

Assessment of nutritional status of gastroenterology patients in Croatia

Malnutrition is a common feature of gastroenterological diseases. In this study, nutritional status of the patients admitted to Department of Gastroenterology at University Hospital Center Zagreb was assessed. Anthropometric, dietetic, biochemical methods and method of Subjective Global Assessment (SGA) was used. The study group included 284 patients admitted to the Hospital. Malnutrition, as defined by SGA, was found in 61.1% of the patients, of whom 75% were moderately and 25% severely malnourished. Those patients classified as moderately and extremely malnourished by SGA were found to have statistically lower values of BMI, albumin, total proteins, calcium, iron, triglycerides, cholesterol, vitamin A and lymphocytes as compared to those who were adequately nourished. The prevalence of malnutrition in hospitalized patients treated at the Department of Gastroenterology is high. The use of nutritional screening with multiple measures would be important in the early identification and treatment of these patients and would help decrease this high prevalence.     

Simian Virus 40 Activates ATR-Δp53 Signaling To Override Cell Cycle and DNA Replication Control

During infection, simian virus 40 (SV40) attempts to take hold of the cell, while the host responds with various defense systems, including the ataxia-telangiectasia mutated/ATM-Rad3 related (ATM/ATR)-mediated DNA damage response pathways. Here we show that upon viral infection, ATR directly activates the p53 isoform Δp53, leading to upregulation of the Cdk inhibitor p21 and downregulation of cyclin A-Cdk2/1 (AK) activity, which force the host to stay in the replicative S phase. Moreover, downregulation of AK activity is a prerequisite for the generation of hypophosphorylated, origin-competent DNA polymerase α-primase (hypo-Polα), which is, unlike AK-phosphorylated Polα (P-Polα), recruited by SV40 large T antigen (T-Ag) to initiate viral DNA replication. Prevention of the downregulation of AK activity by inactivation of ATR-Δp53-p21 signaling significantly reduced the T-Ag-interacting hypo-Polα population and, accordingly, SV40 replication efficiency. Moreover, the ATR-Δp53 pathway facilitates the proteasomal degradation of the 180-kDa catalytic subunit of the non-T-Ag-interacting P-Polα, giving rise to T-Ag-interacting hypo-Polα. Thus, the purpose of activating the ATR-Δp53-p21-mediated intra-S checkpoint is to maintain the host in S phase, an optimal environment for SV40 replication, and to modulate the host DNA replicase, which is indispensable for viral amplification.Infection of quiescent CV-1 cells with the primate polyomavirus simian virus 40 (SV40) induces cell cycle progression and stimulates host cell DNA replication, which is mandatory for viral amplification. SV40 uses only a single viral protein, T antigen (T-Ag), for its own replication; all other components have to be provided by the host. Initially, a specifically phosphorylated subclass of T-Ag binds to a palindromic sequence in the SV40 origin (43), and in the presence of ATP, T-Ag forms a double-hexamer nucleoprotein complex leading to structural distortion and unwinding of origin DNA sequences (5). In concert with the cellular single-strand DNA binding protein RPA and topoisomerase I, the DNA helicase activity of T-Ag promotes more-extensive origin unwinding, forming a preinitiation complex (pre-RC), resulting in an initiation complex (53). Once the initiation complex forms, the primase activity of the heterotetrameric DNA polymerase α-primase (Polα) complex, consisting of the p180 catalytic subunit, the p70 regulatory subunit, and the p48/58 primase subunits, synthesizes a short RNA primer on each template strand, which is extended by the DNA polymerase activity of Polα (6, 17). Immediately after the first nascent RNA/DNA primer is synthesized, the complete replication machinery is assembled, and elongation at both forks by the processive DNA polymerase δ ensues (62). Thus, during the initiation of SV40 replication, T-Ag performs many of the functions attributed to the eukaryotic pre-RC complex proteins, including Orc, Cdc6, Cdt1, and kinase-independent cyclin E, which facilitates loading of the putative replication helicase Mcm2-7 onto the eukaryotic origin (4, 18). Biochemical evidence shows that initiation of SV40 and eukaryotic DNA replication occurs by the physical interaction of Polα with the appropriate pre-RC in the immediate vicinity of the origin. In SV40, Polα is loaded onto the origin by direct physical contact between the helicase T-Ag and its p180 N-terminal domain C (14, 15, 16). In eukaryotes, Cdc45, Mcm10, and And-1 cooperate to recruit Polα to the origin-initiation complex, thereby tethering the replicase to the origin-loaded Mcm2-7 helicase (34, 61).Although SV40 and chromosomal DNA replication share the same essential replication factors that are recruited to the appropriate pre-RC, there are noticeable differences between the SV40 and eukaryotic replication systems. The viral system allows unregulated multiple firing of the origin, whereas in the eukaryotic system, origin-dependent initiation of replication is regulated and restricted to firing only once per cell cycle. Reinitiation at origins within a cell cycle is prevented by the inactivation of pre-RC components in S and G₂. The cyclin-dependent kinases (Cdks) play a central role in establishing a block to rereplication through phosphorylation of each of the components. At present, several proteins of the mammalian pre-RC, such as Orc1, Cdt1, Cdc6, and the Mcm complex are phosphorylated by cyclin A (cycA)-Cdk2/1 (AK) and, as a result, are degraded or inactivated (1, 26, 30, 33, 40). Nevertheless, not all of the pre-RC components mentioned above are utilized by SV40, and accordingly, not all are involved in viral initiation control. However, in both replication systems, DNA synthesis is initiated by Polα and its initiation activity is regulated by Cdks (55). Moreover, AK-phosphorylated Polα is not recruited to mammalian origins in vivo (13) and is unable to initiate SV40 replication in vitro (47, 57, 58). Considering that cellular mechanisms blocking the rereplication of DNA act by AK phosphorylation of the replication factors mentioned above, especially Polα, it is feasible to suggest that downregulation or even inhibition of this kinase activity promotes dysregulation of replication control in SV40-infected cells.One pathway that leads to downregulation of AK activity in response to cellular stress is the intra-S checkpoint, which employs the novel p53 isoform Δp53 (45). In UV-damaged S-phase cells, ATR (ataxia-telangiectasia mutated [ATM]-Rad3 related)-activated Δp53 upregulates the Cdk inhibitor p21, resulting in a transient reduction in AK activity and decelerated S-phase progression (45). Here we demonstrate that SV40 lytic infection triggers the ATR signaling cascade, leading to the activation of Δp53 and accordingly a p21-mediated drop in AK activity to prevent AK-catalyzed inactivation of the hypophosphorylated, T-Ag-interacting Polα (hypo-Polα) subclass, which is essential for the initiation of viral DNA replication.