Counterpoint. Cancer vaccines: single-epitope anti-idiotype vaccine versus multiple-epitope antigen vaccine

Anti-idiotype (Id) vaccine therapy has been tested and shown to be effective, in several animal models, for triggering the immune system to induce specific and protective immunity against bacterial, viral and parasitic infections. The administration of anti-Id antibodies as surrogate tumor-associated antigens (TAA) also represents another potential application of the concept of the Id network. Limited experience in human trials using anti-Id to stimulate immunity against tumors has shown promising results. In this “counterpoint” article, we discuss our own findings showing the potential of anti-Id antibody vaccines to be novel therapeutic approaches to various human cancers and also discuss where anti-Id vaccines may perform better than traditional multiple-epitope antigen vaccines. Received: 27 December 1999 / Accepted: 27 January 2000     

A direct interaction between proliferating cell nuclear antigen (PCNA) and Cdk2 targets PCNA-interacting proteins for phosphorylation

Proliferating cell nuclear antigen is best known as a DNA polymerase accessory protein but has more recently also been shown to have different functions in important cellular processes such as DNA replication, DNA repair, and cell cycle control. PCNA has been found in quaternary complexes with the cyclin kinase inhibitor p21 and several pairs of cyclin-dependent protein kinases and their regulatory partner, the cyclins. Here we show a direct interaction between PCNA and Cdk2. This interaction involves the regions of the PCNA trimer close to the C termini. We found that PCNA and Cdk2 form a complex together with cyclin A. This ternary PCNA-Cdk2-cyclin A complex was able to phosphorylate the PCNA binding region of the large subunit of replication factor C as well as DNA ligase I. Furthermore, PCNA appears to be a connector between Cdk2 and DNA ligase I and to stimulate phosphorylation of DNA ligase I. Based on our results, we propose the model that PCNA brings Cdk2 to proteins involved in DNA replication and possibly might act as an "adaptor" for Cdk2-cyclin A to PCNA-binding DNA replication proteins.     

Efficient gene transfer to airway epithelium using recombinant Sendai virus

Clinical studies of gene therapy for cystic fibrosis (CF) suggest that the key problem is the efficiency of gene transfer to the airway epithelium. The availability of relevant vector receptors, the transient contact time between vector and epithelium, and the barrier function of airway mucus contribute significantly to this problem. We have recently developed recombinant Sendai virus (SeV) as a new gene transfer agent. Here we show that SeV produces efficient transfection throughout the respiratory tract of both mice and ferrets in vivo, as well as in freshly obtained human nasal epithelial cells in vitro. Gene transfer efficiency was several log orders greater than with cationic liposomes or adenovirus. Even very brief contact time was sufficient to produce this effect, and levels of expression were not significantly reduced by airway mucus. Our investigations suggest that SeV may provide a useful new vector for airway gene transfer.     

A Bacillus subtilis gene-encoding protein homologous to eukaryotic SMC motor protein is necessary for chromosome partition

We have analysed the function of a gene of Bacillus subtilis , the product of which shows significant homology with eukaryotic SMC proteins essential for chromosome condensation and segregation. Two mutant strains were constructed; in one, the expression was under the control of the inducible spac promoter (conditional null) and, in the other, the gene was disrupted by insertion (disrupted null). Both could form colonies at 23°C but not at 37°C in the absence of the expression of the Smc protein, indicating that the B. subtilis smc gene was essential for cell growth at higher temperatures. Microscopic examination revealed the formation of anucleate and elongated cells and diffusion of nucleoids within the elongated cells in the disrupted null mutant grown at 23°C and in the conditional null mutant grown in low concentrations of IPTG at 37°C. In addition, immunofluorescence microscopy showed that subcellular localization of the Spo0J partition protein was irregular in the smc disrupted null mutant, compared with bipolar localization in wild-type cells. These results indicate that the B. subtilis smc gene is essential for chromosome partition. The role of B. subtilis Smc protein in chromosome partition is discussed.     

Involvement of an ABC transporter in a developmental pathway regulating hypocotyl cell elongation in the light

In the dark, plant seedlings follow the skotomorphogenetic developmental program, which results in hypocotyl cell elongation. When the seedlings are exposed to light, a switch to photomorphogenetic development occurs, and hypocotyl cell elongation is inhibited. We have manipulated the expression of the AtPGP1 (for Arabidopsis thaliana P glycoprotein1) gene in transgenic Arabidopsis plants by using sense and antisense constructs. We show that within a certain light fluence rate window, overexpression of the AtPGP1 gene under the control of the cauliflower mosaic virus 35S promoter causes plants to develop longer hypocotyls, whereas expression of the gene in antisense orientation results in hypocotyls shorter than those occurring in the wild type. In the dark, hypocotyls of transgenic and wild-type plants are indistinguishable. Because the AtPGP1 gene encodes a member of the superfamily of ATP binding cassette-containing (ABC) transporters, these results imply that a transport process is involved in a hypocotyl cell elongation pathway active in the light. The AtPGP1 transporter is localized in the plasmalemma, as indicated by immunohistochemical techniques and biochemical membrane separation methods. Analysis of the AtPGP1 expression pattern by using reporter gene constructs and in situ hybridization shows that in wild-type seedlings, AtPGP1 is expressed in both the root and shoot apices.     

A Ca2+‐binding protein with numerous roles and uses: parvalbumin in molecular biology and physiology

Parvalbumins (PVs) are acidic, intracellular Ca²⁺‐binding proteins of low molecular weight. They are associated with several Ca²⁺‐mediated cellular activities and physiological processes. It has been suggested that PV might function as a “Ca²⁺ shuttle” transporting Ca²⁺ from troponin‐C (TnC) to the sarcoplasmic reticulum (SR) Ca²⁺ pump during muscle relaxation. Thus, PV may contribute to the performance of rapid, phasic movements by accelerating the contraction–relaxation cycle of fast‐twitch muscle fibers. Interestingly, PVs promote the generation of power stroke in fish by speeding up the rate of relaxation and thus provide impetus to attain maximal sustainable speeds. However, immunological monitoring of diverse tissues demonstrated that PVs are also present in non‐muscle cells. The axoplasmic transport and various intracellular secretory mechanisms including the endocrine secretions seem to be controlled by the Ca²⁺ regulation machinery. Any defect in the Ca²⁺ handling apparatus may cause several clinical problems; for instance, PV deficiency alters the neuronal activity, a key mechanism leading to epileptic seizures. Moreover, atypical relaxation of the heart results in diastolic dysfunction, which is a major cause of heart failure predominantly among the aged people. PV may offer a unique potential to correct defective relaxation in energetically compromised failing hearts through PV gene transfer. Consequently, PV gene transfer may present a new therapeutic approach to correct cellular disturbances in Ca²⁺ signaling pathways of diseased organs. Hence, PVs appear to be amazingly useful candidate proteins regulating a variety of cellular functions through action on Ca²⁺ flux management.     

Lovastatin and (+)‐geodin production by Aspergillus terreus from crude glycerol

The use of pure substrate represents a significant proportion of the cost of manufacturing a drug such as lovastatin. This study explores the production of lovastatin and (+)‐geodin by Aspergillus terreus ATCC 20542 using biodiesel‐derived crude glycerol (CG) as a feedstock. Shake flask experiments showed reduced lovastatin production and glycerol consumption in the presence of 10–50 g/L CG with respect to pure glycerol controls. At 50 g/L, lovastatin and (+)‐geodin production was significantly reduced by 82 and 73%, respectively. The lowest lovastatin inhibition was detected in 30 g/L of CG (48%), which was accompanied by a significant rise in (+)‐geodin production (338%). Further investigation was performed on three major impurities found in CG, namely methanol (MeOH), sodium chloride (NaCl), and fatty acids (oleic acid and palmitic acid (PA), soap). None was particularly inhibitory for lovastatin, except soap and PAs, which reduced its production by more than 50% at all concentrations tested. In contrast, (+)‐geodin was inhibited in the presence of MeOH and PA by up to 46 and 91%, respectively. These observations indicate that partial purification of CG would be potentially useful in improving production of lovastatin and (+)‐geodin by A. terreus.     

Deletion of Metabotropic Glutamate Receptors 2 and 3 (mGlu2 & mGlu3) in Mice Disrupts Sleep and Wheel-Running Activity,and Increases the Sensitivity of the Circadian System to Light

Sleep and/or circadian rhythm disruption (SCRD) is seen in up to 80% of schizophrenia patients. The co-morbidity of schizophrenia and SCRD may in part stem from dysfunction in common brain mechanisms, which include the glutamate system, and in particular, the group II metabotropic glutamate receptors mGlu2 and mGlu3 (encoded by the genes Grm2 and Grm3). These receptors are relevant to the pathophysiology and potential treatment of schizophrenia, and have also been implicated in sleep and circadian function. In the present study, we characterised the sleep and circadian rhythms of Grm2/3 double knockout (Grm2/3^-/-) mice, to provide further evidence for the involvement of group II metabotropic glutamate receptors in the regulation of sleep and circadian rhythms. We report several novel findings. Firstly, Grm2/3^-/- mice demonstrated a decrease in immobility-determined sleep time and an increase in immobility-determined sleep fragmentation. Secondly, Grm2/3^-/- mice showed heightened sensitivity to the circadian effects of light, manifested as increased period lengthening in constant light, and greater phase delays in response to nocturnal light pulses. Greater light-induced phase delays were also exhibited by wildtype C57Bl/6J mice following administration of the mGlu2/3 negative allosteric modulator RO4432717. These results confirm the involvement of group II metabotropic glutamate receptors in photic entrainment and sleep regulation pathways. Finally, the diurnal wheel-running rhythms of Grm2/3^-/- mice were perturbed under a standard light/dark cycle, but their diurnal rest-activity rhythms were unaltered in cages lacking running wheels, as determined with passive infrared motion detectors. Hence, when assessing the diurnal rest-activity rhythms of mice, the choice of assay can have a major bearing on the results obtained.