Immunogenicity of synthetic conjugates of Lewisy oligosaccharide with proteins in mice: towards the design of anticancer vaccines

 Many human carcinomas overexpress the Lewis^y (Le^y) blood-group epitope [Fucα1→2Galβ1→4 (Fucα1→3)GlcNAcβ1→3Gal-]. With a view to developing Le^y based vaccines we have examined the immunogenicity of Le^y-protein conjugates in mice. Le^y pentasaccharide was synthesized as its allyl glycoside and coupled to keyhole limpet hemocyanin (KLH) by reductive amination or by a novel method utilizing a maleido-derivitized alkyl carboxyhydrazide as a bridging group to 2-iminothiolane-derivitized KLH. Le^y oligosaccharide was also coupled to bovine serum albumin by reductive amination. Immunization of groups of mice with the three conjugates, together with the immunological adjuvant QS21, showed that Le^y oligosaccharide directly coupled to KLH was the most efficient conjugate for eliciting IgG and IgM antibody responses to naturally occurring forms of Le^y epitopes carried on mucins and glycolipids. These antibodies were also reactive with and cytotoxic to a human breast cancer cell line expressing Le^y (MCF-7). These experiments suggest that Le^y-KLH antigen and QS21 adjuvant could be considered as an immunogenic therapeutic vaccine in carcinoma patients. Received: 28 March 1997 / Accepted: 2 September 1997     

A novel method for characterisation of microbial growth kinetics on volatile organic compounds

A novel method for the determination of microbial growth kinetics on hydrophobic volatile organic compounds (VOC) has been developed. A stirred tank reactor was operated as a fed-batch system to which the VOC was continuously fed via the gas phase, assuring a constant VOC concentration in the mineral medium. A flow of air was saturated with the VOC, and then mixed with a further flow of air, to obtain a predetermined VOC concentration. Thus, different VOC concentrations in the mineral medium could be obtained by altering the VOC concentration in the feed gas. The growth kinetics of Xanthobacter autotrophicus GJ10 on 1,2-dichloroethane (DCE) and of Pseudomonas sp. strain JS150 on MonoChloroBenzene (MCB) were assessed using this method. The growth of strain JS150 was strongly inhibited at MCB concentrations higher than 160 mg l⁻¹, and the results were fitted using a piecewise function. The growth kinetics of strain GJ10 were described by the Luong model where maximum growth rate μ_max = 0.12 h⁻¹, substrate saturation constant K _S = 7.8 mg l⁻¹, and maximum substrate concentration S _m (above which growth is completely inhibited) = 1080 mg l⁻¹. Varying nitrogen and oxygen flows enabled the effect of oxygen concentration on the growth kinetics of Pseudomonas JS150 to be determined. Received: 30 November 1998 / Received revision: 19 March 1999 / Accepted: 20 March 1999     

Lurking in the shadows: emerging rodent infectious diseases

Rodent parvoviruses, Helicobacter spp., murine norovirus, and several other previously unknown infectious agents have emerged in laboratory rodents relatively recently. These agents have been discovered serendipitously or through active investigation of atypical serology results, cell culture contamination, unexpected histopathology, or previously unrecognized clinical disease syndromes. The potential research impact of these agents is not fully known. Infected rodents have demonstrated immunomodulation, tumor suppression, clinical disease (particularly in immunodeficient rodents), and histopathology. Perturbations of organismal and cellular physiology also likely occur. These agents posed unique challenges to laboratory animal resource programs once discovered; it was necessary to develop specific diagnostic assays and an understanding of their epidemiology and transmission routes before attempting eradication, and then evaluate eradication methods for efficacy. Even then management approaches varied significantly, from apathy to total exclusion, and such inconsistency has hindered the sharing and transfer of rodents among institutions, particularly for genetically modified rodent models that may not be readily available. As additional infectious agents are discovered in laboratory rodents in coming years, much of what researchers have learned from experiences with the recently identified pathogens will be applicable. This article provides an overview of the discovery, detection, and research impact of infectious agents recently identified in laboratory rodents. We also discuss emerging syndromes for which there is a suspected infectious etiology, and the unique challenges of managing newly emerging infectious agents.     

Synthesis of sialyl Lewis<Superscript>a</Superscript> (sLe<Superscript>a</Superscript>, CA19-9) and construction of an immunogenic sLe<Superscript>a</Superscript> vaccine

Sialyl Lewis^a (sLe^a), also termed CA19-9 antigen, is recognized by murine mAb19-9 and is expressed on the cancer cell surface as a glycolipid and as an O-linked glycoprotein. It is highly expressed in a variety of gastrointestinal epithelial malignancies including colon cancer and pancreatic cancer, and in breast cancer and small cell lung cancer, but has a limited expression on normal tissues. sLe^a is known to be the ligand for endothelial cell selectins suggesting a role for sLe^a in cancer metastases and adhesion. For these reasons, sLe^a may be a good target for antibody mediated immunotherapy including monoclonal antibodies and tumor vaccines. However, sLe^a is structurally similar to sLe^x and other blood group related carbohydrates which are widely expressed on polymorphonucleocytes and other circulating cells, raising concern that immunization against sLe^a will induce antibodies reactive with these more widely expressed autoantigens. We have shown previously both in mice and in patients that conjugation of a variety of carbohydrate cancer antigen to keyhole limpet hemocyanin (KLH) and administration of this conjugate mixed with saponin adjuvants QS-21 or GPI-0100 are the most effective methods for induction of antibodies against these cancer antigens. We describe here for the first time the total synthesis of pentenyl glycoside of sLe^a hexasaccharide and its conjugation to KLH to construct a sLe^a-KLH conjugate. Groups of five mice were vaccinated subcutaneously four times over 6 weeks. Sera were tested against sLe^a-HSA by ELISA and against sLe^a positive human cell lines adenocarcinoma SW626 and small cell lung cancer (SCLC) DMS79 by FACS. As expected, mice immunized with unconjugated sLe^a plus GPI-0100 or unconjugated sLe^a mixed with KLH plus GPI-0100 failed to produce antibodies against sLe^a. However, mice immunized with sLe^a-KLH conjugate without GPI-0100 produced low levels of antibodies and mice immunized with sLe^a-KLH plus GPI-0100 produced significantly higher titer IgG and IgM antibodies against sLe^a by ELISA. These antibodies were highly reactive by FACS and mediated potent complement mediated cytotoxicity against sLe^a positive SW626 and DMS79 cells. They showed no detectable cross reactivity against a series of other blood group-related antigens, including Le^y, Le^x, and sLe^x by dot blot immune staining. This vaccine is ready for testing as an active immunotherapy for treating sLe^a positive cancer in clinical settings. Govind Ragupathi and Philip O. Livingston are paid consultants and shareholders in MabVax Therapeutics, Inc., San Diego, CA 92121. The sLe^a vaccine is licensed to MabVax.     

Proof-of-concept human laboratory study for protracted abstinence in alcohol dependence: effects of gabapentin

There is a need for safe medications that can effectively support recovery by treating symptoms of protracted abstinence that may precipitate relapse in alcoholics, e.g. craving and disturbances in sleep and mood. This proof-of-concept study reports on the effectiveness of gabapentin 1200 mg for attenuating these symptoms in a non-treatment-seeking sample of cue-reactive, alcohol-dependent individuals. Subjects were 33 paid volunteers with current Diagnostic and Statistical Manual of Mental Disorders-IV alcohol dependence and a strength of craving rating 1 SD or greater for alcohol than water cues. Subjects were randomly assigned to gabapentin or placebo for 1 week and then participated in a within-subjects trial where each was exposed to standardized sets of pleasant, neutral and unpleasant visual stimuli followed by alcohol or water cues. Gabapentin was associated with significantly greater reductions than placebo on several measures of subjective craving for alcohol as well as for affectively evoked craving. Gabapentin was also associated with significant improvement on several measures of sleep quality. Side effects were minimal, and gabapentin effects were not found to resemble any major classes of abused drugs. Results suggest that gabapentin may be effective for treating the protracted abstinence phase in alcohol dependence and that a randomized clinical trial would be an appropriate next step. The study also suggests the value of cue-reactivity studies as proof-of-concept screens for potential antirelapse drugs.     

C-Terminal Flap Endonuclease (rad27) Mutations: Lethal Interactions With a DNA Ligase I Mutation (cdc9-p) and Suppression by Proliferating Cell Nuclear Antigen (POL30) in Saccharomyces cerevisiae

During lagging-strand DNA replication in eukaryotic cells primers are removed from Okazaki fragments by the flap endonuclease and DNA ligase I joins nascent fragments. Both enzymes are brought to the replication fork by the sliding clamp proliferating cell nuclear antigen (PCNA). To understand the relationship among these three components, we have carried out a synthetic lethal screen with cdc9-p, a DNA ligase mutation with two substitutions (F43A/F44A) in its PCNA interaction domain. We recovered the flap endonuclease mutation rad27-K325* with a stop codon at residue 325. We created two additional rad27 alleles, rad27-A358* with a stop codon at residue 358 and rad27-pX8 with substitutions of all eight residues of the PCNA interaction domain. rad27-pX8 is temperature lethal and rad27-A358* grows slowly in combination with cdc9-p. Tests of mutation avoidance, DNA repair, and compatibility with DNA repair mutations showed that rad27-K325* confers severe phenotypes similar to rad27Δ, rad27-A358* confers mild phenotypes, and rad27-pX8 confers phenotypes intermediate between the other two alleles. High-copy expression of POL30 (PCNA) suppresses the canavanine mutation rate of all the rad27 alleles, including rad27Δ. These studies show the importance of the C terminus of the flap endonuclease in DNA replication and repair and, by virtue of the initial screen, show that this portion of the enzyme helps coordinate the entry of DNA ligase during Okazaki fragment maturation.CELLULAR maintenance of genomic integrity is essential for the continued viability of all organisms. The fidelity of DNA replication has to be maintained and DNA insults have to be repaired to ensure that deleterious mutations are not passed on to progeny or cause cancerous growth. A number of cellular proteins have multiple roles in DNA replication, mutation avoidance, and repair. In Saccharomyces cerevisiae, the flap endonuclease, proliferating cell nuclear antigen (PCNA), and DNA ligase I encoded by RAD27, POL30, and CDC9, respectively, are all required for proper replication and also function to avoid mutation and to facilitate repair.The flap endonuclease, FEN-1 in humans, is a highly conserved structure-specific nuclease that has both endonuclease and 5′–3′ exonuclease activity. During lagging-strand replication these activities function to remove primers from Okazaki fragments, either by endonucleolytic cleavage of a flap made by strand displacement (Liu et al. 2004) or by sequential exonucleolytic removal of single nucleotides at the 5′ end of the primer (Murante et al. 1994).While deletion of RAD27 is not lethal to yeast cells, the rad27Δ mutant exhibits temperature-sensitive growth, is a mutator, and undergoes genomic instability (Johnson et al. 1995; Reagan et al. 1995; Tishkoff et al. 1997b; Chen and Kolodner 1999). In addition, its sensitivity to low doses of the methylating agent methylmethane sulfonate (MMS) implicates the participation of the enzyme in base excision repair (BER) (Reagan et al. 1995; Wu and Wang 1999). rad27Δ mutants have been reported to be either mildly sensitive to UV light or not sensitive to UV light (Reagan et al. 1995; Sommers et al. 1995). In the strain background that the mutant is mildly sensitive, its combination with rad2Δ yields a double mutant more sensitive than each single mutant, implying that the enzyme does not participate in RAD2-mediated nucleotide excision repair (NER) (Reagan et al. 1995). The flap endonuclease has also been implicated in double-strand break (DSB) repair by virtue of the incompatibility of rad27Δ with mutations of the DSB repair pathways (Tishkoff et al. 1997b; Symington 1998). In addition, either the yeast enzyme or its human ortholog has been shown to participate in reactions of homologous recombination, nonhomologous end joining, and telomere maintenance (Parenteau and Wellinger 1999, 2002; Wu et al. 1999; Wang et al. 2004; Kikuchi et al. 2005). Curiously, the rad27Δ mutant is not sensitive to gamma radiation but is sensitive to high doses of MMS that are thought to act as a radiomimetic agent (Reagan et al. 1995; Sommers et al. 1995).PCNA is the replicative clamp that acts as a scaffold to facilitate the loading of DNA replication and repair proteins, including DNA ligase I and the flap endonuclease to DNA (Warbrick 2000, 2006; Maga and Hubscher 2003). PCNA (POL30) is essential for cell viability, which is indicative of its central role in DNA metabolism. Biochemical characterization of its effect on the flap endonuclease shows that it stimulates its activity ∼50-fold, evidencing the productive nature of the interaction (Gomes and Burgers 2000; Tom et al. 2000; Frank et al. 2001; Stucki et al. 2001). The ability of DNA ligase to efficiently catalyze the formation of phosphodiester bonds in the DNA backbone may also be facilitated by its binding to PCNA. Tom et al. (2001) showed that, in vitro, PCNA enhances the ligation reaction 5-fold and that the stable association of DNA ligase with nicked duplex DNA requires PCNA.Both DNA ligase and the flap endonuclease bind to PCNA via their respective PCNA interactive peptide domains (PIP box). The PIP box is a conserved sequence motif of the amino acids QXXLXXFF. The PIP box fits into the interdomain connector loop (IDCL) of PCNA to provide a protein–protein interaction surface (Gomes and Burgers 2000; Chapados et al. 2004; Sakurai et al. 2005; Pascal et al. 2006). Mutations in the PIP box or the IDCL that impair the interaction of DNA ligase and the flap endonuclease to PCNA lead to genomic instability (Amin and Holm 1996; Eissenberg et al. 1997; Gary et al. 1999; Refsland and Livingston 2005; Subramanian et al. 2005). We have reported that the double mutants made by combinations of cdc9-p, rad27-p, and pol30-90—mutations with alterations of the PIP box or the IDCL in the respective proteins—have synergistic phenotypes with respect to MMS sensitivity and to trinucleotide repeat instability (Refsland and Livingston 2005). These results suggest that the two enzymes function in a concerted manner that is facilitated by PCNA.The precise nature of how PCNA coordinates the entry of the flap endonuclease and DNA ligase into the replication fork is not well understood. Biochemical and structural studies have begun to elucidate a possible ordering of these PCNA-mediated interactions. The possibility of such an ordering is underscored by the observation that DNA ligase adopts a toroidal conformation by completely encircling duplex DNA while interacting with PCNA (Pascal et al. 2004). Moreover, both PCNA and DNA ligase may be loaded onto the DNA in a mechanism utilizing the replication clamp loader replication factor C (RFC) (Levin et al. 2004; Vijayakumar et al. 2009), again suggesting a complete encirclement of the DNA by DNA ligase as well as by PCNA. PCNA and DNA ligase are similar in size and their interaction is likely to extend along the face of PCNA in a manner that would prevent other proteins such as the flap endonuclease from binding to the IDCL (Pascal et al. 2004, 2006). A biochemical study with purified yeast proteins showed that the two enzymes cannot bind simultaneously to PCNA (Subramanian et al. 2005). These studies suggest that a coordinated sequential interaction among PCNA, DNA ligase, and the flap endonuclease is important for replication and repair.Alternatively, both the flap endonuclease and DNA ligase may bind to the same molecule of PCNA. Since PCNA is a homotrimer, DNA ligase can potentially bind to one monomer while the flap endonuclease binds to another, using its extended C-terminal tail in a conformation allowing it to be tethered to PCNA concurrently with DNA ligase (Gomes and Burgers 2000; Sakurai et al. 2005). DNA ligase could also bind to PCNA in an extended conformation while the flap endonuclease cleaves the DNA. Sulfolobus solfataricus DNA ligase has been shown to have an open, extended conformation while binding to PCNA (Pascal et al. 2006). Presumably, once the flap endonuclease has removed the 5′ flap, DNA ligase acquires a closed, ring-shaped conformation to catalyze the joining of Okazaki fragments (Pascal et al. 2006).Exactly how the interaction of these enzymes with PCNA is coordinated in vivo, whether singly or concurrently, is not well understood. To further elucidate how the interaction of DNA ligase with PCNA is ordered, we performed a genetic screen to identify mutations that are synthetically lethal with cdc9-p (F44A/F35A), an allele of DNA ligase that has impaired binding to PCNA (Refsland and Livingston 2005; Subramanian et al. 2005). We postulated that genes recovered from this screen would function in DNA repair, replication, and recombination or would be involved in ordering the DNA ligase–PCNA interaction. From the screen we recovered a truncated allele of RAD27, rad27-K325*. This allele encodes a protein that lacks the PIP box and the entire C-terminal domain of the enzyme but retains the N terminus containing the nuclease activities. We have characterized this allele and compared it to two other rad27 alleles in which we have created different alterations of the C-terminal end of the flap endonuclease.     

SEPALLATA3: the 'glue' for MADS box transcription factor complex formation

Background  

Plant MADS box proteins play important roles in a plethora of developmental processes. In order to regulate specific sets of target genes, MADS box proteins dimerize and are thought to assemble into multimeric complexes. In this study a large-scale yeast three-hybrid screen is utilized to provide insight into the higher-order complex formation capacity of the Arabidopsis MADS box family. SEPALLATA3 (SEP3) has been shown to mediate complex formation and, therefore, special attention is paid to this factor in this study.     

Virus-Induced Chaperone-Enriched (VICE) Domains Function as Nuclear Protein Quality Control Centers during HSV-1 Infection

Virus-Induced Chaperone-Enriched (VICE) domains form adjacent to nuclear viral replication compartments (RC) during the early stages of HSV-1 infection. Between 2 and 3 hours post infection at a MOI of 10, host protein quality control machinery such as molecular chaperones (e.g. Hsc70), the 20S proteasome and ubiquitin are reorganized from a diffuse nuclear distribution pattern to sequestration in VICE domains. The observation that VICE domains contain putative misfolded proteins suggests that they may be similar to nuclear inclusion bodies that form under conditions in which the protein quality control machinery is overwhelmed by the presence of misfolded proteins. The detection of Hsc70 in VICE domains, but not in nuclear inclusion bodies, indicates that Hsc70 is specifically reorganized by HSV-1 infection. We hypothesize that HSV-1 infection induces the formation of nuclear protein quality control centers to remodel or degrade aberrant nuclear proteins that would otherwise interfere with productive infection. Detection of proteolytic activity in VICE domains suggests that substrates may be degraded by the 20S proteasome in VICE domains. FRAP analysis reveals that GFP-Hsc70 is dynamically associated with VICE domains, suggesting a role for Hsc70 in scanning the infected nucleus for misfolded proteins. During 42°C heat shock, Hsc70 is redistributed from VICE domains into RC perhaps to remodel viral replication and regulatory proteins that have become insoluble in these compartments. The experiments presented in this paper suggest that VICE domains are nuclear protein quality control centers that are modified by HSV-1 to promote productive infection.     

Management strategies for controlling endemic and seasonal mouse parvovirus infection in a barrier facility

Despite improved diagnostic and rederivation capabilities, research facilities still struggle to manage parvovirus infections (e.g., mouse parvovirus (MPV) and minute virus of mice) in mouse colonies. Multi-faceted approaches are needed to prevent adventitious organisms such as MPV from breaching a barrier facility. In this article, the authors document recent changes to the Salk Institute's animal care program that were intended to help manage mouse parvovirus in the barrier facility. Specifically, the Institute started to use a new disinfectant and to give mice irradiated feed. The authors found an association between these modifications and a reduction in MPV incidence and prevalence in endemically infected colonies. These data suggest that using irradiated feed and appropriate disinfectants with contemporary management practices can be an effective plan for eradicating or controlling MPV infection in a research facility. The authors recommend further study of the environmental risk factors for parvovirus infection and of potential biological interactions associated with the use of irradiated feed.