Paucity of functional CTL epitopes in the E7 oncoprotein of cervical cancer associated human papillomavirus type 16

Many specific antiviral and antitumour immune responses have been attributed to the protective effects of antigen-specific CD8+ cytotoxic T lymphocytes (CTL). Recognition of virus infected or tumour cells by CTL requires presentation of at least one peptide epitope from a virus or tumour-specific antigen by the relevant MHC Class I molecule. Viral genes with mutations which remove CTL epitopes may thus be favoured for survival. Human cervical cancers are caused by papillomavirus infection, and these cancers consistently express the E7 protein of the oncogenic papillomavirus. We therefore investigated the MHC Class I restricted T cell epitopes of the human papillomavirus type 16 E7 oncoprotein using mice of five different genetic backgrounds, and an IFN-gamma ELISPOT assay, to determine the frequency with which MHC Class I epitopes might be expected in this small oncoprotein (98 amino acids). No MHC Class I restricted responses were detected in E7 immunized BALB/c (H-2d), CBA/CaH (H-2 k), FVB/N (H-2q) or A2KbH2b human HLA2.1 transgenic mice. In C57BL/6 J (H-2b) mice, a previously identified single antigenic epitope was detected. Therefore, we conclude that there is a paucity of MHC Class I restricted T cell epitopes in HPV16 E7 protein because of its small size. This might be advantageous to the virus. Furthermore here we present a quick and easy method to exhaustively determine CD8 T cell epitopes in proteins using a unique set of overlapping 8, 9 and 10 mer synthetic peptides.     

Preparation of soybean seed samples for FT-IR microspectroscopy

Typical preparation of seed samples for infrared (IR) microspectroscopy involves imbibition of the seed for varying time periods followed by cryosectioning. Imbibition, however, may initiate germination even at 4° C with associated changes in the chemistry of the sample. We have found that it is possible to section seeds that are sufficiently hard, such as soybeans, on a standard laboratory microtome without imbibition. The use of dry sectioning of unimbibed seeds is reported here, as well as a comparison of different mounting media and modes of analysis. Glycerol, Tissue-Tek, and ethanol were used as mounting media, and the quality of the resulting spectra was assessed. Ethanol was the preferred mountant, because it dried quickly with no residue and thus did not interfere with the spectrum of interest. Analysis in transmission mode using barium fluoride windows to hold the samples was compared with transmission-reflection analysis with sections mounted on special infrared-reflecting slides. The two modes of analysis performed well in different regions of the spectrum. The mode of analysis (transmission vs. transmission-reflection) should be based on the components of greatest interest in the sample.     

Sporicidal efficacy of pH-adjusted bleach for control of bioburden on production facility surfaces

pH-adjusted bleach was one of the agents used to disinfect contaminated public buildings in the USA following the 2001 bioterrorist attack with Bacillus anthracis spores. A USEPA fact sheet describes the preparation of pH-adjusted bleach by combining diluted sodium hypochlorite (NaOCl) with a controlled amount of 5 % acetic acid. This paper reports a modification of this procedure to qualify the use of pH-adjusted bleach for routine disinfection of cleanroom surfaces in pharmaceutical manufacturing facilities whenever a short contact time is desirable or there is a need for enhanced germicidal or sporicidal activity. Adjustment of pH was obtained reproducibly with either acetic acid or HCl, confirming the feasibility of developing standard procedures for the controlled addition of acid to diluted NaOCl solutions without compromising operator safety and convenience. Efficacy testing using spores from an in-house isolate of Bacillus pumilus confirmed that NaOCl solutions in the pH 5–8 range have much greater sporicidal activity on surfaces than do unadjusted alkaline solutions (pH > 11). With a contact time of 0.5 min, the log₁₀ reduction in spore viable counts was >5.4 for the five representative surfaces tested relative to untreated controls. Solutions of pH-adjusted NaOCl are known to be less stable than unadjusted alkaline solutions. Stability studies were performed by monitoring sporicidal efficacy, level of free available chlorine (FAC), and pH. Testing included several NaOCl concentrations and adjustment to different starting pHs. The efficacy of pH-adjusted solutions persisted in open containers for at least 12 h even though some FAC degradation occurred. In addition, solutions of 0.29 or 0.50 % NaOCl stored at room temperature protected from light retained efficacy for at least 4 weeks, indicating that short-term storage of solutions is possible following pH adjustment. The inorganic chemical degradation of pH-adjusted NaOCl solutions generates chlorate ion, an undesirable by-product. A comparison of chemical stability for 0.12, 0.25, and 0.50 % NaOCl solutions adjusted to different initial pHs indicated that the least chlorate formation occurred with 0.12 % NaOCl.     

HPV16-E7 Expression in Squamous Epithelium Creates a Local Immune Suppressive Environment via CCL2- and CCL5- Mediated Recruitment of Mast Cells

Human Papillomavirus (HPV) 16 E7 protein promotes the transformation of HPV infected epithelium to malignancy. Here, we use a murine model in which the E7 protein of HPV16 is expressed as a transgene in epithelium to show that mast cells are recruited to the basal layer of E7-expressing epithelium, and that this recruitment is dependent on the epithelial hyperproliferation induced by E7 by inactivating Rb dependent cell cycle regulation. E7 induced epithelial hyperplasia is associated with increased epidermal secretion of CCL2 and CCL5 chemokines, which attract mast cells to the skin. Mast cells in E7 transgenic skin, in contrast to those in non-transgenic skin, exhibit degranulation. Notably, we found that resident mast cells in E7 transgenic skin cause local immune suppression as evidenced by tolerance of E7 transgenic skin grafts when mast cells are present compared to the rejection of mast cell-deficient E7 grafts in otherwise competent hosts. Thus, our findings suggest that mast cells, recruited towards CCL2 and CCL5 expressed by epithelium induced to proliferate by E7, may contribute to an immunosuppressive environment that enables the persistence of HPV E7 protein induced pre-cancerous lesions.     

Can a Threshold Value Be Used to Classify Chondrichthyan Reproductive Modes: Systematic Review and Validation Using an Oviparous Species

The maternal-embryonic nutritional relationship in chondrichthyans has been poorly explored. Consequently, accurately discerning between their different reproductive modes is difficult; especially lecithotrophy and incipient histotrophy. This present study is the first to assess changes in mass throughout embryonic development of an oviparous chondrichthyan other than Scyliorhinus canicula. Heterodontus portusjacksoni egg cases were collected and used to quantify the gain or loss of wet mass, dry mass, water content, inorganic and organic matter from freshly deposited eggs (without macroscopically visible embryos) to near full-term embryos. A loss in organic mass of ∼40% found from this study is approximately double the values previously obtained for S. canicula. This raises concerns for the validity of the current threshold value used to discern between lecithotrophic and matrotrophic species. Accordingly, 26 studies published in the primary literature between 1932 and 2012 addressing the maternal-embryonic nutritional relationship in sharks were reviewed. Values for changes in mass reported for over 20 different shark species were synthesised and recalculated, revealing multiple typographical, transcribing, calculation and rounding errors across many papers. These results suggest that the current threshold value of −20% established by previous studies is invalid and should be avoided to ascertain the reproductive mode of aplacental viviparous species.     

A Gram-Negative Anaerobic Bacterium That Utilizes O-Methyl Substituents of Aromatic Acids

A novel strain of gram-negative anaerobic rods which utilized O-methyl substituents of monoaromatic acids as a sole organic source of carbon was isolated from municipal sewage sludge. Energy for growth seemed to be generated by an acetate formation pathway. The growth yield in defined medium was 7.9 g (dry weight) of cells per mol of ferulate utilized. This isolate and other O-demethylating anaerobes may play a role in the turnover of acetate and the metabolism of highly methoxylated lignaceous materials in anaerobic environments.     

Importance of tetrahydrofolate and ATP in the anaerobic O-demethylation reaction for phenylmethylethers.

DL-Tetrahydrofolate (THF) and ATP were necessary for the anaerobic O-demethylation of phenylmethylethers in cell extracts of the type strain (ATCC 29683) of the homoacetogen Acetobacterium woodii. The reactants for this enzymatic activity have not been previously demonstrated in any system, nor has the mediating enzyme been studied. An assay using reaction mixtures containing 1 mM THF, 2 mM ATP, and 2 mM hydroferulate (i.e., 4-hydroxy,3-methoxyphenylpropionate) was developed and was performed under stringent anaerobic conditions. Pyridine nucleotides and several other possible cofactors were tested but had no effect on the activity. After centrifugation of disrupted cells at 27,000 x g, the activity was found primarily in the supernatant, which had a specific activity of 14.2 +/- 0.5 nmol/min/mg of protein. At saturating levels of each of the other two substrates, apparent Km values for the variable substrate were 0.65 mM hydroferulate, 0.27 mM ATP, and 0.17 mM THF. Activity was significantly decreased when extract was preincubated at 60 degrees C and was completely lost after preincubation in air for 30 min. Thus, the soluble anaerobic O-demethylating enzyme system of A. woodii is oxygen sensitive. The THF- and ATP-dependent activity measurable in the soluble fraction of cell extracts constituted about 34% of the activity seen with intact cells.     

SDS-PAGE characterization of the proteins in equine seminal plasma

The aims of this project were to document the protein profile of equine seminal plasma and determine the variability between stallions in the relative composition of proteins in the ejaculate. A single ejaculate was obtained from 14 stallions of varying breed and age. The gel fraction was removed by an in-line filter. The semen was centrifuged and the supernatant seminal plasma aspirated without disturbing the sperm pellet. The seminal plasma was recentrifuged and stored in cryovials at -70 degrees C. Samples were thawed, recentrifuged, assayed for protein concentration (BCA protein assay), divided into aliquots, then stored at -70 degrees C. A standard protein concentration of 50 microg was loaded in each 10 microl sample. SDS-PAGE was performed using 15% polyacrylamide and a mixture of molecular weight standards. The electrophoresed gel was stained for proteins with Coomassie blue, air-dried, then scanned by a megapixel camera interfaced to a computer. Image analysis software calculated integrated optical density (IOD) values for each lane, and bands within a lane. Each band IOD was expressed as a percentage of the total lane IOD, thus reflecting the relative concentration of each protein within the ejaculate. A total of 14 bands were identified, ranging from a large 120 kDa protein down to a small 14 kDa protein. No sample contained all 14 protein bands. Seven protein bands (101 kDa, 32 kDa, 26 kDa, 22 kDa, 18 kDa, 16 kDa, 14 kDa) were present in all samples, however the relative concentrations of protein within those bands varied between stallions. We demonstrated that although there is a characteristic equine seminal plasma protein profile on SDS-PAGE gels, there is between stallion variability in the relative amounts of each protein.     

Testicular prosthesis in a Quarterhorse stallion: a case report

A testicular prosthesis was surgically removed from the scrotum of a Quarterhorse stallion presented for evaluation of a large, firm, unilateral scrotal mass. The prosthesis was constructed from methyl methacrylate moulded around a roll of fiberglass casting tape. The prosthesis had been surgically implanted in the scrotum approximately 4 yr prior to presentation in order to give the appearance of 2 testicles in the scrotum for showing and breeding purposes. The horse had been used to successfully breed mares prior to presentation and produced 4.046 x 10(9) progressively motile, morphologically normal spermatozoa in an ejaculate collected 4 mo after surgery to remove the prosthesis. Ethical issues raised by this case are discussed.     

Two-dimensional polyacrylamide gel electrophoresis of bovine semen after cryopreservation in half-milliliter straws

One of the problems encountered with two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) is the streaking of proteins so that individual spot identification is compromised. This study was conducted to determine whether a low loading dose (50 microg) of protein would permit resolution of more discrete protein spots using megapixel camera technology, and if so, to present a nomenclature for future comparisons of the identified proteins. If the major proteins could be identified in a 50-microg sample we aimed to determine whether they could be identified in the supernatant (seminal plasma plus extender) of cryopreserved semen. Two ejaculates were obtained from each of 6 bulls and bovine seminal plasma (BSP) protein concentration was standardized to 50 microg/10 microl. Isoelectric points (pI) and molecular weights (MWt) of BSP proteins were determined by measuring spot mobility on 2-D PAGE (15% polyacrylamide). Three distinct protein spot constellations (a,b,c) could be readily seen by the naked eye and a faintly stained constellation "d" was identified by the megapixel camera. The image analysis software located 6 protein spots in both constellation "a" (MWt 26 kDa; pI 4.2 to 4.8) and "b" ( MWt 27 kDa; pI 6.6 to 8.0). Constellation "c" contained 13 protein spots distributed in a right-angled triangle with its base towards the acidic end of the gel (MWt 14.7 to 18.8 kDa; pI 5.3 to 7.4). Only spots c(2), c(3), c(5), c(8), and c(13) were present in all 12 samples. Streaking can be eliminated by using 50 microg protein for 2-D PAGE, and the major protein spots are readily identified by megapixel camera technology. Protein spots c(3), c(5), c(13) and constellation "a" appear to correspond with Manjunath's proteins (BSP-A(1), -A(2); -A(3); -30 kDa). Killian's 2 low fertility proteins may lie in the "c" constellation, and 1 of the high fertility proteins may lie in the "b" constellation. The 3 major BSP proteins can be visualized in the supernatant of cryopreserved semen. We believe that the technique may prove useful for retrospective analysis of processed semen batches that achieve less than satisfactory results in the field.