A physicochemical analysis of conjugation in Tetrahymena pyriformis

The process of conjugation in Tetrahymena pyriformis is a useful model system for investigating mechanisms of cellular recognition, adhesion and fusion. As a first step in the biochemical analysis of this process, we have examined the effects of (a) nutrients; (b) metal ions; (c) several pharmacological agents (actinomycin D, cycloheximide, colchicine, theophylline, dithiothreitol and caffeine); and (d) temperature. We find that:

1. 1. While the complete nutrient medium inhibits conjugation, no single compound or group of compounds of the defined medium [1]produces any inhibition.

2. 2. At least trace amounts of Ca²⁺ are required.

3. 3. All of the pharmacological agents tested, except actinomycin D, inhibit both the preparations for conjugation and pair formation itself, indicating a requirement for both protein synthesis and low intracellular levels of cAMP, as well as the involvement of microtubules.

4. 4. While actinomycin D inhibits the preparations for conjugation, its addition after cells have begun to pair does not block further pairing. This result suggests that a stable RNA which is required for conjugation is produced during the preparations for conjugation.

5. 5. Paired cells may be disrupted for the first i h after pairing by proteose peptone, cycloheximide, theophylline, and dithiothreitol. The cells undergo a transition h after pairing which renders them resistant to these agents.

6. 6. The period of initiation (the time of starvation required to make cells competent to conjugate, the period of costimulation (the lag time preceding cell pairing after competent cells are mixed), and the rate of cell pairing are all temperature sensitive. Large changes in these parameters occur over narrow temperature ranges, possibly as a result of temperature-induced changes in membrane lipid composition or structure.

     

Influence of Post-Traumatic Stress Disorder on Neuroinflammation and Cell Proliferation in a Rat Model of Traumatic Brain Injury

Long-term consequences of traumatic brain injury (TBI) are closely associated with the development of severe psychiatric disorders, such as post-traumatic stress disorder (PTSD), yet preclinical studies on pathological changes after combined TBI with PTSD are lacking. In the present in vivo study, we assessed chronic neuroinflammation, neuronal cell loss, cell proliferation and neuronal differentiation in specific brain regions of adult Sprague-Dawley male rats following controlled cortical impact model of moderate TBI with or without exposure to PTSD. Eight weeks post-TBI, stereology-based histological analyses revealed no significant differences between sham and PTSD alone treatment across all brain regions examined, whereas significant exacerbation of OX6-positive activated microglial cells in the striatum, thalamus, and cerebral peduncle, but not cerebellum, in animals that received TBI alone and combined TBI-PTSD compared with PTSD alone and sham treatment. Additional immunohistochemical results revealed a significant loss of CA3 pyramidal neurons in the hippocampus of TBI alone and TBI-PTSD compared to PTSD alone and sham treatment. Further examination of neurogenic niches revealed a significant downregulation of Ki67-positive proliferating cells, but not DCX-positive neuronally migrating cells in the neurogenic subgranular zone and subventricular zone for both TBI alone and TBI-PTSD compared to PTSD alone and sham treatment. Comparisons of levels of neuroinflammation and neurogenesis between TBI alone and TBI+PTSD revealed that PTSD did not exacerbate the neuropathological hallmarks of TBI. These results indicate a progressive deterioration of the TBI brain, which, under the conditions of the present approach, was not intensified by PTSD, at least within our time window and within the examined areas of the brain. Although the PTSD manipulation employed here did not exacerbate the pathological effects of TBI, the observed long-term inflammation and suppressed cell proliferation may evolve into more severe neurodegenerative diseases and psychiatric disorders currently being recognized in traumatized TBI patients.     

Fusion Peptides from Oncogenic Chimeric Proteins as Putative Specific Biomarkers of Cancer

Chromosomal translocations encoding chimeric fusion proteins constitute one of the most common mechanisms underlying oncogenic transformation in human cancer. Fusion peptides resulting from such oncogenic chimeric fusions, though unique to specific cancer subtypes, are unexplored as cancer biomarkers. Here we show, using an approach termed fusion peptide multiple reaction monitoring mass spectrometry, the direct identification of different cancer-specific fusion peptides arising from protein chimeras that are generated from the juxtaposition of heterologous genes fused by recurrent chromosomal translocations. Using fusion peptide multiple reaction monitoring mass spectrometry in a clinically relevant scenario, we demonstrate the specific, sensitive, and unambiguous detection of a specific diagnostic fusion peptide in clinical samples of anaplastic large cell lymphoma, but not in a diverse array of benign lymph nodes or other forms of primary malignant lymphomas and cancer-derived cell lines. Our studies highlight the utility of fusion peptides as cancer biomarkers and carry broad implications for the use of protein biomarkers in cancer detection and monitoring.A cancer biomarker is generally an analyte that indicates the presence or extent of a specific form of cancer. A useful cancer biomarker should reliably distinguish between benign and malignant states and, ideally, distinguish one form of cancer from other, related differential diagnoses. Many human cancers contain recurrent chromosomal translocations and chimeric gene fusions that could be exploited as cancer-specific biomarkers (1, 2). Indeed, several structural aberrations are specific and pathognomonic for distinct types of cancer (3). Moreover, as new molecular therapies increasingly target oncogenic fusion proteins, the detection and quantitation of these proteins may also provide important, direct therapeutic guidance (4–6). Although genomic techniques targeting fusion partner genes are routinely used for diagnosing cancers, fusion peptides resulting from oncogenic chimeric fusions are unexplored as biomarker candidates for cancer detection. The specificity and qualitative/binary nature (i.e. present or absent) of fusion proteins in specific tumor types make these analytes attractive candidates for cancer detection.Advances in mass spectrometry permit the direct and unbiased interrogation of proteins and peptides in complex mixtures with unambiguous identification of specific proteins (7, 8). Multiple reaction monitoring (MRM)¹ via mass spectrometry is a powerful approach for the targeted detection of biomarker candidates in a complex background (9). MRM involves the focused interrogation of specific m/z windows for the precursor analyte, as well as selected fragment ions, following MS/MS analysis. By focusing only on specific m/z windows, one increases the sensitivity of detection dramatically, and within the context of a complex mixture there is the potential for a reproducible dynamic range spanning ≥4 orders of magnitude (10, 11).Despite their enormous potential as biomarkers, fusion peptides resulting from oncogenic chimeric fusions have not been exploited for the specific and sensitive detection of cancer. Here we demonstrate the detection of unique fusion peptides that are specific for various forms of cancer. To demonstrate applicability in a clinically relevant scenario, we show the utility of our MRM-based MS approach combined with an innovative double stable isotope strategy for the identification of nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) fusion peptide arising from the corresponding chimeric fusion protein for the identification of NPM-ALK-positive anaplastic large cell lymphoma (ALCL). We show the exquisite specificity and sensitivity of this fusion peptide (FP) MRM approach and the extraordinary accuracy of its application with clinical biopsy material.     

TCR-dependent and -independent activation underlie liver-specific regulation of NKT cells

The fate of invariant NKT (iNKT) cells following activation remains controversial and unclear. We systemically examined how iNKT cells are regulated following TCR-dependent and -independent activation with α-galactosylceramide (αGC) or IL-18 plus IL-12, respectively. Our studies reveal activation by αGC or IL-18 plus IL-12 induced transient depletion of iNKT cells exclusively in the liver that was independent of caspase 3-mediated apoptosis. The loss of iNKT cells was followed by repopulation and expansion of phenotypically distinct cells via different mechanisms. Liver iNKT cell expansion following αGC, but not IL-18 plus IL-12, treatment required an intact spleen and IFN-γ. Additionally, IL-18 plus IL-12 induced a more prolonged expansion of liver iNKT cells compared with αGC. iNKT cells that repopulate the liver following αGC had higher levels of suppressive receptors PD-1 and Lag3, whereas those that repopulate the liver following IL-18 plus IL-12 had increased levels of TCR and ICOS. In contrast to acute treatment that caused a transient loss of iNKT cells, chronic αGC or IL-18 plus IL-12 treatment caused long-term systemic loss requiring an intact thymus for repopulation of the liver. This report reveals a previously undefined role for the liver in the depletion of activated iNKT cells. Additionally, TCR-dependent and -independent activation differentially regulate iNKT cell distribution and phenotype. These results provide new insights for understanding how iNKT cells are systemically regulated following activation.     

Effects of chronic nicotine on heteromeric neuronal nicotinic receptors in rat primary cultured neurons

Nicotine increases the number of neuronal nicotinic acetylcholine receptors (nAChRs) in brain. This study investigated the effects of chronic nicotine treatment on nAChRs expressed in primary cultured neurons. In particular, we studied the chronic effects of nicotine exposure on the total density, surface expression and turnover rate of heteromeric nAChRs. The receptor density was measured by [12?I]epibatidine ([12?I]EB) binding. Untreated and nicotine-treated neurons were compared from several regions of embryonic (E19) rat brain. Twelve days of treatment with 10 μM nicotine produced a twofold up-regulation of nAChRs. Biotinylation and whole-cell binding studies indicated that up-regulation resulted from an increase in the number of cell surface receptors as well as intracellular receptors. nAChR subunit composition in cortical and hippocampal neurons was assessed by immunoprecipitation with subunit-selective antibodies. These neurons contain predominantly α4, β2 and α5 subunits, but α2, α3, α6 and β4 subunits were also detected. Chronic nicotine exposure yielded a twofold increase in the β2-containing receptors and a smaller up-regulation in the α4-containing nAChRs. To explore the mechanisms of up-regulation we investigated the effects of nicotine on the receptor turnover rate. We found that the turnover rate of surface receptors was > 2 weeks and chronic nicotine exposure had no effect on this rate.     

Pasteurellaceae from Colorado bighorn sheep herds

We compared phenotypic and genotypic characterizations of 88 Pasteurellaceae isolates from bighorn sheep (Ovis canadensis) in the course of exploring epizootiologic relationships. Based on our observations, adopting contemporary taxonomic conventions and using molecular methods to detect and compare Pasteurellaceae may help improve understanding of bighorn respiratory disease epizootiology and management.     

Chronic sazetidine‐A maintains anxiolytic effects and slower weight gain following chronic nicotine without maintaining increased density of nicotinic receptors in rodent brain

Chronic nicotine administration increases the density of brain α4β2* nicotinic acetylcholine receptors (nAChRs), which may contribute to nicotine addiction by exacerbating withdrawal symptoms associated with smoking cessation. Varenicline, a smoking cessation drug, also increases these receptors in rodent brain. The maintenance of this increase by varenicline as well as nicotine replacement may contribute to the high rate of relapse during the first year after smoking cessation. Recently, we found that sazetidine‐A (saz‐A), a potent partial agonist that desensitizes α4β2* nAChRs, does not increase the density of these receptors in brain at doses that decrease nicotine self‐administration, increase attention in rats, and produce anxiolytic effects in mice. Here, we investigated whether chronic saz‐A and varenicline maintain the density of nAChRs after their up‐regulation by nicotine. In addition, we examined the effects of these drugs on a measure of anxiety in mice and weight gain in rats. After increasing nAChRs in the rodent brain with chronic nicotine, replacing nicotine with chronic varenicline maintained the increased nAChR binding, as well as the α4β2 subunit proteins measured by western blots. In contrast, replacing nicotine treatments with chronic saz‐A resulted in the return of the density of nAChRs to the levels seen in saline controls. Nicotine, saz‐A and varenicline each demonstrated anxiolytic effects in mice, but only saz‐A and nicotine attenuated the gain of weight over a 6‐week period in rats. These findings suggest that apart from its modest anxiolytic and weight control effects, saz‐A, or drugs like it, may be useful in achieving long‐term abstinence from smoking.

     

Improved sperm cryosurvival in diluents containing amides versus glycerol in the Przewalski’s horse (Equus ferus przewalskii)

Two studies were conducted to understand sperm cryosensitivity in an endangered equid, the Przewalski’s horse (Equus ferus przewalski), while testing the cryoprotectant ability of formamides. The first assessed the toxicity of permeating cryoprotectants (glycerol, methylformamide [MF] and dimethylformamide [DMF]) to Przewalski’s horse spermatozoa during liquid storage at 4 °C. The second examined the comparative influence of three diluents (with or without formamides) on cryosurvival of sperm from the Przewalski’s versus domestic horse. When Przewalski’s horse spermatozoa were incubated at 4 °C in INRA 96 with differing concentrations of glycerol, MF or DMF or a combination of these amides, cells tolerated all but the highest concentration (10% v/v) of MF alone or in combination with DMF, both of which decreased (P < 0.05) motility traits. There was no effect of cryoprotectants on sperm acrosomal integrity. In the cryosurvival study, average sperm motility and proportion of cells with intact acrosomes in fresh ejaculates were similar (P > 0.05) between the Przewalski’s (67%, 84%, respectively) and domestic (66%, 76%) horse donors. Sperm from both species were diluted in lactose–EDTA–glycerol (EQ), Botu-Crio (BOTU; a proprietary product containing glycerol and MF) or SM (INRA 96 plus 2% [v/v] egg yolk and 2.5% [v/v] MF and DMF) and then frozen over liquid nitrogen vapor. After thawing, the highest values recovered for total and progressive sperm motility, acrosomal integrity and mitochondrial membrane potential were 42.4%, 21.8%, 88.7% and 25.4 CN (CN = mean JC-1 fluorescence intensity/cell on a channel number scale), respectively, in the Przewalski’s and 49.3%, 24.6%, 88.9% and 25.8 CN, respectively, in the domestic horse. Although sperm progressive motility and acrosome integrity did not differ (P > 0.05) among treatments across species, mitochondrial membrane potential was higher (P < 0.05) in both species using EQ compared to BOTU or SM media. Additionally, Przewalski’s stallion sperm expressed higher (P < 0.05) post-thaw total motility in BOTU and SM compared to EQ, whereas there were no differences among freezing diluents in the domestic horse. In summary, Przewalski’s stallion sperm benefit from exposure to either MF or DMF as an alternative cryoprotectant to glycerol. Overt sperm quality appears similar between the Przewalski’s and domestic horse, although the total motility of cells from the former appears more sensitive to certain freezing diluents. Nonetheless, post-thaw motility and acrosomal integrity values for Przewalski’s horse spermatozoa mimic findings in the domestic horse in the presence of INRA 96 supplemented with 2% (v/v) egg yolk and a combined 2.5% concentration of MF and DMF.