A STUDY OF GLUCO-CORTICOSTEROID-INDUCED PYKNOSIS IN THE THYMUS AND LYMPH NODE OF THE ADRENALECTOMIZED RAT

Pyknotic nuclei, observed in the thymus of steroid-treated rats, are dense, homogeneous, intensely basophilic and Feulgen positive. Under the electron microscope, the image is that of a complete segregation of the chromatin from the nuclear sap producing a margin or crescent of condensed chromatin. Approximately 30% of all small thymocytes appeared to undergo this type of degeneration within 3–4 hr after administration of the synthetic corticosteroid, dexamethasone. At this time, pyknotic thymocytes were observed in clusters, probably as a result of the activity of dense reticular cells and macrophages. Topographical and experimental data suggest the existence of a select population of steroid-sensitive thymic cells. Furthermore, on the basis of thymidine-³H incorporation studies, it appears that the steroid-sensitive population of thymocytes does not correspond to "aged" cells. In addition, many plasma cells became pyknotic after the same steroid treatment, indicating an unexpected similarity between their nuclei and those of lymphocytes. Finally, steroid failed to induce pyknosis of thymocytes in a variety of in vitro experiments, suggesting that the in vivo effect of steroid is of an indirect nature. The results are discussed in terms of (a) the nature of the nuclear changes characterizing pyknosis, (b) the hypothetical mechanism whereby steroids trigger such changes, and (c) the population of cells susceptible to steroid-induced pyknosis.     

Involvement of poly(ADP-ribose) polymerase in base excision repair

Poly(ADP-ribose) polymerase (PARP) is a zinc-finger DNA binding protein that detects and signals DNA strand breaks generated directly or indirectly by genotoxic agents. In response to these lesions, the immediate poly(ADP-ribosylation) of nuclear proteins converts DNA interruptions into intracellular signals that activate DNA repair or cell death programs. To elucidate the biological function of PARP in vivo, the mouse PARP gene was inactivated by homologous recombination to generate mice lacking a functional PARP gene. PARP knockout mice and the derived mouse embryonic fibroblasts (MEFs) were acutely sensitive to monofunctional alkylating agents and gamma-irradiation demonstrating that PARP is involved in recovery from DNA damage that triggers the base excision repair (BER) process. To address the issue of the role of PARP in BER, the ability of PARP-deficient mammalian cell extracts to repair a single abasic site present on a circular duplex plasmid molecule was tested in a standard in vitro repair assay. The results clearly demonstrate, for the first time, the involvement of PARP in the DNA synthesis step of the base excision repair process.     

The link between the PDL1 costimulatory pathway and Th17 in fetomaternal tolerance

Fetomaternal tolerance has been shown to depend both on regulatory T cells (Tregs) and negative signals from the PD1-PDL1 costimulatory pathway. More recently, IL-17-producing T cells (Th17) have been recognized as a barrier in inducing tolerance in transplantation. In this study, we investigate the mechanisms of PDL1-mediated regulation of fetomaternal tolerance using an alloantigen-specific CD4(+) TCR transgenic mouse model system (ABM-tg mouse). PDL1 blockade led to an increase in embryo resorption and a reduction in litter size. This was associated with a decrease in Tregs, leading to a lower Treg/effector T cell ratio. Moreover, PDL1 blockade inhibited Ag-specific alloreactive T cell apoptosis and induced apoptosis of Tregs and a shift toward higher frequency of Th17 cells, breaking fetomaternal tolerance. These Th17 cells arose predominantly from CD4(+)Foxp3(-) cells, rather than from conversion of Tregs. Locally in the placenta, similar decrease in regulatory and apoptotic markers was observed by real-time PCR. Neutralization of IL-17 abrogated the anti-PDL1 effect on fetal survival rate and restored Treg numbers. Finally, the adoptive transfer of Tregs was also able to improve fetal survival in the setting of PDL1 blockade. This is to our knowledge the first report using an alloantigen-specific model that establishes a link between PDL1, Th17 cells, and fetomaternal tolerance.     

The changing incidence of human papillomavirus-associated oropharyngeal cancer using multiple imputation from 2000 to 2010 at a Comprehensive Cancer Centre

Introduction Human papillomavirus (HPV) is a risk and prognostic factor for oropharyngeal cancer (OPC). Determining whether the incidence of HPV-associated OPC is rising informs health policy. Methods HPV status was ascribed using p16 immunohistochemistry in 683/1474 OPC patients identified from the Princess Margaret Hospital's Cancer Registry (from 2000 to 2010). Missing p16 data was estimated using multiple (n = 100) imputation (MI) and validated using an independent OPC cohort (n = 214). Non-OPC head and neck squamous cell carcinoma (HNSCC) (n = 3262) were also used for time-trend comparison. Regression was used to compare HNSCC subsets and time-trends. The c-index was used to measure the predictive ability of MI. Results The incidence of OPC rose from 23.3% of all HNSCC in 2000 to 31.2% in 2010 (p = 0.002). In the subset of OPC tested for p16, there was no change in p16 positivity over time (p = 0.9). However, p16 testing became more frequent over time (p < 0.0001), but was nonetheless biased, favouring never-smokers [OR 1.87 (95% CI 1.29–2.70)] and tumors of the tonsil [OR 2.30 (1.52–3.47)] or base-of-tongue [OR 1.72 (1.10–2.70)]. These same factors were also associated with p16-positivity [ORs 3.22 (1.27–8.16), 7.26 (3.50–15.1), 5.83 (2.70–12.7), respectively]. Following MI and normalization, the proportion of OPC that was p16-associated rose from 39.8% in 2000 to 65.0% in 2010, p = 0.002, fully explaining the rise in OPC in our patient population. Conclusion The rise in HNSCC referrals seen from 2000 to 2010 at our institution was driven primarily by p16-associated OPC. MI was necessary to derive reliable conclusions when cases with missing data are considerable.     

Technical considerations for assessing alterations in skeletal muscle sarcoplasmic reticulum Ca++-sequestration functionin vitro

A multiple measurement system for assessing sarcoplasmic reticulum (SR) Ca⁺⁺-ATPase activity and Ca⁺⁺-uptake was used to examine the effects of SR fractionation and quick freezing on rat white (WG) and red (RG) gastrocnemius muscle.In vitro measurements were performed on whole muscle homogenates (HOM) and crude microsomal fractions (CM) enriched in SR vesicles before and after quick freezing in liquid nitrogen. Isolation of the CM fraction resulted in protein yields of 0.96±0.1 and 0.99±0.1 mg/g in WG and RG, respectively. The percent Ca⁺⁺-ATPase recovery for CM compared to HOM was 14.5% (WG) and 10.1% (RG). SR Ca⁺⁺-activated Ca⁺⁺-ATPase activity was not affected by quick freezing of HOM or CM, but basal ATPase was reduced (P<0.05) in frozen HOM (5.12±0.18–3.98±0.20 mole/g tissue/min in WG and from 5.39±0.20–4.48±0.24 mole/g tissue/min in RG). Ca⁺⁺-uptake was measured at a range of physiological free [Ca⁺⁺] using the Ca⁺⁺ fluorescent dye Indo-1. Maximum Ca⁺⁺-uptake rates when corrected for initial [Ca⁺⁺]_f were not altered in HOM or CM by quick freezing but uptake between 300 and 400nM free Ca⁺⁺ was reduced (P<0.05) in quick frozen HOM (1.30±0.1–0.66±0.1 mole/g tissue/min in WG and 1.04±0.2–0.60±0.1 mole/g tissue/min in RG). Linear correlations between Ca⁺⁺-uptake and Ca⁺⁺-ATPase activity measured in the presence of the Ca⁺⁺ ionophore A23187 were r=+0.25, (P<0.05) and r=+0.74 (P<0.05) in HOM and CM preparations, respectively, and were not altered by freezing. The linear relationships between HOM and CM maximum Ca⁺⁺-uptake (r=+0.44, P<0.05) and between HOM and CM Ca⁺⁺-ATPase activity (r=+0.34, P<0.05) were also not altered by tissue freezing. These data suggest that alterations in maximal SR Ca⁺⁺-uptake function and maximal Ca⁺⁺-ATPase activity may be measured in both HOM and CM fractions following freezing and short term storage. (Mol Cell Biochem139, 41–52, 1994)     

Solution 1H NMR investigation of the seating and rotational "hopping" of centrosymmetric etioheme-I in myoglobin: effect of globin origin and its oxidation/spin state on heme dynamics

Solution 1H NMR spectroscopy was used to investigate the heme active-site structure and dynamics of rotation about the Fe-His bond of centrosymmetric etioheme-I reconstituted into sperm whale and horse myoglobin (Mb). Comparison of the NOESY cross-peak pattern and paramagnetic relaxation properties of the cyanomet complexes confirm a heme pocket that is essentially the same as Mb with either native protoheme or etioheme-I. Dipolar contacts between etioheme and the conserved heme pocket residues establish a unique seating of etioheme that conserves the orientation of the N-Fe-N vector relative to the axial His plane, with ethyl groups occupying the vinyl positions of protoheme. Saturation transfer between methyls on adjacent pyrroles in etioheme-reconstituted horse Mb in all accessible oxidation/spin states reveals rotational hopping rates that decrease dramatically with either loss of ligands or reduction of the heme, and correlate qualitatively with expectations based on the Fe-His bond strength and the rate of heme dissociation from Mb. The rate of hopping for etioheme in metMbCN, in contrast to hemes with propionates, is the same in the sperm whale and horse proteins.     

Genome Sequence of Legionella tunisiensis Strain LegMT,a New Legionella Species Isolated from Hypersaline Lake Water

Legionella tunisiensis is a gammaproteobacterium from the class Legionellaceae, growing in amoebae. We sequenced the genome from strain LegM^T. It is composed of 3,508,121 bp and contains 4,747 protein-coding genes and 38 RNA genes, including 3 rRNA genes.     

Habitat and landscape effects on abundance of Missouri's grassland birds

Of 6 million ha of prairie that once covered northern and western Missouri, <36,500 ha remain, with planted, managed, and restored grasslands comprising most contemporary grasslands. Most grasslands are used as pasture or hayfields. Native grasses largely have been replaced by fescue (Festuca spp.) on most private lands (almost 7 million ha). Previously cropped fields set aside under the Conservation Reserve Program (CRP) varied from a mix of cool-season grasses and forbs, or mix of native warm-season grasses and forbs, to simple tall-grass monocultures. We used generalized linear mixed models and distance sampling to assess abundance of 8 species of breeding grassland birds on 6 grassland types commonly associated with farm practices in Missouri and located in landscapes managed for grassland-bird conservation. We selected Bird Conservation Areas (BCAs) for their high percentage of grasslands and grassland-bird species, and for <5% forest cover. We used an information-theoretic approach to assess the relationship between bird abundance and 6 grassland types, 3 measures of vegetative structure, and 2 landscape variables (% grassland and edge density within a 1-km radius). We found support for all 3 levels of model parameters, although there was less support for landscape than vegetation structure effects likely because we studied high-percentage-grassland landscapes (BCAs). Henslow's sparrow (Ammodramus henslowii) counts increased with greater percentage of grassland, vegetation height-density, litter depth, and shrub cover and lower edge density. Henslow's sparrow counts were greatest in hayed native prairie. Dickcissel (Spiza americana) counts increased with greater vegetation height-density and were greatest in planted CRP grasslands. Grasshopper sparrow (A. savannarum) counts increased with lower vegetation height, litter depth, and shrub cover. Based on distance modeling, breeding densities of Henslow's sparrow, dickcissel, and grasshopper sparrow in the 6 grassland types ranged 0.9–2.6, 1.4–3.2, and 0.1–1.5 birds/ha, respectively. We suggest different grassland types and structures (vegetation height, litter depth, shrub cover) are needed to support priority grassland-bird species in Missouri. © 2011 The Wildlife Society.     

Ganglioside-induced differentiation associated protein 1 is a regulator of the mitochondrial network: new implications for Charcot-Marie-Tooth disease

Mutations in GDAP1 lead to severe forms of the peripheral motor and sensory neuropathy, Charcot-Marie-Tooth disease (CMT), which is characterized by heterogeneous phenotypes, including pronounced axonal damage and demyelination. We show that neurons and Schwann cells express ganglioside-induced differentiation associated protein 1 (GDAP1), which suggest that both cell types may contribute to the mixed features of the disease. GDAP1 is located in the mitochondrial outer membrane and regulates the mitochondrial network. Overexpression of GDAP1 induces fragmentation of mitochondria without inducing apoptosis, affecting overall mitochondrial activity, or interfering with mitochondrial fusion. The mitochondrial fusion proteins, mitofusin 1 and 2 and Drp1(K38A), can counterbalance the GDAP1-dependent fission. GDAP1-specific knockdown by RNA interference results in a tubular mitochondrial morphology. GDAP1 truncations that are found in patients who have CMT are not targeted to mitochondria and have lost mitochondrial fragmentation activity. The latter activity also is reduced strongly for disease-associated GDAP1 point mutations. Our data indicate that an exquisitely tight control of mitochondrial dynamics, regulated by GDAP1, is crucial for the proper function of myelinated peripheral nerves.