Defining components of early thyroid hormone signalling through temperature-mediated activation of molecular memory in cultured Rana [lithobates] catesbeiana tadpole back skin.

Thyroid hormones (THs) are essential signalling molecules for the postembryonic development of all vertebrates. THs are necessary for the metamorphosis from tadpole to froglet and exogenous TH administration precociously induces metamorphosis. In American bullfrog (Rana [Lithobates] catesbeiana) tadpoles, the TH-induced metamorphosis observed at a warm temperature (24 °C) is arrested at a cold temperature (4 °C) even in the presence of exogenous THs. However, when TH-exposed tadpoles are shifted from cold to warm temperatures (4 â†’ 24 °C), they undergo TH-dependent metamorphosis at an accelerated rate even when the initial TH signal is no longer present. Thus, they possess a "molecular memory" of TH exposure that establishes the TH-induced response program at the cold temperature and prompts accelerated metamorphosis after a shift to a warmer temperature. The components of the molecular memory that allow the uncoupling of initiation from the execution of the metamorphic program are not understood. To investigate this, we used cultured tadpole back skin (C-Skin) in a repeated measures experiment under 24 °C only, 4 °C only, and 4 â†’ 24 °C temperature shifted regimes and reverse transcription quantitative polymerase chain reaction (RT-qPCR) and RNA-sequencing (RNA-seq) analyses. RNA-seq identified 570, 44, and 890 transcripts, respectively, that were significantly changed by TH treatment. These included transcripts encoding transcription factors and proteins involved in mRNA structure and stability. Notably, transcripts associated with molecular memory do not overlap with those identified previously in cultured tail fin (C-fin) except for TH-induced basic leucine zipper-containing protein (thibz) suggesting that thibz may have a central role in molecular memory that works with tissue-specific factors to establish TH-induced gene expression programs.

Large deletions and other gross forms of chromosome imbalance compatible with viability and fertility in the mouse.

Large deletions and other gross forms of chromosome imbalance are known in man but have rarely been found in the mouse. By screening progeny of spermatogonially irradiated male mice for a combination of runting and other phenotypic effects, we have identified animals that have large deletions comprising from 2.5-30 percent of the length of individual chromosomes, or other major chromosome changes, which are compatible with viability and fertility. Certain chromosome regions appear particularly susceptible to the generation of viable deletions and this has implications for radiation mutagenesis studies. Correlations with human deletions are also indicated.

The meiotic checkpoint monitoring synapsis eliminates spermatocytes via p53-independent apoptosis.

Evidence is accumulating that meiosis is subject to 'checkpoints' that monitor the quality of this complex process. In yeast, unresolved double strand breaks (DSBs) in DNA are thought to trigger a 'recombination checkpoint' that leads to pachytene arrest. In higher eukaryotes, there is evidence for a checkpoint that monitors chromosome synapsis and in mammals the most compelling evidence relates to the sex chromosomes. In normal male mice, there is synapsis between the X and Y pseudoautosomal regions; in XSxr(a)O mice, with a single asynaptic sex chromosome, there is arrest at the first meiotic metaphase, the arrested cells being eliminated by apoptosis (our unpublished data). Satisfying the requirement for pseudoautosomal synapsis by providing a pairing partner for the XSxr(a) chromosome avoids this arrest. We have considered that this 'synapsis checkpoint' may be a modification of the yeast 'recombination checkpoint' with unresolved DSBs (a corollary of asynapsis) providing the trigger for apoptosis. DSBs induced by irradiation are known to trigger apoptosis in a number of cell types via a p53-dependent pathway, and we now show that irradiation-induced spermatogonial apoptosis is also p53-dependent. In contrast, the apoptotic elimination of spermatocytes with synaptic errors proved to be p53-independent.

A candidate mouse model for Prader-Willi syndrome which shows an absence of Snrpn expression.

The best examples of imprinting in humans are provided by the Angelman and Prader-Willi syndromes (AS and PWS) which are associated with maternal and paternal 15q11-13 deletions, respectively, and also with paternal and maternal disomy 15. The region of the deletions has homology with a central part of mouse chromosome 7, incompletely tested for imprinting effects. Here, we report that maternal duplication for this region causes a murine imprinting effect which may correspond to PWS. Paternal duplication was not associated with any detectable effect that might correspond with AS. Gene expression studies established that Snrpn is not expressed in mice with the maternal duplication and suggest that the closely-linked Gabrb-3 locus is not subject to imprinting. Finally, an additional new imprinting effect is described.

Quantum monitoring of cellular metabolic activities in single mitochondria.

Free radicals play a vital role in all kinds of biological processes including immune responses. However, free radicals have short lifetimes and are highly reactive, making them difficult to measure using current methods. Here, we demonstrate that relaxometry measurement, or T1, inherited from the field of diamond magnetometry can be used to detect free radicals in living cells with subcellular resolution. This quantum sensing technique is based on defects in diamond, which convert a magnetic signal into an optical signal, allowing nanoscale magnetic resonance measurements. We functionalized fluorescent nanodiamonds (FNDs) to target single mitochondria within macrophage cells to detect the metabolic activity. In addition, we performed measurements on single isolated mitochondria. We were able to detect free radicals generated by individual mitochondria in either living cells or isolated mitochondria after stimulation or inhibition.

Cytological methods for the study of meiotic properties in mice.

Cytological methods now available produce excellent preparations of many of the meiotic stages in both male and female mice. Large samples of these stages can be obtained from males, limited samples from females. Some of these stages are useful for the identification of structural heterozygosity. As yet, the potentially valuable stage of pachytene can be used only for looking at specific chromosome bivalents and cannot be used for extensive genome analysis. The later stages of diakinesis and first metaphase have proved most rewarding for study and have enabled us to identify the meiotic configurations resulting from several different kinds of translocations and from paracentric inversions. It is possible, however, that many smaller rearrangements between and within chromosomes escape detection.

Narrowing the critical regions for mouse t complex transmission ratio distortion factors by use of deletions.

Previously a deletion in mouse chromosome 17, T(22H), was shown to behave like a t allele of the t complex distorter gene Tcd1, and this was attributed to deletion of this locus. Seven further deletions are studied here, with the aim of narrowing the critical region in which Tcd1 must lie. One deletion, T(30H), together with three others, T(31H), T(33H), and T(36H), which extended more proximally, caused male sterility when heterozygous with a complete t haplotype and also enhanced transmission ratio of the partial t haplotype t(6), and this was attributed to deletion of the Tcd1 locus. The deletions T(29H), T(32H), and T(34H) that extended less proximally than T(30H) permitted male fertility when opposite a complete t haplotype. These results enabled narrowing of the critical interval for Tcd1 to between the markers D17Mit164 and D17Leh48. In addition, T(29H) and T(32H) enhanced the transmission ratio of t(6), but significantly less so than T(30H). T(34H) had no effect on transmission ratio. These results could be explained by a new distorter located between the breakpoints of T(29H) and T(34H) (between T and D17Leh66E). It is suggested that the original distorter Tcd1 in fact consists of two loci: Tcd1a, lying between D17Mit164 and D17Leh48, and Tcd1b, lying between T and D17Leh66E.

Relationship of platelet retention in glass bead columns to the rate of aggregation with adenosine diphosphate and thrombin.

The initial rate of platelet aggregation induced by adenosine diphosphate and thrombin correlated with the number of platelets retained when samples of citrated blood from the same normal donors were passed through glass bead columns. The results support the view that the glass bead column method of measuring platelet ;adhesiveness' is influenced by platelet aggregation.

A clinical and experimental study of platelet function in chronic renal failure.

Coagulation and platelet function studies were performed on 24 normal subjects and 29 patients with chronic renal failure due to various causes. Thrombocytopenia was uncommon in the uraemic patients but there was reduced platelet retention in glass bead columns and platelet aggregation with adenosine diphosphate (ADP) and thrombin was slower and less complete than normal. The rate of platelet disaggregation in uraemic patients was significantly reduced. The abnormalities tended to be more severe in more uraemic subjects. In normal subjects no inter-relationships were observed between the various measurements of platelet activity. In patients there were significant interrelationships between the measurements of platelet aggregation with ADP and thrombin and between the measurements of aggregation and retention in glass bead columns. It is suggested that if a common pathway is involved in these reactions it is adversely affected in uraemia. Plasma coagulation defects were uncommon and present in only five of the uraemic subjects. Impaired prothrombin consumption apparently due to defective platelet function was present in half the patients but was not detected by a kaolin activation method. Although platelet coagulation function was activated during ADP aggregation and disaggregation in normal and uraemic subjects, it did not correlate in the latter with impairment of aggregation. It is suggested that aggregation and activation of platelet coagulant activity are not necessarily related aspects of platelet function. An effect of uraemic plasma on normal platelets was demonstrated by mixing experiments consistent with a humoral cause for the uraemic platelet defects.