Development of an immortalised, post-pubertal gonadotrophin-releasing hormone neuronal cell line.

Gonadotrophin-releasing hormone (GnRH) is important in reproduction, although some of the mechanisms for its synthesis and release remain elusive. Progress in understanding the GnRH neurone has been hampered by the limited number and diffuse distribution of the neurone in the mammalian brain. Several stable GnRH-expressing cell lines have been developed using in vivo expression of the simian virus 40 T Antigen (TAg), and they have been helpful for the study of gene expression and neuronal function. However, expression of an immortalising gene may interfere with normal cellular function. We developed a novel GnRH-secreting cell line transgenic mouse model suitable for targeted transformation in post-pubertal mice using a tetracycline-regulated TAg transgene. This clonal cell line, GRT, expresses neuronal markers and GnRH. GRT cells grown in medium containing tetracycline-free serum express increasing mRNA levels of GnRH associated with declining levels of TAg expression. The novelty and ultimately the usefulness of this cell line is that TAg expression, which could affect the GnRH neuronal phenotype, can be regulated by tetracycline.

Temporal and spatial regulation of CRE recombinase expression in gonadotrophin-releasing hormone neurones in the mouse.

Gonadotrophin-releasing hormone (GnRH) neurones located within the brain are the final neuroendocrine output regulating the reproductive hormone axis. Their small number and scattered distribution in the hypothalamus make them particularly difficult to study in vivo. The Cre/loxP system is a valuable tool to delete genes in specific cells and tissues. We report the production of two mouse lines that express the CRE bacteriophage recombinase in a GnRH-specific manner. The first line, the GnRH-CRE mouse, contains a transgene in which CRE is under the control of the murine GnRH promoter and targets CRE expression specifically to GnRH neurones in the hypothalamus. The second line, the GnRH-CRETeR mouse, uses the same murine GnRH promoter to target CRE expression to GnRH neurones, but is modified to be constitutively repressed by a tetracycline repressor (TetR) expressed from a downstream tetracycline repressor gene engineered within the transgene. GnRH neurone-specific CRE expression can therefore be induced by treatment with doxycycline which relieves repression by TetR. These GnRH-CRE and GnRH-CRETeR mice can be used to study the function of genes expressed specifically in GnRH neurones. The GnRH-CRETeR mouse can be used to study genes that may have distinct roles in reproductive physiology during the various developmental stages.

Finite difference modelling of the temperature rise in non-linear medical ultrasound fields.

Non-linear propagation of ultrasound can lead to increased heat generation in medical diagnostic imaging due to the preferential absorption of harmonics of the original frequency. A numerical model has been developed and tested that is capable of predicting the temperature rise due to a high amplitude ultrasound field. The acoustic field is modelled using a numerical solution to the Khokhlov-Zabolotskaya-Kuznetsov (KZK) equation, known as the Bergen Code, which is implemented in cylindrical symmetric form. A finite difference representation of the thermal equations is used to calculate the resulting temperature rises. The model allows for the inclusion of a number of layers of tissue with different acoustic and thermal properties and accounts for the effects of non-linear propagation, direct heating by the transducer, thermal diffusion and perfusion in different tissues. The effect of temperature-dependent skin perfusion and variation in background temperature between the skin and deeper layers of the body are included. The model has been tested against analytic solutions for simple configurations and then used to estimate temperature rises in realistic obstetric situations. A pulsed 3 MHz transducer operating with an average acoustic power of 200 mW leads to a maximum steady state temperature rise inside the foetus of 1.25 degrees C compared with a 0.6 degree C rise for the same transmitted power under linear propagation conditions. The largest temperature rise occurs at the skin surface, with the temperature rise at the foetus limited to less than 2 degrees C for the range of conditions considered.

Proton ATPases in bacteria: comparison to Escherichia coli F1F0 as the prototype.

The F1F0 ATP synthase complex of Escherichia coli functions reversibly in coupling proton translocation to ATP synthesis or hydrolysis. The structural organization and subunit composition corresponds to that seen in many other bacteria, i.e. a membrane extrinsic F1 sector with five subunits in an alpha 3 beta 3 gamma delta epsilon stoichiometry, and a membrane-traversing F0 sector with three subunits in an a1b2c12 stoichiometry. The structure of much of the F1 sector is known from a X-ray diffraction model. During function, The gamma subunit is known to rotate within a hexameric ring of alternating alpha and beta subunits to promote sequential substrate binding and product release from catalytic sites on the three beta subunits. Proton transport through F0 must be coupled to this rotation. Subunit c folds in the membrane as a hairpin to two alpha helices to generate the proton-binding site in F0. Its structure was determined by NMR, and the structure of the c oligomer was deduced by cross-linking experiments and molecular mechanics calculations. The implications of the oligomeric structure of subunit c will be considered and related to the H+/ATP pumping ratio, P/O ratios and the cation-binding site in other types of F0. The possible limits of the structure in changing the ion-binding specificity, stoichiometry and routes of proton entrance/exit to the binding site will be considered.