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1.  Gene Expression Profiles of Intracellular and Membrane Progesterone Receptor Isoforms in the Mediobasal Hypothalamus during Pro-oestrus 
Journal of neuroendocrinology  2009;21(12):993-1000.
Progesterone action is mediated by its binding to specific receptors. Two progesterone receptor (PR) isoforms (PRA and PRB), three membrane progesterone receptor (mPR) isoforms (mPRα, mPRβ and mPRγ), and at least one progesterone membrane-binding protein - PR membrane component 1 (PRmc1) have been identified in reproductive tissues and brain of various species. In this study, we examined gene expression patterns for PR isoforms, mPR isoforms and PRmc1 in the rat mediobasal hypothalamus (MBH) during pro-oestrus. The mRNA level for each receptor subtype was quantified by real-time RT-PCR at the following time points: 1300h on dioestrous day 2, 0900h, 1300h, 1700h and 2200h on pro-oestrus and 1300h on oestrus. For PR, one primer set amplified PRA+PRB, whereas a second primer set amplified PRB. As expected, PRA+PRB mRNA expression was greater than PRB in MBH tissue. PRB mRNA levels increased throughout the day on pro-oestrus with the highest levels observed at 1700h. PRB mRNA levels in the MBH were increased by 2.4- and 3.0-fold at 1300h and 1700h, respectively, on pro-oestrus as compared to 1300h on dioestrous day 2. There were differential mRNA expression levels for mPRs and PRmc1 in the MBH, with highest expression for PRmc1 and the lowest for mPRγ. The mPRα mRNA contents at 1300h and 1700h on pro-oestrus were increased by 1.5-fold as compared to that at 1300h on dioestrous day 2. The mPRβ mRNA levels at 1300h and 1700h on pro-oestrus were 2.5- and 2.4- fold higher than that at 1300h on dioestrus day 2, respectively. PRA+PRB, mPRγ and PRmc1 mRNA levels did not vary on pro-oestrus. These findings suggest that the higher expression of PRB, mPRα and mPRβ in the MBH on pro-oestrous afternoon may influence both genomic and non-genomic mechanisms of progesterone action during the critical preovulatory period.
doi:10.1111/j.1365-2826.2009.01920.x
PMCID: PMC2788049  PMID: 19807848
oestradiol; oestrous cycle; arcuate nucleus; dopamine; prolactin
2.  Analysis of partner of inscuteable (mPins) expression in the developing mouse eye 
Molecular Vision  2008;14:2575-2596.
Purpose
Asymmetric cell division (ACD) is the fundamental mechanism underlying the generation of cellular diversity in invertebrates and vertebrates. During Drosophila neuroblast division, this process involves stabilization of the apical complex and interaction between the Inscuteable (Insc) and Partner of inscuteable (Pins) proteins. Both cell-intrinsic factors and cell–cell interactions seem to contribute to cell fate decisions in the retina. The Pins protein is known to play a major role in the asymmetric segregation of cell fate determinants during development of the central nervous system in general, but its role in asymmetric cell divisions and retinoblast cell fate has never been explored. The primary aim of this study was to determine the spatial distribution and time course of mouse homolog of Drosophila Partner of Inscuteable (mPins) expression in the developing and adult mouse eye.
Methods
The expression pattern of mPins was studied in the mouse eye from embryonic (E) stage E11.5 until adulthood, by semiquantitative RT–PCR, in situ hybridization, and immunohistochemistry. In addition, variations in mRNA and protein levels for mPins were analyzed in the developing postnatal and adult lens, by semiquantitative RT–PCR, western blot analysis, in situ hybridization, and immunohistochemistry.
Results
We detected mPins mRNA at early stages of mouse embryonic eye development, particularly in the neuroblastic layer. In early postnatal development, mPins mRNA was still detected in the neuroblastic layer, but also began to be detectable in the ganglion cell layer. Thereafter, mPins mRNA was found throughout the retina. This pattern was maintained in differentiated adult retina. Immunohistochemical studies showed that mPins protein was present in the neuroblastic layer and the ganglion cell layer during the early stages of postnatal retinal development. At these stages, mPins protein was colocalized with Numb protein, a marker of the ACD. At later postnatal stages, mPins protein was present in all retinal nuclear layers and in the inner plexiform layer. It continued to be detected in these layers in the differentiated retina; the outer plexiform layer and the photoreceptor inner segments also began to display positive immunostaining for mPins. In the adult retina, mPins was also detected in the retinal pigment epithelium and choroidal melanocytes. Throughout development, mPins protein was detected in nonretinal tissues, including the cornea, ciliary body, and lens. We focused our attention on lens development and showed that mPins protein was first detected at E14.5. The most striking results obtained concerned the lens, in which mPins protein distribution switched from the anterior to the posterior region of the lens during embryonic development. Interestingly, in the postnatal and adult lens, mPins protein was detected in all lens cells and fibers.
Conclusions
We provide the first demonstration that mPins protein is expressed from embryonic stages until adulthood in the mouse eye. These results suggest that mPins plays important roles in eye development. This work provides preliminary evidence strongly supporting a role for mPins in the asymmetric division of retinoblasts, and in the structure and functions of adult mouse retina. However, the link between the presence of mPins in different ocular compartments and the possible occurrence of asymmetric cell divisions in these compartments remains to be clarified. Further studies are required to elucidate the in vitro and in vivo functions of mPins in the developing and adult human eye.
PMCID: PMC2613078  PMID: 19122831
3.  Gamma-D crystallin gene (CRYGD) mutation causes autosomal dominant congenital cerulean cataracts 
Journal of Medical Genetics  2003;40(4):262-267.
Congenital cataracts are a major cause of bilateral visual impairment in childhood. We mapped the gene responsible for autosomal congenital cerulean cataracts to chromosome 2q33–35 in a four generation family of Moroccan descent. The maximum lod score (7.19 at recombination fraction θ=0) was obtained for marker D2S2208 near the γ-crystallin gene (CRYG) cluster. Sequencing of the coding regions of the CRYGA, B, C, and D genes showed the presence of a heterozygous C>A transversion in exon 2 of CRYGD that is associated with cataracts in this family. This mutation resulted in a proline to threonine substitution at amino acid 23 of the protein in the first of the four Greek key motifs that characterise this protein. We show that although the x ray crystallography modelling does not indicate any change of the backbone conformation, the mutation affects a region of the Greek key motif that is important for determining the topology of this protein fold. Our data suggest strongly that the proline to threonine substitution may alter the protein folding or decrease the thermodynamic stability or solubility of the protein. Furthermore, this is the first report of a mutation in this gene resulting in autosomal dominant congenital cerulean cataracts.
doi:10.1136/jmg.40.4.262
PMCID: PMC1735438  PMID: 12676897
4.  Effect of inhibition of protein synthesis on lipid metabolism in Lactobacillus plantarum. 
Journal of Bacteriology  1975;123(3):962-971.
In Lactobacillus plantarum 17-5, lipid synthesis appears to be correlated with protein synthesis. Inhibition of protein synthesis by chloramphenicol (50 mug/ml) caused the nearly simultaneous inhibition of incorporation of radioactive oleic acid into polar lipids before the cessation of growth. In addition, de novo fatty acid synthesis, as determined by the incorporation of radioactive acetate into cellular lipids, was also inhibited. Removal of the antibiotic resulted in the resumption of growth, protein synthesis, and polar lipid synthesis. Inhibition of protein synthesis by leucine deprivation also produced a marked reduction in the incorporation of radioactive oleic acid into the total polar lipids at about the same time that growth stopped (30 to 60 min after the removal of leucine). However, the different classes of lipids behaved differently. For example, the incorporation of oleic acid into cardiolipin was inhibited immediately upon removal of leucine from the cultures, whereas incorporation into phosphatidyl-glycerol was maintained at near normal rates for 60 min after the removal of leucine and then ceased. In contrast, the accumulation of radioactive oleic acid in a neutral lipid identified as diglyceride occurred to a much greater extent in leucine-deprived cultures than in control (+ leucine) cultures. Upon addition of leucine to leucine-deprived cultures, the rates of synthesis of phosphatidyl-glycerol and cardiolipin returned to normal; the amount of radioactivity in the diglyceride fraction decreased to normal levels concomitantly with increased phospholipid synthesis.
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PMCID: PMC235820  PMID: 1158852
5.  Gene Expression Profiles of Intracellular and Membrane Progesterone Receptor Isoforms in the Mediobasal Hypothalamus During Pro-Oestrus 
Journal of Neuroendocrinology  2009;21(12):993-1000.
Progesterone action is mediated by its binding to specific receptors. Two progesterone receptor (PR) isoforms (PRA and PRB), three membrane progesterone receptor (mPR) subtypes (mPRα, mPRβ and mPRγ) and at least one progesterone membrane-binding protein [PR membrane component 1 (PRmc1)] have been identified in reproductive tissues and brain of various species. In the present study, we examined gene expression patterns for PR isoforms, mPR subtypes and PRmc1 in the rat mediobasal hypothalamus (MBH) during pro-oestrus. The mRNA level for each receptor subtype was quantified by a real-time reverse transcriptase-polymerase chain reaction (RT-PCR) at the time points: 13.00 h on dioestrous day 2; 09.00, 13.00, 17.00 and 22.00 h on pro-oestrus; and 13.00 h on oestrus. For PR, one primer set amplified PRA+PRB, whereas a second primer set amplified PRB. As expected, PRA+PRB mRNA expression was greater than PRB in MBH tissue. PRB mRNA levels increased throughout the day on pro-oestrus, with the highest levels being observed at 17.00 h. PRB mRNA levels in the MBH were increased by 2.4- and 3.0-fold at 13.00 and 17.00 h, respectively, on pro-oestrus compared to 13.00 h on dioestrous day 2. There were differential mRNA expression levels for mPRs and PRmc1 in the MBH, with the highest expression for PRmc1 and the lowest for mPRγ. The mPRα mRNA contents at 13.00 and 17.00 h on pro-oestrus were increased by 1.5-fold compared to that at 13.00 h on dioestrous day 2. The mPRβ mRNA levels at 13.00 and 17.00 h on pro-oestrus were 2.5- and 2.4-fold higher compared to that at 13.00 h on dioestrous day 2, respectively. PRA+PRB, mPRγ and PRmc1 mRNA levels did not vary on pro-oestrus. These findings suggest that the higher expression of PRB, mPRα and mPRβ in the MBH on pro-oestrous afternoon may influence both genomic and nongenomic mechanisms of progesterone action during the critical pre-ovulatory period.
doi:10.1111/j.1365-2826.2009.01920.x
PMCID: PMC2788049  PMID: 19807848
oestradiol; oestrous cycle; arcuate nucleus; dopamine; prolactin

Results 1-5 (5)