Experimental evidence for the enhanced and reduced stopping regimes for protons propagating through hot plasmas.

Our understanding of the dynamics of ion collisional energy loss in a plasma is still not complete, in part due to the difficulty and lack of high-quality experimental measurements. These measurements are crucial to benchmark existing models. Here, we show that such a measurement is possible using high-flux proton beams accelerated by high intensity short pulse lasers, where there is a high number of particles in a picosecond pulse, which is ideal for measurements in quickly expanding plasmas. By reducing the energy bandwidth of the protons using a passive selector, we have made proton stopping measurements in partially ionized Argon and fully ionized Hydrogen plasmas with electron temperatures of hundreds of eV and densities in the range 1020-1021 cm-3. In the first case, we have observed, consistently with previous reports, enhanced stopping of protons when compared to stopping power in non-ionized gas. In the second case, we have observed for the first time the regime of reduced stopping, which is theoretically predicted in such hot and fully ionized plasma. The versatility of these tunable short-pulse laser based ion sources, where the ion type and energy can be changed at will, could open up the possibility for a variety of ion stopping power measurements in plasmas so long as they are well characterized in terms of temperature and density. In turn, these measurements will allow tests of the validity of existing theoretical models.

A marked paucity of granule cells in the developing cerebellum of the Npc1(-/-) mouse is corrected by a single injection of hydroxypropyl-ß-cyclodextrin.

In this study we show that postnatal development of cerebellar granule neurons (GNs) is defective in Npc1(-/-) mice. Compared to age-matched wild-type littermates, there is an accelerated disappearance of the external granule layer (EGL) in these mice. This is due to a premature exit from the cell cycle of GN precursors residing at the level of the EGL. As a consequence, the size of cerebellar lobules of these mice displays a 20%-25% reduction compared to that of age-matched wild-type mice. This size reduction is detectable at post-natal day 28 (PN28), when cerebellar GN development is completed while signs of neuronal atrophy are not yet apparent. Based on the analysis of EGL thickness and the determination of proliferating GN fractions at increasing developmental times (PN8-PN14), we trace the onset of this GN developmental defect during the second postnatal week. We also show that during this developmental time Shh transcripts undergo a significant reduction in Npc1(-/-) mice compared to age-matched wild-type mice. In light of the mitogenic activity of Shh on GNs, this observation further supports the presence of defective GN proliferation in Npc1(-/-) mice. A single injection of hydroxypropyl-ß-cyclodextrin at PN7 rescues this defect, restoring the normal patterns of granule neuron proliferation and cerebellar lobule size. To our knowledge, these findings identify a novel developmental defect that was underappreciated in previous studies. This defect was probably overlooked because Npc1 loss-of-function does not affect cerebellar foliation and causes the internal granule layer and molecular layer to decrease proportionally, giving rise to a normally appearing, yet harmoniously smaller, cerebellum.

Monochromatic short pulse laser produced ion beam using a compact passive magnetic device.

High-intensity laser accelerated protons and ions are emerging sources with complementary characteristics to those of conventional sources, namely high charge, high current, and short bunch duration, and therefore can be useful for dedicated applications. However, these beams exhibit a broadband energy spectrum when, for some experiments, monoenergetic beams are required. We present here an adaptation of conventional chicane devices in a compact form (10 cm × 20 cm) which enables selection of a specific energy interval from the broadband spectrum. This is achieved by employing magnetic fields to bend the trajectory of the laser produced proton beam through two slits in order to select the minimum and maximum beam energy. The device enables a production of a high current, short duration source with a reproducible output spectrum from short pulse laser produced charged particle beams.

Reduced transfusion requirements with a closed cardiopulmonary bypass system.

AIM: The aim of this investigation is to reduce blood transfusion in cardiac surgery patients with preoperative conditions predictive for transfusion requirements. We compared the amount of blood transfused in two groups of patients undergoing cardiopulmonary bypass (CPB) with two different circuit systems. METHODS: Sixty patients undergoing cardiac surgery were randomly assigned to two groups: in group A (N=30) cardiopulmonary bypass was accomplished with an open circuit and in group B (N=30) with a closed circuit. The open circuit consisted of a cardiotomy reservoir, a membrane oxygenator and an arterial line filter, while the closed circuit was made up of a collapsible venous reservoir, a membrane oxygenator, an arterial line filter and a cardiotomy reservoir. The amount of transfused packed red cells in each patient was measured until discharge from the hospital. RESULTS: Groups were similar regarding age, gender, body surface area (BSA), New York Heart Association (NYHA) class and comorbidity risk factors. Moreover, there were no significant differences between groups regarding the type of procedures, CPB and aortic cross-clamp times, total amount of cardioplegia and urinary output during CPB. Priming volume was 1180+/-84 mL (group A) and 760+/-72 mL (group B) (P<0.001). Significant differences in transfusion requirements emerged in the two groups: the total volume of packed red cells transfused for each patient was significantly higher in the open system group compared to the closed system group (717+/-486 mL versus 378+/-364 mL) (P=0.003). Clinical outcomes were similar in both groups. CONCLUSION: In patients with preoperative conditions predictive for the need of transfusions, the use of a closed cardiopulmonary bypass circuit can diminish the amount of transfused blood products.

Growing dictyate oocytes, but not early preimplantation embryos, of the mouse display high levels of DNA homologous recombination by single-strand annealing and lack DNA nonhomologous end joining.

We have investigated the ability of growing dictyate oocytes and early preimplantation embryos of the mouse to process extrachromosomal DNA molecules with free ends by intranuclearly microinjecting DNA fragments containing a region of homology of various extent at either the 5' or 3' terminus. Homologous recombination of these fragments by single-strand annealing (SSA), but not other DNA recombination/joining mechanisms, resulted in the formation of a full-length hsp-lacZ-pA fusion gene that was transcriptionally activated by heat shock in growing oocytes and spontaneously at the early two-cell stage in the embryos, making it possible to quantitatively evaluate SSA activities of these cells by the beta-galactosidase produced. SSA activities of oocytes and embryos were similar in their general properties and in the activity levels observed with saturating amounts of DNA. However, embryo SSA was almost one order of magnitude less effective than that of oocytes. Oocyte and embryo 5' --> 3' exonuclease (a key function of the SSA pathway) and DNA nonhomologous end joining (NHEJ) activities were also investigated using an asymmetric PCR assay. Results showed that NHEJ is lacking in oocytes and is very prominent in the embryos, where it competes with SSA for the injected DNA.

A developmentally regulated GAGA box-binding factor and Sp1 are required for transcription of the hsp70.1 gene at the onset of mouse zygotic genome activation.

We have investigated the onset of zygotic genome transcription in early two-cell mouse embryos by analyzing the regulation of hsp70.1, one of the first genes expressed after fertilization. The transcriptional activation of both an episomic hsp70 promoter and the endogenous hsp70.1 gene requires the contiguity of the GC box proximal to the TATA box with a GAGA box and involves GC box- and GAGA box-binding factors. In vivo transcription factor titrations with double-stranded oligodeoxyribonucleotides and antibodies pinpoint these factors as Sp1 and a novel murine GAGA box-binding factor, which is structurally related to the Drosophila GAGA factor and acts as transcriptional coactivator/potentiator of Sp1. Mouse unfertilized eggs and one-cell and two-cell embryos display a GAGA box-binding activity of maternal origin that disappears at the four-cell stage and is also abundant in the gonads, but is barely detectable in other adult tissues. In light of the well-established nucleosome-disruption role of the Drosophila GAGA factor, these findings suggest a novel mechanism of enhancer-independent gene derepression in early mouse embryos.

Quantitative RT-PCR amplification of RNA in single mouse oocytes and preimplantation embryos.

We describe a simple whole-cell method for quantitative reverse transcription (RT) PCR amplification of RNA that consistently allows the analysis of trace amounts of RNA, such as those carried by a fraction of a single mouse oocyte or preimplantation embryo, without organic extraction. The method is based on a preliminary genomic DNA digestion by DNase I in the presence of Mn++ and a subsequent RT step with rTth Reverse Transcriptase at 70 degrees C with the same buffer components, which also has the effect to irreversibly denature DNase I activity. Because of the completeness of genomic DNA digestion and RNA recovery, this procedure makes it possible to quantitatively amplify any target RNA, including those coded by intronless genes or genes whose intron-exon boundaries are unknown. By taking mRNAs of beta-actin, heat-shock protein HSP70.1 and ribosomal protein S16 as experimental models, we demonstrate the effectiveness of genomic DNA digestion by DNase I-Mn++ and of DNase I heat-denaturation and the quantitative properties of our method. We also show that this procedure is useful for transcriptional analyses during development that are hindered by paucity of biological material.

Parthenogenetic activation of mouse eggs by microinjection of a truncated c-kit tyrosine kinase present in spermatozoa.

A truncated form of the c-kit tyrosine kinase receptor, corresponding to the phosphotransferase portion of the cytoplasmic catalytic domain and the carboxyterminus (tr-kit), is accumulated during late mouse spermiogenesis. Here we report that tr-kit is specifically localized in the residual sperm cytoplasm, with maximal accumulation in the midpiece of the flagellum, suggesting that it can enter the egg during fertilization. Microinjection of extracts from COS cells expressing a recombinant tr-kit protein into metaphase II-arrested mouse oocytes caused complete oocyte activation, including cortical granule exocytosis, completion of the 2nd meiotic division, formation of a parthenogenetic pronucleus and progression through cleavage stages. No activation above background levels was obtained with extracts from mock-transfected COS cells. Similar results were obtained by microinjection of in vitro synthesized tr-kit mRNA into metaphase II-arrested oocytes. Tr-kit-induced parthenogenetic egg activation was completely inhibited by oocyte preincubation with the Ca2(+)-chelating agent BAPTA-AM or with a specific inhibitor of phospholipase C activity. Tr-kit-induced egg activation was associated with a decrease in activity of mitogen-activated protein kinase, an essential component of the cytostatic factor. These results candidate tr-kit as a putative sperm factor required for triggering activation of mouse eggs at fertilization.

Developmental activation of an episomic hsp70 gene promoter in two-cell mouse embryos by transcription factor Sp1.

To investigate the control of zygotic genome expression in two-cell mouse embryos, we studied transcription factors required for transient expression of microinjected DNA constructs driven by the promoter of one of the earliest genes activated after fertilization in this system, the heat shock gene hsp70. Cis-acting elements required for hsp70 activation were first investigated by mutational analysis. Mutation of the TATA box and a proximal GC box strongly inhibited construct expression, while that of a CCAAT box had no effect. Transcription factors binding the wild-type hsp70 promoter were then titrated in vivo by coinjecting the construct with double-stranded oligodeoxyribonucleotides containing definite consensus sequences. Wild-type GC box oligonucleotides strongly inhibited construct expression, while those containing mutated GC boxes, wild-type CCAAT boxes, and heat shock elements had no effects. Finally, construct expression was challenged by coinjecting antibodies to specific transcription factors. Antibodies to factor Sp1 depressed construct expression in a dose-dependent manner, while those to Sp2, HSF1 and HSF2 were ineffective. These results pinpoint the Sp1 transcription factor as an absolute requirement for activation of the hsp70 gene promoter in two-cell mouse embryos, and make this factor a candidate for a major regulator of the onset of murine zygotic genome expression.

Stage-specific regulation of murine Hsp68 gene promoter in preimplantation mouse embryos.

In early mouse embryos, the major inducible heat shock gene, hsp68, is spontaneously and transiently activated at the two-cell stage and becomes heat-inducible around blastocyst stage. We have probed mouse embryo's ability to activate the promoter of this gene during preimplantation development by expression analysis of DNA constructs containing a reporter lacZ gene driven by hsp68 (hsp70A1) 5'-regulatory sequences of various length: (i) a full-length promoter (construct phsplacZ); (ii) a heat shock element (HSE)-deleted promoter (p delta 1hsplacZ); and (iii) a minimal, proximal promoter (p delta 2hsplac Z). When analyzed in transfected L-cells, phsplacZ was heat-inducible, while neither p delta 1hsplacZ nor p delta 2hsplacZ was. Developmental activity of the full-length construct was first analyzed after genome integration in transgenic embryos and found to follow endogenous hsp68 expression in terms of spontaneous activation at the 2-cell stage, down-regulation at the 4-cell stage, and acquisition of heat inducibility at the 16/32-cell stage. In transient expression experiments, injected phsplacZ, p delta 1hsplacZ, and p delta 2hsplacZ were expressed at similar levels by 2-cell embryos, independently of construct topology and injection stage. At the 4-cell stage, however, phsplacZ and p delta 1hsplacZ were expressed at similar levels, while p delta 2hsplacZ was inactive. Only phsplacZ became heat-inducible in late morulas. We conclude that in early mouse embryos, developmental activity of episomic hsp68 promoter depends on proximal sequences at the 2-cell stage and on putative enhancer sequences at the 4-cell stage, while HSEs appear dispensable during early cleavage.

Effects of water deprivation on renal hydroelectrolytic excretion in chronically Trypanosoma cruzi-infected rats

The effect of an 8 hour-period of water deprivation on fluid and electrolyte renal excretion was investigated in male Wistar rats infected with the strain Sao Felipe (12SF) of Trypanosoma cruzi, in comparison with age and sex matched non-infected controls. The median percent reductions in the urinary flow (-40 per cent v -63 per cent) and excretion of sodium (-57 per cent v - 79 per cent) were smaller in chagasic than in control rats, respectively. So, chagasic rats excereted more than controls. On the other hand, the median percent decrement in the clearance of creatinine was higher in chagasic (-51 per cent) than in controls (-39 per cent). Thus, chagasic rats showed some disturbed renal hydroelectrolytic responses to water deprivation, expressed by smaller conservation, or higher excretion of water and sodium in association with smallerglotmerular filtration rate. This fact denoted an elevation in the fractional excretion of sodium and water.

Effects of water deprivation on renal hydroelectrolytic excretion in chronically Trypanosoma cruzi-infected rats.

The effect of an 8 hour-period of water deprivation on fluid and electrolyte renal excretion was investigated in male Wistar rats infected with the strain São Felipe (12SF) of Trypanosoma cruzi, in comparison with age and sex matched non-infected controls. The median percent reductions in the urinary flow (-40% v-63%) and excretion of sodium (-57% v -79%) were smaller in chagasic than in control rats, respectively. So, chagasic rats excreted more than controls. On the other hand, the median percent decrement in the clearance of creatinine was higher in chagasic (-51%) than in controls (-39%). Thus, chagasic rats showed some disturbed renal hydroelectrolytic responses to water deprivation, expressed by smaller conservation, or higher excretion of water and sodium in association with smaller glomerular filtration rate. This fact denoted an elevation in the fractional excretion of sodium and water.

Activity of a microinjected inducible murine hsp68 gene promoter depends on plasmid configuration and the presence of heat shock elements in mouse dictyate oocytes but not in two-cell embryos.

After fertilization in the mouse, the zygotic genome is activated in two-cell embryos by the spontaneous expression, among other genes, of the major inducible heat shock gene, hsp68, in the absence of heat-inducibility of heat shock genes. To obtain information on this phenomenon, we have probed one- and two-cell embryo's ability to express microinjected reporter DNA constructs, containing the Escherichia coli lacZ gene driven by promoters from early SV40 genes, the human beta-actin gene, and the normal or HSE-deleted mouse hsp68 gene. Activity of these promoters was also tested in mouse granulosa cells and dictyate oocytes, as a function of circular/linear construct configuration and occurrence of heat shock. The hsp68 promoter was heat-inducible in both granulosa cells and oocytes. Its heat activation required the presence of HSEs and, in the oocytes, of construct linear configuration. In the embryos however, this promoter was expressed independently of the presence of HSEs and of construct configuration, and its activity was not affected by heat shock. When constructs with early SV40 and beta-actin promoters were injected into one-cell embryos, they appeared to be inactivated with the first embryonic cleavage, in agreement with previous observations [Wiekowski et al., 1992]. By contrast, both normal and HSE-deleted hsp68 promoters maintained their activity through the first cleavage, providing the first evidence of a gene escaping such transcriptional repression. Present results confirm previous findings on hsp68 expression during early mouse development, and suggest that this activation is mediated by a factor(s) other than HSF.

Genetic manipulation of mammalian dictyate oocytes: factors affecting transient expression of microinjected DNA templates.

Transcription of exogenous DNA templates in mouse ovarian oocytes was investigated by microinjecting constructs encoding for the Escherichia coli lacZ gene under control of promoters from: 1) the mouse hsp68 gene; 2) the human beta-actin gene; and 3) simian virus 40 (SV40) early genes. Various amounts of circular or linear DNA constructs were injected into dictyate oocyte nuclei at different stages of follicle growth, and the beta-galactosidase activity was then cytochemically evaluated in single cells. In middle-sized growing oocytes, expression of circular constructs was observed with amounts of DNA ranging from 50 to 10(3) plasmid copies/nucleus and was first observed 10-12 hr after injection. Maximal expression levels were reached by 17 hr after injection and were specific for the constructs used. Circular constructs containing the hsp68 and early SV40 promoters were expressed at similar levels in small- and middle-sized growing oocytes, while the construct carrying the beta-actin promoter was expressed only in small-sized cells. In contrast to growing oocytes, these constructs were never expressed in fully grown oocytes. DNA linearization depressed construct activity regardless of the site of cleavage. These results show that: 1) lacZ is a valuable reporter gene in the analysis of eukaryotic promoter activity in dictyate mouse oocytes; 2) transient construct expression requires the injection of DNA in circular form; and 3) the expression efficiency of different DNA templates is dependent on the presence of a specific promoter and on the differentiation stage of oocytes analyzed.

Hyperthermia specifically inhibits bivalent chromosome disjunction in maturing mouse oocytes.

The effect of hyperthermia on mammalian oocyte maturation was studied by allowing preovulatory mouse oocytes to mature spontaneously for 17 h in vitro under controlled temperature conditions. At the end of culture, oocytes were screened for their maturation stage and for chromosome morphology and number. Mild hyperthermic conditions (38.5-40.0 degrees C) during maturation specifically disturbed the process of bivalent chromosome disjunction, but not other maturation steps, by blocking oocytes at the metaphase I stage and preventing cells from entering subsequent maturation steps. Some oocytes that had reached metaphase II under hyperthermic conditions had chromosome imbalance. Oocytes matured at 40.0 degrees C displayed chromosome morphological abnormalities, including altered sister chromatid separation and nucleus/nuclei formation, at a frequency significantly higher than oocytes matured at 37.0-39.0 degrees C. When incubation temperature was raised above 40.0 degrees C, increasing fractions of oocytes were inhibited from entering initial maturation steps. We conclude that hyperthermia during mammalian oocyte maturation specifically damages the process of bivalent chromosome disjunction and induces the appearance of chromosome structural defects and imbalance in unfertilized eggs.

Granulosa cell-oocyte interactions: the phosphorylation of specific proteins in mouse oocytes at the germinal vesicle stage is dependent upon the differentiative state of companion somatic cells.

The role of granulosa cells in the regulation of mouse ovarian oocyte metabolism was investigated. Fully grown antral oocytes, isolated from surrounding cumulus cells, were cultured on monolayers of preantral granulosa cells in the presence of dbcAMP to prevent the resumption of meiosis. Under these conditions metabolic cooperativity was established between the two cell types as early as 1 hr after seeding. Moreover, cocultured oocytes phosphorylated two polypeptides of 74 and 21 kDa which are normally phosphorylated in follicle-enclosed growing oocytes but not in cumulus cell-enclosed fully grown oocytes at the germinal vesicle stage. When cocultured oocytes were allowed to resume meiosis, the 74 and 21 kDa proteins were synthesized but no longer phosphorylated even though intercellular coupling between the two cell types was maintained during radiolabeling. It appears therefore: a) that the different protein kinase activity of growing and fully grown germinal vesicle-stage mouse oocytes is related to the differentiative state of granulosa cells, and b) that the regulation of oocyte protein phosphorylation activity by granulosa cells is dependent on the meiotic stage of the oocyte.

Developmental regulation of heat-shock response in mouse oogenesis: identification of differentially responsive oocyte classes during Graafian follicle development.

The response to heat (hs response) of dictyate mouse oocytes at various differentiation stages was analyzed in vitro, by determining patterns of oocyte heat-shock (hs) gene expression and heat-shock protein (HSP) synthesis, under both normal conditions and after an hs. Growing oocytes constitutively synthesized HSP89 and HSC70, and, in contrast to preovulatory oocytes which do not display an hs response, displayed a heat-elicited, transcription-dependent synthesis of two HSP68 isoforms, but not of other inducible HSPs. To determine the developmental schedule of hs response disappearance during oogenesis, fully grown oocytes from Graafian follicles were morphologically sorted into three discrete classes related to the follicle development, namely, loosely associated with granulosa cells (LA oocytes, from small Graafian follicles), intermediately associated with granulosa cells (IA oocytes, from medium-sized Graafian follicles), and cumulus-associated (CA oocytes, from mature follicles). LA oocytes displayed an hs response qualitatively similar to, but smaller in extent than, that of growing oocytes, and were able to resume and complete spontaneous meiotic maturation in vitro at a high rate after hs. We conclude that hs response of mouse dictyate oocytes is maximal during growth period, significantly declines with acquisition of full oocyte size and antrum formation within the follicle, and is finally shut off with oocyte/follicle terminal differentiation.

Effects of protein kinase C stimulation and free Ca2+ rise in mammalian egg activation.

Protein phosphorylation activity, chromosome segregation, and cortical granule exocytosis (CGE) have been studied in mouse eggs activated parthenogenetically by specific PKC stimulators such as 4 beta-phorbol 12-myristate 13-acetate (PMA) and 1-oleyl-2-acetylglycerol (OAG), or by agents inducing an immediate increase in cytosolic calcium concentration ([Ca2+]i) such as ethanol and Ca-ionophore A23187. When protein phosphorylation activity of mouse eggs was analyzed 10 min after different activation treatments, the phosphorylation of a 32 kDa polypeptide was a feature common to all different parthenogenetic agents used. The appearance of such labeling was independent of an increasing [Ca2+]i, as indicated by direct measurements of 1) cytosolic Ca2+ concentration with fura-2 and 2) exogenous Ca2+ entrance into activated eggs. Emission of the second polar body was blocked in PMA-elicited parthenogenones, whereas it was apparently normal in OAG-treated eggs, unless the eggs were continuously exposed to OAG. CGE was almost immediate in ethanol-activated eggs, but in PMA-treated cells, it occurred significantly later, with a timing corresponding to that found for the appearance of sustained Ca2+ oscillations in this system. Here, we propose that in mammalian eggs 1) PKC stimulation represents an early regulatory step in egg activation; 2) this kinase activity is turned off before the second meiotic cleavage; and 3) cytosolic free Ca2+ rise is essential for CGE occurrence.

Somatic cell-oocyte interactions in mouse oogenesis: stage-specific regulation of mouse oocyte protein phosphorylation by granulosa cells.

The relative rate of synthesis of a number of proteins and the protein phosphorylation pattern of growing and fully grown oocytes were influenced by the presence of granulosa cells. In particular, a 74-kDa phosphorylated protein was detected only in granulosa cell-enclosed growing mouse oocytes. When reaggregated with granulosa cells, the growing oocyte displayed the phosphorylated form of the 74-kDa protein but when oocytes were cultured on Sertoli cell monolayers or in granulosa cell-conditioned medium the 74-kDa protein was not phosphorylated. We propose that (1) granulosa cells regulate protein phosphorylation in mouse oocytes; (2) a 74-kDa protein is phosphorylated only in growing oocytes when surrounded by granulosa cells; and (3) granulosa cells, but not Sertoli cells, are competent to send the appropriate "signal" to the growing oocyte.