High-field terahertz bulk photovoltaic effect in lithium niobate.

The terahertz (THz) response of the ferroelectric prototype material lithium niobate (LiNbO3) is studied in the nonperturbative regime of light-matter interaction. Applying two-dimensional THz spectroscopy with few-cycle pulses of an amplitude E≈100 kV/cm and a center frequency of 2 THz, we dissect the overall nonlinear response into different orders in the electric field. The underlying nonlinear current is of interband character and consists of a strong low-frequency shift current (SC) and higher harmonics of the THz fundamental. The SC component originates from the lack of inversion symmetry and the strong interband decoherence for long electron trajectories in k space as shown by theoretical calculations.

High-field transport in an electron-hole plasma: transition from ballistic to drift motion.

The time evolution of high-field carrier transport in bulk GaAs is studied with intense femtosecond THz pulses. While ballistic transport of electrons occurs in an n-type sample, a transition from ballistic to driftlike motion is observed in an electron-hole plasma. This onset of friction is due to the holes, which are heated by THz absorption. Theoretical calculations, which reproduce the data quantitatively, show that both electron-hole scattering and local-field effects in the electron-hole plasma are essential for the time-dependent friction.

Differential cellular compartmentalization of the nuclear receptor SpSHR2 splicing variants in early sea urchin embryos.

SpSHR2 is a member of the nuclear receptor superfamily, expressed in embryos, larvae, and adult tissues of sea urchin. During embryonic development, two receptor isoforms are produced via alternative splicing. One exhibits the typical structure of nuclear receptors (SpSHR2-full length), whereas the other is missing the entire LBD (SpSHR2-splice variant). DNA-constructs encoding these isoforms and two additional in vitro generated deletion mutants were engineered in an expression vector carrying the myc-tag. Expression of the tagged isoforms in S. purpuratus embryos showed that the exogenous SpSHR2 full-length protein displays a similar subcellular localization as the endogenous receptor. In early cleavage stages (4-cells), the full-length isoform is predominantly localized in the nucleus, whereas two cell divisions later (16-cells) protein accumulations are detected in both the nucleus and cytoplasm. To the contrary, the SpSHR2-splice variant is confined in the embryonic nuclei both at 4- and 16-cell stage embryos. Analysis of the intracellular distribution of two receptor mutants, one having a deletion within the DBD (DeltaP) and the other a truncation of the C-terminal F-domain (DeltaF), revealed that DeltaP is localized similarly to full-length receptor, whereas DeltaF is maintained in the nucleus, similar to the SpSHR2 splice variant. Investigation of the DNA binding and dimerization properties of the two SpSHR2 isoforms demonstrated that they recognize and bind to a DR1-element as monomers, whereas DeltaP does not bind DNA and DeltaF binds to DR1 poorly. These results suggest that the receptor's putative LBD is responsible for the differential subcellular localization of the two natural SpSHR2-isoforms in early development.

Subcellular trafficking of the nuclear receptor COUP-TF in the early embryonic cell cycle.

The nuclear receptor SpCOUP-TF is the highly conserved sea urchin homologue of the COUP family of transcription factors. Previous results from our laboratory demonstrated that SpCOUP-TF transcripts are localized in the egg and asymmetrically distributed in the early embryonic blastomeres (A. Vlahou et al., 1996, Development 122, 521-526). To examine the subcellular localization of SpCOUP-TF protein, polyclonal antibodies were separately raised against the divergent N-terminus as well as the conserved DNA-binding and ligand-binding domains. Immunohistochemical analyses suggest that SpCOUP-TF is a maternal protein residing in the cytoplasm of the unfertilized egg. After fertilization, and as soon as the two-cell-stage embryo, most of the receptor translocates from the cytoplasm to the cell nuclei. During the rapid embryonic cell division, SpCOUP-TF was found to shuttle from the interphase nuclear periphery to the condensed chromosomes in mitosis, in a cell-cycle-dependent manner. In an attempt to confirm these observations, the subcellular localization of myc-tagged human COUP-TF I introduced into the sea urchin embryo by RNA injection of fertilized eggs was examined. The pattern of human COUP-TF I subcellular localization, detected with a monoclonal myc antibody, recapitulated the essential features described for the endogenous SpCOUP-TF trafficking. Replacement of the N-terminus of the human receptor with the unique sea urchin N-terminus enhanced its localization to the nuclear rim during interphase. Deletion of the DNA-binding domain of human COUP-TF I resulted in loss of all aspects of nuclear periphery and chromosomal localization. Taken together these data suggest that SpCOUP-TF transcriptional activity is keyed on a cell-cycle-dependent mechanism that regulates chromosomal protein traffic.

Optical limitation in resonant Faraday media.

Optical limitation in resonant Faraday media sandwiched between crossed polarizers is analyzed both theoretically and experimentally. In this technique resonant Faraday rotation permits a net transmission at low intensity levels, whereas the setup transmission is quenched at higher intensities when the active transition is completely saturated. This technique offers many advantages, such as a very low fluence threshold (~12 nJ/cm(2) in cesium vapor), a high intensity range (more than 4 orders of magnitude for polarizer losses of 10(-5)), and quasi invulnerability to optical damage, which make this technique attractive for the protection of fixed-wavelength operated detectors.

Magneto-optic parametric oscillation.

We develop a model of large-signal steady-state magneto-optic parametric oscillation in the Faraday configuration of a singly resonant cavity. The conversion efficiency and the threshold and phase-matching conditions are discussed, and we show that tunable phase matching can be achieved by use of a static magnetic field, eliminating any walk-off effects.

Optical parametric generation and phase matching in magneto-optic media.

We derive the equations and main characteristics of optical parametric generation in magneto-optic media. The key performances are discussed in terms of Stokes parameters and conservation laws of electromagnetic linear and angular momentum.

Embryonic and post-embryonic utilization and subcellular localization of the nuclear receptor SpSHR2 in the sea urchin.

SpSHR2 (Strongylocentrotus purpuratus steroid hormone receptor 2) is a nuclear receptor, encoded by a maternal RNA in the sea urchin embryo. These maternal SpSHR2 transcripts, which are present in all cells, persist until the blastula stage and then are rapidly turned over. A small fraction of the embryonic SpSHR2 protein is maternal, but the majority of this nuclear receptor in the embryo is the product of new synthesis, presumably from the maternal RNA after fertilization. In agreement with the mRNA distribution, the SpSHR2 protein is also detected in all embryonic cells. Contrary to the RNA though, the SpSHR2 protein persists throughout embryonic development to the pluteus stage, long after the mRNA is depleted. Following fertilization and as soon as the 2-cell stage, the cytoplasmic SpSHR2 protein enters rapidly into the embryonic nuclei where it appears in the form of speckles. During subsequent stages (from fourth cleavage onward), SpSHR2 resides in speckled form in both the nucleus and the cytoplasm of the embryonic cells. The cytoplasmic localization of SpSHR2 differs between polarized and non-polarized cells, maintaining an apical position in the ectoderm and endoderm versus a uniform distribution in mesenchyme cells. Following the end of embryonic development (pluteus stage), the SpSHR2 protein is depleted from all tissues. During the ensuing four weeks of larval development, the SpSHR2 is not detected in either the larval or the rudiment cells which will give rise to the adult. Just prior to metamorphosis, at about 35 days post-fertilization, the protein is detected again but in contrast to the uniform distribution in the early embryo, the larval SpSHR2 is specifically expressed in cells of the mouth epithelium and the epaulettes. In adult ovaries and testes, SpSHR2 is specifically detected in the myoepithelial cells surrounding the ovarioles and the testicular acini. Nuclear SpSHR2 in blastula extracts binds to the C1R hormone response element in the upstream promoter region of the CyIIIb actin gene indicating that the latter may be a target of this nuclear receptor in the sea urchin embryo.

An extracellular matrix response element in the promoter of the LpS1 genes of the sea urchin Lytechinus pictus.

The extracellular matrix (ECM) has been shown to play an important role in development and tissue-specific gene expression, yet the mechanism by which genes receive signals from the ECM is poorly understood. The aboral ectoderm-specific LpS1-alpha and -beta genes of Lytechinus pictus , members of the Spec gene family, provide an excellent model system to study ECM- mediated gene regulation. Disruption of the ECM by preventing collagen deposition using the lathrytic agent beta-aminopropionitrile (BAPN) inhibits LpS1 gene transcription. LpS1 transcription resumes after removal of BAPN and subsequent collagen reformation. Using a chloramphenicol acetyltransferase (CAT) reporter gene assay, we show that a 125 bp region of the LpS1-beta promoter from -108 to +17 contains an ECM response element (ECM RE). Insertion of the 125 bp region into the promoter of the metallothionein gene of L. pictus, a gene unaffected by ECM disruption, caused the fused promoter to become ECM dependent. As with the endogenous LpS1 genes, CAT activity directed by the fused LpS1-beta promoter resumed in embryos recovered from ECM disruption. A mutation in a cis -acting element called the proximal G-string, which lies in the 125 bp region, caused CAT activity levels in ECM-disrupted embryos to equal that of the wild-type LpS1-bet apromoter in ECM-intact embryos. These results suggest that the intact ECM normally transmits signals to inhibit repressor activity at the proximal G-string in aboral ectoderm cells. Consistent with these results were our findings which showed that in addition to expression in the aboral ectoderm, the proximal G-string mutation caused expression of the CAT gene in oral ectoderm cells. These studies suggested that the proximal G-string serves as a binding site for negative regulation of the LpS1 genes in oral ectoderm during development. We also examined trans -acting factors binding the proximal G-string following ECM disruption. Band shift gels revealed a predominant set of slower migrating nuclear proteins from ECM-disrupted embryos which bound the proximal G-string. This work suggested that ECM disruption initiates signaling that induces a repressor to bind the ECM RE and/or modifies ECM RE binding proteins, which in turn represses LpS1 gene activity.

Phase-modulation-induced two-wave mixing in semiconductors.

Two-wave mixing amplification of a weak probe beam interfering with a powerful pump beam of the same linear polarization is demonstrated when the probe beam enters the semiconductor sample before the pump pulse. This mixing of two beams of the same frequency is made possible by the difference between self-phase modulation and cross-phase modulation, which modify differently the instantaneous frequencies of both beams. Net gain coefficients up to 5.5 cm (-1) are obtained for experiments performed in CdTe. Theoretical calculations quantitatively confirm this interpretation and predict much higher gain for more resonant configurations or higher pump energies, making this process of potential interest for optical processing.

Distal cis-acting elements restrict expression of the CyIIIb actin gene in the aboral ectoderm of the sea urchin embryo.

The distal region of the S. purpuratus actin CyIIIb gene, between -400 and -1400 nucleotides, contains at least three distinct cis-acting elements (C1R, C1L and E1) which are necessary for correct expression of fusion reporter genes in transgenic sea urchin embryos. The contribution of these elements in the temporal and spatial regulation of the gene was analyzed by single and double site-directed mutagenesis in fusion constructs which carry the bacterial chloramphenicol acetyl transferase (CAT) gene as a reporter. Following microinjection of the transgenes in sea urchin embryos, the activity of the mutants was compared to the wild type in time and space by measuring CAT activity at the blastula and pluteus embryonic stages and by in situ hybridization to the CAT mRNA at pluteus stage. Our results indicate that E1 is involved in the temporal regulation of CyIIIb and that all three elements are necessary and sufficient to confer aboral (dorsal) ectoderm specificity to the proximal promoter. This is achieved by suppressing the promoter's activity in all other tissues by the cooperative interaction of the cis-acting elements. The C1R element, binding site of the nuclear receptors SpCOUP-TF and SpSHR2, is by itself sufficient to restrict expression in the ectoderm, whereas the aboral ectoderm restricted expression requires in addition the presence of both C1L and E1. It is therefore evident, that the actin CyIIIb gene is exclusively expressed in the aboral ectoderm by a combinatorial repression in all other cell lineages of the developing embryo.

Very early and transient vegetal-plate expression of SpKrox1, a Krüppel/Krox gene from Stronglyocentrotus purpuratus.

All endodermal and mesenchymal cells of the sea urchin embryo descend from the vegetal plate, a thickened epithelium of approximately 50 cells arising at the early blastula stage. Cell types that derive from the vegetal plate are specified conditionally by inductive interactions with underlying micromeres, but the molecular details of vegetal-plate specification remain unresolved. In a search for regulatory proteins that have roles in vegetal-plate specification, a screen was performed to clone Krüppel/Krox-related genes from a Strongylocentrotus purpuratus embryo cDNA library. One newly identified clone, named SpKrox1, contained four zinc fingers and a leucine zipper domain. SpKrox1 expression was low in unfertilized eggs, increased severalfold to the early blastula stage and decreased between the early gastrula and pluteus stages. SpKrox1 mRNA was first seen in macromeres of 16-cell stage embryos and was restricted to cells of the developing vegetal plate thereafter. Vegetal-plate expression corresponded to a ring of cells around the blastopore and overlapped the expression patterns of other genes with potential roles in vegetal plate-specification. As the vegetal-plate cells invaginated into the blastopore, SpKrox1 expression was lost, suggesting that its role was not in endoderm differentiation per se but rather in the initial establishment of the vegetal plate.

A novel sea urchin nuclear receptor encoded by alternatively spliced maternal RNAs.

Screening of a genomic library from the sea urchin Strongylocentrotus purpuratus with a human COUP-TF I cDNA probe revealed the presence of a novel gene member of the steroid-thyroid-retinoic acid receptor superfamily, which was named SpSHR2 (S. purpuratus Steroid Hormone Receptor 2). Sequence analysis of the isolated genomic clone revealed that the DNA binding domain of this orphan receptor is most homologous to the human TR2 receptor. Using this sea urchin genomic fragment as probe, a S. purpuratus embryonic cDNA library was screened and two distinct but homologous cDNA clones were isolated. The two cDNAs encode the same DNA binding domain as the SpSHR2 gene and carry an almost identical 3'-untranslated sequence. One of the clones, however, is missing an entire region of about 1100 nt which includes the putative ligand binding domain. Genomic DNA hybridization suggests that SpSHR2 is a single-copy gene in the S. purpuratus genome. Exon skipping during splicing of a single primary transcript appears to be the reason for the differently sized mRNAs. RNA blot hybridization results suggest that SpSHR2 transcripts are stored as maternal RNA in the egg and are not detected beyond the blastula stage. In vitro transcription and translation of the full-length cDNA produced a polypeptide which specifically binds to the hormone response element in the 5'-flanking region of the sea urchin actin CyIIIb gene. In vivo labeling of proteins synthesized by cleavage stage embryos followed by immune precipitation of the SpSHR2 protein using specific antibodies reveals that the maternal SpSHR2 mRNA is being translated during early embryonic development.

Maternal mRNA encoding the orphan steroid receptor SpCOUP-TF is localized in sea urchin eggs.

The SpCOUP-TF gene is a highly conserved sea urchin homologue of the vertebrate COUP-TFs and the Drosophila seven up subfamily of transcription factors, which are members of the orphan steroid hormone receptors. Whole-mount in situ hybridization experiments, using three sea urchin species, detect the maternal SpCOUP-TF mRNA deposited unevenly in the oocytes, mature eggs and the blastomeres of the early embryo. The localization pattern indicates that, in all three sea urchin species, the maternal SpCOUP-TF mRNA is placed in the egg in a fixed position relative to the embryonic axes, i.e. lateral to the animal-vegetal and at 45 degree angle to the oral-aboral (ventral-dorsal) axis. The embryonic expression of the SpCOUP-TF gene is spatially restricted in the oral ectoderm of the early embryo and, at later stages, in the cells of the ciliated band, the neurogenic cell lineage of the sea urchin embryo. The SpCOUP-TF mRNA, the first sea urchin maternal mRNA encoding a transcription factor that is specifically localized with respect to both embryonic axes in the egg, could be involved in early specification events in the sea urchin embryo.

Purification of a high-mobility-group 1 sea-urchin protein and cloning of cDNAs.

The isolation of the sea urchin high-mobility-group 1 (HMG1) protein, the cloning of corresponding cDNA clones and the similarity to the human homologue are described. Sea urchin HMG1 was purified as one of the nuclear embryonic proteins which associate with an upstream regulatory element (E1) of the Strongylocentrotus purpuratus (Sp) CyIIIb actin-encoding gene. Using a synthetic oligodeoxyribonucleotide (oligo) which includes the E1 cis-acting element in a DNA affinity chromatography purification, the most prominent of the binding proteins was isolated and the N terminus sequenced. cDNA clones were isolated by screening an embryonic cDNA library with a synthetic oligo derived from the amino acid (aa) sequence. Comparison of the cDNAs ORF to known proteins revealed a 50% aa identity to the mammalian HMG1 and all the structural characteristics of this group of proteins. The sea urchin protein, SpHMG1, was synthesized in bacteria, as well as translated in vitro. Binding assays carried out with the recombinant SpHMG1 protein did not produce specific in vitro complexes with E1.

Characterization of T-cell receptor V beta repertoire in ovarian tumour-reacting CD3+ CD8+ CD4- CTL lines.

T cells from tumour infiltrating lymphocytes (TIL) cultured in media containing IL-2 were shown to mediate in vitro and in vivo antitumor responses. To characterize the T-cell antigen receptor (TCR) V beta expression in autologous cytotoxic effectors we isolated CD3+ CD8+ CD4- cells from cultures of TIL and tumour-associated lymphocytes (TAL) and analysed the TCR V beta repertoire of CD3+ CD8+ CD4- lines of known HLA-A, -B and -C phenotype, using polymerase chain reaction (PCR). These lines showed preferential lysis of autologous tumours and lysed, to a much lesser extent, NK and LAK cell-sensitive targets. Tumour lysis was inhibited by antibodies to CD3 and MHC class I antigens indicating that they are cytotoxic T lymphocytes (CTL). These CD8+ CTL lines expressed a broad distribution of TCR V beta repertoire which was dominated by particular groups of V beta families in each CTL line. However, no predominant expression of one or the same V beta segment in all CTL lines was observed although statistical correlations between V beta family usage and magnitude of the antitumour cytolytic response were found. These results suggest that certain TCR V beta families may be selected by antigen in ovarian tumour-reactive T cells and this selection may be affected by Ag expression, and/or host factors. To our knowledge, this is the first documentation of TCR V beta repertoire of human ovarian tumour-reactive CD3+ CD8+ CD4- CTL from different individuals of known HLA types.

Differential expression of cuticle-epidermis proteins in the shrimp Penaeus vannamei during molting.

High-resolution mini-two-dimensional polyacrylamide gel electrophoresis (mini-2D-PAGE) was used to analyze silver-stained, soluble proteins from the cuticle-epidermis of Penaeus vannamei during molting. The 2D-PAGE patterns of epidermis polypeptides from metecdysis and anecdysis/proecdysis molt stages demonstrated similarities as well as several quantitative and qualitative differences. Quantitative modulation in polypeptide expression was noted in at least seven prevalent polypeptides during molting. A 50 kDa protein is specifically expressed in anecdysis/proecdysis tissue samples. Quantitative and qualitative differences were also noted in proteins migrating mainly in the molecular mass ranges of 26-32 kDa. An overall increase in polypeptide expression was noted in this molecular mass range at metecdysis as compared to anecdysis/proecdysis epidermis tissues. These results indicate modulation of cuticle-epidermis proteins in Penaeus vannamei shrimps during molting.