Few-cycle Yb laser source at 20 kHz using multidimensional solitary states in hollow-core fibers.

We demonstrate ultrashort pulse compression from 300 fs down to 17 fs at a repetition rate of 20 kHz and 160-µJ output pulse energy (3.2 W of average power) using multidimensional solitary states (MDSS) in a 1-meter hollow-core fiber (HCF) filled with N2O. Under static pressure, thermal limitations at this repetition rate annihilate the MDSS with suppression of spectral broadening. The results obtained in differential pressure configuration mitigate thermal effects and significantly increase the range of repetition rate over which MDSS can be used to compress sub-picosecond laser pulses.

70 mJ nonlinear compression and scaling route for an Yb amplifier using large-core hollow fibers.

In this Letter, we investigate the energy-scaling rules of hollow-core fiber (HCF)-based nonlinear pulse propagation and compression merged with high-energy Yb-laser technology, in a regime where the effects such as plasma disturbance, optical damages, and setup size become important limiting parameters. As a demonstration, 70 mJ 230 fs pulses from a high-energy Yb laser amplifier were compressed down to 40 mJ 25 fs by using a 2.8-m-long stretched HCF with a core diameter of 1 mm, resulting in a record peak power of 1.3 TW. This work presents a critical advance of a high-energy pulse (hundreds of mJ level) nonlinear interactions platform based on high energy sub-ps Yb technology with considerable applications, including driving intense THz, X-ray pulses, Wakefield acceleration, parametric wave mixing and ultraviolet generation, and tunable long-wavelength generation via enhanced Raman scattering.

High energy redshifted and enhanced spectral broadening by molecular alignment.

We demonstrate an efficient approach for enhancing the spectral broadening of long laser pulses and for efficient frequency redshifting by exploiting the intrinsic temporal properties of molecular alignment inside a gas-filled hollow-core fiber (HCF). We find that laser-induced alignment with durations comparable to the characteristic rotational time scale TRotAlign enhances the efficiency of redshifted spectral broadening compared to noble gases. The applicability of this approach to Yb lasers with (few hundred femtoseconds) long pulse duration is illustrated, for which efficient broadening based on conventional Kerr nonlinearity is challenging to achieve. Furthermore, this approach proposes a practical solution for high energy broadband long-wavelength light sources, and it is attractive for many strong field applications.

Hypoxia-induced enrichment and mutagenesis of cells that have lost DNA mismatch repair.

Loss of DNA mismatch repair (MMR) increases the risk of spontaneous mutations. We sought to determine whether there was an interaction between hypoxia and MMR deficiency that might contribute to the phenomenon of tumor progression. Human colon carcinoma HCT116+ch2 (MMR-deficient) and HCT116+ch3 (MMR-proficient) sublines were exposed for varying periods of time to an environment of <0.1% O2 and pH as low as 6.1. When a population containing 5% MMR-deficient cells and 95% MMR-proficient cells was subjected to hypoxia for 72 h, the MMR-deficient cells were enriched by a factor of 2-fold in the surviving population, whereas no enrichment was detected in cells maintained under aerobic conditions. The potential of hypoxia to destabilize the genome was determined by measuring the frequency of clones in the surviving population resistant to very high concentrations of 6-thioguanine or cisplatin. A 72-h exposure to hypoxia did not increase the frequency of resistant clones in the MMR-proficient cells but produced a 7.8-fold increase in 6-thioguanine-resistant clones and a 2.5-fold increase in cisplatin-resistant clones in the MMR-deficient cells. Loss of MMR increased the frequency of mutations in a reporter vector sensitive to frameshift mutations in a microsatellite sequence. Exposure to hypoxia for a time period as short as 48 h further increased the number of mutations in both cell types, but the absolute number of mutants was higher in the MMR-deficient cells. These results indicate that hypoxia and its accompanying low pH enrich for MMR-deficient cells and that loss of MMR renders human colon carcinoma cells hypersensitive to the ability of hypoxia to induce microsatellite instability and generate highly drug-resistant clones in the surviving population.

A target of the HoxB5 gene from the mouse nervous system.

An epitope-specific antibody against the protein product of the murine HoxB5 gene was used to select an enriched library of Hox target sequences. Genomic DNA was purified by immunoaffinity chromatography, using glutaraldehyde-cross-linked chromatin from CNS of mouse embryos at gestational day 15. Screening was done by colony hybridization with TAAT-containing oligonucleotides, filter DNA-protein binding, and gel mobility shift assay. Nucleotide sequencing identified a 910 bp DNA fragment, containing a consensus Antennapedia-like binding site, and identical in 640 bps at 3' end of the clone to the promoter of the SPI3 gene, which encodes a serine protease inhibitor protein [Sun, J., Rose, J.B. and Bird, P., J. Biol. Chem., 270 (1995) 16089-16096]. In situ hybridization experiments were performed to see if a correlation could be found between the expression patterns the SPI3 and the HoxB5 genes. Using a 120 bp cDNA fragment as probe, SPI3 expression was detected mainly in the CNS of 15 day mouse embryos, a pattern which is similar to that of the HoxB5 gene at this stage [Hogan, B.L., Holland, P.W. and Lumsden, A., Cell Diff. Dev., 25 Suppl. (1988) 39-44; Sakach, M. and Safaei, R., Int. J. Dev. Neurosci., 14 (1996) 567-573]. In conclusion, data presented here suggest that the SPI3 gene is a candidate target of the HoxB5 gene in vertebrate embryos.

Localization of the HoxB5 protein in the developing CNS of late gestational mouse embryos.

We cloned a full-length HoxB5 cDNA from the human neuroblastoma cell line, SHSY5Y, and used it to raise specific antibodies against a divergent domain of its protein. The antibodies then were used to immunolocalize and determine the expression pattern of the HoxB5 homeodomain protein in the late gestational mouse embryos. With particular reference to the structures of the developing nervous system, relatively high levels of the HoxB5 protein were detected in 15- and 18-day-old gestational mouse embryos. Expression domains were similar in both stages and were limited almost exclusively to the central nervous system. Expression of the HoxB5 protein was found in areas that extended from the rostral medulla oblongata to the caudal parts of the spinal cord. In all regions of the CNS, expression levels were higher in the rostral and ventral regions compared to the caudal and dorsal areas, respectively. These findings suggest that the function of the HoxB5 protein is not limited to the early stages of neural development and extends into later stages as well. These findings suggest also that the HoxB5 protein may play a role in the organization of the neural structures along both of the dorsoventral and craniocaudal axes of the developing fetus in vertebrate species.

Modulation of HOX2 gene expression following differentiation of neuronal cell lines.

The expression of the genes in the human HOX2 locus has been studied during differentiation of two human neuroblastoma (SH-SY5Y and Kelly), a human glioblastoma (251-MG), and the murine F9 embryonal carcinoma cell lines. Cells were differentiated with retinoic acid (RA), or with RA together with dibutyral cyclic AMP (db-cAMP) and nerve growth factor (NGF) in order to assess the changes in the expression patterns of these homeobox genes during neuronal differentiation. We show that the genes of the HOX2 locus are expressed in a complex transcription pattern that varies with cell type. The two uninduced neuroblastoma cell lines show a similar pattern of expression for a number of HOX2 genes although the levels of expression are different for individual cell lines. The embryonal carcinoma cell line F9 expresses low levels of several HOX2 genes which is restricted to the 5' region of the HOX2 cluster. The glioblastoma cell line, 251-MG expresses almost all of the genes of the HOX2 locus. Differentiation of these cells modulates the expression of the HOX2 genes in a manner that is dependent upon the cell type as well as the differentiation factor. Differentiation affects both the level of HOX2 gene expression and the distribution of transcript sizes. In conclusion, our analysis reveals a complex pattern of expression for the genes of the HOX2 locus in neuronal and glial cells and suggests that the cell-specific expression of these genes may be correlated with the phenotypic differences that are observed between different neuronal and glial cell populations within the nervous system.

Regulation of microtubule associated protein 2 (MAP2) expression by nerve growth factor in PC12 cells.

In the presence of nerve growth factor (NGF), PC12 cells cease to divide and differentiate, extending long microtubule-containing neurites. We showed by immunoblot analysis that MAP2 was detectable in PC12 after 4 days of NGF treatment and that its levels increased five- to sevenfold after 12 days of NGF treatment. The apparent molecular weight of MAP2 in PC12 cells was similar to that of rat brain MAP2 (280,000), with a doublet representing the MAP2 isoforms. However, the relative levels of MAP2 in differentiated PC12 cells were 5-10% of those found in rat brain. Immunofluorescence analysis of NGF-treated PC12 cells revealed that MAP2 co-localized with tubulin and was present in cell bodies and neurites. Northern blot analysis showed that the levels of MAP2 mRNA increased in PC12 cells during NGF-treatment in a pattern that paralleled the protein levels, suggesting that MAP2 expression is transcriptionally regulated.

Turnover of cytoskeletal proteins in vivo.

The turnover of the microtubule-associated proteins 1B and 2 (MAP1B and MAP2), tubulin, high molecular weight neurofilament protein (NF-H), and spectrin were studied by in vivo labeling. Radiolabeled [35S]methionine was injected intracranially to 10-day-old rats and the rate of turnover was measured for total and specific brain proteins. The turnover of total brain proteins was biphasic and consisted of a fast and a slow component with half lives of 6.5 +/- 0.4 and 14.2 +/- 0.7 (mean +/- S.E.M.) days, respectively. The turnover of individual cytoskeletal brain proteins was also biphasic. The fast decay rates of MAP1B, MAP2, tubulin and spectrin were 5.8 +/- 0.7, 6.9 +/- 0.3, 4.8 +/- 0.5 and 4.9 +/- 0.4 days, respectively, while the slow decay rates of these proteins were 12.0 +/- 1.3, 12.4 +/- 1.7, 15.0 +/- 0.5 and 16.0 +/- 1.2 days, respectively. In addition, the Triton X-100 insoluble fraction of MAP1B, tubulin, spectrin and NF-H showed monophasic decay rates of 29.0 +/- 2.3, 15.0 +/- 1.4, 16.0 +/- 0.9 and 18.5 +/- 1.5 days, respectively, which were similar to their slow decay rates in whole brain homogenates, suggesting that incorporation of these proteins into the cytoskeletal lattice increases their stability.

Isolation and sequence of lambda gt11 cDNA clones encoding the 5B4 antigen expressed on sprouting neurons.

Monoclonal antibody (mAb) 5B4 recognizes a developmentally regulated membrane glycoprotein (Mr approximately 185,000-255,000) expressed on sprouting neurons. The amino acid sequence deduced from lambda gt11 cDNA clones encoding the transmembrane and cytoplasmic domains of the 5B4 antigen is co-linear with that of chick NCAM-ld. The significant level of overall sequence identity (75%) demonstrates that the 5B4 antigen is rat brain NCAM. The 5B4 epitope maps to the carboxy-terminus common to both NCAM-ld and NCAM-sd.

Cloning of a cDNA encoding MAP1B in rat brain: regulation of mRNA levels during development.

This article describes the isolation of a microtubule-associated protein 1B (MAP1B) cDNA clone from a rat brain lambda gt11 library and the study of MAP1B mRNA expression during brain development. On Northern blots, the cDNA hybridized with an mRNA of greater than 10 kilobases which was present only in the brain. The identity of the cDNA was confirmed by the characterization of the antiserum against the fusion protein, and also by comparing both the original antibody and the anti-fusion protein antiserum with a panel of well-studied monoclonal antibodies against different forms of MAP1 and MAP2. The regulation of MAP1B mRNA during development was studied in whole brain, cerebral cortex, hypothalamus, brainstem, and olfactory bulbs. The steady-state levels of MAP1B mRNA in all tissues examined were relatively low in the adult compared to developing brains. This decrease varied in different brain regions, and its time course appeared to coincide with the pattern of postnatal developmental and morphological events. The developmental patterns of the MAP1B mRNA and protein in the brain were similar, suggesting that expression of this protein is under transcriptional control. The RNA blots were also probed with beta-actin and beta-tubulin to compare the levels of MAP1B mRNA with other cytoskeletal elements and as controls for the quality of the RNA.

Regulation of microtubule-associated protein 2 (MAP2) mRNA expression during rat brain development.

The expression of MAP2 during rat brain development was studied by using specific antibodies and cDNA probes. MAP2 cDNAs were isolated from a rat brain lambda gt11 library, and their identity was confirmed by the reactivity of their fusion proteins with several independent monoclonal antibodies that recognize MAP2. Northern blot analyses of the RNA prepared from whole brains, cerebral cortex, hypothalamus, brain stem, olfactory bulbs, and cerebellum showed that the levels of MAP2 mRNA increase during the initial phase of development, reach a maximum between postnatal weeks 2 and 3, and then decrease in the adult. The time course and the kinetics of this change varied between different brain regions and appeared to reflect the pattern of morphological changes in these regions. RNA blots were also analyzed with beta-tubulin and beta-actin cDNA probes to ensure the quality and the quantity of the RNA. The levels of MAP2 mRNA and protein showed similar changes during the initial part of brain development and suggested a transcriptional control. However, while MAP2 protein levels remained high throughout development, MAP2 mRNA levels decreased in adulthood. We suggest that the increased stability of the MAP2 molecule may be a contributing factor in the developmental regulation of steady-state levels of MAP2.

Thyroid hormone binding and regulation of adrenergic enzymes in two neuroblastoma cell lines.

Two neuroblastoma cell lines were cultured in control (euthyroid) and hypothyroid media and examined for protein, RNA and DNA content, activity of the catecholaminergic enzymes tyrosine hydroxylase (TH, EC 1.14.16.2) and monoamine oxidase-A (MAO-A, EC 1.4.3.4), and for L-triiodothyronine (T3) nuclear receptors. In the hypothyroid condition, the rate of cell division and the levels of RNA and protein as well as the activities of TH and MAO were lower than in the euthyroid condition, the reduction being more marked in the E than in the A2(1) cell line. T3 nuclear receptors, unaltered in affinity, were increased in number in the hypothyroid medium, possibly as a regulatory response to hormonal deficiency. Examination of a possible relationship between T3 occupancy and TH activity in the E cells, most sensitive to thyroid hormone deficiency, revealed that induction of TH activity by T3 is dose-dependent and correlates with the number of nuclear sites occupied by the hormone. When neuroblastoma cells were induced to differentiate by the addition of sodium butyrate to the medium, parameters of cell growth (protein, RNA) and enzyme activity (TH and MAO-A) increased in both cell lines irrespective of the presence of thyroid hormones. These data indicate that thyroid hormones, through their nuclear receptors, directly affect the activity of catecholaminergic enzymes in cultured, immature (undifferentiated) neurons.