Ray tracing analysis of a two-dimensional rectangular optical cavity for enhancing detector efficiency.

High detector efficiency has broad appeal and includes such diverse fields as quantum optics and solar energy. An optical resonator can improve detector efficiency by employing multiple re-reflections to the detector. This short paper uses geometric ray tracing to examine-for a given entry port size-the probability that a photon will escape from an ideal perfectly reflective two-dimensional cavity.

Nest building is a novel method for indexing severity of alcohol withdrawal in mice.

Withdrawal after chronic ethanol (EtOH) affects body temperature, goal-directed behavior and motor function in mice and increases general central nervous system excitability. Nest-building tests have been used to assay these states but to this point have not been employed as measures of EtOH withdrawal severity. We first refined nest-scoring methods using a genetically heterogeneous stock of mice (HS/Npt). Mice were then made physically dependent following three days of chronic EtOH vapor inhalation to produce average blood EtOH concentrations (BECs) of 1.89 mg/mL. EtOH withdrawal affected the progression of nest building over time when mice were tested 2-4 days after removal from three days of chronic exposure to EtOH. In a separate group of mice, chronic EtOH vapor inhalation (BECs 1.84 mg/mL) suppressed nest building over days 1-2 but not days 2-3 of withdrawal. In a following experiment, EtOH withdrawal dose-dependently slowed recovery of nest building for up to 32 h. Finally, we determined that long-lasting nest-building deficits extend to mice undergoing withdrawal from a high dose (4 g/kg) of acute EtOH. Sex differences for nest building were absent following EtOH exposure. In mice naïve to EtOH treatments, male mice had lower pre-test body temperatures and increased nest scores across a two-day testing period compared to females. These results suggest that nest building can be used to assess chronic and acute EtOH withdrawal severity in mice.

Weak value amplification of an off-resonance Goos-Hänchen shift in a Kretschmann-Raether surface plasmon resonance device.

During the past two decades there has been increased interest in the optical excitation of surface plasmon resonance (SPR) at a metal-dielectric interface. This is due in large part to its potential applications in such areas as medical diagnostics and pharmaceutical research. Also occurring during this time has been a growing recognition by the quantum physics community that weak value amplification (WVA) can serve as a valuable metrological research resource. Recently WVA has been used to amplify very small optical Goos-Hänchen (GH) shifts in glass and it has also been shown that SPR can greatly enhance optical GH shifts at the metal/air interface in Kretschmann-Raether (KR) devices. This paper demonstrates experimentally the WVA of an off-resonance GH shift in a KR device and explains why WVA of sufficiently SPR enhanced optical GH shifts cannot be achieved.

Progress in a replicated selection for elevated blood ethanol concentrations in HDID mice.

Drinking in the dark (DID) is a limited access ethanol-drinking phenotype in mice. High Drinking in the Dark (HDID-1) mice have been bred for 27 selected generations (S27) for elevated blood ethanol concentrations (BECs) after a 4-h period of access to 20% ethanol. A second replicate line (HDID-2) was started later from the same founder population and is currently in S20. An initial report of response to selection in HDID-1 was published after S11. This article reports genetic and behavioral characteristics of both lines in comparison with the HS controls. Heritability is low in both replicates (h(2) = 0.09) but the lines have shown 4-5 fold increases in BEC since S0; 80% of HDID-1 and 60% of HDID-2 mice reach BECs greater than 1.0 mg/ml. Several hours after a DID test, HDID mice show mild signs of withdrawal. Although not considered during selection, intake of ethanol (g/kg) during the DID test increased by approximately 80% in HDID-1 and 60% in HDID-2. Common genetic influences were more important than environmental influences in determining the similarity between BEC and intake for HDID mice. Analysis of the partitioning of intake showed that 60% of intake is concentrated in the last 2 h of the 4 h session. However, this has not changed during selection. Hourly BECs during the DID test reach peak levels after 3 or 4 h of drinking. HDID mice do not differ from HS mice in their rate of elimination of an acute dose of alcohol.

Weak value amplification of an optical Faraday differential refraction effect.

In the presence of a longitudinal magnetic field B, a beam of linearly polarized light incident from a Faraday medium of Verdet constant V refracts at its interface with a medium of negligible Verdet constant and emerges as two opposite circularly polarized beams that are separated by a small divergence angle δ that is proportional to the product BV. Judicious postselection of the polarization state of the emergent light can be used to amplify the measured value of δ by several orders of magnitude. This technique makes it possible to optically measure either very small V values when B is known or small magnetic fields when V is known.

Detection of growth factor receptor RNA in individual hematopoietic cells by in situ RT-PCR; comparison with RT-PCR.

The ability to detect changes in RNA expression in single cells would greatly enhance understanding of the molecular basis of biological responses to positive and negative growth regulators. To this end, we compared expression of RNA encoding the receptors for interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-6, leukemia inhibitory factor (LIF) and stem cell factor (SCF) in populations of primitive hematopoietic progenitors (lineage marker negative, Lin(-), and Lin(-) c-Kit(+)) by RT-PCR and in situ RT-PCR. Both Lin(-) and Lin(-) c-Kit(+) progenitors expressed all receptors by RT-PCR. However, RT-PCR could not distinguish between the possibility that all cells expressed growth factor receptor RNA, or the possibility that only a proportion of cells expressed RNA. Therefore, we used in situ RT-PCR to examine growth factor receptor mRNA expression in individual cells. In contrast to RT-PCR, we observed that only 40-80% of Lin(-) cells and 75-100% of Lin(-) c-Kit(+) cells were positive for expression of the growth factor receptor subunits, demonstrating that not all cells were receptor positive. We found that in situ RT-PCR could also be used to measure induction or repression of receptor RNA expression in these cell populations. Specifically, the percentage of cells expressing IL-6alpha receptor RNA decreased from 88% positive in freshly harvested cells to 9% in Lin(-) c-Kit(+) cells cultured in IL-3 for 18 h. Thus, in situ RT-PCR can be used to detect and quantify the number of individual cells that express growth factor receptor mRNA, and may also be useful to measure changes in expression of other endogenous genes or genes introduced by transfection and gene therapy vectors.

Redirected infection of directly biotinylated recombinant adenovirus vectors through cell surface receptors and antigens.

The inability of adenovirus to infect primitive hematopoietic cells presents an obstacle to the use of adenovirus vectors for gene transfer to these cell types. Therefore, expanding the tropism of adenovirus vectors to unique cell surface antigens would be an important development for gene therapy protocols. In this study, we sought to redirect infection of adenovirus vectors to primitive human hematopoietic cells that universally express the c-Kit receptor on their cell surface. To accomplish this, a vector was constructed by covalently linking biotin molecules to recombinant adenovirus, followed by addition of the biotinylated ligand for the c-Kit receptor, stem cell factor (SCF), through an avidin bridge. Gene transfer was directed specifically to c-Kit-positive hematopoietic cell lines, resulting in up to a 2,440-fold increase in luciferase expression with frequencies equivalent to recombinant virus infection of permissive cells. Substitution of biotinylated antibodies directed against c-Kit, CD34 (binds L-selectin), and CD44 (hyaluronate receptor) receptors for biotinylated SCF resulted in 50-, 8-, and 260-fold increases in reporter gene expression, respectively, demonstrating that infection also could be redirected through antibody-antigen interactions and through antigens other than growth factor receptors. The versatility of this vector was demonstrated further by infection of primary T cells with vectors targeted with antibodies to CD44 (resting and activated T cells) and biotinylated IL-2 (activated T cells only). Taken together, directly biotinylated adenovirus vectors represent a versatile and efficient method for redirection of virus infection to specific cells.

Functional characterization of a novel hematopoietic stem cell and its place in the c-Kit maturation pathway in bone marrow cell development.

While the majority of purified pluripotential hematopoietic stem cells (PHSC) express c-Kit, the receptor for steel factor, we have phenotypically and functionally separated a distinct class of PHSC that does not express c-Kit. In contrast to c-Kit-positive (c-Kit(pos)) PHSC, the c-Kit-negative (c-Kit(neg)) PHSC do not proliferate in response to multiple hematopoietic growth factors in vitro and do not radioprotect or form macroscopic spleen colonies (CFU-s) when transplanted into lethally irradiated recipients. However, the c-Kit(neg) PHSC show delayed or slow reconstitution kinetics when cotransplanted with radioprotective bone marrow cells. c-Kit(neg) PHSCs cells can give rise to c-Kit(pos) cells with CFU-s activity, radioprotective activity, and PHSC activity. Thus, constitutive hematopoiesis is maintained by c-Kit(pos) PHSCS cells that are recruited from a more primitive quiescent c-Kit(neg) PHSC population, which represents a critical developmental stage in definitive hematopoiesis.

Engraftment of ex vivo expanded and cycling human cord blood hematopoietic progenitor cells in SCID mice.

The ability of human hematopoietic cells to engraft SCID mice provides a useful model in which to study the efficiency of retroviral gene transfer and expression in primitive stem cells. In this regard, it is necessary to determine whether SCID mice can be engrafted by cycling human hematopoietic progenitor cells. Human cord blood cells from 12 different donors were cultured in vitro for 6 days with interleukin-3 and stem cell factor. Phenotypic analysis indicated that hematopoietic cells were induced to cycle and the number of progenitors was expanded, thus making them targets for retroviral gene transfer. The cells were then transferred to SCID mice. Human hematopoietic progenitor cell engraftment was assessed up to 7 weeks later by growth of human progenitor cells in soft agar. After in vitro culture under conditions used for retroviral gene transfer, human cord blood hematopoietic cells engrafted the bone marrow and spleen of SCID mice. Interestingly, cultured cord blood cells engrafted after intraperitoneal but not after intravenous injection. Furthermore, engraftment of cord blood cells was observed in mice receiving no irradiation before transfer of the human cells, suggesting that competition for space in the marrow is not a limiting factor when these cells have been cultured. Administration of human cytokines after transfer of human cord blood cells to SCID mice was also not required for engraftment. Thus, engraftment of SCID mice with human hematopoietic cells cultured under conditions suitable for gene transfer may provide an in vivo assay for gene transfer to early human hematopoietic progenitor cells.

Pw1, a novel zinc finger gene implicated in the myogenic and neuronal lineages.

The cellular and molecular processes leading to the establishment of the skeletal muscle lineage in the vertebrate are not well understood. The MyoD-related family of myogenic regulatory factors (MRFs) are expressed during somitogenesis although cells with myogenic capacity are present prior to gastrulation. We propose that regulatory genes exist that guide the skeletal muscle lineage during early development. In an effort to identify these regulatory genes, we performed a differential screening to isolate transcripts that are present in myogenic cells and in the embryo prior to MRF expression but absent in nonmyogenic fibroblasts. We report here the identification of Pw1. The Pw1 transcript is approximately 8.5 kb long and encodes a large protein containing 12 widespread C2H2 zinc fingers and 3 motifs containing periodic prolines and acidic residues. Consistent with the possibility that Pw1 is a transcription factor, we observe nuclear localization of the protein. Pw1 is strongly expressed upon gastrulation and subsequently becomes restricted to skeletal muscle and subregions of the central nervous system. Pw1 is initially expressed in all mesodermal cells early in development; however, its maintained expression in adult differentiated muscle suggests a specific role in the skeletal muscle lineage. Pw1 expression is cell cycle specific with levels highest during late M-phase. The gene is intronless which may facilitate transcription during cell division. At present, the precise function of Pw1 is not understood; however, we note that Pw1 maps to the proximal region of chromosome 7 near the axial segmentation mutant pudgy which shows severe perturbation of axial skeletal and muscle structures.

Enhancement of vincristine cytotoxicity in drug-resistant cells by simultaneous treatment with onconase, an antitumor ribonuclease.

BACKGROUND: Onconase, a protein isolated from oocytes and early embryos of the frog Rana pipiens, shares extensive homology with bovine pancreatic ribonuclease (RNase A) and possesses similar enzyme activity. Onconase is cytotoxic toward cancer cells in vitro and exhibits antitumor activity in animal models. In addition, Onconase has been shown to enhance the cytotoxic activity of some chemotherapeutic agents in vitro. PURPOSE: We studied interactions between the cytotoxic effects of Onconase and the chemotherapeutic agent vincristine (VCR) in the treatment of drug-sensitive and multidrug-resistant human colon carcinoma cells in vitro and in mice. METHODS: Transplantable human colon carcinoma cells (HT-29par cells) were infected with a retrovirus containing human mdr1 (also known as MDR1 and PGY1) complementary DNA (encoding P-glycoprotein [P-gp]), and clones that were cross-resistant to colchicine, doxorubicin, and vinblastine were selected (HT-29mdr1 cells). Drug-resistant HT-29mdr1 cells and drug-sensitive HT-29par parental cells were treated with Onconase and/or VCR in vitro at varying concentrations to measure the effects on protein synthesis and cell viability. The impact of Onconase on VCR accumulation in both types of cells was determined in the presence or absence of MRK-16, an anti-P-gp monoclonal antibody capable of reversing the multidrug-resistant phenotype. The antitumor effects of Onconase and/or VCR treatment were assessed in nude mice bearing established HT-29par or HT-29mdr1 intraperitoneal tumors. IC50 values (drug concentrations resulting in 50% inhibition of protein synthesis or cell viability) for Onconase and VCR were determined from semilogarithmic dose-response curves; interactions between the cytotoxic effects of these two agents were evaluated using data from protein synthesis inhibition experiments and a two-way analysis of variance. Survival distributions from in vivo experiments were compared using Cox proportional hazards models. RESULTS: The combination of Onconase and VCR yielded enhanced cytotoxicity in vitro that was independent of P-gp expression. Evaluation of the effects of these two compounds on protein synthesis over a wide range of drug concentrations indicated possible synergistic interactions (i.e., greater than additive effects) in both drug-resistant and drug-sensitive cells. The enhancement of VCR cytotoxicity was dependent on Onconase enzyme activity and was not associated with increased intracellular levels of VCR. Simultaneous treatment of mice bearing HT-29par tumors with Onconase and VCR did not extend their median survival time (MST) significantly (MST with VCR = 66 days; MST with VCR plus Onconase = 69 days; two-tailed P = .57); however, the MST of mice with HT-29mdr1 tumors was extended significantly by this treatment (MST with VCR = 44 days; MST with VCR plus Onconase = 66 days; two-tailed P<.001). CONCLUSION: Combined administration of Onconase and VCR yields enhanced cytotoxicity in vitro and in vivo against human colon carcinoma cells that overexpress the mdr1 gene.

Targeted gene transfer to human hematopoietic progenitor cell lines through the c-kit receptor.

In this report, we describe a novel gene therapy approach for hematopoietic stem/progenitor cells using a specific receptor-mediated gene transfection procedure to target c-kit+ cell lines. The vector consists of plasmid DNA containing a luciferase reporter gene that is condensed by electrostatic forces with polylysine (PL) covalently linked to streptavidin (binds biotinylated ligand) and PL covalently linked to adenovirus (AD; to achieve endosomal lysis) with the final addition of biotinylated steel factor (SLF-biotin). Targeted transfection of growth factor-dependent hematopoietic progenitor cell lines that express c-kit showed specific luciferase gene expression over cell lines that did not express c-kit. This effect was dependent on the dose of SLF-biotin and was competed by excess SLF or with monoclonal antibodies that recognize c-kit and block the binding of SLF to its receptor. Maximum transfection efficiency (> 90%) requires a 2-hour incubation period of the vector with the cells, and maximum gene expression occurred 30 hours later. Removal of the endosomalytic agent, AD, from the vector resulted in the loss of gene expression. Vector targeting was versatile and could be changed by the addition of other biotinylated ligands. In principle, this vector should be broadly applicable to deliver genes to hematopoietic stem/progenitor cells in vitro and in vivo.

Engraftment of SCID mice by human bone marrow hematopoietic cells cultured in vitro: an in vivo model for human gene transfer.

Demonstration of the ability of fresh human hematopoietic cells to engraft severe combined immuno-deficient (scid) mice has provided an in vivo assay for expansion and maturation of early human progenitor cells. However, engraftment of cultured hematopoietic cells has been difficult to achieve. We wished to further develop this model as an in vivo assay for efficiency of retroviral gene transfer and expression in the differentiated progeny of adult human bone marrow progenitor cells. Human bone marrow cells were cultured in vitro for six days under conditions suitable for infection by retroviral vectors prior to transfer to irradiated scid mice. Cultured human bone marrow cells introduced by both intravenous (i.v.) and intraperitoneal (i.p.) injection persisted in the bone marrow, spleen and peritoneum of recipient animals up to four weeks after transfer. Following irradiation scid mice receiving cultured human bone marrow cells by either i.v. or i.p. routes demonstrated engraftment of the bone marrow and spleen as determined by the growth of human hematopoietic progenitors in soft agar. By flow cytometric analysis human cells were also detected in the peritoneum of mice receiving cultured human bone marrow cells i.p. These results suggest that the transfer of cultured human bone marrow cells to scid mice with the subsequent engraftment of these cells in the bone marrow, spleen and peritoneum of recipients can routinely occur. This provides an in vivo model for retroviral gene transfer to human cells.

First impressions count: a controlled investigation of social skill following closed head injury.

This study examined social skills in 14 closed head injured (CHI) patients who were assessed at four months post-injury and compared with 19 orthopaedic control (OC) patients. Social skills deficits were found to be more common in the CHI patients, of whom over half were classified as socially unskilled. CHI patients displayed poorer social skills in the earlier (but not later) part of an extended social interaction, indicating that they make a poor first impression. It is suggested that poor initial impression formation skills may be one reason why CHI patients fail to establish and maintain friendships. Implications of these findings for patient management are discussed. Close others of CHI patients reported higher levels of mood disturbance. Higher levels of close other hostility were associated with social skills deficits in CHI patients.

Genetic localization of Hao-1, blind-sterile (bs), and Emv-13 on mouse chromosome 2.

The blind-sterile (bs) mutation in the mouse was localized on Chromosome 2 between Hao-1 and Emv-13. N2 progeny from a backcross between congenic female 129.AKR-bs Emv-13 mice and (129.AKR-bs/bs x Mus musculus molossinus) F1 male mice were typed by analysis of isozyme variants for Hao-1, visible inspection for bs, and restriction fragment length polymorphism for Emv-13 and Emv-15. Comparison between markers on mouse Chromosome 2 and corresponding markers on human chromosomes suggest that the human homolog of bs will be located on 20q11-q13.

Spontaneous germ line virus infection and retroviral insertional mutagenesis in eighteen transgenic Srev lines of mice.

SWR/J-RF/J hybrid mice spontaneously acquire new germ line ecotropic proviruses at high frequency. In the studies described here, we used these hybrids to produce 18 transgenic mouse lines, each carrying a single newly acquired Srev locus (SWR/J-RF/J ecotropic proviral locus). All of the newly acquired proviruses identified in mosaic founder SWR/J-RF/J mice that could be transmitted through the germ line were also present in somatic tissues, demonstrating that viral integration occurred before the germ line was set aside from the somatic lineages. Quantitative analysis of proviral DNA copy numbers in somatic and germinal tissues of mosaic founder parents combined with structural analysis of Srev loci indicated that these proviruses are acquired after multiple rounds of somatic viral reinfection and that most of these viral integration events occurred after DNA replication in the zygote and before DNA replication in the four-cell embryo. The frequency of provirus acquisition in Srev lines that expressed the infectious ecotropic virus was similar to that in SWR.RF mice carrying Emv-16 and Emv-17, suggesting that the chromosomal integration site of the parental locus is not an important determinant for high-frequency provirus acquisition. The frequency of recessive lethal mutations induced by spontaneous viral integration was 5%, which was similar to that induced by preimplantation embryo infection. This approach represents a simple and viable strategy for inducing and studying mutations that affect mammalian development.

Localization and characterization of members of a family of repetitive sequences in the goat beta globin locus.

We have characterized a family of repetitive DNA elements in the beta-globin locus of the goat. These sequences are structurally analogous to the Alu families of repeats of other mammals. Repetitive elements are located both in the intervening sequences and in the intergenic regions of the goat beta-globin locus. Nucleotide sequence analysis of five repetitive elements located within the large intervening sequence of the beta-like globin genes, and four repeats located 5' to the major developmentally regulated beta-globin genes has resulted in the definition of a consensus sequence for this family of repeats.

Organization, structure, and expression of the goat globin genes.

Several hemoglobin switches occur during the development of the goat, making this a useful animal for the study of globin gene expression. In order to help understand the basis for these switches, we have isolated the beta-globin genes of the goat by recombinant DNA technology and characterized these genes with respect to linkage, nucleotide sequence, and expression. The linkage arrangement so far established is epsilon I-epsilon II-psi beta X-beta C-epsilon III-epsilon IV-psi beta Z-beta A-epsilon V. It is proposed that epsilon V is followed by epsilon VI-psi beta-gamma, but so far this linkage has not been established. Several conclusions can be drawn from our findings to date. First, the beta- and gamma-globin genes of the goat have a very different evolutionary history from the beta- and gamma-globin genes of humans. While the beta and gamma genes of the human can be traced to a duplication of the ancestral epsilon/beta-globin gene before the mammalian radiation, the goat beta and gamma genes have arisen much later, and are probably the results of a duplication of a four-gene set, namely the epsilon-epsilon-psi beta-beta primordial linkage group. The beta C gene probably arose from a similar, even later duplication of the non-gamma quadruplet. Because the beta C, beta A, and gamma genes of the goat have diverged much more recently in evolution, they are much more homologous than the equivalent genes in other species. In fact, there are large regions of these genes that share identical sequences. This is meaningful in that regions of sequence identity define areas that cannot be involved in the developmental regulation of these genes.(ABSTRACT TRUNCATED AT 250 WORDS)

Five nucleotide changes in the large intervening sequence of a beta globin gene in a beta+ thalassemia patient.

A beta globin gene from a patient with homozygous beta+ thalassemia has been cloned and completely sequenced. No changes from normal are found in the 200 nucleotides 5' to the cap site, in the 3' untranslated region up to the poly A addition site, in the small intervening sequence (IVS 1), or in the coding sequence except for a third base change in codon 2. The only other differences are in the large intervening sequence (IVS 2). One of these, at a position 16 nucleotides from the 5' end of IVS 2, has been reported previously in normal individuals, and is probably a polymorphism. Four other changes, at positions 74, 81, 666, and 705 are also seen in IVS 2. Abnormal beta globin mRNA precursors detected in the bone marrow cells of this patient, and abnormal beta globin RNA splicing observed when this gene is transcribed in a tissue culture system taken together with these IVS 2 changes, suggest that the beta+ thalassemia phenotype is produced by a decrease in normal beta globin mRNA processing.