Velocity-tunable beam of continuously decelerated polar molecules for cold ion-molecule reaction studies.

Producing high densities of molecules is a fundamental challenge for low-temperature, ion-molecule reaction studies. Traveling-wave Stark decelerators promise to deliver high density beams of cold, polar molecules but require non-trivial control of high-voltage potentials. We have overcome this experimental challenge and demonstrate continuous deceleration of ND3 from 385 to 10 m/s, while driving the decelerator electrodes with a 10 kV amplitude sinewave. In addition, we test an alternative slowing scheme, which increases the time delay between decelerated packets of ND3 and non-decelerated molecules, allowing for better energy resolution of subsequent reaction studies. We characterize this source of neutral, polar molecules suitable for energy-resolved reaction studies with trapped ions at cold translational temperatures. We also propose a combined apparatus consisting of the traveling-wave decelerator and a linear ion trap with a time-of-flight mass spectrometer and discuss to what extent it may achieve cold, energy-resolved, ion-neutral reactions.

Collisions between Ultracold Molecules and Atoms in a Magnetic Trap.

We prepare mixtures of ultracold CaF molecules and Rb atoms in a magnetic trap and study their inelastic collisions. When the atoms are prepared in the spin-stretched state and the molecules in the spin-stretched component of the first rotationally excited state, they collide inelastically with a rate coefficient k_{2}=(6.6±1.5)×10^{-11} cm^{3}/s at temperatures near 100 µK. We attribute this to rotation-changing collisions. When the molecules are in the ground rotational state we see no inelastic loss and set an upper bound on the spin-relaxation rate coefficient of k_{2}<5.8×10^{-12} cm^{3}/s with 95% confidence. We compare these measurements to the results of a single-channel loss model based on quantum defect theory. The comparison suggests a short-range loss parameter close to unity for rotationally excited molecules, but below 0.04 for molecules in the rotational ground state.

Long Rotational Coherence Times of Molecules in a Magnetic Trap.

Polar molecules in superpositions of rotational states exhibit long-range dipolar interactions, but maintaining their coherence in a trapped sample is a challenge. We present calculations that show many laser-coolable molecules have convenient rotational transitions that are exceptionally insensitive to magnetic fields. We verify this experimentally for CaF where we find a transition with sensitivity below 5 Hz G^{-1} and use it to demonstrate a rotational coherence time of 6.4(8) ms in a magnetic trap. Simulations suggest it is feasible to extend this to more than 1 s using a smaller cloud in a biased magnetic trap.

Deep Laser Cooling and Efficient Magnetic Compression of Molecules.

We introduce a scheme for deep laser cooling of molecules based on robust dark states at zero velocity. By simulating this scheme, we show it to be a widely applicable method that can reach the recoil limit or below. We demonstrate and characterize the method experimentally, reaching a temperature of 5.4(7) µK. We solve a general problem of measuring low temperatures for large clouds by rotating the phase-space distribution and then directly imaging the complete velocity distribution. Using the same phase-space rotation method, we rapidly compress the cloud. Applying the cooling method a second time, we compress both the position and velocity distributions.

A high-voltage amplifier for traveling-wave Stark deceleration.

Controlling high voltage is a critical aspect of Stark deceleration, a technique that uses electric fields to produce slow molecules. Traditionally, Stark deceleration required only commercial high-voltage switches to operate. However, a new continuous version of Stark deceleration, which promises significantly improved performance, requires chirped sinusoidal voltages. The complexity of the electronics needed to run this new decelerator has restricted the implementation for all but a few groups. The challenge is to create high-voltage amplifiers that have a frequency range of tens of kilohertz down to DC and that can source and sink enough current (∼1 A) to drive the capacitive load of the decelerator. We present a new high-voltage linear amplifier for driving in-vacuum electrodes for traveling-wave Stark deceleration. The amplifier has a gain of 12 000, output voltages up to ±10 kV, instantaneous currents up to 1.5 A, and a frequency range from 30 kHz down to DC. This makes the amplifier suitable for traveling-wave Stark deceleration of a supersonic molecular beam down to rest.

Magnetic Trapping and Coherent Control of Laser-Cooled Molecules.

We demonstrate coherent microwave control of the rotational, hyperfine, and Zeeman states of ultracold CaF molecules, and the magnetic trapping of these molecules in a single, selectable quantum state. We trap about 5×10^{3} molecules for almost 2 s at a temperature of 70(8) µK and a density of 1.2×10^{5} cm^{-3}. We measure the state-specific loss rate due to collisions with background helium.

Laser Cooled YbF Molecules for Measuring the Electron's Electric Dipole Moment.

We demonstrate one-dimensional sub-Doppler laser cooling of a beam of YbF molecules to 100 µK. This is a key step towards a measurement of the electron's electric dipole moment using ultracold molecules. We compare the effectiveness of magnetically assisted and polarization-gradient sub-Doppler cooling mechanisms. We model the experiment and find good agreement with our data.

Principles and Design of a Zeeman-Sisyphus Decelerator for Molecular Beams.

We explore a technique for decelerating molecules using a static magnetic field and optical pumping. Molecules travel through a spatially varying magnetic field and are repeatedly pumped into a weak-field seeking state as they move towards each strong field region, and into a strong-field seeking state as they move towards weak field. The method is time-independent and so is suitable for decelerating both pulsed and continuous molecular beams. By using guiding magnets at each weak field region, the beam can be simultaneously guided and decelerated. By tapering the magnetic field strength in the strong field regions, and exploiting the Doppler shift, the velocity distribution can be compressed during deceleration. We develop the principles of this deceleration technique, provide a realistic design, use numerical simulations to evaluate its performance for a beam of CaF, and compare this performance to other deceleration methods.

Large effects of electric fields on atom-molecule collisions at millikelvin temperatures.

Controlling interactions between cold molecules using external fields can elucidate the role of quantum mechanics in molecular collisions. We create a new experimental platform in which ultracold rubidium atoms and cold ammonia molecules are separately trapped by magnetic and electric fields and then combined to study collisions. We observe inelastic processes that are faster than expected from earlier field-free calculations. We use quantum scattering calculations to show that electric fields can have a major effect on collision outcomes, even in the absence of dipole-dipole interactions.

Experimental demonstration of classical Hamiltonian monodromy in the 1:1:2 resonant elastic pendulum.

The 1:1:2 resonant elastic pendulum is a simple classical system that displays the phenomenon known as Hamiltonian monodromy. With suitable initial conditions, the system oscillates between nearly pure springing and nearly pure elliptical-swinging motions, with sequential major axes displaying a stepwise precession. The physical consequence of monodromy is that this stepwise precession is given by a smooth but multivalued function of the constants of motion. We experimentally explore this multivalued behavior. To our knowledge, this is the first experimental demonstration of classical monodromy.

Human brain cytochrome P450 1B1: immunohistochemical localization in human temporal lobe and induction by dimethylbenz(a)anthracene in astrocytoma cell line (MOG-G-CCM).

CYP1B1, a new member of human cytochrome P450 family 1, is involved in the xenobiotic detoxification metabolism and possibly activation of numerous procarcinogens and promutagens. Localization of CYP1B1 in human temporal lobe and its induction in astrocytoma cell line (MOG-G-CCM) by 7,12-dimethylbenz(a)anthracene (DMBA) was investigated using antibodies against human CYP1B1. A single band of approximately 58 kDa size in both human temporal lobe and in MOG-G-CCM was detected by Western blot analysis. Treatment of MOG-G-CCM cells with DMBA resulted in approximately 2.8-fold induction of CYP1B1. CYP1B1 immunoreactivity was detected at the blood-brain interface areas of the temporal lobe as evidenced by co-localization with CD34 antigen. These results suggest that this enzyme may be important in brain xenobiotic metabolism acting as an enzymatic barrier.

In vivo compartmentalization of human immunodeficiency virus: evidence from the examination of pol sequences from autopsy tissues.

High rates of mutation and replication of human immunodeficiency virus (HIV) allow for the continuous generation of diverse genetic variants in vivo. Selective pressures within the microenvironments of different anatomic compartments result in the emergence of dominant quasispecies which can be distinguished by their envelope sequences. It is not known whether comparable tissue-specific selective pressures lead to the independent evolution of pol sequences within different tissue compartments, nor is it known how differing rates of virus turnover in tissues might affect the pace of such evolution. These issues are of importance for the formulation of a model for the emergence of drug resistance in vivo and for a general understanding of virus trafficking and virus turnover. Regions of the HIV type 1 reverse transcriptase (RT) which carry the majority of the known resistance codons to RT inhibitors (700 nucleotides from each clone) were cloned and sequenced directly from autopsied brain, spleen, and lymph node specimens from four subjects who had received zidovudine therapy. Clones from proviral DNA (143) and from viral cDNA (14) were analyzed. In three of four subjects, a discordance in distribution of resistance codons was noted. Moreover, brain-derived sequences appeared to be phylogenetically distinct from spleen- and lymph node-derived sequences even after exclusion of resistance codons from analysis. In each case, evidence for differential immune selective pressure, based on comparison of inferred amino acid sequences corresponding to known major histocompatibility complex class I cytotoxic T-lymphocyte epitopes, was found. These observations support the concept of anatomically distinct, independently evolving quasispecies (virodemes).

Human cysteine dioxygenase type I (CDO-I; EC 1.13.11.20): 5' flanking region and intron-exon structure of the gene.

AIM: The elucidation of the structure of the 5' flanking region and the exonic organisation of the human cysteine dioxygenase type I gene. METHODS: Material for sequence studies was generated by polymerase chain reaction (PCR) methods using human genomic DNA, a cosmid clone containing the entire gene, and a commercial gene walking kit. RESULTS AND CONCLUSIONS: The gene was found to be--12 kb in length and made up of five exons (418, 78, 155, 170, and 731 base pairs, respectively). The immediate 5' flanking region did not contain the canonical TATAA box. One DNA source (Clontech promoter finder kit) contained a liver specific nuclear factor response element approximately 550 base pairs from the transcription start site, whereas a second source did not. This and other cis acting factors in the 5' flanking region were identified by computer analysis. The physiological significance of such elements requires detailed experimental evaluation.

Producer-cell modification of human immunodeficiency virus type 1: Nef is a virion protein.

Type 1 human immunodeficiency viruses encoding mutated nef reading frames are 10- to 30-fold less infectious than are isogenic viruses in which the nef gene is intact. This defect in infectivity causes nef-negative viruses to grow at an attenuated rate in vitro. To investigate the mechanism of Nef-mediated enhancement of viral growth rate and infectivity, a complementation analysis of nef mutant viruses was performed. To provide Nef in trans upon viral infection, a CEM derivative cell line (designated CLN) that expresses Nef under the control of the viral long terminal repeat was constructed. When nef-negative virus was grown in CLN cells, its growth rate was restored to wild-type levels. However, the output of nef-negative virus during the first 72 h after infection of CLN cells was not restored, suggesting that provision of Nef within the newly infected cell does not enhance the productivity of a nef-negative provirus. The genetically nef-negative virions produced by the CLN cells, however, were restored to wild-type levels of infectivity as measured in a syncytium formation assay in which CD4-expressing HeLa cells were targets. These trans-complemented, genetically nef-negative virions yielded wild-type levels of viral output following a single cycle of replication in primary CD4 T cells as well as in parental CEM cells. To define the determinants for producer cell modification of virions by Nef, the role of myristoylation was investigated. Virus that encodes a myristoylation-negative nef was as impaired in infectivity as was virus encoding a deleted nef gene. Because myristoylation is required for both membrane association of Nef and optimal viral infectivity, the possibility that Nef protein is included in the virion was investigated. Wild-type virions were purified by filtration and exclusion chromatography. A Western blot (immunoblot) of the eluate fractions revealed a correlation between peak Nef signal and peak levels of p24 antigen. Although virion-associated Nef was detected in part as the 27-kDa full-length protein, the majority of immunoreactive protein was detected as a 20-kDa isoform. nef-negative virus lacked both 27- and 20-kDa immunoreactive species. Production of wild-type virions in the presence of a specific inhibitor of the human immunodeficiency virus type 1 protease resulted in virions which contained only 27-kDa full-length Nef protein. These data indicate that Nef is a virion protein which is processed by the viral protease into a 20-kDa isoform within the virion particle.

The effects of ethanol, 3,5,3'-triiodothyronine (T3) and oestradiol (E2) on thyroid hormone nuclear receptor expression in human hepatocyte primary cultures.

The possibility that ethanol regulated the expression of thyroid hormone receptor mRNA levels was explored in a model involving human hepatocyte primary culture. Ethanol (20, 50 and 100 mM for 24 h was found to have no effect on the steady-state levels of either triiodothyronine (T3) receptor alpha 1 or alpha 2 mRNAs. In contrast both T3 (1, 10 and 100 nM) and oestradiol (0.1, 1 and 10 ng/ml) treatments affected either one or both of these mRNA levels in a complex manner, showing that the model was capable of responding to other stimuli. Triiodothyronine receptor beta 1 mRNA was not assayed. The hypermetabolic effects of long-term ethanol consumption in humans appears not to be due to the direct effect of ethanol on the regulation of these receptors.

Cell surface CD4 downregulation and resistance to superinfection induced by a defective provirus of HIV-1.

Proviral sequences encoding defective HIV-1 regulatory genes have been detected previously in infected individuals; however, the role of these defective genomes in pathogenesis is unclear. The hypothesis that such replication-defective genomes might induce downregulation of the cellular receptor for HIV-1 (CD4) was tested. CEM cells were stably transfected with a provirus that contains a mutation in the splice site immediately 5' of the rev coding sequence. This mutant expresses early HIV-1 mRNAs but is defective for replication. Cells expressing this defective provirus displayed markedly reduced surface CD4. This downregulation of CD4 was dependent on an intact nef gene and was sufficient to cause resistance to superinfection by wild-type virus. These findings indicate that Nef as expressed by replication-defective HIV-1 can downregulate CD4. This perturbation of the T lymphocyte cell membrane is a potential basis for pathogenicity of defective HIV-1.

Optimal infectivity in vitro of human immunodeficiency virus type 1 requires an intact nef gene.

The replication competence of human immunodeficiency virus type 1 genomes containing mutations in the nef open reading frame was evaluated in continuous cell lines. Mutants that contained a deletion in the nef open reading frame, premature termination codons, or missense mutations in the N-terminal myristoylation signal were constructed. The replication of these mutants was tested in three ways. First, plasmid genomes were used to transfect T-lymphoblastoid cells. Second, low-passage posttransfection supernatants were used to infect cells with a relatively low virus input. Third, high-titer virus stocks were used to infect cells with a relatively high virus input. These experiments demonstrated a 100- to 10,000-fold decrement in p24 production by the nef mutants compared with that by the wild-type virus. The greatest difference was obtained after infection with the lowest virus input. The myristoylation signal was critical for this positive effect of nef. To investigate the mechanism of the positive influence of nef, nef-positive and nef-minus viruses were compared during a single cycle of replication. These single-cycle experiments were initiated both by infection with high-titer virus stocks and by transfection with viral DNA. Single-cycle infection yielded a three- to fivefold decrement in p24 production by nef-minus virus. Single-cycle transfection yielded equal amounts of p24 production. These results implied that nef does not affect replication after the provirus is established. In support of these results, viral production from cells chronically infected with nef-positive or nef-minus viruses was similar over time. To determine whether the effect of nef was due to infectivity, end point titrations of nef-positive and nef-minus viruses were performed. nef-positive virus had a greater infectivity per picogram of HIV p24 antigen than nef-minus virus. These data indicated that the positive influence of nef on viral growth rate is due to an infectivity advantage of virus produced with an intact nef gene.

The structure of the human thyroxine binding globulin (TBG) gene.

A portion of the human X-chromosome (> 5 kb) encoding the translated portion of the thyroxine-binding globulin (TBG) gene was sequenced. The primary templates for sequencing were isolated from a human X-chromosome library (two positive plaques from 400,000 screened initially with a TBG cDNA probe) or were produced by PCR amplification using leucocyte genomic DNA as the amplification template. Potential hormone response elements (HREs) were identified at either end of the gene. These HREs have sequences based on the consensus half-site of thyroid hormone response elements, although it is unclear whether the structures are functional HREs. Other potential regulatory elements also were identified towards the 3' end of the gene.

Familial nephropathic non-neuropathic amyloidosis: clinical features, immunohistochemistry and chemistry.

Classification of familial amyloidosis by the chemical nature of the fibrillar protein has become possible. Most such amyloidogenic proteins so far recognized are variant transthyretins, but two kindreds with the same apolipoprotein AI modification have been reported. We describe the clinical features of another such family in whom petechial skin rash appeared to be a marker for the disease, which was non-neuropathic and of the Ostertag-type. Immunohistochemistry showed the protein to be apolipoprotein AI, but allele-specific DNA amplification indicated that it was not the Arg26 variant previously identified.

An inherited non-amyloidogenic transthyretin variant, [Ser6]-TTR, with increased thyroxine-binding affinity, characterized by DNA sequencing.

Amplification and sequencing of the four transthyretin (TTR) exons of a subject with a variant TTR with four-fold increased affinity for thyroxine revealed a heterozygous G to A point mutation at base 7 of exon 2. This results in a serine for glycine change at residue 6 of the mature TTR monomer. No other mutations were found in any exon. Amplification and MspI digestion of TTR exon 2 from the leucocyte DNA of eight members of the subject's family revealed that all but one member were also heterozygous for [Ser6]-TTR.