Search

We report integration of an InAs quantum well micro-Hall magnetic sensor with microfluidics and real-time detection of moving superparamagnetic beads. Beads moving within and around the Hall cross area result in positive and negative Hall voltage signals respectively. Relative magnitudes and polarities of the signals measured for a random distribution of immobilized beads over the sensor are in good agreement with calculated values and explain consistently the shape of the dynamic signal.

Induced pluripotent stem cells (iPSCs) may be obtained by direct reprogramming of different somatic cells to a pluripotent state by forced expression of a handful of transcription factors. It was generally assumed that iPSCs are functionally equivalent to their embryonic stem cell (ESC) counterparts. Recently, a number of research groups have demonstrated that this is not the case, showing that iPSCs retain 'epigenetic memory' of the donor tissue from which they were derived and display skewed differentiation potential. This raises the question whether such cells are fit for experimental, diagnostic or therapeutic purpose. A brief survey of the literature illustrates that differences at both epigenetic and transcriptome level are observed between various pluripotent stem cell populations. Interestingly, iPSC populations with perceived 'anomalies' can be coaxed to a more ESC-like cellular state either by continuous passaging--which attenuates these epigenetic differences--or treatment with small molecules that target the machinery responsible for remodelling the genome. This suggests that the establishment of an epigenetic status approximating an ESC counterpart is largely a passive process. The mechanisms responsible remain to be established. Meanwhile, other areas of reprogramming are rapidly evolving such as, trans-differentiation of one somatic cell type to another by the forced expression of key transcription factors. When it comes to assessing their practical usefulness, the same question will also apply.

We have measured the room temperature response of nanoscale semiconductor Hall crosses to local applied magnetic fields under various local electric gate conditions using scanning probe microscopy. Near-surface quantum wells of AlSb/InAs/AlSb, located just 5 nm from the heterostructure surface, allow very high sensitivity to localized electric and magnetic fields applied near the device surfaces. The Hall crosses have critical dimensions of 400 and 100 nm, while the mean free path of the carriers is about 160 nm; hence the devices nominally span the transition from diffusive to quasi-ballistic transport. With certain small gate voltages (V(g)) the devices of both sizes are strongly responsive to the local magnetic field at the center of the cross, and the results are well described using finite element modeling. At high V(g), the response to local magnetic fields is greatly distorted by strong electric fields applied near the cross corners. However we observe no change in behavior with the size of the device.

Human ribosomal gene repeats are distributed among five nucleolar organizer regions (NORs) on the p arms of acrocentric chromosomes. On exit from mitosis, nucleoli form around individual active NORs. As cells progress through the cycle, these mini-nucleoli fuse to form large nucleoli incorporating multiple NORs. It is generally assumed that nucleolar incorporation of individual NORs is dependent on ribosomal gene transcription. To test this assumption, we determined the nuclear location of individual human acrocentric chromosomes, and their associated NORs, in mouse> human cell hybrids. Human ribosomal genes are transcriptionally silent in this context. Combined immunofluorescence and in situ hybridization (immuno-FISH) on three-dimensional preserved nuclei showed that human acrocentric chromosomes associate with hybrid cell nucleoli. Analysis of purified nucleoli demonstrated that human and mouse NORs are equally likely to be within a hybrid cell nucleolus. This is supported further by the observation that murine upstream binding factor can associate with human NORs. Incorporation of silent NORs into mature nucleoli raises interesting issues concerning the maintenance of the activity status of individual NORs.

Transcription of Xenopus ribosomal genes by RNA polymerase I is directed by a stable transcription complex that forms on the gene promoter. This complex is comprised of the HMG box factor UBF and the TBP-containing complex Rib1. Repeated sequence elements found upstream of the ribosomal gene promoter act as RNA polymerase I-specific trans-criptional enhancers. These enhancers function by increasing the probability of a stable transcription complex forming on the adjacent promoter. UBF is required for enhancer function. This role in enhancement is distinct from that at the promoter and does not involve translocation of UBF from enhancer repeats to the promoter. Here we utilize an in vitro system to demonstrate that a combination of the dimerization domain of UBF and HMG boxes 1-3 are sufficient to specify its role in enhancement. We also demonstrate that the acidic C-terminus of UBF is primarilyresponsible for its observed interaction with Rib1. Thus, we have uncoupled the Rib1 interaction and enhancer functions of UBF and can conclude that direct interaction with Rib1 is not a prerequisite for the enhancer function of UBF.

Repeated sequence elements found upstream of the ribosomal gene promoter in Xenopus function as RNA polymerase I-specific transcriptional enhancers. Here we describe an in vitro system in which these enhancers function in many respects as in vivo. The principal requirement for enhancer function in vitro is the presence of a high concentration of upstream binding factor (UBF). This system is utilized to demonstrate that enhancers function by increasing the probability of a stable transcription complex forming on the adjacent promoter. Species differences in UBF are utilized to demonstrate that enhancers do not act by recruiting UBF to the promoter, rather UBF performs its own distinct role at the enhancers. UBF function in enhancement differs from that at the promoter, as it is flexible with respect to both the species of UBF and the enhancer element employed. Additionally, we identify a potential role for the mammalian UBF splice variant, UBF2, in enhancer function. We demonstrate that the TATA box binding protein (TBP)-containing component of Xenopus RNA polymerase I transcription, Rib1, can interact with an enhancer-UBF complex. This suggests a model in which enhancers act by recruiting Rib1 to the promoter.

The quadtree data structure is commonly used in image coding to decompose an image into separate spatial regions to adaptively identify the type of quantizer used in various regions of an image. The authors describe the theory needed to construct quadtree data structures that optimally allocate rate, given a set of quantizers. A Lagrange multiplier method finds these optimal rate allocations with no monotonicity restrictions. They use the theory to derive a new quadtree construction method that uses a stepwise search to find the overall optimal quadtree structure. The search can be driven with either actual measured quantizer performance or ensemble average predicted performance. They apply this theory to the design of a motion compensated interframe video coding system using a quadtree with vector quantization.

We present an estimation-theoretic analysis of motion compensation that, when used with fields of block-based motion vectors, leads to the development of overlapped block algorithms with improved compensation accuracy. Overlapped block motion compensation (OBMC) is formulated as a probabilistic linear estimator of pixel intensities given the limited block motion information available to the decoder. Although overlapped techniques have been observed to reduce blocking artifacts in video coding, this analysis establishes for the first time how (and why) OBMC can offer substantial reductions in prediction error as well, even with no change in the encoder's search and no extra side information. Performance can be further enhanced with the use of state variable conditioning in the compensation process. We describe the design of optimized windows for OBMC. We also demonstrate how, with additional encoder complexity, a motion estimation algorithm optimized for OBMC offers further significant gains in compensation accuracy. Overall mean-square prediction improvements in the range of 16 to 40% (0.8 to 2.2 dB) are demonstrated.

A pruning algorithm of P.A. Chou et al. (1989) for designing optimal tree structures identifies only those codebooks which lie on the convex hull of the original codebook's operational distortion rate function. The authors introduce a modified version of the original algorithm, which identifies a large number of codebooks having minimum average distortion, under the constraint that, in each step, only modes having no descendents are removed from the tree. All codebooks generated by the original algorithm are also generated by this algorithm. The new algorithm generates a much larger number of codebooks in the middle- and low-rate regions. The additional codebooks permit operation near the codebook's operational distortion rate function without time sharing by choosing from the increased number of available bit rates. Despite the statistical mismatch which occurs when coding data outside the training sequence, these pruned codebooks retain their performance advantage over full search vector quantizers (VQs) for a large range of rates.

A repetitive element from the genome of Leptospira interrogans serovar hardjo type hardjo-bovis ('L. hardjo-bovis') was identified, cloned and sequenced. Similar sequences were shown by hybridization to be encoded by a further eight of 32 other leptospiral serovars tested. An undefined number of repetitive elements were located in the L. hardjo-bovis genome; sequence degeneracy of the elements was observed and no significant open reading frames were identified within the AT-rich (60%) 1467 bp repetitive element. The termini encoded a GC-rich 8 bp repeat motif and two variants showed rearrangements centred on these motifs. The nucleotide sequences of the chromosomal regions flanking the repetitive elements were determined but showed no similarities, with one exception which had a GAAC repeat directly adjacent to both termini. Similar hybridization patterns were shown by Southern transfers of L. hardjo-bovis total genomic digests probed with the repetitive element. Oligonucleotide primer pairs designed from sequences internal to the repetitive element and adjacent chromosomal regions were used in polymerase chain reaction experiments. With one primer pair all L. hardjo-bovis isolates, but no other serovar, gave identical amplified products. Evidence that the repetitive element may have derived from an acquired insertion sequence that is now inactive and chromosomally fixed is discussed.

Previous studies established that Mycoplasma arthritidis produces a soluble T cell mitogen (MAM), and that response of murine T cells to MAM is genetically restricted. MAM appeared predominantly in the supernatants of senescent cultures, but was not extracted in significant amounts from whole cells. A quantitative assay of MAM activity was devised. MAM formed noncovalent complexes with nucleic acids and uncharacterized high m.w. constituents of sera and of complex media. Partially purified MAM was adsorbed or denatured by glass and plastic surfaces. MAM was protease-labile, had pI greater than or equal to 9, and had Mr ca 15,000 according to gel filtration experiments. MAM was a very minor component of culture supernatant proteins, and even after 200- to estimated 5 X 10(4)-fold purification was not identified as a stainable or ultraviolet-absorbing entity in electrophoretigrams or chromatograms. It was estimated that MAM was half-optimally active at less than 1000th the half-optimal concentration of concanavalin A or phytohemagglutinin. Culture supernatants and highly purified MAM exhibited the same haplotype specificity (H-2k-dependent response) for stimulated proliferation of lymphocytes and for induction of interferon in vitro.

The mitogenic property of supernatants from M. arthritidis cultures (MAS) is shown to be nonsedimentable, nondialyzable, labile to 56 degrees C for 1 hr, and active against spleen cells from both normal and germfree mice. Both viable M. arthritidis and MAS were active for T lymphocytes because anti-Thy-1 antiserum and C eliminated responsiveness. Spleen cells enriched for T lymphocytes by passage over nylon columns lost responsiveness unless supplemented with a radioresistant adherent cell population that was shown to bear Ia antigens. Evidence is also presented that the genetic control of T lymphocyte responses to the mycoplasma mitogen was exercised at the level of the Ia-bearing adherent cells. Thus adherent cells from positive responder mouse strains, but not those from nonresponder mouse strains, restored the responses of T cells from F1 hybrids between responder and nonresponder strains.

A comparison of splenic cells from various inbred rat strains indicated that DA, Lewis, Buffalo, August, Wistar Furth, and (LEW X BN)F1 all responded well to the Mycoplasma arthritidis T cell mitogen, phytohemagglutinin and concanavalin A, but cells from BN and MAXX rats were very weakly or nonresponsive. Cells from congenic strains expressing nonresponder background genes, and responder haplotypes at RT1 (BN.1L(LEW), RT1; BN.1A(DA), RT1av1) failed to respond significantly to the mitogens. Rats expressing responder background genes but the nonresponder haplotype at RT1 at RT1 (WF.1N-(MAXX), RT1n) exhibited high responses to all mitogens. The controlling role of non-RT1 genes was confirmed by testing tissue-typed (DA X BN)F2 progeny and (DA X BN)F1 X DA and (DA X BN)F1 X BN progeny. No association was seen between the expression of a/a, a/n, or n/n at RT1 and the degree of response to the mitogens. In contrast, as the proportion of DA non-RT1 genes increased, so did the degree of mitogenic responsiveness. Similar results were obtained by using a partially purified preparation of the mycoplasma T cell mitogen. The results indicated that in the (DA X BN)F1 hybrids, responsiveness to all mitogens was recessive: this contrasts with the (LEW X BN)F1 hybrids in which responsiveness was dominant. Finally, we showed that both responder and nonresponder splenic cells were capable of binding the M. arthritidis mitogen. The data contrast with those obtained with nonresponder mouse strains the cells of which failed to bind mitogen due to the absence of the E alpha chain of the I-E-coded molecule.

Previous work using inbred, congenic and recombinant mouse strains showed a positive association with expression of E alpha and the ability of splenic cells to bind to and undergo proliferation in response to a T cell mitogen present in culture supernatants of Mycoplasma arthritidis (MAS). Studies described in the present manuscript confirm this association because lymphocytes from mice expressing H-2a, H-2d, H-2j, H-2k, H-2p, H-2u, and H-2v all of which possess E alpha responded to MAS, whereas those expressing H-2b, H-2f, H-2q, and H-2s, which lack E alpha, failed to respond. One exception was noted in that the inbred RIIIS mouse (H-2r) that expresses E alpha failed to respond to MAS but responded normally to concanavalin A, and phytohemagglutinin. In contrast, the congenic B10.RIII (H-2r) mouse did respond to MAS, suggesting the presence of an MAS nonresponsive, non-major histocompatibility complex (MHC) gene(s) in the RIIIS mouse. MAS nonresponsiveness in the RIIIS mouse was recessive because the lymphocytes from F1 crosses with responder B10.RIII (H2r) and C3H (H2k) mice responded to MAS. Analysis of (RIIIS X B10.RIII)F1 X RIIIS or B10.RIII parental test cross progeny confirmed that nonresponsiveness to MAS was associated with a recessive, non-MHC gene(s). Evidence was also found that a non-MHC, MAS-nonresponsive gene(s) is also present in the inbred SWR (H-2q) and SJL (H-2s) strains, because lymphocytes from F1 crosses between these strains and the RIIIS mouse failed to respond to MAS. Both RIIIS and B10.RIII splenic cells bound the mitogen in MAS to a similar degree, confirming the presence of the binding site in both mice. In contrast, C3H.SW (H-2b) splenic cells that do not express E alpha failed to bind the mitogen. The nonresponsiveness of RIIIS lymphocytes to MAS was exercised at the level of the T cell rather than the accessory cell. Thus RIIIS T cells failed to respond to MAS presented by RIIIS, B10.RIII, or (RIIIS X B10.RIII)F1 accessory cells. In contrast, B10.RIII and (RIIIS X B10.RIII)F1 T cells responded to MAS when presented by RIIIS, B10.RIII, or F1 accessory cells. Similar observations were made using SWR and SJL T cells, which failed to respond to MAS irrespective of the source of accessory cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Spleen lymphocytes from Lewis and Buffalo rats and peripheral blood lymphocytes from 10 human donors exhibited high levels of transformation when exposed to Mycoplasma arthritidis supernatants. In contrast, spleen lymphocytes from rabbits and guinea pigs and peripheral blood lymphocytes from sheep and calves failed to transform when exposed to M. arthritidis supernatants. The lymphocytes from all hosts were transformed in the presence of phytohemagglutinin or concanavalin A or both Serological studies failed to provide evidence that the responding hosts were presensitized against M. arthritidis antigens.

Syngeneic and allogeneic target cells were killed in the presence of CBA mouse lymphocytes and viable Mycoplasma arthritidis. Medium supplementation had no effect on the response. Nonviable M. arthritidis was also capable of stimulating lymphocytotoxicity, although to a much lesser extent. Cytotoxicity was shown to be largely dependent upon the lymphocytes, since lymphocytes preincubated with mycoplasmas and treated to remove remaining organisms were highly toxic to target cells, whereas supernatants prepared from lymphocyte/mycoplasma mixtures exhibited minimal effects. A 6-h exposure of lymphocytes to mycoplasmas at a ratio of 100:1 was sufficient for commitment to target cell killing. Functional lymphocytes were required for the reaction, since gamma-irradiated lymphocytes did not develop cytotoxic potential despite the fact that the mycoplasmas replicated equally well in the presence of these and untreated lymphocytes. Furthermore, lymphocytes already activated with mycoplasmas lost cytotoxic potential after disruption. The kinetics and degree of lymphocytotoxicity induced by M. arthritidis and phytohemagglutinin toward 51Cr-labeled syngeneic fibroblasts were similar. Removal of most B cells and other adherent cells by column separation did not abrogate the cytotoxic effect. Lymphocyte suspensions treated with anti-Thy 1 antiserum and complement exhibited a marked decrease in their cytotoxic potential when added to labeled target cells in the presence of M. arthritidis. We conclude that the cytotoxic reaction is dependent upon the T-lymphocyte subpopulation.

A T-cell mitogen present in culture supernatants of Mycoplasma arthritidis (MAS) is known to exhibit an absolute dependence on E alpha-bearing accessory cells (AC), which appear to function by binding the mitogen. We therefore compared the specificity and nature of the AC requirements for MAS and antigen-induced production of IL 2 in T hybridoma cell lines originating from a fusion by using hen egg-white lysozyme (HEL)-specific, H-2d-restricted T blasts. A marked specificity was noted in the ability of the hybridoma lines to become activated by Con A, MAS, or HEL antigen. Thus all three lines produced IL 2 in response to Con A without the addition of B lymphoma AC. Two lines responded to MAS, but only in the presence of AC, and only one line responded to HEL antigen in the presence of AC. Using the HEL responsive T hybridoma line, we demonstrated that disrupted AC and AC membranes could present MAS but not HEL. MAS rapidly associated with AC at 4 degrees C, whereas HEL failed to do so. Paraformaldehyde-fixed AC could absorb the mitogen in MAS and present it to T hybridoma cells within several minutes, whereas HEL antigen could only be presented by fixed AC if there was a prolonged period of incubation (greater than 30 min) at 37 degrees C before fixation. The combined data indicate that metabolically active cells are not required for the association of MAS with AC or for presentation of MAS to T hybridomas. In contrast, HEL antigen requires metabolically active cells for both of these processes. Thus, the mitogen in MAS can bind to AC without any processing requirements, and it is likely that the resulting complex of mitogen and Ia molecules can directly activate T hybridoma cells.