Gene for improved nutritional value in barley seed protein.

Genetically dependent 20 to 30 percent increase in lysine per 16 grams of nitrogen results in improved nutritional values in feeding trials with mice and rats. The recessive gene was selected from the World Barley Collection. Other amino acids are also influenced by the gene. Protein content segregates independently of the changed amino acid pattern. The gene putatively influences the matrix proteins, which characteristically adhere to the starch grains in meal preparations. The morphological character permits rapid microscopic screening of single seeds without affecting viability. Low yield is considerably improved by crossing and selection.

Expression profiling across many samples via manifold-assisted mRNA processing.

Analysis of mRNA provides a condensed view of gene structure, and quantitative analyses can reveal induction of physiological or pathological gene expression programs. One of the main hurdles for routine mRNA analyses is the need to prepare large sets of samples in a rapid and standardized manner. We describe here a procedure for mRNA isolation and cDNA synthesis using manifold devices, consisting of a set of prongs that project into individual reaction wells. The prongs have a high binding capacity for the polyA-tails of mRNA and the captured mRNA is directly used to synthesize cDNA on the supports, followed by amplification. The convenience and reproducibility of the procedure allows profiling of gene expression over time, by comparing many different samples. Using the device mRNA was simultaneously isolated and accurately measured from up to 96 different samples of anywhere between 10 and 200 000 cells. The amounts of a leukemia-specific transcript could be measured when the malignant cells represented

Molecular genetic applications of streptavidin-coated manifold supports.

Practical problems of handling large numbers of samples limit the application of molecular genetic procedures in clinical settings and in research. In the present review we describe a multipronged manifold support, coated with streptavidin, that offers distinct advantages in preparative and diagnostic applications. In order to increase the surface available on the manifold, porous Sepharose particles conjugated with streptavidin were attached to the plastic support. This procedure increased the surface by almost three orders of magnitude, permitting sufficient streptavidin to be coupled to the support for most routine applications. The manifold supports have been used for sample preparation and in a number of genetic assays, including allele discrimination assays and DNA sequencing, In all these assay formats the manifold supports allow large numbers of samples to be processed in parallel.

Dependence of shunt on cardiac output in unilobar oleic acid edema. Distribution of ventilation and perfusion.

OBJECTIVE: In acute respiratory failure, increased cardiac output (Qt) increases shunt (Qs/Qt). We have tested if this is caused by: 1) a redistribution of blood flow towards edematous regions, or 2) a decrease of regional ventilation in the edematous region. DESIGN: Oleic acid edema was induced in the left lower lobe (LLL) of 11 pigs. Qt was varied with bleeding and infusion of blood and dextran. Blood flow to the LLL was measured at low and high Qt with electromagnetic low probes in 6 animals and with a gamma camera in 5. In the gamma camera pigs regional ventilation was also measured. MEASUREMENTS AND RESULTS: Qt was increased by 45% (electromagnetic flow probes) and 73% (gamma camera). Qs/Qt increased from 24.9-31.3% (p < 0.05) and from 17.6-28.8% (p < 0.001) respectively. No change in fractional perfusion of LLL could be seen, neither with flow probes nor with gamma camera. A decrease in ventilation of LLL, 2.6%, was observed when Qt was increased (p < 0.05). CONCLUSION: Theoretically a small decrease in ventilation can explain the increase in shunt, if regions with low ventilation/perfusion (VA/Q) ratio are transformed to shunt. This is, however, unlikely since earlier studies have shown that blood flow is distributed either to regions with normal VA/Q ratio or to shunt regions. We conclude that the cardiac output dependent shunt is not caused by redistribution of blood flow between lobes or by decreased ventilation in the edematous region. We cannot exclude that blood flow is redistributed within the edematous lobe.

Multiphase patterns in periodically forced oscillatory systems.

Periodic forcing of an oscillatory system produces frequency locking bands within which the system frequency is rationally related to the forcing frequency. We study extended oscillatory systems that respond to uniform periodic forcing at one quarter of the forcing frequency (the 4:1 resonance). These systems possess four coexisting stable states, corresponding to uniform oscillations with successive phase shifts of pi/2. Using an amplitude equation approach near a Hopf bifurcation to uniform oscillations, we study front solutions connecting different phase states. These solutions divide into two groups: pi fronts separating states with a phase shift of pi and pi/2 fronts separating states with a phase shift of pi/2. We find a type of front instability where a stationary pi front "decomposes" into a pair of traveling pi/2 fronts as the forcing strength is decreased. The instability is degenerate for an amplitude equation with cubic nonlinearities. At the instability point a continuous family of pair solutions exists, consisting of pi/2 fronts separated by distances ranging from zero to infinity. Quintic nonlinearities lift the degeneracy at the instability point but do not change the basic nature of the instability. We conjecture the existence of similar instabilities in higher 2n:1 resonances (n=3,4, em leader) where stationary pi fronts decompose into n traveling pi/n fronts. The instabilities designate transitions from stationary two-phase patterns to traveling 2n-phase patterns. As an example, we demonstrate with a numerical solution the collapse of a four-phase spiral wave into a stationary two-phase pattern as the forcing strength within the 4:1 resonance is increased.

Phase dynamics of nearly stationary patterns in activator-inhibitor systems

The slow dynamics of nearly stationary patterns in a FitzHugh-Nagumo model are studied using a phase dynamics approach. A Cross-Newell phase equation describing slow and weak modulations of periodic stationary solutions is derived. The derivation applies to the bistable, excitable, and Turing unstable regimes. In the bistable case stability thresholds are obtained for the Eckhaus and zigzag instabilities and for the transition to traveling waves. Neutral stability curves demonstrate the destabilization of stationary planar patterns at low wave numbers to zigzag and traveling modes. Numerical solutions of the model system support the theoretical findings.

Beta-globin mRNA increases rapidly during erythropoietin treatment.

Recombinant human erythropoietin (r-HuEpo) has an important role in the treatment of anaemic patients. Because of the high cost of r-HuEpo treatment, an early indicator of whether a patient is responding to the therapy would be valuable. Although measurement of gene expression is a promising new tool, it has not yet been established in clinical practice. The response pattern of a possible new marker, beta-globin mRNA, is compared with reticulocyte count, levels of haemoglobin, transferrin receptor and ferritin after r-HuEpo treatment. Eight healthy volunteers were stimulated with erythropoietin three times a week for four weeks and compared with five untreated control subjects. Blood samples were collected before each erythropoietin injection. Quantitative measurement of beta-globin mRNA was performed by poly(A) selection onto a manifold plastic support, coated with oligo(dT). The mRNA was reverse transcribed, followed by quantitative analysis using PCR via the 5' nuclease assay. The individuals treated with rHuEpo showed a more distinct increase in beta-globin mRNA levels than all other laboratory measurements. Beta-globin mRNA levels are therefore promising as a marker for the response to treatment with Epo.

Induced mutations and barley improvement.

Seven barley varieties, originating from three X-ray induced mutations, have been officially approved in Sweden since 1958. Some have gained a wide area of cultivation. The list is as follows: Pallas, isolated 1947, approved 1958, mutant ert-k (32) of Bonus barley. - Mari, isolated 1950, approved 1960, mutant mat-a (8) of Bonus. - Hellas, approved 1967, mutant cross of Pallas × Herta. - Kristina, approved 1969, mutant cross of Domen × Mari. - Visir, approved 1970, Pallas × Long Glumes back-crossed to Pallas. - Mona, approved 1970, mutant cross of Mari × Monte Cristo back-crossed to Mari. - Gunilla, approved 1970, hybrid cross of the mutant 44/3 arisen from Gull barley in 1939; evolved in a series of steps, using one six-row and four two-row varieties, with mutant characters prevailing and Gull genes reiterated. - After the first approval of Pallas in 1958, 12 more years have led to the approval of a second mutant case and five mutant crosses. In addition, chromosome translocations, induced by irradiation in Bonus, have been instrumental in the production of hybrid barley in USA and are used in the barley improvement program of Sweden, as well as for theoretical analysis in numerous countries.

Decreased levels of pathogenic IgG anti-DNA antibodies in autoimmune mice after linomide treatment.

Treatment with a novel immunomodulator called linomide resulted in highly reduced levels of pathogenic anti-DNA antibodies in MRL-lpr/lpr mice compared to untreated mice. This reduction occurred without altering the total levels of serum immunoglobulins, implying that the compound does not have any direct B-cell suppressive effect. The possible role of T cells in regulating autoreactive B-cell clones is discussed.

Front propagation and pattern formation in anisotropic bistable media

The effects of diffusion anisotropy on pattern formation in bistable media are studied using a FitzHugh-Nagumo reaction-diffusion model. A relation between the normal velocity of a front and its curvature is derived and used to identify distinct spatiotemporal patterns induced by the diffusion anisotropy. In a wide parameter range anisotropy is found to have an ordering effect: initial patterns evolve into stationary or breathing periodic stripes parallel to one of the principal axes. In a different parameter range, anisotropy is found to induce spatiotemporal chaos confined to one space dimension, a state we term "stratified chaos."

Amelioration of intrathymic T cell development and peripheral T cell reactivities in autoimmune mice undergoing therapy with a novel immunomodulator.

MRL lpr/lpr mice develop a generalized autoimmune disease associated with a massive accumulation in the peripheral lymphoid organs of abnormal, phenotypically immature T cells. Both the lymphoadenopathy and the autoimmunity are thymus-dependent and likely to arise from an aberrant pathway of intrathymic differentiation. We here present the marked beneficial effects acquired in MRL lpr/lpr mice after in vivo administration of a novel immunomodulator called linomide. The highly altered pattern of thymic subpopulations in MRL lpr/lpr mice is normalized after linomide-treatment. Concomitantly, the peripheral T cell compartment, which in MRL lpr/lpr is highly deficient in producing and responding to IL-2, gains substantial functional reactivities. We propose that linomide acts by correcting the abnormal T cell development in autoimmune MRL lpr/lpr mice. This new immunomodulator may be a useful tool for providing insight into both the pathogenesis of autoimmune disorders and intrathymic differentiation.

Analysis of T cell receptor V beta gene usage in primary mixed lymphocyte reactions: evidence for directive usage by different antigen-presenting cells and Mls-like determinants on T cell blasts.

The usage of four different T cell receptor (TcR) V beta gene families within normal, non-primed T cell populations in response to various types of antigen-presenting cells (APC) in primary mixed lymphocyte reaction has been studied. We demonstrate that distinct patterns of V beta gene usage are obtained within a given T cell population in response to different types of APC with the same allo-H-2. When responder T cells are stimulated with one type of allogeneic APC, from various H-2-disparate mice, the same V beta gene preference is observed. Furthermore, when H-2- and Mls-mismatched APC gene used as stimulators, the Mls-associated V beta 6 and V beta 8.1 gene families are highly elevated in response to both B and T cell blasts from certain Mls-positive strains. The results demonstrate that different types of allogeneic APC have the capacity to generate biases in TcR V beta gene usage and imply that functional Mls-like determinants are presented by T cell blasts. The findings are discussed with respect to TcR-major histocompatibility complex interactions in allostimulation.

Manifold-assisted reverse transcription-PCR with real-time detection for measurement of the BCR-ABL fusion transcript in chronic myeloid leukemia patients.

BACKGROUND: BCR-ABL fusion mRNA expression in bone marrow or peripheral blood can be used as a measure of minimal residual disease in patients with chronic myeloid leukemia (CML). METHODS: We used an oligo(dT)-coated manifold support to capture the mRNA directly from the cell lysate. After reverse transcription, the cDNA was eluted from the manifold support, and BCR-ABL and GAPDH mRNAs were quantified in real time using the TaqMan fluorogenic detection system. RESULTS: The detection limit of the method was one positive K562 cell among 10(5) negative cells. GAPDH was chosen as a reference gene based on the low variation between samples from different stages of the disease and the low signal in the absence of reverse transcription. The day-to-day variation of the method (CV) was 32%. In 43 blood samples from 13 CML patients, mRNA quantification agreed well with cytogenetic data. CONCLUSIONS: The proposed procedure constitutes a reproducible and sensitive BCR-ABL mRNA quantification method and is suitable to monitor minimal residual disease in CML patients.

Four-phase patterns in forced oscillatory systems

We investigate pattern formation in self-oscillating systems forced by an external periodic perturbation. Experimental observations and numerical studies of reaction-diffusion systems and an analysis of an amplitude equation are presented. The oscillations in each of these systems entrain to rational multiples of the perturbation frequency for certain values of the forcing frequency and amplitude. We focus on the subharmonic resonant case where the system locks at one-fourth the driving frequency, and four-phase rotating spiral patterns are observed at low forcing amplitudes. The spiral patterns are studied using an amplitude equation for periodically forced oscillating systems. The analysis predicts a bifurcation (with increasing forcing) from rotating four-phase spirals to standing two-phase patterns. This bifurcation is also found in periodically forced reaction-diffusion equations, the FitzHugh-Nagumo and Brusselator models, even far from the onset of oscillations where the amplitude equation analysis is not strictly valid. In a Belousov-Zhabotinsky chemical system periodically forced with light we also observe four-phase rotating spiral wave patterns. However, we have not observed the transition to standing two-phase patterns, possibly because with increasing light intensity the reaction kinetics become excitable rather than oscillatory.