Test of charge conjugation invariance.

We report on the first determination of upper limits on the branching ratio (BR) of eta decay to pi0pi0gamma and to pi0pi0pi0gamma. Both decay modes are strictly forbidden by charge conjugation (C) invariance. Using the Crystal Ball multiphoton detector, we obtained BR(eta-->pi0pi0gamma)<5 x 10(-4) at the 90% confidence level, in support of C invariance of isoscalar electromagnetic interactions of the light quarks. We have also measured BR(eta-->pi0pi0pi0gamma)<6 x 10(-5) at the 90% confidence level, in support of C invariance of isovector electromagnetic interactions.

Observation of the charge symmetry breaking d+d-->4He+pi(0) reaction near threshold.

We report the first observation of the charge symmetry breaking d+d-->4He+pi(0) reaction near threshold. Measurements using a magnetic channel (gated by two photons) of the 4He scattering angle and momentum (from time of flight) permitted reconstruction of the pi(0) "missing mass," the quantity used to separate 4He+pi(0) events from the continuum of double radiative capture 4He+gamma+gamma events. We measured total cross sections for neutral pion production of 12.7+/-2.2 pb at 228.5 MeV and 15.1+/-3.1 pb at 231.8 MeV. The uncertainty is dominated by statistical errors. These cross sections arise fundamentally from the down-up quark mass difference and quark electromagnetic effects that contribute in part through meson mixing (e.g., pi(0)-eta) mechanisms.

Dynamics of the pi(-)p-->pi(0)pi(0)n reaction for p(pi(-))<750 MeV/c.

Data are presented for the reaction pi(-)p-->pi(0)pi(0)n in the range from threshold to p(pi(-))=750 MeV/c. The systematics of the data and multipole analyses are examined for sensitivity to a f(0)(600) ("sigma") meson. A one-pion-exchange mechanism is found to be very weak, or absent. The reaction appears to become dominated by sequential pi(0) decays through the Delta(1232) resonance as the beam momentum increases, along with substantial interference effects from several competing mechanisms.

Properties of the Lambda(1670)(1-)/2 resonance.

Recently the Crystal Ball Collaboration measured precise new data for the near-threshold reaction K(-)p-->etaLambda, which is dominated by formation of the Lambda(1670)1 / 2(-). In this Letter, we present results of a unitary, multichannel analysis that incorporates the new Crystal Ball data. For our preferred fit, we obtain mass M = 1673+/-2 MeV, width Gamma = 23+/-6 MeV, and elasticity x = 0.37+/-0.07. This elasticity is significantly larger than previously recognized. Resonance parameters of our preferred fit are in striking agreement with the quark-model predictions of Koniuk and Isgur.

Determination of the quadratic slope parameter in eta-->3pi(0) decay.

We have determined the quadratic slope parameter alpha for eta-->3pi(0) to be alpha = -0.031(4) from a 99% pure sample of 10(6)eta-->3pi(0) decays produced in the reaction pi(-)p-->n(eta) close to the eta threshold using the Crystal Ball detector at the AGS. The result is four times more precise than the present world data and disagrees with current chiral perturbation theory calculations by about four standard deviations.

Search for the CP forbidden decay eta-->4pi(0)

We report the first determination of the upper limit for the branching ratio of the CP forbidden decay eta-->4pi(0). No events were observed in a sample of 3.0x10(7) eta decays. The experiment was performed with the Crystal Ball multiphoton spectrometer installed in a separated pi(-) beam at the AGS (Alternating Gradient Synchrotron). At the 90% confidence limit, B(eta-->4pi(0))

Measurement of pi(0)pi(0) production in the nuclear medium by pi(-) interactions at 0.408 GeV/c.

We report on an investigation of the (pi(-),pi(0)pi(0)) reaction by means of measurements of the pi(0)pi(0) invariant mass distributions from pi(-) interactions on H, D, C, Al, and Cu targets at p(pi(-)) = 0.408 GeV/c. The sharp, strong peak in the pi(+)pi(-) invariant mass near 2m(pi) reported by the CHAOS Collaboration is not seen in our pi(0)pi(0) data. However, we do observe a change in the shape of the pi(0)pi(0) invariant mass spectrum for the different targets, indicating that the pi(0)pi(0) interaction diminishes in the nuclear medium as represented by nuclei D, C, Al, and Cu, compared to hydrogen.

Distinct neurodevelopmental patterns of bcl-2 and bcl-x expression are altered in glioneuronal hamartias of the human temporal lobe.

Bcl-2 and bcl-xL are homologous proteins that inhibit cell death and are expressed in the nervous system. We tested the hypothesis that aberrant expression of such "death suppressor" molecules may promote the survival of abnormal cells in glioneuronal lesions associated with temporal lobe epilepsy. The normal pattern of bcl-2 and bcl-x expression was studied in postmortem human fetal and adult temporal lobes. Formalin-fixed, paraffin-embedded tissue sections were probed for bcl-2 and bcl-x in immunohistochemical studies using well-characterized primary antibodies that had been raised against epitopes that are not shared by these proteins. Strong staining for both proteins was observed in the ventricular zone and in migrating, postmitotic and postmigratory young neurons of the neocortex, hippocampus, and entorhinal cortex from 6 to 20 weeks gestational age (GA). However, bcl-2 immunoreactivity gradually decreased to weak or nondetectable levels between 20 and 39 weeks GA, while strong bcl-x staining of neurons persisted throughout fetal development and into adulthood. Twenty-eight temporal lobe resections from children and adults ranging in age from 1 to 45 years (mean=19 years) with intractable epilepsy were then screened for differences in the pattern of bcl-2 and bcl-x expression compared to normal controls. Bcl-2 (but not bcl-x) was strongly immunoreactive in small, immature-appearing cells that were components of microscopic glioneuronal aggregates (hamartias) and that have been shown previously to express an embryonic form of the neural cell adhesion molecule. These immature cells were immunonegative for standard markers of neuronal and glial lineage and were negative for Ki67, suggesting that they are post-mitotic. The persistent expression of bcl-2 and apparent downregulation of bcl-x in these cells represent deviations from the normal ontogeny of these molecules in the human nervous system. These data suggest that dysregulation of bcl-2 and related proteins may be involved in the pathogenesis of some temporal lobe malformative lesions.

Phosphorylation of microtubule-associated proteins MAP2 and MAP4 by the protein kinase p110mark. Phosphorylation sites and regulation of microtubule dynamics.

The phosphorylation of microtubule-associated proteins (MAPs) is thought to be a key factor in the regulation of microtubule stability. We have shown recently that a novel protein kinase, termed p110 microtubule-affinity regulating kinase ("MARK"), phosphorylates microtubule-associated protein tau at the KXGS motifs in the region of internal repeats and causes the detachment of tau from microtubules (Drewes, G., Trinczek, B., Illenberger, S., Biernat, J., Schmitt-Ulms, G., Meyer, H.E., Mandelkow, E.-M., and Mandelkow, E. (1995) J. Biol. Chem. 270, 7679-7688). Here we show that p110mark phosphorylates analogous KXGS sites in the microtubule binding domains of the neuronal MAP2 and the ubiquitous MAP4. Phosphorylation in vitro leads to the dissociation of MAP2 and MAP4 from microtubules and to a pronounced increase in dynamic instability. Thus, the phosphorylation of the repeated motifs in the microtubule binding domains of MAPs by p110mark might provide a mechanism for the regulation of microtubule dynamics in cells.

A muscle-specific variant of microtubule-associated protein 4 (MAP4) is required in myogenesis.

Microtubule-associated protein 4 (MAP4) transcripts vary in different mouse tissues, with striated muscle (skeletal and cardiac) expressing 8- and 9-kb transcripts preferentially to the more widely distributed 5.5- and 6.5-kb transcripts (West, R. W., Tenbarge, K. M. and Olmsted, J. B. (1991). J. Biol. Chem. 266, 21886-21896). Cloning of the sequence unique to the muscle transcripts demonstrated that these mRNAs vary from the more ubiquitous ones by a single 3.2-kb coding region insertion within the projection domain of MAP4. During differentiation of the myogenic cell line, C2C12, muscle-specific MAP4 transcripts appear within 24 hours of growth in differentiation medium, and a larger MAP4 isotype (350 X 10(3) Mr) accumulates to high levels by 48 hours of differentiation. In situ hybridization analyses of transcript distribution in mouse embryos demonstrated that muscle-specific transcripts appear early in myogenesis. To block the expression of the muscle-specific MAP4, stable lines of C2C12 were generated bearing an antisense construct with the muscle-specific MAP4 sequence. Myoblast growth was unaffected whereas myotube formation was severely perturbed. Fusion occurred in the absence of the muscle MAP4 isotype, but the multinucleate syncytia were short and apolar, microtubules were disorganized and normal anisotropic myofibrils were absent. The patterns of expression of the muscle-specific transcripts and the antisense experiments indicated that this unique structural form of MAP4 plays a critical role in the formation and maintenance of muscle.

Analysis of MAP 4 function in living cells using green fluorescent protein (GFP) chimeras.

MAP 4 is a ubiquitous microtubule-associated protein thought to play a role in the polymerization and stability of microtubules in interphase and mitotic cells. We have analyzed the behavior of protein domains of MAP 4 in vivo using chimeras constructed from these polypeptides and the green fluorescent protein (GFP). GFP-MAP 4 localizes to microtubules; this is confirmed by colocalization of GFP-MAP 4 with microtubules that have incorporated microinjected rhodamine-tubulin, and by loss of localized fluorescence after treatment of cells with anti-microtubule agents. Different subdomains of MAP 4 have distinct effects on microtubule organization and dynamics. The entire basic domain of MAP 4 reorganizes microtubules into bundles and stabilizes these arrays against depolymerization with nocodazole. Within the basic domain, the PGGG repeats, which are conserved with MAP 2 and tau, have a weak affinity for microtubules and are dispensable for microtubule binding, whereas the MAP 4-unique PSP region can function independently in binding. The projection domain shows no microtubule localization, but does modulate the association of various binding subdomains with microtubules. The acidic carboxy terminus of MAP 4 strongly affects the microtubule binding characteristics of the other domains, despite constituting less than 6% of the protein. These data show that MAP 4 association with microtubules is modulated by sequences both within and outside the basic domain. Further, our work demonstrates that GFP chimeras will allow an in vivo analysis of the effects of MAPs and their variants on microtubule dynamics in real time.

Stable expression of heterologous microtubule-associated proteins (MAPs) in Chinese hamster ovary cells: evidence for differing roles of MAPs in microtubule organization.

To study the effects of microtubule-associated proteins (MAPs) on in vivo microtubule assembly, cDNAs containing the complete coding sequences of a Drosophila 205-kD heat stable MAP, human MAP 4, and human tau were stably transfected into CHO cells. Constitutive expression of the transfected genes was low in most cases and had no obvious effects on the viability of the transfected cell lines. High levels of expression, as judged by Western blots, immunofluorescence, and Northern blots, could be induced by treating cells with sodium butyrate. High levels of MAPs were maintained for at least 24-48 h after removal of the sodium butyrate. Immunofluorescence analysis indicated that all three MAPs bound to cellular microtubules, but only the transfected tau caused a rearrangement of microtubules into bundles. Despite high levels of expression of these exogenous MAPs and the bundling of microtubules in cells expressing tau, transfected cells had normal levels of assembled and unassembled tubulin. With the exception of the tau-induced bundles, microtubules in transfected cells showed the same sensitivity as control cells to microtubule depolymerization by Colcemid. Further, all three MAPs were ineffective in reversing the taxol-dependent phenotype of a CHO mutant cell line. The absence of a quantitative effect of any of these heterologous proteins on the assembly of tubulin suggests that these MAPs may have different roles in vivo from those inferred previously from in vitro experiments.

Intrauterine injection of recombinant ovine interferon-tau extends the interestrous interval in sheep.

The ability of recombinant ovine interferon-tau (roIFNtau) to extend the interestrous interval (IEI) in sheep was studied. Ewes were fitted with bilateral uterine catheters 7 or 8 days post estrus and were assigned to receive either 10 or 20 million antiviral (AV) units/day i.u. ( approximately 100 or 200 ug) of roIFNtau or ovine conceptus secretory proteins containing equivalent AV units of native oIFNtau (noIFNtau; 4 ewes/treatment). Four control ewes received ovine serum proteins (SP). Total protein injected was 6 mg per day, half at 0700 hours and half at 1730 hours. The treatments were administered from Day 11.5 (estrus=Day 0) to Day 16. Blood samples were collected by jugular vienipuncture daily from Day 11 until ewes returned to estrus. Concentrations of progesterone (P) in plasma were determined by RIA. Treatment with either noIFNtau or roIFNtau extended IEI beyond that of SP-treated ewes (19.1 vs 31.2+/-3.4 days P<0.03). Of the ewes receiving 100 mug/day of oIFNtau, 2 of 4 receiving noIFNtau (23.6+/-5.2 days) and 3 of 4 receiving roIFNtau (34.2+/-5.2 days) had an extended IEI. All ewes receiving 200 mug/day of noIFNtau or roIFNtau had an extended IEI (28.8 and 38.5+/-5.2 days. respectively). Ewes receiving roIFNtau had a longer IEI than those receiving noIFNtau (36.7 vs 26.2+/-3.4 days; P=0.07). Ewes with an extended IEI had functional corpora lutea, as assessed by P production. The results demonstrate that 10 or 20 million AV units ( approximately 100 or 200 ug) of roIFNtau extends the IEI and that the length of the IEI is longer for ewes receiving roIFNtau than noIFNtau following injection of equivalent AV units.

Mouse microtubule-associated protein 4 (MAP4) transcript diversity generated by alternative polyadenylation.

Mouse microtubule-associated protein 4 (MAP4) is a protein that co-locates with microtubules in vivo. It is encoded by a single-copy gene that expresses multiple transcripts in most cell types [West et al., J. Biol. Chem. 266 (1991) 21886-21896]. This report describes the identification of two distinct 3'-untranslated regions (UTR) for MAP4 transcripts. The 3'-UTRs of the transcripts are identical up to the site of polyadenylation of the shorter mRNA. The longer transcript contains an additional 775 nucleotides after the first polyadenylation site. Both poly(A) tails follow the canonical polyadenylation site motif, AAUAAA. These data show that two different UTRs arise as a result of alternative polyadenylation site usage. Northern blots of RNA from different tissues probed with coding sequence show hybridization to the common 5.5- and 6.5-kb transcripts, whereas blots probed with sequence unique to the longer 3'-UTR show hybridization only to the 6.5-kb band. Both transcripts are found within the same cell type. In addition, muscle contains additional transcripts of 8 and 9 kb, of which only the 9-kb transcript hybridizes to the longer 3'-UTR probe.

Cytoskeletal integrity in interphase cells requires protein phosphatase activity.

Phosphorylation by protein kinases has been established as a key factor in the regulation of cytoskeletal structure. However, little is known about the role of protein phosphatases in cytoskeletal regulation. To assess the possible functions of protein phosphatases in this respect, we studied the effects of the phosphatase inhibitors calyculin A, okadaic acid, and dinophysistoxin 1 (35-methylokadaic acid) on BHK-21 fibroblasts. Within minutes of incubation with these inhibitors, changes are seen in the structural organization of intermediate filaments, followed by a loss of microtubules, as assayed by immunofluorescence. These changes in cytoskeletal structure are accompanied by a rapid and selective increase in vimentin phosphorylation on interphase-specific sites, and they are fully reversible after removal of calyculin A. The results indicate that there is a rapid phosphate turnover on cytoskeletal intermediate filaments and further suggest that protein phosphatases are essential for the maintenance and structural integrity of two major cytoskeletal components.

A model for microtubule-associated protein 4 structure. Domains defined by comparisons of human, mouse, and bovine sequences.

cDNAs encoding human and mouse microtubule-associated protein 4 (MAP 4) were isolated. MAP 4 is encoded by a single gene. Multiple MAP 4 mRNAs are transcribed that are differentially expressed among mouse tissues. Open reading frames for the human and mouse MAP 4 clones indicate three distinct regions consisting of related sequences with different motifs. Approximately 30% of the protein is tandem related repeats of approximately 14 amino acids. Another region contains clusters of serine and proline. Four 18-mer repeats characteristic of the microtubule-binding domains of MAP 2 and tau are located at the carboxyl-terminal portion of MAP 4. Amino acid sequence analysis revealed that human and mouse MAP 4 are homologs of the bovine 190-kDa MAP/MAP U (Aizawa, H., Emori, Y., Murofushi, H., Kawasakai, H., Sakai, H., and Suzuki, K. (1990) J. Biol. Chem. 265, 13849-13855). Mouse and human MAP 4 and the bovine 190-kDa MAP are approximately 75% similar, indicating that these proteins are all members of the same class. Domains with extremely high conservation (greater than or equal to 88%) are: 1) the extreme amino terminus; 2) a proline-rich region between the KDM and S,P domains; 3) the microtubule-binding domain; and 4) the extreme carboxyl terminus.

Non-motor microtubule-associated proteins.

Cloning of primary sequences has generated information on the structures of the non-motor microtubule-associated proteins and their relationship to one another. Questions about how classes of microtubule-associated proteins interact are starting to be addressed in vitro and, in vivo, tests of function are being pursued using a variety of cellular and molecular biological strategies.

On the validity of Shannon-information calculations for molecular biological sequences.

The usefulness of information-theoretic measures of the Shannon-Weaver type, when applied to molecular biological systems such as DNA or protein sequences, has been critically evaluated. It is shown that entropy can be re-expressed in dimensionless terms, thereby making it commensurate with information. Further, we have identified processes in which entropy S and information H change in opposite directions. These processes of opposing signs for delta S and delta H demonstrate that while the Second Law of Thermodynamics mandates that entropy always increases, it places no such restrictions on changes in information. Additionally, we have developed equations permitting information calculations, incorporating conditional occurrence probabilities, on DNA and protein sequences. When the results of such calculations are compared for sequences of various general types, there are no informational content patterns. We conclude that information-theoretic calculations of the present level of sophistication do not provide any useful insights into molecular biological sequences.