A protein arginine N-methyltransferase 1 (PRMT1) and 2 heteromeric interaction increases PRMT1 enzymatic activity

Protein arginine N-methyltransferases (PRMTs) act in signaling pathways and gene expression by methylating arginine residues within target proteins. PRMT1 is responsible for most cellular arginine methylation activity and can work independently or in collaboration with other PRMTs. In this study, we demonstrate a direct interaction between PRMT1 and PRMT2 using co-immunoprecipitation, bimolecular fluorescence complementation, and enzymatic assays. As a result of this interaction, PRMT2 stimulated PRMT1 activity, affecting its apparent V(max) and K(M) values in vitro and increasing the production of methylarginines in cells. Active site mutations and regional deletions from PRMT1 and -2 were also investigated, which demonstrated that complex formation required full-length, active PRMT1. Although the inhibition of methylation by adenosine dialdehyde prevented the interaction between PRMT1 and -2, it did not prevent the interaction between PRMT1 and a truncation mutant of PRMT2 lacking its Src homology 3 (SH3) domain. This result suggests that the SH3 domain may mediate an interaction between PRMT1 and -2 in a methylation-dependent fashion. On the basis of our findings, we propose that PRMT1 serves as the major methyltransferase in cells by forming higher-order oligomers with itself, PRMT2, and possibly other PRMTs.     

A rapid and sensitive intracellular flow cytometric assay to identify Theileria parva infection within target cells

Theileria parva is an intracellular protozoan parasite transmitted by ticks that causes a fatal lymphoproliferative disease of cattle known as East Coast Fever. Vaccination against the disease currently relies on inoculation of the infective sporozoite stage of the parasite and simultaneous treatment with long-acting formulations of oxytetracycline. Sporozoites are maintained as frozen stabilates of triturated infected ticks and the method requires accurate titration of stabilates to determine appropriate dose rates. Titration has traditionally been undertaken in cattle and requires large numbers of animals because of individual variation in susceptibility to infection. An alternative tissue culture-based method is laborious and time consuming. We have developed a flow cytometric method for quantifying the infectivity of sporozoite stabilates in vitro based on the detection of intracellular parasite antigen. The method allows clear identification of parasitized cells with a high degree of sensitivity and specificity. Analysis of infected cells between 48 and 72 h post-infection clearly defines the potential transforming capability of different stabilates.     

Temporal requirement of the alternative-splicing factor Sfrs1 for the survival of retinal neurons

Alternative splicing is the primary mechanism by which a limited number of protein-coding genes can generate proteome diversity. We have investigated the role of the alternative-splicing factor Sfrs1, an arginine/serine-rich (SR) protein family member, during mouse retinal development. Loss of Sfrs1 function during embryonic retinal development had a profound effect, leading to a small retina at birth. In addition, the retina underwent further degeneration in the postnatal period. Loss of Sfrs1 function resulted in the death of retinal neurons that were born during early to mid-embryonic development. Ganglion cells, cone photoreceptors, horizontal cells and amacrine cells were produced and initiated differentiation. However, these neurons subsequently underwent cell death through apoptosis. By contrast, Sfrs1 was not required for the survival of the neurons generated later, including later-born amacrine cells, rod photoreceptors, bipolar cells and Müller glia. Our results highlight the requirement of Sfrs1-mediated alternative splicing for the survival of retinal neurons, with sensitivity defined by the window of time in which the neuron was generated.     

Polyelectrolyte behavior of carrageenans in aqueous solutions

Osmotic coefficients of sodium, potassium, and calcium counterions have been determined in aqueous solutions on kappa-, iota-, and lambda-carrageenans at 25°C. The experimental results are correlated with the calculated ones from the limiting law of Manning. An orderd secondary structure exists in kappa- and iota-carrageenans. Its stability is discussed as a function of temperature, ionic strength, and the nature of the counterions.     

In vitro dissociation-recombination of malate dehydrogenase subunits in Corydalis solida

Summary Two allelic forms of NAD specific malate dehydrogenase were found in samples of a wild population of Corydalis solida. The dimeric nature and the origin of the heterodimeric form has been demonstrated by in vitro dissociation and recombination of the subunits detected by subsequent electrophoresis. The method is applicable for polyacrylamide gel electrophoresis of crude leaf extracts of individual MDH isozyme forms.     

Root gravitropism requires lateral root cap and epidermal cells for transport and response to a mobile auxin signal

Re-orientation of Arabidopsis seedlings induces a rapid, asymmetric release of the growth regulator auxin from gravity-sensing columella cells at the root apex. The resulting lateral auxin gradient is hypothesized to drive differential cell expansion in elongation-zone tissues. We mapped those root tissues that function to transport or respond to auxin during a gravitropic response. Targeted expression of the auxin influx facilitator AUX1 demonstrated that root gravitropism requires auxin to be transported via the lateral root cap to all elongating epidermal cells. A three-dimensional model of the root elongation zone predicted that AUX1 causes the majority of auxin to accumulate in the epidermis. Selectively disrupting the auxin responsiveness of expanding epidermal cells by expressing a mutant form of the AUX/IAA17 protein, axr3-1, abolished root gravitropism. We conclude that gravitropic curvature in Arabidopsis roots is primarily driven by the differential expansion of epidermal cells in response to an influx-carrier-dependent auxin gradient.     

Shortened seasonal photoperiodic cycles accelerate aging of the diurnal and circadian locomotor activity rhythms in a primate

The gray mouse lemur (Microcebus murinus), a prosimian primate, exhibits seasonal rhythms strictly controlled by photoperiodic variations. Previous studies indicated that longevity can be altered by long-term acceleration of seasonal rhythms, providing a model for assessing various aspects of aging. To assess the effect of aging and accelerated aging on the circadian system of this primate, we compared the circadian rhythm of the locomotor activity in adult mouse lemurs (2-4.5 years, n = 9), aged mouse lemurs (5-9 years, n = 10), and adult mouse lemurs that had been exposed from birth to a shortened seasonal photoperiodic cycle (2-4.5 years, n = 7). Compared to adult animals, aged mouse lemurs showed a significant increase in intradaily variability and an advanced activity onset. Aging was characterized by a decrease in amplitude, with both a decrease in nocturnal activity and an increase in daytime activity. When maintained in constant dim red light, aged animals exhibited a shortening of the free-running period (22.8 +/- 0.1 h) compared to adult animals (23.5 +/- 0.1 h). A 3- to 5-year exposure to an accelerated seasonal photoperiodic rhythm ("annual" duration of 5 months) in accelerated mouse lemurs produced disturbances of the locomotor activity rhythm that resembled those of aged mouse lemurs, whether animals were studied in entrained or in free-running conditions. The present study demonstrated a weakened and fragmented locomotor activity rhythm during normal aging in this primate. Increasing the number of expressed seasonal cycles accelerated aging of parameters related to circadian rhythmicity in adult animals.     

Kinetics and subunit composition of NMDA receptors in respiratory-related neurons

NMDA receptors are involved in a variety of brainstem functions. The excitatory postsynaptic NMDA currents of pre-Botzinger complex interneurons and hypoglossal motoneurons, which are located in the medulla oblongata, show remarkably fast deactivation kinetics of approximately 30 ms compared with NMDA receptors in other types of neurons. Because structural heterogeneity might be the basis for physiological properties, we examined the expression of six NMDA receptor subunits (NMDAR1, NR2A-2D, and NR3A) plus eight NMDR1 splice variants in pre-Botzinger complex, hypoglossal and, for comparison, neurons from the nucleus of the solitary tract in young rats using single cell multiplex RT-PCR. Expression of NR2A, NR2B, and NR2D was observed in all three cell types while NR3A was much more abundant in pre-Botzinger complex interneurons, which belong to the rhythm generator of respiratory activity. In hypoglossal neurons, the NMDAR1 splice variants NMDAR1-4a and NMDAR1-4b were found. In neurons of the nucleus of the solitary tract, instead of NMDAR1-4b, the NMDAR1-2a splice variant was detected. This differential expression of modulatory splice variants might be the molecular basis for the characteristic functional properties of NMDA receptors, as neurons expressing a special NMDAR1 splice variant at the mRNA level show fast kinetics compared with neurons lacking this splice variant.