Identification of dopamine D1-D3 receptor heteromers. Indications for a role of synergistic D1-D3 receptor interactions in the striatum

The function of dopamine D(3) receptors present in the striatum has remained elusive. In the present study evidence is provided for the existence of dopamine D(1)-D(3) receptor heteromers and for an intramembrane D(1)-D(3) receptor cross-talk in living cells and in the striatum. The formation of D(1)-D(3) receptor heteromers was demonstrated by fluorescence resonance energy transfer and bioluminescence resonance energy transfer techniques in transfected mammalian cells. In membrane preparations from these cells, a synergistic D(1)-D(3) intramembrane receptor-receptor interaction was observed, by which D(3) receptor stimulation enhances D(1) receptor agonist affinity, indicating that the D(1)-D(3) intramembrane receptor-receptor interaction is a biochemical characteristic of the D(1)-D(3) receptor heteromer. The same biochemical characteristic was also observed in membrane preparations from brain striatum, demonstrating the striatal co-localization and heteromerization of D(1) and D(3) receptors. According to the synergistic D(1)-D(3) intramembrane receptor-receptor interaction, experiments in reserpinized mice showed that D(3) receptor stimulation potentiates D(1) receptor-mediated behavioral effects by a different mechanism than D(2) receptor stimulation. The present study shows that a main functional significance of the D(3) receptor is to obtain a stronger dopaminergic response in the striatal neurons that co-express the two receptors.     

Underivatized polyamine analysis in plant samples by ion pair LC coupled with electrospray tandem mass spectrometry

Polyamines are key regulators of cell development and many plant responses to environmental challenges, however, their functions still remain unclear in complex interactions with other hormones and in biotic or abiotic stress. This lack of knowledge derives from the difficulties on measuring natural polyamines in plants. Here, we present a fast multiresidue method for putrescine (Put), 1,3-diaminopropane (DAP), l-ornithine, spermidine (Spd) and spermine (Spn) measurements in plant samples. Polyamine determination is based on a perchloric acid extraction followed by a simple filtration procedure without previous derivatization. Polyamines are resolved by HPLC in a C18 common column and quantified by electrospray ionization tandem mass spectrometry. ¹³C₄-putrescine and 1,7-diaminoheptane standards were added prior to sample extraction to achieve an accurate quantification in a single run. Chromatography of polyamines presents poor retention when reverse phase C18 common columns are used, because they are very polar compounds and contain several positive charges. To circumvent this problem ionic pairing technique has been used successfully with heptafluorobutyric acid (HFBA) at 1 mM in the aqueous phase and 25 mM in the sample. Improvement of the signal depleted by HFBA has been achieved by adding 1% of propionic acid to the aqueous and organic eluents. All together, gives a method accurate enough to determine polyamines in plants. To demonstrate the usefulness of the method it has been validated in Arabidopsis thaliana samples and polyamines have been determined in several genotypes that over express (35S::ADC2 line 3.6) or are disrupted (adc2) in the Arginine Decarboxylase2 (ADC2) gene.     

Subcutaneous Fat Shows Higher Thyroid Hormone Receptor‐α1 Gene Expression Than Omental Fat

The aims of this work were to evaluate thyroid hormone receptor‐α (TRα), TRα1, and TRα2 mRNA gene expression and TRα1:TRα2 ratio, identified as candidate factors for explaining regional differences between human adipose tissue depots. TRα, TRα1, and TRα2 mRNA levels, and the gene expressions of arginine–serine‐rich, splicing factor 2 (SF2), heterogeneous nuclear ribonucleoprotein H1 (hnRNP H1), heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1), and Spot 14 (S14) were evaluated in 76 paired adipose tissue samples obtained from a population of 38 women who varied widely in terms of obesity and body fat distribution. Gene expression for these factors was also studied in stromal‐vascular cells (SVCs) and mature adipocytes (MAs) from eight paired fat depots. TRα gene and TRα1 mRNA expression were increased 1.46‐fold (P = 0.006) and 1.80‐fold (P < 0.0001), respectively, in subcutaneous (SC) vs. visceral fat. These differences in gene expression levels were most significant in the obese group, in which the TRα1:TRα2 ratio was 2.24‐fold (P < 0.0001) higher in SC vs. visceral fat. S14 gene expression was also increased by 2.42‐fold (P < 0.0001) and correlated significantly with TRα and TRα1 gene expression and with the TRα1:TRα2 ratio. In agreement with these findings, hnRNP A1:SF2 ratio was decreased by 1.39‐fold (P = 0.001). TRα and S14 levels were 2.1‐fold (P < 0.0001) and 112.4‐fold (P < 0.0001), respectively, higher in MAs than in SVCs from both fat depots. In summary, genes for TR‐α, their upstream regulators, and downstream effectors were differentially expressed in SC vs. omental (OM) adipose tissue. Our findings suggest that TRα1 could contribute to SC adipose tissue expandability in obese subjects.     

Two Chromatin Remodeling Activities Cooperate during Activation of Hormone Responsive Promoters

Steroid hormones regulate gene expression by interaction of their receptors with hormone responsive elements (HREs) and recruitment of kinases, chromatin remodeling complexes, and coregulators to their target promoters. Here we show that in breast cancer cells the BAF, but not the closely related PBAF complex, is required for progesterone induction of several target genes including MMTV, where it catalyzes localized displacement of histones H2A and H2B and subsequent NF1 binding. PCAF is also needed for induction of progesterone target genes and acetylates histone H3 at K14, an epigenetic mark that interacts with the BAF subunits by anchoring the complex to chromatin. In the absence of PCAF, full loading of target promoters with hormone receptors and BAF is precluded, and induction is compromised. Thus, activation of hormone-responsive promoters requires cooperation of at least two chromatin remodeling activities, BAF and PCAF.     

Comparative use patterns of plant resources in rural areas of South Africa and Zimbabwe

Documentation of use patterns of plants across national boundaries is of relevance in understanding the importance of plant resources to livelihood strategies of different ethnic groups. Plant resources have gained prominence as a natural asset through which families derive food, firewood, income, medicines and timber, enabling particularly poor communities to achieve self-sufficiency. The objective of this study was to investigate the trends in plant usage in South Africa and Zimbabwe. An ethnobotanical investigation was conducted between January 2012 and January 2013 in the Limpopo Province, South Africa and the Midlands Province, Zimbabwe. The study used questionnaire surveys and interviews with a total of 143 participants to explore plant use patterns in South Africa and Zimbabwe. A total of 98 plant species were identified, with Zimbabwe contributing 70 species and 47 species from South Africa. The uses were classified into 15 categories, major use categories were firewood, food plants, medicine and timber. Food plant was a major plant use category in Zimbabwe, contributing 55.1%, followed by medicinal plants (36.8%), firewood (35.7%) and timber (31.6%). In contrast, firewood was the major plant use category in South Africa, contributing 18.4%, followed by food plants (17.3%), medicinal (14.3%) and timber (1.0%). Comparison of the two countries demonstrated remarkable differences in plant use patterns. The results showed that rural households in Zimbabwe were more reliant on plant resources than their counterparts in South Africa. Such a trend could be attributed to a close relationship between the local people, and their natural and agricultural environment leading to a rich knowledge base on plants, plant use and related practices. This comparative analysis strengthens the firm belief that utilization of plant resources represents an important shared heritage, preserved over the centuries, which must be exploited in order to provide further new and useful body of ethnobotanical knowledge.     

Reduced proportions of natural killer T cells are present in the relatives of lupus patients and are associated with autoimmunity

Introduction

Systemic lupus erythematosus is a genetically complex disease. Currently, the precise allelic polymorphisms associated with this condition remain largely unidentified. In part this reflects the fact that multiple genes, each having a relatively minor effect, act in concert to produce disease. Given this complexity, analysis of subclinical phenotypes may aid in the identification of susceptibility alleles. Here, we used flow cytometry to investigate whether some of the immune abnormalities that are seen in the peripheral blood lymphocyte population of lupus patients are seen in their first-degree relatives.

Methods

Peripheral blood mononuclear cells were isolated from the subjects, stained with fluorochrome-conjugated monoclonal antibodies to identify various cellular subsets, and analyzed by flow cytometry.

Results

We found reduced proportions of natural killer (NK)T cells among 367 first-degree relatives of lupus patients as compared with 102 control individuals. There were also slightly increased proportions of memory B and T cells, suggesting increased chronic low-grade activation of the immune system in first-degree relatives. However, only the deficiency of NKT cells was associated with a positive anti-nuclear antibody test and clinical autoimmune disease in family members. There was a significant association between mean parental, sibling, and proband values for the proportion of NKT cells, suggesting that this is a heritable trait.

Conclusions

The findings suggest that analysis of cellular phenotypes may enhance the ability to detect subclinical lupus and that genetically determined altered immunoregulation by NKT cells predisposes first-degree relatives of lupus patients to the development of autoimmunity.