An Alternative Form of Replication Protein A Expressed in Normal Human Tissues Supports DNA Repair

Replication protein A (RPA) is a heterotrimeric protein complex required for a large number of DNA metabolic processes, including DNA replication and repair. An alternative form of RPA (aRPA) has been described in which the RPA2 subunit (the 32-kDa subunit of RPA and product of the RPA2 gene) of canonical RPA is replaced by a homologous subunit, RPA4. The normal function of aRPA is not known; however, previous studies have shown that it does not support DNA replication in vitro or S-phase progression in vivo. In this work, we show that the RPA4 gene is expressed in normal human tissues and that its expression is decreased in cancerous tissues. To determine whether aRPA plays a role in cellular physiology, we investigated its role in DNA repair. aRPA interacted with both Rad52 and Rad51 and stimulated Rad51 strand exchange. We also showed that, by using a reconstituted reaction, aRPA can support the dual incision/excision reaction of nucleotide excision repair. aRPA is less efficient in nucleotide excision repair than canonical RPA, showing reduced interactions with the repair factor XPA and no stimulation of XPF-ERCC1 endonuclease activity. In contrast, aRPA exhibits higher affinity for damaged DNA than canonical RPA, which may explain its ability to substitute for RPA in the excision step of nucleotide excision repair. Our findings provide the first direct evidence for the function of aRPA in human DNA metabolism and support a model for aRPA functioning in chromosome maintenance functions in nonproliferating cells.     

Tomato linalool synthase is induced in trichomes by jasmonic acid

Tomato (Lycopersicon esculentum) plants emit a blend of volatile organic compounds, which mainly consists of terpenes. Upon herbivory or wounding, the emission of several terpenes increases. We have identified and characterized the first two tomato monoterpene synthases, LeMTS1 and LeMTS2. Although these proteins were highly homologous, recombinant LeMTS1 protein produced (R)-linalool from geranyl diphosphate (GPP) and (E)-nerolidol from farnesyl diphosphate (FPP), while recombinant LeMTS2 produced β-phellandrene, β-myrcene, and sabinene from GPP. In addition, these genes were expressed in different tissues: LeMTS1 was expressed in flowers, young leaves, stems, and petioles, while LeMTS2 was strongest expressed in stems and roots. LeMTS1 expression in leaves was induced by spider mite-infestation, wounding and jasmonic acid (JA)-treatment, while LeMTS2 did not respond to these stimuli. The expression of LeMTS1 in stems and petioles was predominantly detected in trichomes and could be induced by JA. Because JA treatment strongly induced emission of linalool and overexpression of LeMTS1 in tomato resulted in increased production of linalool, we propose that LeMTS1 is a genuine linalool synthase. Our results underline the importance of trichomes in JA-induced terpene emission in tomato.     

An overview of the serpin superfamily

Serpins are a broadly distributed family of protease inhibitors that use a conformational change to inhibit target enzymes. They are central in controlling many important proteolytic cascades, including the mammalian coagulation pathways. Serpins are conformationally labile and many of the disease-linked mutations of serpins result in misfolding or in pathogenic, inactive polymers.     

High visceral fat mass and high liver fat are associated with resistance to lifestyle intervention

Objective: High visceral adipose tissue (VAT) and high liver fat (LF) are associated with the metabolic syndrome and diabetes. We studied changes in these two fat depots during weight loss and analyzed whether VAT and LF at baseline predict the response to lifestyle intervention. Research Methods and Procedures: One hundred twelve subjects (48 men and 64 women; age, 46 ± 11 years; BMI, 29.2 ± 4.4 kg/m²) were studied after a follow up‐time of 264 ± 60 (SD) days. Insulin sensitivity was estimated from the oral glucose tolerance test. Body fat depots were quantified using magnetic resonance imaging and spectroscopy. Results: Cross‐sectionally high VAT (r = ?0.22, p = 0.02) and high LF (r = ?0.36, p < 0.0001) were independently associated with low insulin sensitivity. With intervention, BMI (?3.0%), VAT (?12.0%), and LF (?33.0%) were reduced (all p < 0.001). Insulin sensitivity was improved (+17%, p < 0.01). The changes in BMI (r = ?0.41), VAT (r = ?0.28), and LF (r = ?0.39) were associated with the increase in insulin sensitivity (all p < 0.01). High VAT (r = ?0.28, p = 0.01) and high LF (r = ?0.38, p < 0.01) at baseline were associated with a lesser increase in insulin sensitivity. Discussion: Baseline values and changes in BMI, VAT, and LF are related to changes in insulin sensitivity during lifestyle intervention. Subjects with high VAT and LF have a lower chance of profiting from lifestyle intervention and may require intensified lifestyle prevention strategies or even pharmacological approaches to improve insulin sensitivity.     

High-magnitude, virus-specific CD4 T-cell response in the central nervous system of coronavirus-infected mice

The neurotropic JHM strain of mouse hepatitis virus (MHV) causes acute encephalitis and chronic demyelinating encephalomyelitis in rodents. Previous results indicated that CD8 T cells infiltrating the central nervous system (CNS) were largely antigen specific in both diseases. Herein we show that by 7 days postinoculation, nearly 30% of the CD4 T cells in the acutely infected CNS were MHV specific by using intracellular gamma interferon (IFN-gamma) staining assays. In mice with chronic demyelination, 10 to 15% of the CD4 T cells secreted IFN-gamma in response to MHV-specific peptides. Thus, these results show that infection of the CNS is characterized by a large influx of CD4 T cells specific for MHV and that these cells remain functional, as measured by cytokine secretion, in mice with chronic demyelination.     

Regional heterogeneity of rat brain phencyclidine (PCP) receptors revealed by photoaffinity labeling with [3H] azido phencyclidine

Photoaffinity labeling of rat brain phencyclidine (PCP) receptors with [3H] azido phencyclidine ([3H]AZ-PCP) reveals the existence of five polypeptides which are specifically labeled by the affinity probe (Mr's 90,000, 62,000, 49,000, 40,000 and 33,000). These labeled components are unevenly distributed in rat brain. In the frontal cortex, thalamus and olfactory bulb, the major bands labeled are the Mr's 90 K and 62 K polypeptides; in the cerebellum most of the labeling is in the 90 K and 33 K bands; and in the hippocampus all but the Mr 40 K band are heavily labeled. Together with dexoxadrol/[3H]PCP competition binding data, which indicated the existence of high and low affinity dexoxadrol/PCP binding sites, these results suggest regional heterogeneity of PCP receptors. The regional distribution of the high affinity dexoxadrol binding sites correlates best with that of the Mr 90 K polypeptide.     

Induction of a leaf specific geranylgeranyl pyrophosphate synthase and emission of (E,E)-4,8,12-trimethyltrideca-1,3,7,11-tetraene in tomato are dependent on both jasmonic acid and salicylic acid signaling pathways

Two cDNAs encoding geranylgeranyl pyrophosphate (GGPP) synthases from tomato (Lycopersicon esculentum) have been cloned and functionally expressed in Escherichia coli. LeGGPS1 was predominantly expressed in leaf tissue and LeGGPS2 in ripening fruit and flower tissue. LeGGPS1 expression was induced in leaves by spider mite (Tetranychus urticae)-feeding and mechanical wounding in wild type tomato but not in the jasmonic acid (JA)-response mutant def-1 and the salicylic acid (SA)-deficient transgenic NahG line. Furthermore, LeGGPS1 expression could be induced in leaves of wild type tomato plants by JA- or methyl salicylate (MeSA)-treatment. In contrast, expression of LeGGPS2 was not induced in leaves by spider mite-feeding, wounding, JA- or MeSA-treatment. We show that emission of the GGPP-derived volatile terpenoid (E,E)-4,8,12-trimethyltrideca-1,3,7,11-tetraene (TMTT) correlates with expression of LeGGPS1. An exception was MeSA-treatment, which resulted in induction of LeGGPS1 but not in emission of TMTT. We show that there is an additional layer of regulation, because geranyllinalool synthase, catalyzing the first dedicated step in TMTT biosynthesis, was induced by JA but not by MeSA.Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at .