Severity of influenza A 2009 (H1N1) pneumonia is underestimated by routine prediction rules. Results from a prospective, population-based study.

BACKGROUND: Characteristics of patients with community-acquired pneumonia (CAP) due to pandemic influenza A 2009 (H1N1) have been inadequately compared to CAP caused by other respiratory pathogens. The performance of prediction rules for CAP during an epidemic with a new infectious agent are unknown. METHODS: Prospective, population-based study from November 2008-November 2009, in centers representing 70% of hospital beds in Iceland. Patients admitted with CAP underwent evaluation and etiologic testing, including polymerase chain reaction (PCR) for influenza. Data on influenza-like illness in the community and overall hospital admissions were collected. Clinical and laboratory data, including pneumonia severity index (PSI) and CURB-65 of patients with CAP due to H1N1 were compared to those caused by other agents. RESULTS: Of 338 consecutive and eligible patients 313 (93%) were enrolled. During the pandemic peak, influenza A 2009 (H1N1) patients constituted 38% of admissions due to CAP. These patients were younger, more dyspnoeic and more frequently reported hemoptysis. They had significantly lower severity scores than other patients with CAP (1.23 vs. 1.61, P= .02 for CURB-65, 2.05 vs. 2.87 for PSI, P<.001) and were more likely to require intensive care admission (41% vs. 5%, P<.001) and receive mechanical ventilation (14% vs. 2%, P= .01). Bacterial co-infection was detected in 23% of influenza A 2009 (H1N1) patients with CAP. CONCLUSIONS: Clinical characteristics of CAP caused by influenza A 2009 (H1N1) differ markedly from CAP caused by other etiologic agents. Commonly used CAP prediction rules often failed to predict admissions to intensive care or need for assisted ventilation in CAP caused by the influenza A 2009 (H1N1) virus, underscoring the importance of clinical acumen under these circumstances.

Identification and characterization of Grainyhead-like epithelial transactivator (GET-1), a novel mammalian Grainyhead-like factor.

LMO-4 is an LIM-only factor that is highly expressed in many epithelial cells, including those of the epidermis and hair follicles. Because LMOs may function by interacting with DNA-binding proteins, we have used the yeast two-hybrid system to screen mouse skin libraries for LMO-4-interacting DNA-binding proteins. In this screen, we isolated a novel LMO-4-interacting factor highly related to the Drosophila gene Grainyhead. Grainyhead is epidermally expressed and carries out important functions in cuticular formation in the fly embryo. With the identification of this novel mammalian Grainyhead-like gene, referred to as Grainyhead-like epithelial transactivator 1 (GET-1), the known members of the mammalian Grainyhead-like gene family are extended to six, falling into two classes based on sequence homology. Of interest, the expression pattern of GET-1 is similar to that of Drosophila Grainyhead with highest expression in the somatic ectoderm/epidermis, but GET-1 is additionally expressed in epithelial cells of gastrointestinal, genitourinary, and respiratory tracks. The GET-1 protein localizes to the nucleus and binds to at least one Grainyhead DNA-binding site. The GET-1 DNA-binding domain maps to a region containing homology to the Drosophila Grainyhead DNA-binding domain. GET-1 homodimerizes in solution by means of a short C-terminally located domain that is homologous to other Grainyhead-like genes. A short domain located between amino acids 100 and 190, which bears no homology to known transactivation domains, is sufficient to confer transactivation to the heterologous GAL4 DNA-binding domain. In addition, GET-1 appears to contain repression domains consistent with the observation that Grainyhead and other mammalian Grainyhead-like genes can act both as activators and repressors. These data suggest that GET-1 is a transcriptional regulator that may perform important functions in epithelial tissues of mammals.

Role of heteroduplex joints in the functional interactions between human Rad51 and wild-type p53.

Our previous work (Dudenhöffer et al., 1999) unveiled a link between the capacity of p53 to regulate homologous recombination processes and to specifically bind to heteroduplex junction DNAs. Here, we show that p53 participates in ternary complex formation after preassembly of nucleoproteins, consisting of the human recombinase hRad51 and junction DNA. The cancer-related mutant p53(273H), which is defective in inhibiting recombination processes, displays a reduced capacity to associate with hRad51-DNA complexes, even under conditions which support DNA-binding. This suggests that hRad51-p53 contacts play a role in targeting p53 to heteroduplex joints and indicates an involvement in recombination immediately following hRad51-mediated strand transfer. To study the initial phase of strand exchange, when heteroduplex joints arise, we applied oligonucleotide based strand transfer assays. We observed that hRad51 stimulates exonucleolytic DNA degradation by p53, when it generates strand transfer intermediates. In agreement with this observation, artificial 3-stranded junction DNAs, designed to mimic nascent recombination intermediates, were found to represent preferred exonuclease substrates, especially when comprising a mismatch within the heteroduplex part. From our data, we propose a model according to which, p53-dependent correction of DNA exchange events is triggered by high-affinity binding to joint molecules and by stabilizing contacts with hRad51 oligomers. Oncogene (2000) 19, 4500 - 4512.

Dissociation of the recombination control and the sequence-specific transactivation function of P53.

Recently, we described a new biological function of p53 in inhibiting recombination processes when encountering mismatches in heteroduplexes (Dudenhöffer et al., 1998). Here, we characterized protein domains of p53 participating in this process by in vitro analysis of mutated p53 proteins, and by applying our SV40-based assay system on monkey cells, which express different p53 variants. We present evidence that both binding of artificial recombination intermediates and p53-dependent recombination control require an intact p53 core and the oligomerization domain, strongly suggesting that the recognition of DNA undergoing recombination represents an essential step of this genomic surveillance mechanism. Further analyses indicated a role of the C-terminus in negatively regulating recombination control, an effect which can be neutralized by concurrent mismatch recognition. p53 lacking the oligomerization domain totally lost its ability to suppress homologous recombination. The cancer-related mutant p53(273H) was also significantly defective in this function, although we observed only twofold reductions in the corresponding transactivation activities on p53-response elements in episomal constructs. HDM2, an inhibitor of p53's transcriptional and growth regulatory activities, interfered with the inhibition of DNA exchange processes by p53 only weakly. Thus, functions of p53 in recombination control can be structurally dissociated from p53-dependent transcriptional transactivation.

The dual role model for p53 in maintaining genomic integrity.

The tumour suppressor p53 is a potent mediator of cellular responses against genotoxic insults. In this review we describe the multiple functions of p53 in response to DNA damage, with an emphasis on p53's role in DNA repair. We summarize data demonstrating that p53 actively participates in various processes of DNA repair and DNA recombination via its ability to interact with components of the repair and recombination machinery, and by its various biochemical activities. An important aspect in evaluating p53 functions is provided by the finding that the core domain of p53 harbours two mutually exclusive biochemical activities, sequence-specific DNA binding required for its transactivation function, and 3'-5' exonuclease activity, possibly involved in aspects of DNA repair. Based on the finding that modifications of p53 which lead to activation of its sequence-specific DNA-binding activity result in inactivation of its 3'-5' exonuclease activity, we propose that p53 exerts its functions as a 'guardian of the genome' at various levels: in its noninduced state, p53 should not be regarded as a 'dead' protein but, for example, via its exonuclease activity might be actively involved in prevention and repair of endogenous DNA damage. Upon induction through exogenous DNA damage, p53 will exert its well-documented functions as a superior response element in various types of cellular stress. This dual role model for p53 in maintaining genomic integrity significantly enhances p53's possibilities as a guardian of the genome.

Surface plasmon resonance measurements reveal stable complex formation between p53 and DNA polymerase alpha.

Surface plasmon resonance measurements were used for detecting and quantifying protein-protein interactions between the tumor suppressor protein p53, the SV40 large T antigen (T-ag), the cellular DNA polymerase alpha-primase complex (pol-prim), and the cellular single-strand DNA binding protein RPA. Highly purified p53 protein bound to immobilized T-ag with an apparent binding constant of 2 x 10(8) M(-1). Binding of p53 to RPA was in the same order of magnitude with a binding constant of 4 x 10(8) M(-1), when RPA was coupled to the sensor chip via its smallest subunit, and 1 x 10(8) M(-1), when RPA was coupled via its p70 subunit. Furthermore, p53 bound human DNA polymerase alpha-primase complex (pol-prim) with a K(A) value of 1 x 10(10) m(-1). Both the p68 subunit and the p180 subunit of pol-prim could interact with p53 displaying binding constants of 2 x 10(10) m1(-1) and 5 X 10(9) M(-1), respectively. Complex formation was also observed with a p180/p68 heterodimer, and again with a binding constant similar. Hence, there was no synergistic effect when p53 bound to higher order complexes of pol-prim. A truncated form of p53, consisting of amino acids 1-320, bound pol-prim by four orders of magnitude less efficiently. Therefore, an intact C-terminus of p53 seems to be important for efficient binding to pol-prim. It was also tried to measure complex formation between p53, pol-prim, and T-ag. However there was no evidence for the existence of a ternary complex consisting of T-ag, pol-prim, and p53.

Different regulation of the p53 core domain activities 3'-to-5' exonuclease and sequence-specific DNA binding.

In this study we further characterized the 3'-5' exonuclease activity intrinsic to wild-type p53. We showed that this activity, like sequence-specific DNA binding, is mediated by the p53 core domain. Truncation of the C-terminal 30 amino acids of the p53 molecule enhanced the p53 exonuclease activity by at least 10-fold, indicating that this activity, like sequence-specific DNA binding, is negatively regulated by the C-terminal basic regulatory domain of p53. However, treatments which activated sequence-specific DNA binding of p53, like binding of the monoclonal antibody PAb421, which recognizes a C-terminal epitope on p53, or a higher phosphorylation status, strongly inhibited the p53 exonuclease activity. This suggests that at least on full-length p53, sequence-specific DNA binding and exonuclease activities are subject to different and seemingly opposing regulatory mechanisms. Following up the recent discovery in our laboratory that p53 recognizes and binds with high affinity to three-stranded DNA substrates mimicking early recombination intermediates (C. Dudenhoeffer, G. Rohaly, K. Will, W. Deppert, and L. Wiesmueller, Mol. Cell. Biol. 18:5332-5342), we asked whether such substrates might be degraded by the p53 exonuclease. Addition of Mg2+ ions to the binding assay indeed started the p53 exonuclease and promoted rapid degradation of the bound, but not of the unbound, substrate, indicating that specifically recognized targets can be subjected to exonucleolytic degradation by p53 under defined conditions.

p53 Protein exhibits 3'-to-5' exonuclease activity.

Highly purified p53 protein from different sources was able to degrade DNA with a 3'-to-5' polarity, yielding deoxynucleoside monophosphates as reaction products. This exonuclease activity was dependent on Mg2+ and inhibited by addition of 5 mM nucleoside monophosphates. This exonuclease activity is intrinsic to the wild-type p53 protein: it copurified with p53 during p53 preparation; only purified wild-type p53, but not identically purified mutant p53 proteins displayed exonuclease activity; the exonuclease activity could be reconstituted from SDS gel-purified and urea-renatured p53 protein and mapped to the core domain of the p53 molecule; and finally, purified p53 protein could be UV-cross-linked to GMP. A p53-intrinsic exonuclease activity should substantially extend our view on the role of p53 as a "guardian of the genome."