Transcription factor EB influences invasion and migration in oral squamous cell carcinomas.

OBJECTIVE: Transcription factor EB (TFEB) is a master regulator of lysosomal biogenesis and plays an important role in various cancers. However, the function of TFEB in oral squamous cell carcinomas has not been examined. The aim of this study was to elucidate the role of TFEB in oral squamous cell carcinomas. MATERIALS AND METHODS: Expression levels of TFEB were examined in six different human oral squamous carcinoma cells: HSC2, HSC3, HSC4, SAS, OSC20, and SCC25. Knockdown of TFEB using small interfering RNA in HSC2 and HSC4 cells was performed. Cell morphology was observed by immunofluorescence microscopy. Cell proliferation, invasion, and adhesion were analyzed. RESULTS: Expression levels of TFEB were high in HSC2, moderate in HSC4 and SCC25, and low in HSC3 and OSC20 cells. Knockdown of TFEB did not affect proliferation of HSC2 and HSC4 cells, but did induced enlargement of lysosomes and endosomes in HSC4 cells. TFEB silencing reduced invasion and migration of these HSC cell squamous carcinoma cells; however, increased cell adhesion was also observed. CONCLUSION: TFEB knockdown reduces invasion and migration of cancer cells, likely through lysosomal regulation. Taken together, TFEB influences cell invasion and migration of oral squamous cell carcinomas.

A sexually dimorphic peptidergic system in the lower spinal cord controlling penile function in non-human primates.

STUDY DESIGN: Experimental animal study. OBJECTIVES: Although a population of gastrin-releasing peptide (GRP) neurons in the lumbar spinal cord has an important role in erection and ejaculation in rats, little information exists on this GRP system in primates. To identify the male-specific GRP system in the primate spinal cord, we studied the lumbosacral cord in macaque monkeys as a non-human primate model. SETTING: University laboratory in Japan. METHODS: To determine the gene sequence of GRP precursors, the rhesus macaque monkey genomic sequence data were searched, followed by phylogenetic analysis. Subsequently, immunocytochemical analysis for GRP was performed in the monkey spinal cord. RESULTS: We have used bioinformatics to identify the ortholog gene for GRP precursor in macaque monkeys. Phylogenetic analysis suggested that primate prepro-GRP is separated from that of other mammalian species and clustered to an independent branch as primates. Immunocytochemistry for GRP further demonstrated that male-dominant sexual dimorphism was found in the spinal GRP system in monkeys as in rodents. CONCLUSION: We have demonstrated in macaque monkeys that the GRP system in the lower spinal cord shows male-specific dimorphism and may have an important role in penile functions not only in rodents but also in primates. SPONSORSHIP: Tissues of Nihonzaru (Japanese macaque monkeys) were provided in part by National Institutes of Natural Sciences (NINS) through the National Bio-Resource Project (NBRP) of the MEXT, Japan. This work was supported in part by KAKENHI from the Japan Society for the Promotion of Science (JSPS) (to KT; 15KK0343, 15J40220 and HS; 15K15202, 15KK0257, 15H05724).

Inter-observer reproducibility of endometrial cytology by the Osaki Study Group method: utilising the Becton Dickinson SurePath™ liquid-based cytology.

OBJECTIVE: The purpose of the present study was to evaluate the reproducibility of the cytological diagnosis of endometrial lesions by the Osaki Study Group (OSG) method of new cytological diagnostic criteria using BD SurePath™ (SP)-liquid-based cytology (LBC). METHODS: This cytological classification using the OSG method consists of six categories: (i) normal endometrium (NE), (ii) endometrial glandular and stromal breakdown (EGBD), (iii) atypical endometrial cells, cannot exclude atypical endometrial hyperplasia or more (ATEC-A), (iv) adenocarcinoma including atypical endometrial hyperplasia or malignant tumour (Malignancy), (v) endometrial hyperplasia without atypia (EH) and (vi) atypical endometrial cells of undetermined significance (ATEC-US). For this study, a total 244 endometrial samplings were classified by two academic cytopathologists as follows: 147 NE cases , 36 EGBD cases , 47 Malignant cases, eight ATEC-A cases, two EH cases and four ATEC-US cases. To confirm the reproducibility of the diagnosis and to study the inter- and intra-observer agreement further, a second review round followed at 3-month intervals, which included three additional cytopathologists. RESULTS: The inter-observer agreement of NE classes improved progressively from 'good to fair' to 'excellent', with values increasing from 0.70 to 0.81. Both EGBD and Malignancy classes improved progressively from 'good to fair' to 'excellent', with values increasing from 0.62-0.63 to 0.84-0.95, respectively. The overall intra-observer agreement between the first and the second rounds was 'good to fair' to 'excellent', with values changing from 0.79 to 0.85. All kappa improvements were significant (P < 0.0001). CONCLUSION: In this study, it seemed that the use of the OSG method as the new diagnostic criteria for SP-LBC preparation, may be a valid method to improve the precision (reproducibility) of endometrial cytology.

Multicentre analysis of long-term outcome after surgical resection for gastric cancer liver metastases.

BACKGROUND: The efficacy of surgical resection for gastric cancer liver metastases (GCLMs) is currently debated. Hitherto, no large-scale clinical studies have been conducted. METHODS: This retrospective multicentre study analysed a database of consecutive patients with either synchronous or metachronous metastases who underwent surgical R0 resection for GCLM between 1990 and 2010. Clinical data were collected from five cancer centres in Japan. Survival curves were assessed, and clinical parameters were evaluated to identify predictors of prognosis. RESULTS: A total of 256 patients were enrolled. The mean(s.d.) number of hepatic tumours resected was 2.0(2.4). The surgical mortality rate was 1.6 per cent. Median follow-up was 65 (range 1-261) months. Recurrences were detected in 192 patients (75.0 per cent). The median interval from hepatic resection to recurrence was 7 (range 1-72) months, and the dominant site of recurrence was the liver (72.4 per cent). Actuarial 1-, 3- and 5-year overall and recurrence-free survival rates were 77.3, 41.9 and 31.1 per cent, and 43.6, 32.4 and 30.1 per cent, respectively. Median overall and recurrence-free survival times were 31.1 and 9.4 months respectively. Multivariable analysis identified serosal invasion of the primary gastric cancer (hazard ratio (HR) 1.50; P = 0.012), three or more liver metastases (HR 2.33; P < 0.001) and liver tumour diameter at least 5 cm (HR 1.62; P = 0.005) as independent predictors of poor survival. CONCLUSION: Clinically resectable GCLM is rare, but strict and careful patient selection can lead to long-term survival following R0 surgical resection.

The gene for Machado-Joseph disease maps to human chromosome 14q.

Machado-Joseph disease (MJD) is an autosomal dominant, multisystem neurodegenerative disorder involving predominantly cerebellar, pyramidal, extrapyramidal, motor neuron and oculomotor systems. Although it was first reported in families of Portuguese-Azorean descent, MJD has also been described in non-Azorean families from various countries, being one of the most common hereditary spinocerebellar degenerations. With the use of highly polymorphic microsatellite DNA polymorphisms, we have assigned the gene for MJD to the long arm of chromosome 14 (14q24.3-q32) by genetic linkage to microsatellite loci D14S55 and D14S48 (multipoint lod score Zmax = 9.719).

IL-17 eliminates therapeutic effects of oral tolerance in murine airway allergic inflammation.

BACKGROUND: Oral tolerance is a classically used strategy for antigen-specific systemic immunotherapy. However, the roles of IL-17 in modification of oral tolerance are not yet understood. OBJECTIVE: To define the effects of IL-17 on the modification of oral tolerance, the effects of transfer of Th17 cells, administration of IL-17 or anti-IL-17 antibody (αIL-17Ab) to a murine allergic airway inflammation model were investigated. METHODS: Mice sensitized to and challenged with OVA, received OVA feeding, followed by OVA challenges. Transfer of Th17 cells, administration of IL-17 or αIL-17Ab were executed during OVA feeding. Airway hyperresponsiveness (AHR), airway inflammation, Th2 cytokine response and lung pathology were assessed. RESULTS: Administration of IL-17 as well as transfer of Th17 cells aggravated AHR and airway allergic inflammation as compared with the findings in mice subjected to OVA feeding alone, whereas administration of αIL-17Ab ameliorated AHR and airway eosinophilia. The effects of Th17 transfer were presumably attributable to augmentation of endogenous IL-6 production in gut. The number of Foxp3-positive regulatory T (Treg) cells in lungs and Payer's patches was increased in the OVA fed mice, whereas the number of these cells was decreased in the mice subjected to OVA feeding + Th17 cell transfer. Neutralization of IL-6 by monoclonal antibody in the mice subjected to OVA feeding + transfer of Th17 cells restored the effects of oral tolerance. CONCLUSIONS AND CLINICAL RELEVANCE: These data suggest that IL-17 may inhibit the induction of tolerance to antigen through, at least in part augmenting IL-6 production, thereby suppressing the expansion of Treg cells.

ADC Level is Related to DWI Reversal in Patients Undergoing Mechanical Thrombectomy: A Retrospective Cohort Study.

BACKGROUND AND PURPOSE: In patients with ischemic stroke, DWI lesions can occasionally be reversed by reperfusion therapy. This study aimed to ascertain the relationship between ADC levels and DWI reversal in patients with acute ischemic stroke who underwent recanalization treatment. MATERIALS AND METHODS: We conducted a retrospective cohort study in patients with acute ischemic stroke who underwent endovascular mechanical thrombectomy with successful recanalization between April 2017 and March 2021. DWI reversal was assessed through follow-up MR imaging approximately 24 hours after treatment. RESULTS: In total, 118 patients were included. DWI reversal was confirmed in 42 patients. The ADC level in patients with reversal was significantly higher than that in patients without reversal. Eighty-three percent of patients with DWI reversal areas had mean ADC levels of ≥520 × 10-6 mm2/s, and 71% of patients without DWI reversal areas had mean ADC levels of <520 × 10-6 mm2/s. The mean ADC threshold was 520 × 10-6 mm2/s with a sensitivity and specificity of 71% and 83%, respectively. In multivariate analysis, the mean ADC level (OR, 1.023; 95% CI, 1.013-1.033; P < .0001) was independently associated with DWI reversal. Patients with DWI reversal areas had earlier neurologic improvement (NIHSS at 7 days) than patients without reversal areas (P < .0001). CONCLUSIONS: In acute ischemic stroke, the ADC value is independently associated with DWI reversal. Lesions with a mean ADC of ≥520 × 10-6 mm2/s are salvageable by mechanical thrombectomy, and DWI reversal areas regain neurologic function. The ADC value is easily assessed and is a useful tool to predict viable lesions.

A DNA functionalized advanced electrochemical biosensor for identification of the foodborne pathogen Salmonella enterica serovar Typhi in real samples.

An efficient platform for the detection of Salmonella enterica serovar Typhi (S. Typhi) is essential for early-stage diagnosis of typhoid to prevent and contain outbreaks. Here, we fabricated an electrochemical DNA biosensor for selective identification of S. Typhi in real samples. The biosensor has been fabricated by immobilizing an amine labelled S. Typhi specific single-strand capture probe on the surface of gold nanoparticles (AuNP) and poly cysteine (P-Cys) modified screen-printed electrode. Differential pulse voltammetry (DPV) of anthraquinone-2-sulfonic acid monohydrate sodium salt (AQMS) as a signal indicator was monitored to detect S. Typhi by hybridization of target DNA with the probe DNA. The fabricated biosensor shows a detection range of 1 × 10-6 to 1 × 10-22 molL-1 with a LOD of 6.8 × 10-25 molL-1 in S. Typhi complementary linear target and 1.8 × 105 to 1.8 CFUml-1 with a LOD of 1 CFUml-1 in a real S. Typhi sample. The biosensor shows excellent discrimination ability to some bases mismatched and different bacterial cultures (same and distant genera). The most beneficial points of the proposed DNA biosensor are the lower limit of detection and the ability to reuse the biosensor more than 6 to 7 times. In addition, the practicability of the biosensor was investigated via detecting S. Typhi in blood, poultry feces, egg, and milk whereby excellent recoveries ranging from 96.54 to 103.47% were demonstrated indicating that this biosensor might be the most promising diagnostic tool for monitoring S. Typhi in clinical and food samples.

The expression of CD36 in vessels with blood-brain barrier impairment in a stroke-prone hypertensive model.

AIMS: The class B scavenger receptor CD36, the receptor for oxidized low-density lipoprotein, mediates free radical production and brain injury in cerebral ischaemia. Free radical production is known to be involved in the remodelling of the cerebral vasculature of stroke-prone spontaneously hypertensive rats (SHRSP). Accordingly, we examined whether the expression of CD36 is increased in the vasculature with blood-brain barrier (BBB) impairment and collagen deposition of SHRSP. METHODS: The gene and protein expression of CD36 was examined in the vessels of the hippocampus of SHRSP with BBB impairment and those of Wistar Kyoto rats without the impairment, by real-time RT-PCR, Western blotting and immunohistochemical techniques. RESULTS: The gene and protein expression of CD36 was increased in the hippocampus of SHRSP compared with that of Wistar Kyoto rats. Confocal microscopic examination revealed CD36 immunoreactivity in perivascular microglial cells immunopositive for ED1. Immunoelectron microscopic examination revealed that the immunosignals for CD36 were located mainly in the cytoplasm of perivascular cells in vessels showing increased vascular permeability and a few in the cytoplasmic membranes of endothelial cells. CONCLUSIONS: These findings indicate that the expression of CD36 was increased in vessels with BBB impairment in the hippocampus of SHRSP and was mainly seen in the cytoplasm of perivascular microglial cells, suggesting a role of CD36 in cerebrovascular injury.

Behavior of pharmaceuticals in waste water treatment plant in Japan.

The fate of pharmaceuticals in a wastewater treatment plant (WWTP) in Kumamoto, Japan with activated sludge treatment is reported. Selected pharmaceuticals were detected in influent. Results from the present study confirmed that Acetaminophen, Amoxicillin, Ampicillin and Famotidine were removed at a high rate (>90% efficiency). In contrast, removal efficiency of Ketoprofen, Losartan, Oseltamivir, Carbamazepine, and Diclofenac was relatively low (<50%). The selected pharmaceuticals were also detected in raw sludge. In digestive process, Indomethacin, Atenolol, Famotidine, Trimethoprim and Cyclofosamide were removed at a high (>70% efficiency). On the other hand, removal of Carbamazepine, Ketoprofen and Diclofenac was not efficient (<50%).

Odontogenic gingival epithelial hamartoma with a pleomorphic histological appearance: a case report and review of the literature.

Odontogenic gingival epithelial hamartoma (OGEH) is a rare odontogenic epitheliallesion. Distinguishing OGEH from peripheral ameloblastoma can pose a differential diagnostic challenge. We report a case of OGEH affecting the lower gingiva in a 78-year-old male patient. The lesion presented as a large, painless, pedunculated mass with a pleomorphic histological appearance. A diagnosis of OGEH was made after the exclusion of peripheral ameloblastoma and peripheral adenomatoid odontogenic tumour. Previous studies have reported differences in sex and age between patients with OGEH and peripheral ameloblastoma; this suggests that peripheral ameloblastoma is not derived from OGEH. Histological findings suggest that the lesions are independent entities.

Therapy of murine tumors with p53 wild-type and mutant sequence peptide-based vaccines.

The BALB/c Meth A sarcoma carries a p53 missense mutation at codon 234, which occurs in a peptide, termed 234CM, capable of being presented to cytotoxic T lymphocytes (CTL) by H-2Kd molecules (Noguchi, Y., E.C. Richards, Y.-T. Chen, and L.J. Old. 1994. Proc. Natl. Acad. Sci. USA. 91:3171-3175). Immunization of BALB/c mice with bone marrow-derived dendritic cells (DC), generated in the presence of granulocyte macrophage colony-stimulating factor and interleukin 4, and prepulsed with the Meth A p53 mutant peptide, induced CTL that specifically recognized peptide-pulsed P815 cells, as well as Meth A cells naturally expressing this epitope. Immunization with this vaccine also protected naive mice from a subsequent tumor challenge, and it inhibited tumor growth in mice bearing day 7 subcutaneous Meth A tumors. We additionally determined that immunization of BALB/c mice with DC pulsed with the p53 peptide containing the wild-type residue at position 234, 234CW, induced peptide-specific CTL that reacted against several methylcholanthrene-induced BALB/c sarcomas, including CMS4 sarcoma, and rejection of CMS4 sarcoma in vaccination and therapy (day 7) protocols. These results support the efficacy of DC-based, p53-derived peptide vaccines for the immunotherapy of cancer. The translational potential of this strategy is enhanced by previous reports showing that DC can readily be generated from human peripheral blood lymphocytes.

Long-term field trial to control the invasive Argentine ant (Hymenoptera: Formicidae) with synthetic trail pheromone.

Previous short-term experiments showed that trail following behavior of the Argentine ant, Linepithema humile (Mayr) (Hymenoptera: Formicidae), can be disrupted by a high concentration of synthetic trail pheromone component (Z)-9-hexadecenal. In this study, a long-term field trial was conducted in 100-m2 plots of house gardens in an urban area of Japan to see whether the control effect on Argentine ants can be obtained by permeating synthetic trail pheromone from dispensers. The dispensers were placed in the experimental plots during the ant's active season (April-November) for 2 yr with monthly renewal. To estimate Argentine ant population density, foraging activity of Argentine ants in the study plots was monitored by monthly bait surveys. Throughout the study period, Argentine ant foraging activity was suppressed in the presence of the dispensers, presumably via trail forming inhibition. In contrast, the level of foraging activity was not different between treatment and no-treatment plots when the dispensers were temporarily removed, suggesting that treatment with pheromone dispensers did not suppress Argentine ant density in the treatment plots. Population decline may be expected with larger-scale treatment that covers a significant portion of the ant colony or with improvement in the potency of the disruptant.

A case of resected retroperitoneal metachronous solitary metastasis from caecal cancer.

Colorectal cancer metastasis to the retroperitoneum, especially solitary metastasis allowing curative resection, is rare. We report a case of complete resection of retroperitoneal metachronous solitary metastasis from caecal cancer without distant metastasis. An 80-year-old woman with caecal cancer underwent laparoscopic ileocaecal resection with regional lymph node dissection. According to the eighth edition of the TNM classification, the pathological diagnosis was stage IIA (T3N0M0). Six months following the surgery, computed tomography revealed a solitary mass of 2cm diameter, dorsal to the right kidney. A second procedure for the removal of the tumour was performed. The lesion was pathologically diagnosed as a metachronous solitary retroperitoneal metastasis from caecal cancer. The patient is surviving and free from recurrence 17 months following the second procedure.

Cross talk between hedgehog and epithelial-mesenchymal transition pathways in gastric pit cells and in diffuse-type gastric cancers.

We previously reported hedgehog (Hh) signal activation in the mucus-secreting pit cell of the stomach and in diffuse-type gastric cancer (GC). Epithelial-mesenchymal transition (EMT) is known to be involved in tumour malignancy. However, little is known about whether and how both signallings cooperatively act in diffuse-type GC. By microarray and reverse transcription-PCR, we investigated the expression of those Hh and EMT signalling molecules in pit cells and in diffuse-type GCs. How both signallings act cooperatively in those cells was also investigated by the treatment of an Hh-signal inhibitor and siRNAs of Hh and EMT transcriptional key regulator genes on a mouse primary culture and on human GC cell lines. Pit cells and diffuse-type GCs co-expressed many Hh and EMT signalling genes. Mesenchymal-related genes (WNT5A, CDH2, PDGFRB, EDNRA, ROBO1, ROR2, and MEF2C) were found to be activated by an EMT regulator, SIP1/ZFHX1B/ZEB2, which was a target of a primary transcriptional regulator GLI1 in Hh signal. Furthermore, we identified two cancer-specific Hh targets, ELK1 and MSX2, which have an essential role in GC cell growth. These findings suggest that the gastric pit cell exhibits mesenchymal-like gene expression, and that diffuse-type GC maintains expression through the Hh-EMT pathway. Our proposed extensive Hh-EMT signal pathway has the potential to an understanding of diffuse-type GC and to the development of new drugs.

The expression of P-glycoprotein is increased in vessels with blood-brain barrier impairment in a stroke-prone hypertensive model.

AIMS: We previously reported that the blood-brain barrier (BBB) function was impaired in vessels in the hippocampus in 3-month-old stroke-prone spontaneously hypertensive rats (SHRSP). In this study, we examined gene and protein expressions of P-glycoprotein, a representative efflux transporter of cerebral vessels, in the BBB-damaged hippocampal vessels of SHRSP and in the vessels of Wistar Kyoto (WKY) rats as controls, to clarify roles of the efflux transporter in the BBB-damaged vessels. METHODS: The expression of P-glycoprotein in hippocampal and cortical samples was examined by real-time quantitative reverse transcriptase-polymerase chain reaction (RT-PCR), Western blotting and immunoelectron microscopic techniques. RESULTS: Real-time RT-PCR and Western blotting analyses revealed that the gene and protein expressions of P-glycoprotein were increased in the hippocampal samples of 3-month-old SHRSP compared with hippocampal samples of 3-month-old WKY rats or with cortical samples of SHRSP. The gene expression of P-glycoprotein was also increased in the hippocampal samples of 4-week-old SHRSP. Immunoelectron microscopic examination revealed that immunosignals of P-glycoprotein were seen in the luminal and ab-luminal cytoplasmic membranes of endothelial cells and the basal lamina, that the labelling density of P-glycoprotein in the vessel wall was higher in the hippocampus of 3-month-old SHRSP than in other groups and that the immunosignals of P-glycoprotein were occasionally co-located with those of albumin. CONCLUSIONS: These findings indicate that the expression of P-glycoprotein is increased in BBB-damaged hippocampal vessels in hypertensive SHRSP compared with those in WKY rats.

Hst-1 (FGF-4) antisense oligonucleotides block murine limb development.

The initiation of limb development depends on the site specific proliferation of the mesenchyme by the signals from the apical ectodermal ridge (AER) in embryonic mouse. We have previously reported that the local expression of Hst-1/Fgf-4 transcripts in AER of the mouse limb bud is developmentally regulated, expressed at 11 and 12 days post coitus (p.c.) embryo. In an effort to further understand the role of Hst-1/FGF-4 in mouse limb development, an antisense oligodeoxynucleotides (ODNs) study was performed. We first established a novel organ culture system to study mouse limb development in vitro. This system allows mouse limb bud at 9.5-10-d p.c. embryo, when placed on a sheet of extracellular matrix in a defined medium, to differentiate into a limb at 12.5-d p.c. embryo within 4.5 d. Using this organ culture system, we have shown that exposure of 9.5-10-d p.c. embryonal limb bud explants to antisense ODNs of Hst-1/FGF-4 blocks limb development. In contrast, sense and scrambled ODNs have no inhibitory effect on limb outgrowth, suggesting that Hst-1/FGF-4 may work as a potent inducing factor for mouse limb development.

Control of doublesex alternative splicing by transformer and transformer-2 in Drosophila.

Sex-specific alternative processing of doublesex (dsx) precursor messenger RNA (pre-mRNA) regulates somatic sexual differentiation in Drosophila melanogaster. Cotransfection analyses in which the dsx gene and the female-specific transformer (tra) and transformer-2 (tra-2) complementary DNAs were expressed in Drosophila Kc cells revealed that female-specific splicing of the dsx transcript was positively regulated by the products of the tra and tra-2 genes. Furthermore, analyses of mutant constructs of dsx showed that a portion of the female-specific exon sequence was required for regulation of dsx pre-messenger RNA splicing.

Cis-trans isomerism in naphthoquinones: interconversion and participation in oxidative phosphorylation.

Synthetic phylloquinone was resolved into cis and trans isomers by thin-layer chromatography. The two isomers had identical ultraviolet spectra characteristic of vitamin K(1) and were differentiated by nuclear-magnetic-resonance spectroscopy on the basis of the displacement of the peak corresponding to the olefinic methyl group in the naphthoquinone side chain. Studies on the restoration of electron transport coupled to phosphorylation in irradiated preparations of Mycobacterium phlei showed that only the trans isomer was active with substrates linked to nicotinamide-adenine dinucleotide. The purified trans phylloquinone was enzymatically converted to the cis isomer. Under similar conditions, cis vitamin K(1) gave rise to the trans-naphthoquinone. The natural naphthoquinone of M. phlei vitamin MK(9)(II-H) was similarly resolved into cis and trans isomers.

High-resolution structure of the oligomerization domain of p53 by multidimensional NMR.

The three-dimensional structure of the oligomerization domain (residues 319 to 360) of the tumor suppressor p53 has been solved by multidimensional heteronuclear magnetic resonance (NMR) spectroscopy. The domain forms a 20-kilodalton symmetric tetramer with a topology made up from a dimer of dimers. The two primary dimers each comprise two antiparallel helices linked by an antiparallel beta sheet. One beta strand and one helix are contributed from each monomer. The interface between the two dimers forming the tetramer is mediated solely by helix-helix contacts. The overall result is a symmetric, four-helix bundle with adjacent helices oriented antiparallel to each other and with the two antiparallel beta sheets located on opposing faces of the molecule. The tetramer is stabilized not only by hydrophobic interactions within the protein core but also by a number of electrostatic interactions. The implications of the structure of the tetramer for the biological function of p53 are discussed.