Iron pill aspiration syndrome: A case report and literature review.

Aspiration of iron pill containing ferrous sulfate into the airway can induce fulminant chemical burn and necrosis of the airway mucosa. Acute chemical burn and inflammatory response can result in life-threatening airway compromise. It can also result in long-term sequelae including but not limited to fibrosis and airway stenosis. Considering the common use of iron supplements, and the potential severity of aspiration related airway injury, clinicians should be fully cognizant of the interaction between aspirated iron and airway passages. Herein, we present a case report with pertinent review of the literature.

The Gulf Coast Tick, Amblyomma maculatum (Ixodida: Ixodidae), and Spotted Fever Group Rickettsia in the Highly Urbanized Northeastern United States.

We report the multi-year collection of the Gulf Coast tick, Amblyomma maculatum Koch (Acaridae: Ixodida: Ixodidae) in Staten Island, New York City (NYC) as well as their detection in Brooklyn, NYC, and in Atlantic and Cumberland counties in southern New Jersey, USA. The first and most common detections were of adults, however in Freshkills Park on Staten Island larvae were also collected in a following year. The presence of larvae indicates that adults are successfully finding hosts in Staten Island. While it is still unknown how A. americanum reached Staten Island, immatures of this species often parasitize migratory birds, which are now often seen in Freshkills Park. We describe the landscape features of the area in Staten Island where populations were highest and larvae were detected, which could have facilitated the establishment of A. maculatum. Notably, we also report the presence of human pathogens Rickettsia parkeri in 5/10 (50%) of adults tested and R. felis in 1/24 (4.17%) of larvae tested. In addition to established populations in Staten Island we found evidence of A. maculatum in NJ and other NYC boroughs, suggesting current or future establishment is possible. The failure thus far to detect established populations in these areas may be due to inherent difficulties in detecting low density, spatially heterogeneous incipient populations, which could require targeted surveillance efforts for this species. We discuss the consequences to public health of the establishment of A. maculatum and detection of two additional rickettsial pathogens in the densely populated northeastern United States.

First glimpse into the origin and spread of the Asian longhorned tick, Haemaphysalis longicornis, in the United States.

Established populations of Asian longhorned ticks (ALT), Haemaphysalis longicornis, were first identified in the United States (US) in 2017 by sequencing the mitochondrial cytochrome c oxidase subunit I (cox1) 'barcoding' locus followed by morphological confirmation. Subsequent investigations detected ALT infestations in 12, mostly eastern, US states. To gain information on the origin and spread of US ALT, we (1) sequenced cox1 from ALT populations across 9 US states and (2) obtained cox1 sequences from potential source populations [China, Japan and Republic of Korea (ROK) as well as Australia, New Zealand and the Kingdom of Tonga (KOT)] both by sequencing and by downloading publicly available sequences in NCBI GenBank. Additionally, we conducted epidemiological investigations of properties near its initial detection locale in Hunterdon County, NJ, as well as a broader risk analysis for importation of ectoparasites into the area. In eastern Asian populations (China/Japan/ROK), we detected 35 cox1 haplotypes that neatly clustered into two clades with known bisexual versus parthenogenetic phenotypes. In Australia/New Zealand/KOT, we detected 10 cox1 haplotypes all falling within the parthenogenetic cluster. In the United States, we detected three differentially distributed cox1 haplotypes from the parthenogenetic cluster, supporting phenotypic evidence that US ALT are parthenogenetic. While none of the source populations examined had all three US cox1 haplotypes, a phylogeographic network analysis supports a northeast Asian source for the US populations. Within the United States, epidemiological investigations indicate ALT can be moved long distances by human transport of animals, such as horses and dogs, with smaller scale movements on wildlife. These results have relevant implications for efforts aimed at minimizing the spread of ALT in the United States and preventing additional exotic tick introductions.

Distinct New York City Aedes albopictus Mosquito Populations Display Differences in Salivary Gland Protein D7 Diversity and Chikungunya Virus Replication.

In an increasingly interconnected world, the exposure and subsequent spread of emergent viruses has become inevitable. This is particularly true for Aedes (Ae.) mosquito-vectored viruses, whose range has increased over the past decade from tropical to temperate regions. However, it is unclear if all populations of Ae. mosquitoes in temperate New York City are able to successfully replicate and transmit arboviruses. To answer this question, we reared Ae. albopictus mosquitoes living in a temperate climate from three locations in New York City. We first sequenced the salivary antiviral protein D7 from individual mosquitoes in each population and found single nucleotide variants that are both shared and unique for each Ae. albopictus population. We then fed each population chikungunya virus (CHIKV) via an artificial blood meal. All three mosquito populations could be infected with CHIKV, yet viral titers differed between populations at 7 days post infection. Moreover, we found that these mosquitoes could transmit CHIKV to mice, and that virus RNA reached the saliva as early as two days post infection. Upon sequencing of the saliva CHIKV genomic RNA, we found mutations at sites correlated with increased transmission and virulence. These studies show that NYC Ae. albopictus populations can be infected with and transmit CHIKV, CHIKV is able to evolve in these mosquitoes, and that host salivary factors display population-specific diversity. Taken together, these studies highlight the need to study how distinct mosquito populations control viral infections, both at the virus and host level.

Environmental Determinants of Aedes albopictus Abundance at a Northern Limit of Its Range in the United States.

Aedes albopictus is a vector of arboviruses with high rates of morbidity and mortality. The northern limit of Ae. albopictus in the northeastern United States runs through New York state (NYS) and Connecticut. We present a landscape-level analysis of mosquito abundance measured by daily counts of Ae. albopictus from 338 trap sites in 12 counties during May-September 2017. During the study period, the mean number of Ae. albopictus caught per day of trapping across all sites was 3.21. We constructed four sets of negative binomial generalized linear models to evaluate how trapping methodology, land cover, as well as temperature and precipitation at multiple time intervals influenced Ae. albopictus abundance. Biogents-Sentinel (BGS) traps were 2.78 times as efficient as gravid traps and 1.49 times as efficient as CO2-baited CDC light traps. Greater proportions of low- and medium-intensity development and low proportions of deciduous cover around the trap site were positively associated with increased abundance, as were minimum winter temperature and March precipitation. The cumulative precipitation within a 28-day time window before the date of collection had a nonlinear relationship with abundance, such that greater cumulative precipitation was associated with increased abundance until approximately 70 mm, above which there was a decrease in abundance. We concluded that populations are established in Nassau, Suffolk, and New York City counties in NYS; north of these counties, the species is undergoing population invasion and establishment. We recommend that mosquito surveillance programs monitoring the northward invasion of Ae. albopictus place BGS traps at sites chosen with respect to land cover.

Enhancement of Risk for Lyme Disease by Landscape Connectivity, New York, New York, USA.

Most tickborne disease studies in the United States are conducted in low-intensity residential development and forested areas, leaving much unknown about urban infection risks. To understand Lyme disease risk in New York, New York, USA, we conducted tick surveys in 24 parks throughout all 5 boroughs and assessed how park connectivity and landscape composition contribute to Ixodes scapularis tick nymphal densities and Borrelia burgdorferi infection. We used circuit theory models to determine how parks differentially maintain landscape connectivity for white-tailed deer, the reproductive host for I. scapularis ticks. We found forested parks with vegetated buffers and increased connectivity had higher nymph densities, and the degree of park connectivity strongly determined B. burgdorferi nymphal infection prevalence. Our study challenges the perspective that tickborne disease risk is restricted to suburban and natural settings and emphasizes the need to understand how green space design affects vector and host communities in areas of emerging urban tickborne disease.

Multistate Infestation with the Exotic Disease-Vector Tick Haemaphysalis longicornis - United States, August 2017-September 2018.

Haemaphysalis longicornis is a tick indigenous to eastern Asia and an important vector of human and animal disease agents, resulting in such outcomes as human hemorrhagic fever and reduction of production in dairy cattle by 25%. H. longicornis was discovered on a sheep in New Jersey in August 2017 (1). This was the first detection in the United States outside of quarantine. In the spring of 2018, the tick was again detected at the index site, and later, in other counties in New Jersey, in seven other states in the eastern United States, and in Arkansas. The hosts included six species of domestic animals, six species of wildlife, and humans. To forestall adverse consequences in humans, pets, livestock, and wildlife, several critical actions are indicated, including expanded surveillance to determine the evolving distribution of H. longicornis, detection of pathogens that H. longicornis currently harbors, determination of the capacity of H. longicornis to serve as a vector for a range of potential pathogens, and evaluation of effective agents and methods for the control of H. longicornis.

A Taxonomic Checklist and Relative Abundance of the Mosquitoes of New York City.

This is the 1st time that a comprehensive checklist of the mosquitoes of New York City has been compiled. This list is based on an arrayed collection of 2.3 million mosquitoes trapped and identified from 1,369 locations in the city between 2000 and 2017. Forty-seven species and 6 subspecies were identified belonging to 9 mosquito genera. Culex pipiens was the most prevalent species, most frequently encountered throughout the city. Over time, species diversity in the genus Aedes has increased from 10 species in the 1930s to 23 species in the recent surveys (2000-17). Invasive species Aedes albopictus and Ae. japonicus japonicus, which were rare in 2000, are now well established in all 5 boroughs of the city.

Investigation of Phytochemical and Antioxidant Properties of Methanol Extract and Fractions of Ballota limbata (Lamiaceae).

Ballota limbata (Lamiaceae) has been used for its antispasmodic, antiulcer, diuretic, vermifuge and sedative effects in folk medicine. However, little is known about how does it work to produce these therapeutic actions. Present research investigated phytochemical components and antioxidant properties of methanol extract and different fractions of Ballota limbata. In this study, phytochemical investigation was done by performing different chemical tests. Here, antioxidant property of the extract and fractions was investigated by using 1,1-diphenyl-2-picryl hydrazyl radical scavenging activity, total antioxidant activity by the phosphomolybdenum method, linoleic acid peroxidation, ferric thiocyanate analysis and ferric-reducing antioxidant power. Methanol extract and fractions showed presence of numerous chemical principles including alkaloids, cardiac glycosides, tannins and flavonoids. The ethyl acetate fraction exhibited higher scavenging activity compared to the other fractions under investigation. This fraction displayed 84.16±1.02% 1,1-diphenyl-2-picryl hydrazyl radical inhibition at a dose of 60 µg/ml. IC50 for 1,1-diphenyl-2-picryl hydrazylradical-scavenging activity was 13.53±0.22 µg/ml, relative to the standard, butylatedhydroxytoluene, having IC50 of 12.33±0.88 µg/ml. Thus, Ballota limbata showed significant antioxidant activity, which may contribute in the mechanism of above pharmacological actions.

Decelerating spread of West Nile virus by percolation in a heterogeneous urban landscape.

Vector-borne diseases are emerging and re-emerging in urban environments throughout the world, presenting an increasing challenge to human health and a major obstacle to development. Currently, more than half of the global population is concentrated in urban environments, which are highly heterogeneous in the extent, degree, and distribution of environmental modifications. Because the prevalence of vector-borne pathogens is so closely coupled to the ecologies of vector and host species, this heterogeneity has the potential to significantly alter the dynamical systems through which pathogens propagate, and also thereby affect the epidemiological patterns of disease at multiple spatial scales. One such pattern is the speed of spread. Whereas standard models hold that pathogens spread as waves with constant or increasing speed, we hypothesized that heterogeneity in urban environments would cause decelerating travelling waves in incipient epidemics. To test this hypothesis, we analysed data on the spread of West Nile virus (WNV) in New York City (NYC), the 1999 epicentre of the North American pandemic, during annual epizootics from 2000-2008. These data show evidence of deceleration in all years studied, consistent with our hypothesis. To further explain these patterns, we developed a spatial model for vector-borne disease transmission in a heterogeneous environment. An emergent property of this model is that deceleration occurs only in the vicinity of a critical point. Geostatistical analysis suggests that NYC may be on the edge of this criticality. Together, these analyses provide the first evidence for the endogenous generation of decelerating travelling waves in an emerging infectious disease. Since the reported deceleration results from the heterogeneity of the environment through which the pathogen percolates, our findings suggest that targeting control at key sites could efficiently prevent pathogen spread to remote susceptible areas or even halt epidemics.

Studies on hibernating populations of Culex pipiens from a West Nile virus endemic focus in New York City: parity rates and isolation of West Nile virus.

A 3-year study was undertaken to examine the parity status, survival, and prevalence of West Nile virus (WNV) in overwintering populations of Culex pipiens pipiens collected from a hibernaculum located in a WNV endemic region in New York City. Nearly 6,000 females were collected from December through April. Parity rates were highest among females collected in December and January, ranging from 12.3% to 21.9%, depending on the year. In each year of the study, the proportion of parous females declined significantly during the course of the winter; the percentage of parous females found in April ranged from 0.9% to 10%. Results provide unequivocal evidence that parous Cx. p. pipiens females from this region of the northeastern US enter hibernacula in the fall in comparatively high proportions not previously recognized for this species, and while these females experience significant mortality during the winter, some survived to April to emerge in the spring. The absence of any detectible blood remnants in overwintering females reaffirms that blood feeding does not occur among diapausing females during the winter. The possibility that a portion of the diapausing population may be autogenous as a result of hybridization with sympatric belowground populations of Cx. p. pipiens "form molestus" is discussed. A single isolation of WNV was obtained in Vero cell culture from a pool of 50 females collected on January 11, 2007, representing an infection prevalence of 0.07% in the overwintering population in 2007 (n = 1,370 mosquitoes, 33 pools). No isolations of WNV were made from mosquitoes collected in 2008 (n = 1,870 mosquitoes, 190 pools) or 2009 (n = 1,767 mosquitoes, 184 pools). Findings provide further evidence for local overwintering of WNV in diapausing Cx. p. pipiens, albeit at very low rates, consistent with the paucity of WNV-positive mosquitoes detected in June and early July despite the emergence of females from hibernacula in early May in this region.

Childhood pyogenic meningitis: clinical and investigative indicators of etiology and outcome.

The relevant parameters of 71 consecutive pediatric admissions for pyogenic meningitis at the University of Ilorin Teaching Hospital, Ilorin, Nigeria, were analyzed to identify possible clinical and nonmicrobiologic investigative clues of disease etiology and mortality. Cerebrospinal fluid (CSF) was Gram-smear positive (GSP) in 41 (57.6%) of the 71 cases. Twenty-three (56.1%) had Gram-positive cocci (GPC), 14 (34.2%) Gram-negative bacilli (GNB) and three (7.3%) Gram-negative diplococci (GND). The respective mean ages of GPC, GNB and GND cases were 4.49 +/- 5.3, 3.06 +/- 4.8 and 4.47 +/-4.9 years. Streptococcus pneumoniae accounted for 22 (78.6%) of the 28 CSF isolates (p=0.00), Haemophilus influenzae for two (7.1%) cases and Neisseria meningitides in one (3.5%). Anemia was significantly more common among GSP cases (p=0.04), as was convulsion among those with GNB-positive smears (p=0.03) and a bulging fontanelle in the Gram-smear-negative category. Otherwise, the prevalence and resolution times of the other clinical parameters were comparable across the etiological categories. There were 30 deaths (42.3%) among which GNB-positive cases had significantly shorter stay (p=0.045). Mortality was significantly higher in those with an abnormal respiratory rhythm at admission (p=0.04), purulent/turbid CSF (p=0.03), CSF protein of >150 mg/dl (p=0.02) and glucose <1 mg/dl (p=0.047). Our findings highlight the inherent limitations of predicting the etiology of pediatric meningitides from the clinical parameters as well as the poor prognostic import of respiratory dysrhythmia and a profoundly deranged CSF protein and glucose. The etiological burden of GPC/S. pneumoniae in childhood meningitides in sub-Saharan Africa, the propensity of GNB/H. influenzae for quick fatality and the need for the relevant preventive vaccines are expounded in the discussion.

Description of a Culex pipiens gynandromorph from New York City.

Morphology and internal anatomy of a Culex pipiens L. bipolar gynandromorph that was collected on September 3, 2005 in a gravid trap at Springfield Park in the borough of Queens, New York City, NY is presented. The head of the mosquito possessed male palpi, compared to the rest of the body, which had female anatomical and morphological characteristics. The relation of morphological characteristics to physiological responses of the gynandromorph is discussed.

Integrated pest management (IPM) and Internet-based information delivery systems / Manejo integrado de pragas (MIP) e sistemas de liberação de informação sediados na Internet

A internet permite compartilhar a colaboração e a informação numa escala sem precedentes. Ela tornou-se um meio inédito para a comunicação em pesquisa e extensão. A rede mundial de computadores (World Wide Web - WWW) torna possível a combinação da informação de diferentes sites de umamaneira contínua. O potencial de uso da rede para integrar todo o tipo de informação, estática ou dinâmica, é unico e inédito. A rede permite fazer a interface para todos os tipos de base de dados interativos e para muitos tipos de análises e processamentos de dados on line. Modelos sediados na rede e sistemas para o apoio de decisões (decision support systems - DSS) estão se tornando populares porque poucos ou nenhum programa de sofware é necessário, dessa forma reduzindo o custo de distribuição e manejo de modelos. Nenhum outro meio oferece tais habilidades, como por exemplo, as informações climáticas em tempo quase real, multimidia, processos analíticos, discussão em vias múltiplas e feedback. O manejo integrado de pragas (MIP) é um sistema intensivo em informação. Ambos, a pesquisa em MIP e a sua implementação, requerem o suprimento de informação em tempo útil. A internet fornece meios para estabelecer acomunicação entre os pesquisadores em MIP e os profissionais da extensão e sua clientela, para maximizar a troca de informações e a transferência de tecnologias. A rede WWW abriu uma amplitude de fontes de dados para pesquisa, extensão, ensino e aprendizado em MIP, não concebida antes do advento da internet. O futuro do uso do MIP pela internet é promissor. A troca de informações baseada na internet está rapidamente se tornando um requisito indispensável para implementação de sistemas de MIP locais, regionais ou em áreas abrangentes e em nível internacional.

The Internet enables collaboration and information sharing on an unprecedented scale. It has become a prime medium for research and extension communication. The World Wide Web (WWW) makes it possible to combine information from many different sites in a seamless fashion. Thepotential for using the web to integrate all types of static and interactive (dynamic) information is unique and unprecedented. The web provides excellent interfaces for all kinds of interactive network databases, and many kinds of online analyses and data processing. Web-based models and decision support systems (DSS) are becoming popular because little or no client software is required, thus reducing software management and distribution costs. No other medium offers such ability as simultaneous real-time weather information, multimedia, analytical processing and multi-way discussion and feedback. IPM is an information-intensive system. Both IPM research and implementation requirethe reliable supply of timely information. The Internet provides the means to establish communication links between IPM researchers and extension professionals and their clientele to expedite multi-way exchange of information and technology transfer. The Internet particularly the WWW, has opened up a rich array of data resources for IPM research, extension, teaching, and learning that was not as readilyaccessible before the advent of the Internet. The future of IPM delivery systems through the Internet is promising; internet-based information exchange is quickly becoming an absolute requirement for local, regional/areawide, and international implementation of IPM systems.

Strategies to circumvent SV40 oncoprotein expression in malignant pleural mesotheliomas.

Although nearly 60% of mesotheliomas contain SV40 early region DNA sequences, the role of T/t antigens in initiating and maintaining the transformed state of mesothelioma cells remains unclear. The majority of mesothelioma cells which contain SV40 early region sequences exhibit extremely low basal expression of SV40 oncoproteins; however, T/t antigen expression can be induced under conditions of cellular stress. Abrogation of SV40 T/t expression by antisense techniques induces apoptosis in part via restoration of p53 function, and enhances chemosensitivity in SV40 (+) MPM cells by mechanisms which have not been fully elucidated. This review briefly summarizes our ongoing efforts to define the role of SV40 oncoproteins in modulating the malignant phenotype of mesothelioma cells, and highlights strategies which may prove efficacious in vivo for circumventing SV40 T/t antigen expression in mesotheliomas.

Antisense to SV40 early gene region induces growth arrest and apoptosis in T-antigen-positive human pleural mesothelioma cells.

Although SV40 oncoproteins have been detected in malignant pleural mesotheliomas (MPMs), their role in the pathogenesis and clinical behavior of these neoplasms remains controversial. In the present study, we sought to define the relevance of SV40 T/t antigen expression in established human mesothelioma cell lines deficient for p16INK4a as well as ARF expression. SV40 early region sequences were readily detected in genomic DNA isolated from pleural mesothelioma lines; however, levels of SV40 T/t antigen expression were highly variable in these cells. An adenoviral vector expressing an antisense transcript to SV40 early region inhibited T antigen expression and mediated significant growth inhibition and apoptosis in T-antigen-positive mesothelioma cells and SV40-transformed COS-7 cells. Abrogation of T/t antigen expression coincided with enhanced p21/WAF-1 expression, suggesting that restoration of p53-mediated pathways may have contributed to the growth inhibition and apoptosis induced by the antisense construct. These effects were not observed after similar treatment of mesothelioma or lung cancer cells containing no SV40 DNA sequences. Collectively, these data suggest that SV40 oncoproteins contribute to the malignant phenotype of pleural mesotheliomas and indicate that interventions designed to abrogate their expression may be efficacious in the treatment of individuals with these neoplasms.

Effect of subcutaneous injection of granulocyte-macrophage colony stimulating factor (GM-CSF) on healing of chronic refractory wounds.

OBJECTIVE: To assess the effect of granulocyte macrophage colony-stimulating factor (GM-CSF) on healing of chronic wounds. DESIGN: Prospective open study. SETTING: 1 cancer hospital and 2 University hospitals, Pakistan. SUBJECTS: 35 patients with chronic wounds (duration 3 months or longer) that had not responded to standard treatment. Patients with malignant ulcers were excluded. INTERVENTION: GM-CSF 10 microg/cm2 was injected subcutaneously along the edges and base of the wound. The treatment was given only once and patients were followed weekly for a minimum of six weeks. MAIN OUTCOME MEASURES: Changes in size, shape, depth, edges and base of the wound, amount and quality of the granulation tissue, and type of discharge. RESULTS: Six patients were excluded from the analysis, 4 who died early of underlying disease and were not evaluable, and 2 who were excluded when histological examination of the wound showed that it was malignant. Although various types of wounds were studied, most (n = 10, 34%) were postoperative. 23 of the wounds were over 2 cm in diameter (mean 4.8 x 4.6 cm) with thin granulation tissue, and almost half were infected. Nine of the 29 wounds healed completely within six weeks while another 11 decreased in size by more than 50%; 7 patients responded slightly. Only two wounds showed no evidence of healing during the observation period. More than half of the 20 wounds that responded had healed by three weeks. Response did not correlate with any clinical variable including the presence of infection. Toxicity was negligible. CONCLUSIONS: We conclude that subcutaneous injection of a single dose of GM-CSF may induce healing in refractory chronic wounds. Trials are necessary to validate these initial observations and to decide the optimal dose and route, and whether any additional benefit may be derived from repeated injections.

Binding of the human cytomegalovirus 80-kDa immediate-early protein (IE2) to minor groove A/T-rich sequences bounded by CG dinucleotides is regulated by protein oligomerization and phosphorylation.

The 80-kDa immediate-early regulatory protein IE2 of human cytomegalovirus (HCMV) functions as an essential positive transactivator of downstream viral promoters, but it also specifically down-regulates transcription from the major immediate-early promoter through a 14-bp DNA target motif known as the cis-repression signal (CRS) located at the transcription start site. The IE2 protein purified from bacteria as a fusion product of either staphylococcal Protein A/IE2(290-579) or glutathione-S-transferase (GST)/IE2(346-579) bound specifically to a [32P]-labeled CRS oligonucleotide probe in an in vitro electrophoretic mobility shift assay (EMSA). In contrast, no direct interaction with the CRS probes could be detected with IE2 wild-type protein in extracts from infected or transfected mammalian cells or when synthesized by in vitro translation. However, in vitro phosphorylation of GST/IE2(346-579) by incubation with either the catalytic subunit of protein kinase A (PKA) or a HeLa cell nuclear extract strongly inhibited its DNA-binding activity. This process required ATP hydrolysis and could be reversed by subsequent incubation with bacterial alkaline phosphatase. Importantly, dephosphorylation of the constitutively expressed native IE2 protein present in a nuclear extract from the U373(A45) cell line unmasked a specific CRS DNA-binding activity that could be supershifted with anti-IE2 monoclonal antibody (mAb). A series of high-molecular-weight hetero-oligomeric DNA-bound structures of intermediate mobility were formed in EMSA assays when a mixture of staphylococcal Protein A/IE2 and GST/IE2 was coincubated with the CRS probe. Coincubation with a DNA-binding negative but dimerization-competent GST/IE2 deletion mutant competitively inhibited DNA-binding by staphylococcal Protein A/IE2, whereas coincubation with a GST/IE2 deletion mutant that lacked the ability to both dimerize and bind to DNA failed to influence the mobility of the DNA-bound staphylococcal Protein A/IE2 protein. Therefore, IE2 appears to bind to DNA as a higher-order oligomer in which the presence of subunits with mutant DNA-binding domains interferes with the overall DNA-binding function. A series of point mutations introduced into each of nine conserved motifs throughout the DNA-binding and dimerization domain, all of which abolish the ability of the transfected intact IE2 protein to autoregulate the MIE promoter, also all lacked the ability to bind to CRS sequences as GST/IE2(346-379) fusion proteins. Detailed analysis of point mutations in the 14-bp CRS target DNA binding motif revealed that IE2 binds in a relatively sequence-independent manner to 10-bp-long A/T-rich DNA elements bounded on each side by CG dinucleotides. Moreover, the A/T-rich minor groove binding agent distamycin, but not the G/C-rich minor groove binding agent chromomycin-A3, actively competed with IE2 for binding to the CRS motif in a dose-dependent fashion. In conclusion, IE2 binds preferentially as multimerized dimers to A/T-rich sequences in the minor groove that are flanked on both sides by appropriately spaced CG dinucleotides, and inhibition of the DNA-binding or oligomerization activity by PKA phosphorylation probably accounts for the inactivity of the mammalian and in vitro translated forms of the protein.

Heat-stable alkaline phosphatase. A putative tumor marker of head and neck squamous cell carcinoma.

Serum total alkaline phosphatase (AP) activity and heat-stable AP (HSAP) were investigated in patients with uncontrolled squamous cell carcinoma of the head and neck before and after treatment. No significant differences in AP activity were seen between normal subjects and cancer patients. However, the HSAP fraction of the total AP activity was significantly elevated prior to treatment and the level declined and remained low during successful treatment, while it increased with tumor progression or recurrences. Heat-stable AP was found to be a useful tumor marker of potential usefulness in the management of patients with squamous cell carcinoma of the head and neck.

Identification and mapping of dimerization and DNA-binding domains in the C terminus of the IE2 regulatory protein of human cytomegalovirus.

The 80-kDa IE2 nuclear phosphoprotein encoded by the human cytomegalovirus (HCMV) major immediate-early (MIE) gene behaves both as a nonspecific transactivator of heterologous reporter genes and as a specific repressor of its own promoter-enhancer region. To begin to examine the biochemical properties of the IE2 protein, we prepared panels of N-terminal and C-terminal truncation mutants by in vitro translation procedures. In cross-linking experiments, the C-terminal half of IE2 (which is sufficient for down-regulation) formed dimers but N-terminal segments did not do so. Cotranslated Oct2/IE2 fusion proteins containing the same IE2 C-terminal region from codons 266 to 579 also formed mixed-subunit DNA-bound oligomeric complexes in gel mobility shift assays. Furthermore, an IE2 domain bounded by codons 388 to 542 proved to immunoprecipitate as heterodimers with cotranslated subunits containing known epitopes for specific antibodies. Deletion up to codon 428 or truncation back to codon 504 prevented this interaction. In direct gel shift DNA-binding assays, a bacterial GST/IE2(346-579) fusion protein bound to a 30-mer oligonucleotide probe encompassing the major immediate-early gene negative cis-regulatory target DNA sequence but failed to bind to a single-base-pair insertion mutant probe (delta CRS). This specific DNA-binding activity was abolished by further deletion up to codon 388 on the N-terminal side or by truncation at codon 542 on the C-terminal side. Therefore, the minimal DNA-binding domain requires additional amino acid motifs on both sides of the dimerization domain. This segment of IE2 is functionally important for both transactivation and down-regulation and contains several highly conserved amino acid motifs that are shared amongst the equivalent HCMV, simian CMV, mouse CMV, rat CMV, and human herpesvirus 6 proteins from other betaherpesviruses.