Response to genotoxic stress can be considered as a multistage process involving initiation of cell-cycle arrest and maintenance of arrest during DNA repair. Although maintenance of G2/M checkpoints is known to involve Chk1, Chk2/Rad53 and upstream components, the mechanisms involved in its initiation are less well defined. Here we report that p38 kinase has a critical role in the initiation of a G2 delay after ultraviolet radiation. Inhibition of p38 blocks the rapid initiation of this checkpoint in both human and murine cells after ultraviolet radiation. In vitro, p38 binds and phosphorylates Cdc25B at serines 309 and 361, and Cdc25C at serine 216; phosphorylation of these residues is required for binding to 14-3-3 proteins. In vivo, inhibition of p38 prevents both phosphorylation of Cdc25B at serine 309 and 14-3-3 binding after ultraviolet radiation, and mutation of this site is sufficient to inhibit the checkpoint initiation. In contrast, in vivo Cdc25C binding to 14-3-3 is not affected by p38 inhibition after ultraviolet radiation. We propose that regulation of Cdc25B phosphorylation by p38 is a critical event for initiating the G2/M checkpoint after ultraviolet radiation.

Melanosome biogenesis and function were studied after purification of early stage melanosomes and characterization of specific proteins sorted to that organelle. Melanosomes were isolated from highly pigmented human MNT1 melanoma cells after disruption and initial separation by sucrose density gradient centrifugation. Low-density sucrose fractions were found by electron microscopy to be enriched in stage I and stage II melanosomes, and these fractions were further separated and purified by free flow electrophoresis. Tyrosinase and dopachrome tautomerase (DCT) activities were found exclusively in stage II melanosomes, even though DCT (and to some extent tyrosinase) proteins were sorted to stage I melanosomes. Western immunoblotting revealed that these catalytic proteins, as well as TYRP1, MART1, and GP100, were cleaved and inactivated in stage I melanosomes. Proteolytic cleavage was critical for the refolding of GP100 within the melanosomal milieu, and subsequent reorganization of amorphous stage I melanosomes into fibrillar, ovoid, and highly organized stage II melanosomes appears to stabilize the catalytic functions of melanosomal enzymes and allows melanin biosynthesis to begin. These results provide a better understanding of the structural features seen during melanosome biogenesis, and they yield further clues as to the physiological regulation of pigmentation.pigment ͉ melanin ͉ tyrosinase ͉ melanoma M ore than 95 distinct genes that play direct or indirect roles in mammalian pigmentation have been identified. Many of these genes encode proteins that are localized in melanosomes, specialized pigment organelles produced only by melanocytes. These gene products alter the quality or quantity of melanin produced and͞or the processing and distribution of melanosomes. The known melanosomal proteins are involved in melanogenesis as catalytic and͞or structural components. These include tyrosinase (TYR), the tyrosinase-related proteins-1 and -2 (TYRP1͞TRP1 and DCT͞TRP2, respectively; refs.

Melanin is a heterogeneous biopolymer produced only by specific cells termed melanocytes, which synthesize and deposit the pigment in specialized membrane-bound organelles known as melanosomes. Although melanosomes have been suspected of being closely related to lysosomes and platelets, the total number of melanosomal proteins is still unknown. Thus far, six melanosome-specific proteins have been identified, and the challenge is to characterize the complete proteome of the melanosome to further understand its mechanism of biogenesis. In this report, we used mass spectrometry and subcellular fractionation to identify protein components of early melanosomes. Using this approach, we have identified all 6 of the known melanosome-specific proteins, 56 proteins that are shared with other organelles, and confirmed the presence of 6 novel melanosomal proteins using western blotting and by immunohistochemistry.

Heterochromatin plays an essential role in the preservation of epigenetic information, the transcriptional repression of repetitive DNA elements and inactive genes and the proper segregation of chromosomes during mitosis. Here we identify KDM2A, a JmjC-domain containing histone demethylase, as a heterochromatin-associated protein that is required to maintain the heterochromatic state, as determined using both a candidate-based approach and an unbiased siRNA library screen. Moreover, we demonstrate that KDM2A represses transcription of small non-coding RNAs that are encoded by clusters of satellite repeats at the centromere. Finally, we show that KDM2A is required to sustain centromeric integrity and genomic stability, particularly during mitosis. Since the disruption of epigenetic control mechanisms contributes to cellular transformation, these results, together with the low levels of KDM2A found in prostate carcinomas, suggest a role for KDM2A in cancer development.

The UspA1 and Hag proteins have previously been shown to be involved in the ability of the Moraxella catarrhalis wild-type strain O35E to bind to human Chang and A549 cells, respectively. In an effort to identify novel adhesins, we generated a plasmid library of M. catarrhalis DNA fragments, which was introduced into a nonadherent Escherichia coli strain. Recombinant E. coli bacteria were subsequently enriched for clones that gained the ability to bind to Chang and A549 cells, yielding the plasmid pELFOS190. The unencapsulated, gram-negative bacterium Moraxella catarrhalis is one of the leading causes of otitis media in young children and of lower respiratory tract infections in adults with chronic obstructive pulmonary disease (COPD). In developed countries, more than 80% of children under the age of 3 years will be diagnosed at least once with otitis media, and M. catarrhalis is responsible for 15 to 25% of all of these cases (6, 7, 10, 12, 26-29, 41, 51, 60). Lower respiratory tract infections (exacerbations) have been shown to contribute to the progression of COPD, and of the approximately 20 million cases of exacerbations documented each year in the United States, up to 35% result from M. catarrhalis infections (3,41,43,54,55).A vaccine that provides protection against M. catarrhalis is highly desirable due to this substantial morbidity as well as the growing concern over antibiotic resistance observed in clinical isolates (30). Toward this end, current research is focused on the identification of potential antigens with emphasis on the outer membrane proteins (OMPs) (26,34,35). While M. catarrhalis OMPs with a wide variety of functions have been identified, adhesins are particularly attractive vaccine candidates because they are surface-exposed antigens. Furthermore, adhesins generally play a crucial role in colonization and pathogenesis. For many bacteria, adherence to epithelial surfaces contributes to the evasion of host clearance mechanisms (4,25,59). Previous studies with M. catarrhalis have identified UspA1 (32, 36), UspA2 (2, 8, 36), Hag (14,15,17,19,24,45,47; M. M. Holm and E. R. Lafontaine, unpublished data), lipooligosaccharide (24), OMPCD (49), and UspA2H (32) as potential adhesins. Of these, only UspA1 and UspA2H have been directly shown to mediate adherence to human cells (32,36). However, while UspA1 is expressed by virtually all strains tested to date, only ϳ20% of clinical isolates contain an uspA2H gene (32, 37).The present study describes the identification of the novel M. catarrhalis OMP McaP. While this protein was identified based on its ability to function as an adhesin, we report that it also displays the enzymatic activity of an esterase and a phospholipase B (PLB). MATERIALS AND METHODSStrains, plasmids, tissue culture (TC) cell lines, and growth conditions. The bacterial strains and plasmids described in this study are listed in Table 1. M. catarrhalis was routinely cultured at 37°C on Todd-Hewitt medium (Difco). When cultured on agar plates, M. catarrhalis was incubated in an at...

In Figure 3B of this article, the VCAP and the control siRNA images are identical. This was the result of a mistake in constructing the figure. The correction to the figure does not affect the conclusion of the paper and the authors would like to apologize for any confusion the error may have caused. The corrected image is printed below.

Coccidioidomycosis is a respiratory disease of humans caused by the desert soil-borne fungal pathogens Coccidioides spp. Recurrent epidemics of this mycosis in the southwestern United States have contributed significantly to escalated health care costs. Clinical and experimental studies indicate that prior symptomatic coccidioidomycosis induces immunity against subsequent infection, and activation of T cells is essential for containment of the pathogen and its clearance from host tissue. Development of a human vaccine against coccidioidomycosis has focused on recombinant T-cell-reactive antigens which elicit a durable protective immune response against pulmonary infection in mice. In this study we fractionated a protective multicomponent parasitic cell wall extract in an attempt to identify T-cell antigens. Immunoblots of electrophoretic separations of this extract identified patient seroreactive proteins which were subsequently excised from two-dimensional polyacrylamide gel electrophoresis gels, trypsin digested, and sequenced by tandem mass spectrometry. The full-length gene which encodes a dominant protein in the immunoblot was identified using established methods of bioinformatics. The gene was cloned and expressed, and the recombinant protein was shown to stimulate immune T cells in vitro. The deduced protein was predicted to contain epitopes that bind to human major histocompatibility complex class II molecules using a TEPITOPE-based algorithm. Synthetic peptides corresponding to the predicted T-cell epitopes induced gamma interferon production by immune T lymphocytes. The T-cell-reactive antigen, which is homologous to secreted fungal aspartyl proteases, protected mice against pulmonary infection with Coccidioides posadasii. We argue that this immunoproteomic/ bioinformatic approach to the identification of candidate vaccines against coccidioidomycosis is both efficient and productive.

Coccidioides posadasii is a fungal respiratory pathogen of humans that can cause disease in immunocompetent individuals. Coccidioidomycosis ranges from a mild to a severe infection. It is frequently characterized either as a persistent disease that requires months to resolve or as an essentially asymptomatic infection that can reactivate several years after the original insult. In this report we describe a mechanism by which the pathogen evades host detection during the pivotal reproductive (endosporulation) phase of the parasitic cycle. A metalloproteinase (Mep1) secreted during endospore differentiation digests an immunodominant cell surface antigen (SOWgp) and prevents host recognition of endospores during the phase of development when these fungal cells are most vulnerable to phagocytic cell defenses. C57BL/6 mice were immunized with recombinant SOWgp and then challenged with a mutant strain of C. posadasii in which the MEP1 gene was disrupted. The animals showed a significant increase in percent survival compared to SOWgp-immune mice challenged with the parental strain. To explain these results, we proposed that retention of SOWgp on the surfaces of endospores of the mutant strain in the presence of high titers of antibody to the immunodominant antigen contributes to opsonization, increased phagocytosis, and killing of the fungal cells. In vitro studies of the interaction between a murine alveolar macrophage cell line and parasitic cells coated with SOWgp showed that the addition of anti-SOWgp antibody could enhance phagocytosis and killing of Coccidioides. We suggest that Mep1 plays a pivotal role as a pathogenicity determinant during coccidioidal infections and contributes to the ability of the pathogen to persist within the mammalian host.