On the formation and role of reactive oxygen species in light-driven LPMO oxidation of phosphoric acid swollen cellulose.

Light-driven activation of lytic polysaccharide monooxygenases (LPMOs) has been attributed to the transfer of high redox potential electrons from excited photopigments to the enzyme. However, due to the formation of reactive oxygen species (ROS) in such a system, not only electrons from the pigments but also ROS could be part of the enzyme mechanism. This work investigates the role of ROS in the oxidation of phosphoric acid swollen cellulose (PASC) by a light-driven LPMO system. Our results clearly show that the addition of superoxide dismutase or catalase to remove ROS did not attenuate the capacity of the light-driven LPMO system to oxidize PASC, as measured by formation of oxidized oligosaccharides. We conclude that ROS are not part of the light-driven LPMO activation; hence, transfer of high redox potential electrons from the excited photopigment to the LPMO remains the most likely mechanism under the conditions tested in this study.

Light-driven oxidation of polysaccharides by photosynthetic pigments and a metalloenzyme.

Oxidative processes are essential for the degradation of plant biomass. A class of powerful and widely distributed oxidative enzymes, the lytic polysaccharide monooxygenases (LPMOs), oxidize the most recalcitrant polysaccharides and require extracellular electron donors. Here we investigated the effect of using excited photosynthetic pigments as electron donors. LPMOs combined with pigments and reducing agents were exposed to light, which resulted in a never before seen 100-fold increase in catalytic activity. In addition, LPMO substrate specificity was broadened to include both cellulose and hemicellulose. LPMO enzymes and pigment derivatives common in the environment of plant-degrading organisms thus form a highly reactive and stable light-driven system increasing the turnover rate and versatility of LPMOs. This light-driven system may find applications in biotechnology and chemical processing.

Dentistry and childhood poverty in the United States.

The proportion and numbers of children living in low income families and without health insurance continues to increase. The magnitude of these problems is considered at localized levels in terms of the impact on the use of dental services.

Toxoplasma gondii gene expression is under the control of regulatory pathways acting through chromatin structure.

The activity state of a gene is determined by a complex regulatory network of co-acting factors affecting the structure of the chromatin into which the gene is embedded. While significant changes of the transcriptome occur during cell differentiation in apicomplexan parasites, basic mechanisms controlling gene expression are still unknown. Recent studies support and expand the concept of the chromatin environment being key factor for the control of transcriptional activity in these lower eukaryotes organisms. Here, we review recent advances in the field of epigenetic gene regulation in Toxoplasma gondii, the model apicomplexan.

Growth inhibition by CDK-cyclin and PCNA binding domains of p21 occurs by distinct mechanisms and is regulated by ubiquitin-proteasome pathway.

The CDK inhibitor, p21WAF1/Cip1 blocks cell cycle progression. In vitro, the N-terminus of p21 binds and inhibits CDK-cyclin kinase activity, whereas the C-terminus binds and inhibits PCNA (proliferating cell nuclear antigen) function. PCNA is essential for processivity of both DNA polymerase delta and epsilon. We have performed a detailed analysis of growth inhibition by the N- and C-terminal regions of p21, and determined whether the N- and C-terminal regions mediate this effect by different mechanisms. Expression of either the N- or the C-terminal region of p21 inhibits DNA synthesis and cell growth, but not as efficiently as full length p21. The effectiveness of the two p21 domains is dependent on their stability which is determined by the ubiquitin-proteasome pathway. The stabilization of the N- and C-terminal region of p21 increases their effectiveness as inhibitors of DNA synthesis to levels comparable to full length p21. Inhibition of DNA synthesis by the N-terminal region of p21 involves suppression of E2F activity. In contrast, inhibition by the C-terminal region of p21 is not accompanied by suppression of E2F activity, but is mediated via PCNA binding. The C-terminal region of p21 therefore inhibits cell growth by a mechanism distinct from that of the N-terminal region containing the CDK-cyclin inhibitory domain.

Growth inhibition by CDK-cyclin and PCNA binding domains of p21 occurs by distinct mechanisms and is regulated by ubiquitin-proteasome pathway.

The CDK inhibitor, p21(WAF1/Cip1) blocks cell cycle progression. In vitro, the N-terminus of p21 binds and inhibits CDK-cyclin kinase activity, whereas the C-terminus binds and inhibits PCNA (proliferating cell nuclear antigen) function. PCNA is essential for processivity of both DNA polymerase delta and epsilon. We have performed a detailed analysis of growth inhibition by the N- and C-terminal regions of p21, and determined whether the N- and C-terminal regions mediate this effect by different mechanisms. Expression of either the N- or the C-terminal region of p21 inhibits DNA synthesis and cell growth, but not as efficiently as full length p21. The effectiveness of the two p21 domains is dependent on their stability which is determined by the ubiquitin-proteasome pathway. The stabilization of the N- and C-terminal region of p21 increases their effectiveness as inhibitors of DNA synthesis to levels comparable to full length p21. Inhibition of DNA synthesis by the N-terminal region of p21 involves suppression of E2F activity. In contrast, inhibition by the C-terminal region of p21 is not accompanied by suppression of E2F activity, but is mediated via PCNA binding. The C-terminal region of p21 therefore inhibits cell growth by a mechanism distinct from that of the N-terminal region containing the CDK-cyclin inhibitory domain.

Association of cyclin A and cdk2 with SV40 DNA in replication initiation complexes is cell cycle dependent.

The cell cycle is driven by the sequential activation of a family of cyclin-dependent kinases (CDK) in association with cyclins. In mammalian cells the timing of activation of cyclin A-associated kinase activity coincides with the onset of DNA synthesis in S-phase. Using in vitro replication of SV40 origin-containing DNA as a model system, we have analyzed the proteins associated with DNA during initiation of DNA replication in S-phase cell extracts. This analysis reveals that, in addition to replication initiation proteins, cyclin A and cdk2 are also specifically associated with DNA. The association of cyclin A and cdk2 with DNA during initiation is cell cycle regulated and occurs specifically in the presence of SV40 origin-containing plasmid and SV40 T antigen (the viral replication initiator protein). The interactions among proteins involved in initiation play an important role in DNA replication. We therefore investigated the ability of cyclin A and cdk2 to associate with replication initiation proteins. Under replication initiation conditions, cyclin A and cdk2 from S-phase extracts specifically associate with SV40 T antigen. Further, the interaction of cyclin A-cdk2 with SV40 T antigen is mediated via cyclin A, and purified recombinant cyclin A associates directly with SV40 T antigen. Taken together, our results suggest that cyclin A and cdk2 are components of the SV40 replication initiation complex, and that protein-protein interactions between cyclin A-cdk2 and T antigen may facilitate the association of cyclin A-cdk2 with the complex.

Gatekeeping through media format: strategies of voice for the HIV-positive via human interest news formats and organizations.

This research examines the extent to which HIV-positive voices are marginalized in the mainstream versus the "alternative" press. The central claim of this research is that news media format considerations, constructed around what has come to be called "media logic," leave persons who are HIV-positive with comparatively little voice in the mainstream press. By utilizing techniques of content analysis, the research examines 535 major HIV/AIDS-specific stories published in two oppositional papers toward an assessment of the level of HIV-positive voice in each outlet. While arguments of "homophobia" have been previously used to explain bias in mainstream HIV/AIDS-coverage, this article asserts that "heterocentric" bias is, in fact, embedded in the routinized practices of mainstream "news production." The article concludes by suggesting that successful future HIV/AIDS-activism demands a recognition of "media logic" and an adoption of its tactics.

Role for cyclin A-dependent kinase in DNA replication in human S phase cell extracts.

Cell cycle progression is regulated by cyclin-dependent kinases. Using in vitro replication of SV40 origin containing DNA as a model system, we have performed a detailed analysis of the dependence on cyclin-associated kinases of mammalian DNA replication. Complete immunodepletion of cyclin A from human S phase cell extracts decreases replication, and replication activity of cyclin A-depleted S phase extracts can subsequently be restored by the addition of purified CDK2-cyclin A kinase. Addition of cyclin A alone reconstitutes both kinase activity and DNA replication, whereas addition of cyclin E or cyclin B reconstitutes neither. We therefore conclude that reconstitution of DNA replication specifically correlates with an increase in kinase activity. By comparison, depletion of cyclin E from S phase cell extracts does not have any significant inhibitory effect on DNA replication. Moreover, specific p21(Waf1) mutants that bind to CDK2-cyclin and inhibit both cyclin A and cyclin E kinase activities, but do not bind to proliferating cell nuclear antigen, inhibit DNA replication to the same extent as cyclin A depletion. Together, these results show that the kinase activity associated with cyclin A, but not with cyclin E, is primarily responsible for activating SV40 plasmid replication in mammalian S phase cell extracts. Finally, we present evidence that the cyclin-dependent kinase does not influence the assembly of initiation complexes but acts at a stage prior to elongation.

p21 contains independent binding sites for cyclin and cdk2: both sites are required to inhibit cdk2 kinase activity.

Cyclin dependent kinases regulate the progression of eukaryotic cells through the cell cycle. p21Cip1/Waf1/Sdi1 is an inhibitor of cdk-cyclin kinase activity, and has been shown to form complexes with cdk-cyclins and with PCNA, an accessory protein of DNA polymerase delta. The kinase inhibitory domain maps to the N-terminus (1-82) and contains the cdk2 binding site (28-82). We have generated a panel of deletion mutants of p21. A functional characterization of p21 mutants in the N-terminal domain reveals that cyclins bind to this domain independently of cdk2. Correlating with these results we find that p21 can associate with cyclin-cdk kinases in two functionally distinct forms, one in which the kinase activity is inhibited and the other in which the kinase is still active. The cdk2 and cyclin binding sites on p21 are both required to inhibit kinase activity. The second type of interaction, in which an active cyclin-cdk complex only interacts with p21 either via the cyclin or the cdk2 binding site but not through both, does not lead to inhibition of cyclin kinase activity. These results thus provide a basis for understanding the mechanism by which p21, and perhaps other cdk-cyclin kinase inhibitory proteins, suppress kinase activity.

Secretory IgA antibodies to Cryptosporidium parvum in AIDS patients with chronic cryptosporidiosis.

Immune mechanisms that may control Cryptosporidium parvum infection remain unknown. The role of T cell-mediated immunity is suggested by the chronic disease observed in AIDS patients and in athymic or CD4+ T cell-depleted mice. The role of specific antibodies is also unclear. This study sought to determine serum and secretory antibodies to C. parvum in patients infected with human immunodeficiency virus type 1 (HIV-1) with or without chronic cryptosporidiosis. C. parvum-specific antibodies and specific secretory antibodies were determined by ELISA in saliva and sera from 50 HIV-1-infected patients, 27 healthy adults, and 21 healthy children. Despite lower CD4+ lymphocyte counts, patients with chronic cryptosporidiosis had increased levels of C. parvum-specific antibodies in saliva and serum and higher specific secretory antibody levels in saliva than did controls. Persistence of protracted diarrhea despite high levels of both serum and secretory antibodies suggests that specific secretory antibodies are not sufficient to control this protozoan parasite infection of intestinal mucosa.

Gatekeeping through media format: strategies of voice for the HIV-positive via human interest news formats and organizations.

This research examines the extent to which HIV-positive voices are marginalized in the mainstream versus the "alternative" press. The central claim of this research is that news media format considerations, constructed around what has come to be called "media logic," leave persons who are HIV-positive with comparatively little voice in the mainstream press. By utilizing techniques of content analysis, the research examines 535 major HIV/AIDS-specific stories published in two oppositional papers toward an assessment of the level of HIV-positive voice in each outlet. While arguments of "homophobia" have been previously used to explain bias in mainstream HIV/AIDS-coverage, this article asserts that "hetero-centric" bias is, in fact, embedded in the routinized practices of mainstream "news production." The article concludes by suggesting that successful future HIV/AIDS activism demands a recognition of "media logic" and an adoption of its tactics.

Cryptosporidium parvum sporozoite staining by propidium iodide.

Modified Ziehl-Neelsen (ZN) acid-fast stain is the usual method for detection of Cryptosporidium oocysts in feces. Propidium iodide permitted us to stain free or intra-oocyst sporozoites. With the ZN method only 3-5% of the oocysts purified from three human and one experimentally infected lamb dichromate-preserved feces were stained by carbol fuchsin. These fuchsin-stained oocysts were free of intact sporozoites as identified by propidium iodide staining. Treatment with 10% formalin or 0.5% sodium hypochlorite increased the percentage of acid-fast stained oocysts and thus the sensitivity of acid-fast staining. Treatment with sodium hypochlorite induced intra-oocyst sporozoite alterations as demonstrated by flow cytometric analysis of the oocysts' DNA content. Propidium iodide staining of fixed oocysts is a simple and rapid method to visualize sporozoites and to assess oocyst preservation after different treatments.

Ovine mononuclear phagocytes in situ: identification by monoclonal antibodies and involvement in experimental pyogranulomas.

In order to characterize in situ the macrophages present in experimental pyogranulomas induced in lambs with Corynebacterium pseudotuberculosis, a set of monoclonal antibodies (MAbs) was produced following immunization of BALB/c mice with alveolar macrophages from healthy sheep. Three MAbs were retained after two steps of screening using alveolar macrophages, peripheral blood lymphocytes, and polymorphonuclear leukocytes as target cells. Their reactivity was tested not only on macrophages in pyogranulomas but also on sections of various organs in steady-state conditions. One MAb, termed OM1, recognized the monocytes and the majority of cells of the mononuclear phagocyte system in lymphoid and nonlymphoid organs. The two other MAbs, OM2 and OM3, reacted with a subpopulation of alveolar macrophages and with other cell types in tissues, in particular with endothelial cells for the MAb OM2. On sections of experimental pyogranulomas that developed in lymph nodes draining the C. pseudotuberculosis-injected sites, MAb OM1 reacted with all the macrophages distributed in a palisade surrounding the necrotic center of the lesion from day 6 to day 28 postinoculation. The two other MAbs, OM2 and OM3, enabled two types of granulomas to be distinguished: one type was characterized by a large number of epithelioid cells stained by OM2; and the other was characterized by a few OM2-positive macrophages, whereas the OM3-positive cells were more numerous. These results show that macrophages are predominant cells in pyogranulomas and suggest two different histological patterns in the evolution of pyogranulomas induced by C. pseudotuberculosis, according to the immunological status of the host.

Transient secretory IgA deficiency in mice after cyclophosphamide treatment.

Cyclophosphamide (Cy), an alkylating agent widely used in chemotherapy of leukemia and cancer, causes a well-documented toxicity on hematopoietic and lymphoid cells. Neutropenia is thought to be the main factor involved in infectious complications following antimitotic chemotherapy. Little is known on the effects of these therapies on the mucosal associated lymphoid system which is one of the main barriers against environmental pathogenic agents. The present study examined the effects of a single administration of Cy (200 mg/kg) on murine T and B cell populations of Peyer's patches (PPs), IgA secretion in the proximal part of the small intestine, and plasma cells of the lamina propria. Cy induced in mice a transient decrease in the T and B cell populations of the PPs with a drastic fall of B cell counts and a profound decrease of intestinal IgA secretion due to a reduction of lamina propria plasma cells. This transient secretory IgA deficiency may contribute to the infectious complications following antimitotic chemotherapy.

Superior sagittal sinus laceration complicating an autogenous calvarial bone graft harvest: report of a case.

A case of laceration of the superior sagittal sinus complicating removal of a calvarial graft is reported. Despite the advantages of using autogenous calvarial bone grafts for craniofacial reconstructive surgery, the potential for serious complications related to graft harvest must be appreciated. Surgeons involved in calvarial graft harvest must be familiar with cranial surgical technique, and should be capable of managing intraoperative complications at the donor site.

Cerebral resuscitation with succinate and fructose-1, 6-diphosphate.

To test the hypotheses that succinate or fructose-1, 6-diphosphate may have a beneficial effect in global cerebral ischemia, we induced complete global cerebral ischemia for 5 minutes in rabbits by occlusion of the ascending aorta and the superior and inferior vena cavae. Fifteen minutes after restoration of cerebral blood flow, animals received an intravenous bolus of either succinate or fructose-1,6-diphosphate followed by continuous infusion. Another group of animals received fructose-1, 6-diphosphate beginning prior to aortic occlusion. Control animals received intravenous glucose by bolus, followed by infusion. Cerebrospinal fluid lactate levels were measured before occlusion and at 2 1/2 hours after occlusion, when the animals were sacrificed. In all animals electrocortical silence was demonstrated for the 5 minutes of global ischemia. The percent change in cerebrospinal fluid lactate levels in all groups was statistically similar. Only two of seven of the control animals recovered electroencephalogram amplitude during the 2 1/2 hour observation period. Time for recovery of amplitude on the electroencephalogram in animals receiving fructose-1, 6-diphosphate either before or after ischemia was statistically similar to controls. In the succinate treated group, all seven animals regained preocclusion levels of electroencephalogram amplitude within 36 minutes of the restoration of cerebral blood flow. Succinate administered after complete global cerebral ischemia resulted in significantly increased recovery of cerebral electrical activity (Fischer's exact test, p less than 0.05).

Primary intracranial plasma-cell granuloma. Case report.

The authors report the fourth case of primary intracranial plasma-cell granuloma. The patient was a 16-year-old girl who presented with loss of vision as the major clinical feature. The tumor resembled a meningioma both preoperatively and grossly at surgery. Because the tumor did not respond to steroid treatment following subtotal surgical excision, radiation therapy was administered to the affected area. Major considerations in the differential diagnosis of this neoplasm are discussed.