Phosphorylation-dependent cleavage regulates von Hippel Lindau proteostasis and function.

Loss of von Hippel Lindau (VHL) protein function is a key driver of VHL diseases, including sporadic and inherited clear cell renal cell carcinoma. Modulation of the proteostasis of VHL, especially missense point-mutated VHL, is a promising approach to augmenting VHL levels and function. VHL proteostasis is regulated by multiple mechanisms including folding, chaperone binding, complex formation and phosphorylation. Nevertheless, many details underlying the regulations of VHL proteostasis are unknown. VHL is expressed as two variants, VHL30 and VHL19. Furthermore, the long-form variant of VHL was often detected as multiple bands by western blotting. However, how these multiple species of VHL are generated and whether the process regulates VHL proteostasis and function are unknown. We hypothesized that the two major species are generated by VHL protein cleavage, and the cleavage regulates VHL proteostasis and subsequent function. We characterized VHL species using genetical and pharmacological approaches and showed that VHL was first cleaved at the N-terminus by chymotrypsin C before being directed for proteasomal degradation. Casein kinase 2-mediated phosphorylation at VHL N-terminus was required for the cleavage. Furthermore, inhibition of cleavage stabilized VHL protein and thereby promoted HIF downregulation. Our study reveals a novel mechanism regulating VHL proteostasis and function, which is significant for identifying new drug targets and developing new therapeutic approaches targeting VHL deficiency in VHL diseases.

Does an evidence-based healthy relationships program for 9th graders show similar effects for 7th and 8th graders? Results from 57 schools randomized to intervention.

Integrating social and emotional learning (SEL) programming throughout curricula to support the development of healthy behaviors and prevent violence is critical for a comprehensive approach to school health. This study used a post-test comparison design to evaluate a healthy relationships program for eighth grade students that applies a SEL approach. The program was adapted from the Fourth R, an evidence-based program for ninth graders, but matches the curriculum and developmental context for eighth graders. Surveys were collected post-intervention from 1012 students within 57 schools randomized to intervention or control conditions. Multivariate multilevel analysis accounted for the nested nature of students within schools. There were significant group differences on three of four outcomes following intervention, including improved knowledge about violence, critical thinking around the impact of violence, and identification of more successful coping strategies. There was no group difference on general acceptance of violence. Overall, students learned relevant information and strategies and were able to apply that knowledge to demonstrate critical thinking, suggesting that adapting an evidence-based approach for use with younger students provided similar benefits. These findings build a case for 2 years of consecutive evidence-based healthy relationships programming in grades 8 and 9, consistent with best practice guidelines.

HOXA1 drives melanoma tumor growth and metastasis and elicits an invasion gene expression signature that prognosticates clinical outcome.

Melanoma is a highly lethal malignancy notorious for its aggressive clinical course and eventual resistance to existing therapies. Currently, we possess a limited understanding of the genetic events driving melanoma progression, and much effort is focused on identifying pro-metastatic aberrations or perturbed signaling networks that constitute new therapeutic targets. In this study, we validate and assess the mechanism by which homeobox transcription factor A1 (HOXA1), a pro-invasion oncogene previously identified in a metastasis screen by our group, contributes to melanoma progression. Transcriptome and pathway profiling analyses of cells expressing HOXA1 reveals upregulation of factors involved in diverse cytokine pathways that include the transforming growth factor beta (TGFß) signaling axis, which we further demonstrate to be required for HOXA1-mediated cell invasion in melanoma cells. Transcriptome profiling also shows HOXA1's ability to potently downregulate expression of microphthalmia-associated transcription factor (MITF) and other genes required for melanocyte differentiation, suggesting a mechanism by which HOXA1 expression de-differentiates cells into a pro-invasive cell state concomitant with TGFß activation. Our analysis of publicly available data sets indicate that the HOXA1-induced gene signature successfully categorizes melanoma specimens based on their metastatic potential and, importantly, is capable of stratifying melanoma patient risk for metastasis based on expression in primary tumors. Together, these validation data and mechanistic insights suggest that patients whose primary tumors express HOXA1 are among a high-risk metastasis subgroup that should be considered for anti-TGFß therapy in adjuvant settings. Moreover, further analysis of HOXA1 target genes in melanoma may reveal new pathways or targets amenable to therapeutic intervention.

Coordinate phosphorylation of multiple residues on single AKT1 and AKT2 molecules.

Aberrant AKT activation is prevalent across multiple human cancer lineages providing an important new target for therapy. Twenty-two independent phosphorylation sites have been identified on specific AKT isoforms likely contributing to differential isoform regulation. However, the mechanisms regulating phosphorylation of individual AKT isoform molecules have not been elucidated because of the lack of robust approaches able to assess phosphorylation of multiple sites on a single AKT molecule. Using a nanofluidic proteomic immunoassay (NIA), consisting of isoelectric focusing followed by sensitive chemiluminescence detection, we demonstrate that under basal and ligand-induced conditions that the pattern of phosphorylation events is markedly different between AKT1 and AKT2. Indeed, there are at least 12 AKT1 peaks and at least 5 AKT2 peaks consistent with complex combinations of phosphorylation of different sites on individual AKT molecules. Following insulin stimulation, AKT1 was phosphorylated at Thr308 in the T-loop and Ser473 in the hydrophobic domain. In contrast, AKT2 was only phosphorylated at the equivalent sites (Thr309 and Ser474) at low levels. Further, Thr308 and Ser473 phosphorylation occurred predominantly on the same AKT1 molecules, whereas Thr309 and Ser474 were phosphorylated primarily on different AKT2 molecules. Although basal AKT2 phosphorylation was sensitive to inhibition of phosphatidylinositol 3-kinase (PI3K), basal AKT1 phosphorylation was essentially resistant. PI3K inhibition decreased pThr451 on AKT2 but not pThr450 on AKT1. Thus, NIA technology provides an ability to characterize coordinate phosphorylation of individual AKT molecules providing important information about AKT isoform-specific phosphorylation, which is required for optimal development and implementation of drugs targeting aberrant AKT activation.

Sds22/PP1 links epithelial integrity and tumor suppression via regulation of myosin II and JNK signaling.

Loss of epithelial integrity often correlates with the progression of malignant tumors. Sds22, a regulatory subunit of protein phosphatase 1 (PP1), has recently been linked to regulation of epithelial polarity in Drosophila. However, its role in tumorigenesis remains obscure. In this study, using Drosophila imaginal tissue as an in vivo model system, we show that sds22 is a new potential tumor suppressor gene in Drosophila. Without sds22, cells lose epithelial architecture, and become invasive and tumorigenic when combined with Ras overexpression; conversely, sds22 overexpression can largely suppress tumorigenic growth of Ras(V12)scrib(-/-) mutant cells. Mechanistically, we show that sds22 prevents cell invasion and metastasis by inhibiting myosin II and Jun N-terminal kinase (JNK) activity downstream of PP1. Loss of this inhibition causes cells to lose epithelial organization and promotes cell invasion. Finally, human Sds22 is focally deleted and downregulated in multiple carcinomas, and this downregulation correlates with tumor progression, suggesting that sds22 inactivation may contribute to tumorigenesis and metastatic potential in human cancers via a similar mechanism.

A prospective study of awareness and recall associated with general anaesthesia for caesarean section.

BACKGROUND: The obstetric population is considered at high risk of awareness and recall when undergoing general anaesthesia for caesarean section. In recent years the incidence may have been altered by developments in obstetric anaesthesia. METHODS: A prospective observational study of general anaesthesia for caesarean section was conducted during 2005 and 2006 in 13 maternity hospitals dealing with approximately 49,500 deliveries per annum in Australia and New Zealand. As a component of this study the frequency of recall of intraoperative events was examined using a structured postoperative interview on two occasions. RESULTS: There were 1095 general anaesthetics surveyed with 47% being performed for urgent fetal delivery. Thiopental was the most common induction agent (83%) with sevoflurane being used for maintenance in 63%. In 32% of cases a depth-of-anaesthesia monitor was used. In 763 cases (70%) there was least one postoperative interview enquiring into dreaming and awareness. There were two cases deemed to be consistent with awareness (incidence 0.26%, CI 0.03-0.9%, or 1 in 382) and three cases of possible awareness. CONCLUSION: Awareness with recall of intraoperative events remains a significant complication of obstetric general anaesthesia but was potentially avoidable in all cases detected in this study.

Difficult and failed intubation in obstetric anaesthesia: an observational study of airway management and complications associated with general anaesthesia for caesarean section.

BACKGROUND: Recent developments in anaesthesia and patient demographics have potentially changed the practice of obstetric general anaesthesia. There are few contemporary data on Australasian practice of general anaesthesia for caesarean section, especially relating to airway management, anaesthetic techniques and complications. METHODS: Using a standardised case record form, a prospective observational study was conducted during 2005-06 in 13 maternity hospitals dealing with approximately 49 500 deliveries per annum. Patient demographics, airway management, anaesthetic techniques and major complications were evaluated in those given general anaesthesia. RESULTS: Data were obtained from 1095 women receiving general anaesthesia for caesarean section, 47% of which were classified as category 1 and 18% as category 4. Tracheal intubation was planned in all cases with rapid-sequence induction used in 97%. A grade 3 or 4 laryngoscopic view was obtained in 3.6 and 0.6% of cases respectively, with 3.3% considered a difficult intubation. There were four failed intubations (0.4%, 95% CI 0.01-0.9%), of which three were subsequently managed using a laryngeal mask airway. Antacid prophylaxis was used in 94% of elective cases and 64% of emergencies. Regurgitation of gastric contents was noted in eight cases (0.7%, 95% CI 0.2-1.2%), with one confirmed case of aspiration (0.1%, 95% CI 0.002-0.5%). There were no cases of serious airway-related morbidity. CONCLUSIONS: General anaesthesia is most commonly used in emergency situations. Tracheal intubation after rapid-sequence induction remains the predominant approach to airway management in Australasia. The incidence of failed intubation is consistent with previous studies. Aspiration prophylaxis is not routinely used for emergency surgery.

FTIR analysis of the SII540 intermediate of sensory rhodopsin II: Asp73 is the Schiff base proton acceptor.

Sensory rhodopsin II (SRII), a repellent phototaxis receptor found in Halobacterium salinarum, has several homologous residues which have been found to be important for the proper functioning of bacteriorhodopsin (BR), a light-driven proton pump. These include Asp73, which in the case of bacteriorhodopsin (Asp85) functions as the Schiff base counterion and proton acceptor. We analyzed the photocycles of both wild-type SRII and the mutant D73E, both reconstituted in Halobacterium salinarum lipids, using FTIR difference spectroscopy under conditions that favor accumulation of the O-like, photocycle intermediate, SII540. At both room temperature and -20 degrees C, the difference spectrum of SRII is similar to the BR-->O640 difference spectrum of BR, especially in the configurationally sensitive retinal fingerprint region. This indicates that SII540 has an all-trans chromophore similar to the O640 intermediate in BR. A positive band at 1761 cm-1 downshifts 40 cm-1 in the mutant D73E, confirming that Asp73 undergoes a protonation reaction and functions in analogy to Asp85 in BR as a Schiff base proton acceptor. Several other bands in the C=O stretching regions are identified which reflect protonation or hydrogen bonding changes of additional Asp and/or Glu residues. Intense bands in the amide I region indicate that a protein conformational change occurs in the late SRII photocycle which may be similar to the conformational changes that occur in the late BR photocycle. However, unlike BR, this conformational change does not reverse during formation of the O-like intermediate, and the peptide groups giving rise to these bands are partially accessible for hydrogen/deuterium exchange. Implications of these findings for the mechanism of SRII signal transduction are discussed.

A preschool immunization project to enhance immunization levels, the public-private relationship, and continuity of care.

This study was conducted to determine whether implementing a program aimed at providing a variety of incentives to physicians who provide immunizations to preschool-aged children would help to improve immunization rates and reduce fragmented care for patients. Twenty physicians from 14 private practices that provide care to preschool-aged children from low income families in suburban Cook County, Illinois participated in the project. A randomly selected subset of patient case records from the physicians' offices were audited after the implementation of the project to determine the immunization status of children in the practices and the nature of services provided. These 310 records of children under three years of age who were treated between 1991-1994 (the intervention sample) were compared to 310 charts from a 1988-1990 cohort of records (baseline sample). The groups did not differ on race or gender; however, significantly more families in the 1988 through 1990 cohort of children under 3 years of age were insured privately when compared to the 1991 through 1994 cohort. Seventy percent (218) of the records in the intervention sample were up to date for age on immunizations compared to 45% (141) of the baseline records, reflecting a statistically significant difference (p < .00001). The intervention sample showed significantly more well child visits where immunizations were given and follow up visits where immunizations were given when compared to the baseline sample. Physicians completed surveys before and after implementation of the project. They were questioned about their knowledge and practices regarding immunizations as well as their opinion of specific project components. All of the physicians viewed the project as an effective means to improve immunization services to low income children. The project demonstrates a potential means of enhancing immunization levels and continuity of care among preschool-aged children. It also highlights the workable nature of the partnership between public and private sectors.

A eukaryotic protein, NOP-1, binds retinal to form an archaeal rhodopsin-like photochemically reactive pigment.

The nop-1 gene from Neurospora crassa is predicted to encode a seven-helix protein exhibiting conservation with the rhodopsins of the archaeon Halobacterium salinarum. In the work presented here we have expressed this gene heterologously in the yeast Pichia pastoris, obtaining a relatively high yield of 2.2 mg of NOP-1 protein/L of cell culture. The expressed protein is membrane-associated and forms with all-trans retinal a visible light-absorbing pigment with a 534 nm absorption maximum and approximately 100 nm half-bandwidth typical of retinylidene protein absorption spectra. Its lambda(max) indicates a protonated Schiff base linkage of the retinal. Laser flash kinetic spectroscopy demonstrates that the retinal-reconstituted pigment undergoes a photochemical reaction cycle with a near-UV-absorbing intermediate that is similar to the M intermediates produced by transient Schiff base deprotonation of the chromophore in the photocycles of bacteriorhodopsin and sensory rhodopsins I and II. The slow photocycle (seconds) and long-lived intermediates (M and O) are most similar to those of the phototaxis receptor sensory rhodopsin II. The results demonstrate a photochemically reactive member of the archaeal rhodopsin family in a eukaryotic cell.

Immunization practices and beliefs of physicians in suburban Cook County, Illinois.

This study was conducted to ascertain the vaccination beliefs and practices of physicians who provide care for low income children. Sixty-two (56.9%) of a sample of 109 physicians in suburban Cook County, Illinois responded to a mail survey. A majority of physicians reported a willingness to immunize during well child care, follow-up, and chronic illness visits; yet, a substantial lack of willingness to immunize given certain acute mild illnesses was reported. Twenty-six percent of providers did not routinely identify children who were behind in immunizations and only 16% had completed a chart audit in the past three years. Seventy-four percent were willing to provide all shots needed at a single visit. Misconceptions regarding true contraindications was found among the group. Missed well child visits were identified as the greatest barrier to complete immunization. Improvements in vaccination rates are expected if physicians utilize all types of medical encounters to monitor the immunization status of patients and provide vaccines using only true medical contraindications.

Enhancing information acquisition for over-the-counter medications by making better use of container surface space.

Most over-the-counter (OTC) pharmaceutical container labels are printed in very small type. Consequently, people with visual impairments (e.g., presbyopia in older adults) have difficult reading the material. Some OTC drugs are packaged in containers with easy-open caps. This design increases the surface area that could be used to enhance the labeling. In Experiment 1, older adults (M = 75.1 years, SD = 8.1) evaluated six container label variants for an actual OTC product. Besides having a multipanel main label, four containers had labels attached to the cap that displayed the most important information in large print but differed in color. Two control containers lacked a cap label; one had only a four-panel main label, and the other had only the front label. Participants ranked the containers on six dimensions (e.g., label noticeability, willingness to read). Results showed greater preference for containers with the cap labels. Experiment 2 again examined preferences but also measured information-acquisition performance after participants (M = 79 years, SD = 5.8) were briefly exposed to a realistic-appearing, but fictitious, OTC medication. Results showed greater knowledge and preference for containers with the cap labels. Experiment 2 showed that one of the cap colors (yellow) that was different from the main label was preferred over the white and orange (the same colors as on the main label), but color distinctiveness as an explanation was not fully supported because the green cap was not significantly different from the other cap labels. Implications for communicating information about OTC drugs using expanded labels are discussed.

Accessibility of cigarettes to minors in suburban Cook County, Illinois.

This study assessed the problem of under age sale of cigarettes, educated vendors about the law restricting the sale of tobacco to minors, and determined the effectiveness of a vendor education intervention. Twenty-one teen volunteers, 14 to 17 years in age, attempted to purchase cigarettes in suburban Cook County from over-the-counter merchants and vending machines. Of the 347 vendors that were checked, the minors were successful in 37.2% of their attempts to purchase cigarettes. After information was sent to each vendor about the State of Illinois law, follow up visits were made to all the vendors who were willing to sell cigarettes to the minors during the first visits. Approximately 50% of the vendors were again willing to sell cigarettes to minors. This study's findings suggest that minors can easily purchase cigarettes in suburban Cook County. The education intervention component of the study had a limited but promising effect on compliance rates of the vendors.

Neuropeptide Y receptor genes mapped in human and mouse: receptors with high affinity for pancreatic polypeptide are not clustered with receptors specific for neuropeptide Y and peptide YY.

Ppyr1, Npy5r, and Npy6r, the genes encoding mouse type 4, type 5, and type 6 members of the neuropeptide Y receptor family, have been mapped by interspecific backcross analysis to conserved linkage groups on mouse Chr 14, Chr 8, and Chr 18, respectively. The human genes, PPYR1 and NPY5R, have been localized to chromosomes 10q and 4q, respectively, by analysis of a panel of rodent-human somatic cell hybrids and yeast artificial chromosomes. These studies complete the mapping of the cloned NPY receptor subtypes in human and mouse and, together with previous studies, establish that the genes encoding receptors with high affinity for pancreatic polypeptide are not clustered with the genes encoding receptors specific for neuropeptide Y and peptide YY. The physical association of these receptor genes correlates with ligand-binding properties, rather than sequence identity, and suggests a complex evolutionary relationship.