Evidence for the earliest structural use of wood at least 476,000 years ago.

Wood artefacts rarely survive from the Early Stone Age since they require exceptional conditions for preservation; consequently, we have limited information about when and how hominins used this basic raw material1. We report here on the earliest evidence for structural use of wood in the archaeological record. Waterlogged deposits at the archaeological site of Kalambo Falls, Zambia, dated by luminescence to at least 476 ± 23 kyr ago (ka), preserved two interlocking logs joined transversely by an intentionally cut notch. This construction has no known parallels in the African or Eurasian Palaeolithic. The earliest known wood artefact is a fragment of polished plank from the Acheulean site of Gesher Benot Ya'aqov, Israel, more than 780 ka (refs. 2,3). Wooden tools for foraging and hunting appear 400 ka in Europe4-8, China9 and possibly Africa10. At Kalambo we also recovered four wood tools from 390 ka to 324 ka, including a wedge, digging stick, cut log and notched branch. The finds show an unexpected early diversity of forms and the capacity to shape tree trunks into large combined structures. These new data not only extend the age range of woodworking in Africa but expand our understanding of the technical cognition of early hominins11, forcing re-examination of the use of trees in the history of technology12,13.

Community engagement interventions for communicable disease control in low- and lower- middle-income countries: evidence from a review of systematic reviews.

BACKGROUND: Community engagement (CE) interventions include a range of approaches to involve communities in the improvement of their health and wellbeing. Working with communities defined by location or some other shared interest, these interventions may be important in assisting equity and reach of communicable disease control (CDC) in low and lower-middle income countries (LLMIC). We conducted an umbrella review to identify approaches to CE in communicable disease control, effectiveness of these approaches, mechanisms and factors influencing success. METHODS: We included systematic reviews that: i) focussed on CE interventions; ii) involved adult community members; iii) included outcomes relevant to communicable diseases in LLMIC; iv) were written in English. Quantitative results were extracted and synthesised narratively. A qualitative synthesis process enabled identification of mechanisms of effect and influencing factors. We followed guidance from the Joanna Briggs Institute, assessed quality with the DARE tool and reported according to standard systematic review methodology. RESULTS: Thirteen systematic reviews of medium-to-high quality were identified between June and July 2017. Reviews covered the following outcomes: HIV and STIs (6); malaria (2); TB (1); child and maternal health (3) and mixed (1). Approaches included: CE through peer education and community health workers, community empowerment interventions and more general community participation or mobilisation. Techniques included sensitisation with the community and involvement in the identification of resources, intervention development and delivery. Evidence of effectiveness of CE on health outcomes was mixed and quality of primary studies variable. We found: i) significantly reduced neonatal mortality following women's participatory learning and action groups; ii) significant reductions in HIV and other STIs with empowerment and mobilisation interventions with marginalised groups; iii) significant reductions in malaria incidence or prevalence in a small number of primary studies; iv) significant reductions in infant diarrhoea following community health worker interventions. Mechanisms of impact commonly occurred through social and behavioural processes, particularly: changing social norms, increasing social cohesion and social capacity. Factors influencing effectiveness of CE interventions included extent of population coverage, shared leadership and community control over outcomes. CONCLUSION: Community engagement interventions may be effective in supporting CDC in LLMIC. Careful design of CE interventions appropriate to context, disease and community is vital.

The changing health priorities of earthquake response and implications for preparedness: a scoping review.

OBJECTIVES: Earthquakes have substantial impacts on mortality in low- and middle-income countries (LMIC). The academic evidence base to support Disaster Risk Reduction activities in LMIC settings is, however, limited. We sought to address this gap by identifying the health and healthcare impacts of earthquakes in LMICs and to identify the implications of these findings for future earthquake preparedness. STUDY DESIGN: Scoping review. METHODS: A scoping review was undertaken with systematic searches of indexed databases to identify relevant literature. Key study details, findings, recommendations or lessons learnt were extracted and analysed across individual earthquake events. Findings were categorised by time frame relative to earthquakes and linked to the disaster preparedness cycle, enabling a profile of health and healthcare impacts and implications for future preparedness to be established. RESULTS: Health services need to prepare for changing health priorities with a shift from initial treatment of earthquake-related injuries to more general health needs occurring within the first few weeks. Preparedness is required to address mental health and rehabilitation needs in the medium to longer term. Inequalities of the impact of earthquakes on health were noted in particular for women, children, the elderly, disabled and rural communities. The need to maintain access to essential services such as reproductive health and preventative health services were identified. Key preparedness actions include identification of appropriate leaders, planning and training of staff. Testing of plans was advocated within the literature with evidence that this is possible in LMIC settings. CONCLUSIONS: Whilst there are a range of health and healthcare impacts of earthquakes, common themes emerged in different settings and from different earthquake events. Preparedness of healthcare systems is essential and possible, in order to mitigate the adverse health impacts of earthquakes in LMIC settings. Preparedness is needed at the community, organisational and system levels.

The health impact of the 2014-15 Ebola outbreak.

OBJECTIVES: The 2014-15 outbreak in West Africa was the largest and deadliest Ebola outbreak recorded; however, there remains uncertainty over its wider health consequences. Our objective was to provide a comprehensive overview of the impact of the Ebola outbreak on population health in the three most affected countries: Sierra Leone, Liberia and Guinea. STUDY DESIGN: Narrative review. METHODS: A narrative overview of the peer-reviewed and grey literature related to the impact and consequences of the Ebola outbreak was conducted, synthesizing the findings of literature retrieved from a structured search of biomedical databases, the Web and references of reviewed articles. RESULTS: The impact of the Ebola outbreak was profound and multifaceted. The health system was severely compromised due to overwhelming demand, healthcare workers deaths, resource diversion and closure of health facilities. Fear of Ebola and healthcare workers led to a breakdown in trust in health systems. Access to healthcare was compromised. Substantial reductions in healthcare utilization were reported including over 80% reductions in maternal delivery care in Ebola-affected areas, 40% national reductions in malaria admissions among children <5 years and substantial reductions in vaccination coverage. Socio-economic impacts included reduced community cohesion, education loss, reduced child protection, widespread job losses and food insecurity. Increased morbidity and mortality and reduced expected life expectancy were reported. CONCLUSIONS: This review highlights the scope and scale of the consequences of the Ebola outbreak on population health. Sustained commitment of the international community is required to support health system re-building and to urgently address unmet population health needs.

The 2015 Nepal earthquake disaster: lessons learned one year on.

INTRODUCTION: The 2015 earthquake in Nepal killed over 8000 people, injured more than 21,000 and displaced a further 2 million. One year later, a national workshop was organized with various Nepali stakeholders involved in the response to the earthquake. The workshop provided participants an opportunity to reflect on their experiences and sought to learn lessons from the disaster. METHODS: One hundred and thirty-five participants took part and most had been directly involved in the earthquake response. They included representatives from the Ministry of Health, local and national government, the armed forces, non-governmental organizations, health practitioners, academics, and community representatives. Participants were divided into seven focus groups based around the following topics: water, sanitation and hygiene, hospital services, health and nutrition, education, shelter, policy and community. Facilitated group discussions were conducted in Nepalese and the key emerging themes are presented. RESULTS: Participants described a range of issues encountered, some specific to their area of expertize but also more general issues. These included logistics and supply chain challenges, leadership and coordination difficulties, impacts of the media as well as cultural beliefs on population behaviour post-disaster. Lessons identified included the need for community involvement at all stages of disaster response and preparedness, as well as the development of local leadership capabilities and community resilience. A 'disconnect' between disaster management policy and responses was observed, which may result in ineffective, poorly planned disaster response. CONCLUSION: Finding time and opportunity to reflect on and identify lessons from disaster response can be difficult but are fundamental to improving future disaster preparedness. The Nepal Earthquake National Workshop offered participants the space to do this. It garnered an overwhelming sense of wanting to do things better, of the need for a Nepal-centric approach and the need to learn the lessons of the past to improve disaster management for the future.

Understanding the non-pharmacological correlates of self-reported efficacy of antidepressants.

OBJECTIVE: To explore the non-pharmacological correlates of the perceived effectiveness of antidepressants (ADs), thereby enhancing understanding of the mechanisms involved in recovery from depression while taking ADs. METHOD: An online survey was completed by 1781 New Zealand adults who had taken ADs in the previous 5 years. RESULTS: All 18 psychosocial variables measured were associated with depression reduction, and 16 with improved quality of life (QoL). Logistic regression models revealed that the quality of the relationship with the prescriber was related to both depression reduction and improved QoL. In addition, depression reduction was related to younger age, higher income, being fully informed about ADs by the prescriber, fewer social causal beliefs for depression and not having lost a loved one in the 2 months prior to prescription. Furthermore, both outcome measures were positively related to belief in 'chemical' rather than 'placebo' effects. CONCLUSION: There are multiple non-pharmacological processes involved in recovery while taking ADs. Enhancing them, for example focusing on the prescriber-patient relationship and giving more information, may enhance recovery rates, with or without ADs.

Medical education and law: withholding/withdrawing treatment from adults without capacity.

BACKGROUND: Law is increasingly involved in clinical practice, particularly at the end of life, but undergraduate and postgraduate education in this area remains unsystematic. We hypothesised that attitudes to and knowledge of the law governing withholding/withdrawing life-sustaining treatment from adults without capacity (the WWLST law) would vary and demonstrate deficiencies among medical specialists. AIMS: We investigated perspectives, knowledge and training of medical specialists in the three largest (populations and medical workforces) Australian states, concerning the WWLST law. METHODS: Following expert legal review, specialist focus groups, pre-testing and piloting in each state, seven specialties involved with end-of-life care were surveyed, with a variety of statistical analyses applied to the responses. RESULTS: Respondents supported the need to know and follow the law. There were mixed views about its helpfulness in medical decision-making. Over half the respondents conceded poor knowledge of the law; this was mirrored by critical gaps in knowledge that varied by specialty. There were relatively low but increasing rates of education from the undergraduate to continuing professional development (CPD) stages. Mean knowledge score did not vary significantly according to undergraduate or immediate postgraduate training, but CPD training, particularly if recent, resulted in greater knowledge. Case-based workshops were the preferred CPD instruction method. CONCLUSIONS: Teaching of current and evolving law should be strengthened across all stages of medical education. This should improve understanding of the role of law, ameliorate ambivalence towards the law and contribute to more informed deliberation about end-of-life issues with patients and families.

Medical practitioners' knowledge and self-reported practices of substitute decision making and implementation of advance care plans.

BACKGROUND: Advance care planning (ACP) provides patients with the ability to make their decisions known about how they would like to be treated if they lose capacity. Medical practitioners have a key role to play in providing information on ACP to their patients. This research explores their knowledge and attitudes to advance care planning and how this affects their practice. AIM: The objective of this study is to assess the NSW medical practitioners' knowledge and self-reported practice of ACP. METHODS: A postal survey of a random sample of 650 general practitioners plus 350 medical specialists from specialties most often involved in end-of-life decisions was conducted. Respondents' work location post codes were subsequently used to assign respondents to one of the eight NSW Area Health Services. The main outcome measures were medical practitioners' knowledge of and practice pertaining to ACP. RESULTS: Thirty-four per cent of specialists (n = 110) and 24% of general practitioners (n = 150) responded; the majority of respondents had heard of all ACP options. However, respondents' understanding of the uses and legal requirements of the relevant ACP options vary widely. CONCLUSIONS: Respect for patient wishes expressed in advance directives is reassuringly high. The findings suggest significant misunderstanding by medical practitioners of terminologies and systems around substitute decision-making for incompetent persons. Further education and standardisation of terminologies and systems across different jurisdictions would assist in addressing these issues. Low response rate, relating to only one legal jurisdiction, means results may not be generalisable.

The housing and support needs of people with an intellectual disability into older age.

BACKGROUND: People with intellectual disabilities (IDs) are growing older as a population cohort. Many live at home with family members who are their carers but who are also becoming older and less able to provide care. The housing and support preferences of people with IDs and their carers into older age are poorly characterised in the literature. METHODS: Focus groups and individual interviews were conducted with 15 people with IDs who work in supported employment and with 10 family members who care for adults with IDs. Data were thematically analysed independently by two researchers. RESULTS: The major themes that emerged were as follows: (1) living arrangements; (2) housing preferences; (3) ageing in place; and (4) transition from informal to formal housing and support services. CONCLUSIONS: Participants with an ID and their carers want housing and support that enable people with an ID to maintain and enhance their social networks with their peers as they grow older and require transition to formal housing and support services, and to be able to 'age in place'. A preference was expressed for models of housing that provide the opportunity for people with an ID to live in close proximity to their peers and in large groups in the community rather than in small, dispersed community housing.

Performance of the Abbott RealTime CT/NG for detection of Chlamydia trachomatis and Neisseria gonorrhoeae.

A multicenter clinical study was conducted to evaluate the performance characteristics of the Abbott RealTime CT/NG assay, a multiplex real-time PCR assay, for simultaneous detection of Chlamydia trachomatis and Neisseria gonorrhoeae. The specimens were collected from a total of 3,832 male and female subjects at 16 geographically diverse sites. Specimens included male and female urine samples, male urethral swabs, female endocervical swabs, and self-collected and clinician-collected vaginal swabs. Specimens were tested with the automated Abbott RealTime CT/NG assay, Aptima Combo 2 assay (Gen-Probe), ProbeTec ET CT/GC assay (Becton Dickinson), and culture for N. gonorrhoeae. The Aptima Combo 2 assay, the ProbeTec assay, and the N. gonorrhoeae culture were used as the reference assays. For each subject, a patient infected status (PIS) was determined based on the combined results from the reference assays. The overall prevalence in female subjects was 8.9% for C. trachomatis and 3.8% for N. gonorrhoeae. The overall male prevalence was 18.2% for C. trachomatis and 16.7% for N. gonorrhoeae. The overall sensitivity and specificity of the Abbott RealTime CT/NG assay were 92.4% and 99.2% for C. trachomatis and 96.9% and 99.7% for N. gonorrhoeae, respectively. In comparison, the sensitivity and specificity, respectively, for the Aptima Combo 2 assay were 94.5% and 99.0% for C. trachomatis and 96.1% and 99.5% for N. gonorrhoeae, and those for the ProbeTec ET assay were 90.3% and 99.5% for C. trachomatis and 92.0% and 97.3% for N. gonorrhoeae in this study. The Abbott RealTime CT/NG assay offers C. trachomatis and N. gonorrhoeae dual detection with high sensitivity and specificity. The automated assay provides a useful alternative nucleic acid amplification assay for clinical laboratories and clinicians.

Tyr527 is phosphorylated in pp60c-src: implications for regulation.

The Rous sarcoma virus oncogene product, pp60v-src, transforms cultured fibroblasts but its corresponding proto-oncogene product, pp60c-src, does not. Both proteins are known to be protein-tyrosine kinases. Published results suggest that the kinase activity of pp60c-src is inhibited relative to that of pp60v-src, due perhaps to phosphorylation of a tyrosine in pp60c-src that is not phosphorylated in pp60v-src. In this study, it was observed that the tyrosine phosphorylated in pp60c-src is Tyr527, six residues from the COOH-terminus of the protein. The region of pp60c-src from residue 515 to the COOH-terminus, including Tyr527, has been replaced with a different sequence in pp60v-src. Thus, the increase in transforming ability and kinase activity that occurred in the genesis of pp60v-src may have resulted from the loss of a tyrosine involved in negative regulation.

RACK1 inhibits colonic cell growth by regulating Src activity at cell cycle checkpoints.

Previously, we showed that Src tyrosine kinases are activated early in the development of human colon cancer and are suppressed as intestinal cells differentiate. We identified RACK1 as an endogenous substrate, binding partner and inhibitor of Src. Here we show (by overexpressing RACK1, depleting Src or RACK1 and utilizing cell-permeable peptides that perturb RACK1's interaction with Src) that RACK1 regulates growth of colon cells by suppressing Src activity at G(1) and mitotic checkpoints, and consequently delaying cell cycle progression. Activated Src rescues RACK1-inhibited growth of HT-29 cells. Conversely, inhibiting Src abolishes growth promoted by RACK1 depletion in normal cells. Two potential mechanisms whereby RACK1 regulates mitotic exit are identified: suppression of Src-mediated Sam68 phosphorylation and maintenance of the cyclin-dependent kinase (CDK) 1-cyclin B complex in an active state. Our results reveal novel mechanisms of cell cycle control in G(1) and mitosis of colon cells. The significance of this work lies in the discovery of a mechanism by which the growth of colon cancer cells can be slowed, by RACK1 suppression of an oncogenic kinase at critical cell cycle checkpoints. Small molecules that mimic RACK1 function may provide a powerful new approach to the treatment of colon cancer.

Influence of physicians' life stances on attitudes to end-of-life decisions and actual end-of-life decision-making in six countries.

AIM: To examine how physicians' life stances affect their attitudes to end-of-life decisions and their actual end-of-life decision-making. METHODS: Practising physicians from various specialties involved in the care of dying patients in Belgium, Denmark, The Netherlands, Sweden, Switzerland and Australia received structured questionnaires on end-of-life care, which included questions about their life stance. Response rates ranged from 53% in Australia to 68% in Denmark. General attitudes, intended behaviour with respect to two hypothetical patients, and actual behaviour were compared between all large life-stance groups in each country. RESULTS: Only small differences in life stance were found in all countries in general attitudes and intended and actual behaviour with regard to various end-of-life decisions. However, with regard to the administration of drugs explicitly intended to hasten the patient's death (PAD), physicians with specific religious affiliations had significantly less accepting attitudes, and less willingness to perform it, than non-religious physicians. They had also actually performed PAD less often. However, in most countries, both Catholics (up to 15.7% in The Netherlands) and Protestants (up to 20.4% in The Netherlands) reported ever having made such a decision. DISCUSSION: The results suggest that religious teachings influence to some extent end-of-life decision-making, but are certainly not blankly accepted by physicians, especially when dealing with real patients and circumstances. Physicians seem to embrace religious belief in a non-imperative way, allowing adaptation to particular situations.

Elevated c-Src tyrosine kinase activity in premalignant epithelia of ulcerative colitis.

Ulcerative colitis (UC) is a chronic inflammatory disease of the colon with a high incidence of colon cancer. Dysplasia is a precursor to carcinoma and a predictor of malignant potential; epithelia containing high-grade or severe dysplasia is most likely to develop cancer. The cellular oncogene c-src and its viral homologue v-src (the transforming gene of Rous sarcoma virus) encode 60-kD cytoplasmic, membrane-associated protein tyrosine kinases. For the viral protein or transforming mutants of the cellular protein (Src), a close correlation exists between elevated tyrosine kinase activity and malignant transformation of cells. Previously, we and others observed elevated Src activity in sporadic colon carcinomas and benign adenomas at greatest risk for developing cancer (those with large size, villous architecture, and/or severe dysplasia). Here we report that Src activity and protein abundance are also elevated in neoplastic UC epithelia. Activity is highest in malignant and severely dysplastic epithelia, and 6-10-fold higher in mildly dysplastic than in nondysplastic epithelia. Thus, Src activity is elevated in premalignant UC epithelia, which is at greatest risk for developing cancer. The data suggest that activation of the src proto-oncogene is an early event in the genesis of UC colon cancer.

Synergistic action of cyclic adenosine monophosphate- and calcium-mediated chloride secretion in a colonic epithelial cell line.

Vasoactive intestinal polypeptide (VIP) and the calcium ionophore A23187 caused dose-dependent changes in the potential difference and the short circuit current (Isc) across confluent T84 cell monolayers mounted in modified Ussing chambers. Both VIP and A23187 stimulated net chloride secretion without altering sodium transport. Net chloride secretion accounted for the increase in Isc. When A23187 was tested in combination with VIP, net chloride secretion was significantly greater than predicted from the calculated sum of their individual responses indicating a synergistic effect. VIP increased cellular cyclic AMP (cAMP) production in a dose-dependent manner, whereas A23187 had no effect on cellular cAMP. We then determined whether VIP and A23187 activated different transport pathways. Earlier studies suggest that VIP activates a basolaterally localized, barium-sensitive potassium channel as well as an apically localized chloride conductance pathway. In this study, stimulation of basolateral membrane potassium efflux by A23187 was documented by preloading the monolayers with 86Rb+. Stimulation of potassium efflux by A23187 was additive to the VIP-stimulated potassium efflux. By itself, 0.3 microM A23187 did not alter transepithelial chloride permeability, and its stimulation of basolateral membrane potassium efflux caused only a relatively small amount of chloride secretion. However, in the presence of an increased transepithelial chloride permeability induced by VIP, the effectiveness of A23187 on chloride secretion was greatly augmented. Our studies suggest that cAMP and calcium each activate basolateral potassium channels, but cAMP also activates an apically localized chloride channel. Synergism results from cooperative interaction of potassium channels and the chloride channel.

pp60c-src activation in human colon carcinoma.

We measured the in vitro protein-tyrosine kinase activity of pp60c-src from human colon carcinoma cell lines and tumors. The activity of pp60c-src from six of nine carcinoma cell lines was higher (on average, fivefold as measured by enolase phosphorylation, or eightfold as measured by autophosphorylation) than that of pp60c-src from normal colonic mucosal cells, or human or rodent fibroblasts. Similarly, the activity of pp60c-src from 13 of 21 primary colon carcinomas was five- or sevenfold higher than that of pp60c-src from normal colonic mucosa adjacent to the tumor. The increased pp60c-src activity did not result solely from an increase in the level of pp60c-src protein, suggesting the specific activity of the pp60c-src kinase is elevated in the tumor cells. pp60c-src from colon carcinoma cells and normal colonic mucosal cells was phosphorylated at similar sites. We used immunoblotting with antibodies to phosphotyrosine to identify substrates of protein-tyrosine kinases in colonic cells. Three phosphotyrosine-containing proteins were detected at significantly higher levels in most colon carcinoma cell lines than in normal colonic mucosal cells or human or rat fibroblasts. All colon carcinoma cell lines with elevated pp60c-src in vitro kinase activity, showed increased phosphorylation of proteins on tyrosine in vivo, suggesting the presence of an activated protein-tyrosine kinase(s).

Src activity increases and Yes activity decreases during mitosis of human colon carcinoma cells.

Src and Yes protein-tyrosine kinase activities are elevated in malignant and premalignant tumors of the colon. To determine whether Src activity is elevated throughout the human colon carcinoma cell cycle as it is in polyomavirus middle T antigen- or F527 Src-transformed cells, and whether Yes activity, which is lower than that of Src in the carcinoma cells, is regulated differently, we measured their activities in cycling cells. We observed that the activities of both kinases were higher throughout all phases of the HT-29 colon carcinoma cell cycle than in corresponding phases of the fibroblast cycle. In addition, during mitosis of HT-29 cells, Src specific activity increased two- to threefold more, while Yes activity and abundance decreased threefold. The decreased steady-state protein levels of Yes during mitosis appeared to be due to both decreased synthesis and increased degradation of the protein. Inhibition of tyrosine but not serine/threonine phosphatases abolished the mitotic activation of Src. Mitotic Src was phosphorylated at novel serine and threonine sites and dephosphorylated at Tyr-527. Two cellular proteins (p160 and p180) were phosphorylated on tyrosine only during mitosis. Tyrosine phosphorylation of several other proteins decreased during mitosis. Thus, Src in HT-29 colon carcinoma cells, similar to Src complexed to polyomavirus middle T antigen or activated by mutation at Tyr-527, is highly active in all phases of the cell cycle. Moreover, Src activity further increases during mitosis, whereas Yes activity and abundance decrease. Thus, Src and Yes appear to be regulated differently during mitosis of HT-29 colon carcinoma cells.

In vivo topological analysis of Ste2, a yeast plasma membrane protein, by using beta-lactamase gene fusions.

Gene fusions were constructed between Ste2, the receptor for the Saccharomyces cerevisiae alpha-factor, and beta la, the secreted form of beta-lactamase encoded by the bla gene of pBR322. The Ste2 and beta la components were linked by a processing fragment (P) from the yeast killer preprotoxin containing a C-terminal lysine-arginine site for cleavage by the Golgi-associated Kex2 protease. Ste2 is predicted to have a rhodopsinlike topology, with an external N terminus and seven transmembrane segments. Fusions to three of the four Ste2 domains predicted to be external resulted in beta la secretion from yeast cells. A fusion at a site just preceding the first transmembrane segment was an exception; the product was cell associated, indicating that the first 44 residues of Ste2 are insufficient to direct secretion of beta la; translocation of this domain presumably requires the downstream transmembrane segment. Expression of fusions located in two domains predicted to be cytoplasmic failed to result in beta la secretion. Following insertion of the preprotoxin signal peptide (S) between the Ste2 and P components of these cytoplasmic fusions, secretion of beta la activity occurred, which is consistent with inversion of the orientation of the beta la reporter. Conversely, insertion of S between Ste2 and P in an external fusion sharply reduced beta la secretion. Complementary information about both cytoplasmic and external domains of Ste2 was therefore provided, and most aspects of the predicted topology were confirmed. The steady-state levels of beta la detected were low, presumably because of efficient degradation of the fusions in the secretory pathway; levels, however, were easily detectable. This method should be valuable in the analysis of in vivo topologies of both homologous and foreign plasma membrane proteins expressed in yeast cells.

Structural and functional modification of pp60c-src associated with polyoma middle tumor antigen from infected or transformed cells.

The polyoma middle tumor antigen (MTAg) associates with the src proto-oncogene product pp60c-src in infected or transformed rodent cells. The tyrosine protein kinase activity of pp60c-src, as measured by in vitro phosphorylation of pp60c-src itself or the exogenous substrate enolase, was increased 10- to 20-fold in cells transformed or infected with transformation-competent polyoma virus compared with controls. pp60c-src associated with MTAg and precipitated with polyoma antitumor serum had a novel site(s) of in vitro tyrosine phosphorylation within its amino-terminal domain. These observations suggest that association of MTAg with pp60c-src alters the accessibility of pp60c-src tyrosine residues for phosphorylation in vitro and increases pp60c-src protein kinase activity. Several transformation-defective mutants of MTAg did not cause amino-terminal tyrosine phosphorylation of pp60c-src in vitro or enhance its protein kinase activity, suggesting that these properties correlate with the transforming ability of MTAg. However, one transformation-defective MTAg mutant, dl1015, did cause amino-terminal tyrosine phosphorylation of pp60c-src in vitro and did enhance its protein kinase activity. This suggests that properties of MTAg, in addition to modifying the structure and function of pp60c-src, may be important for transformation.

Early activation of endogenous pp60src kinase activity during neuronal differentiation of cultured human neuroblastoma cells.

The proto-oncogene product pp60c-src is a tyrosine-specific kinase with a still unresolved cellular function. High levels of pp60c-src in neurons and the existence of a neuronal pp60c-src variant, pp60c-srcN, suggest participation in the progress or maintenance of the differentiated phenotype of neurons. We have previously reported that phorbol esters, e.g., 12-O-tetradecanoylphorbol-13-acetate (TPA), stimulate human SH-SY5Y neuroblastoma cells to neuronal differentiation, as monitored by morphological, biochemical, and functional differentiation markers. In this report, we describe activation of the pp60src (pp60c-src and pp60c-srcN) kinase activity observed at 6 h after induction of SH-SY5Y cells with TPA. This phenomenon coincides in time with neurite outgrowth, formation of growth cone-like structures, and an increase of GAP43 mRNA expression, which are the earliest indications of neuronal differentiation in these cells. The highest specific src kinase activity (a three- to fourfold increase 4 days after induction) was noted in cells treated with 16 nM TPA; this concentration is optimal for development of the TPA-induced neuronal phenotype. During differentiation, there was no alteration in the 1:1 ratio of pp60c-src to pp60c-srcN found in untreated SH-SY5Y cells. V8 protease and trypsin phosphopeptide mapping of pp60src from in vivo 32P-labeled cells showed that the overall phosphorylation of pp60src was higher in differentiated than in untreated cells, mainly because of an intense serine 12 phosphorylation. Tyrosine 416 phosphorylation was not detectable in either cell type, and no change during differentiation in tyrosine 527 phosphorylation was observed.