Factors influencing health promotion activities in midlife and older Australian women with a chronic disease

Background: Chronic diseases including type 2 diabetes are a leading cause of morbidity and mortality in midlife and older Australian women. There are a number of modifiable risk factors for type 2 diabetes and other chronic diseases including smoking, nutrition, physical activity and overweight and obesity. Little research has been conducted in the Australian context to explore the perceived barriers to health promotion activities in midlife and older Australian women with a chronic disease. Aims: The primary aim of this study was to explore women’s perceived barriers to health promotion activities to reduce modifiable risk factors, and the relationship of perceived barriers to smoking behaviour, fruit and vegetable intake, physical activity and body mass index. A secondary aim of this study was to investigate nurses’ perceptions of the barriers to action for women with a chronic disease, and to compare those perceptions with those of the women. Methods: The study was divided into two phases where Phase 1 was a cross sectional survey of women, aged over 45 years with type 2 diabetes who were attending Diabetes clinics in the Primary and Community Health Service of the Metro North Health Service District of Queensland Health (N = 22). The women were a subsample of women participating in a multi-model lifestyle intervention, the ‘Reducing Chronic Disease among Adult Australian Women’ project. Phase 2 of the study was a cross sectional online survey of nurses working in Primary and Community Health Service in the Metro North Health Service District of Queensland Health (N = 46). Pender’s health promotion model was used as the theoretical framework for this study. Results: Women in this study had an average total barriers score of 32.18 (SD = 9.52) which was similar to average scores reported in the literature for women with a range of physical disabilities and illnesses. The leading five barriers for this group of women were: concern about safety; too tired; not interested; lack of information about what to do; with lack of time and feeling I can’t do things correctly the equal fifth ranked barriers. In this study there was no statistically significant difference in average total barriers scores between women in the intervention group and those is the usual care group of the parent study. There was also no significant relationship between the women’s socio-demographic variables and lifestyle risk factors and their level of perceived barriers. Nurses in the study had an average total barriers score of 44.48 (SD = 6.24) which was higher than all other average scores reported in the literature. The leading five barriers that nurses perceived were an issue for women with a chronic disease were: lack of time and interferes with other responsibilities the leading barriers; embarrassment about appearance; lack of money; too tired and lack of support from family and friends. There was no significant relationship between the nurses’ sociodemographic and nursing variables and the level of perceived barriers. When comparing the results of women and nurses in the study there was a statistically significant difference in the median total barriers score between the groups (p < 0.001), where the nurses perceived the barriers to be higher (Md = 43) than the women (Md = 33). There was also a significant difference in the responses to the individual barriers items in fifteen of the eighteen items (p < 0.002). Conclusion: Although this study is limited by a small sample size, it contributes to understanding the perception of midlife and older women with a chronic disease and also the perception of nurses, about the barriers to healthy lifestyle activities that women face. The study provides some evidence that the perceptions of women and nurses may differ and argues that these differences may have significant implications for clinical practice. The study recommends a greater emphasis on assessing and managing perceived barriers to health promotion activities in health education and policy development and proposes a conceptual model for understanding perceived barriers to action.

Towards the biofortification of banana fruit for enhanced micronutrient content

In Uganda, vitamin A deficiency (VAD) and iron deficiency anaemia (IDA) are major public health problems with between 15-32% of children under 5 years of age showing VAD and 73% being anaemic. This is largely due to the fact that the staple food crop of the country, banana, is low in pro-vitamin A and iron, therefore leading to dietary deficiencies. Although worldwide progress has been made to control VAD and IDA through supplementation, food fortification and diet diversification, their long term sustainability and impact in developing countries such as Uganda is limited. The approach taken by researchers at Queensland University of Technology (QUT), Australia, in collaboration with the National Agricultural Research Organization (NARO), Uganda, to address this problem, is to generate consumer acceptable banana varieties with significantly increased levels of pro-vitamin A and iron in the fruit using genetic engineering techniques. Such an approach requires the use of suitable, well characterised genes and promoters for targeted transgene expression. Recently, a new banana phytoene synthase gene (APsy2a) involved in the synthesis of pro-vitamin A (pVA) carotenoids was isolated from a high â-carotene banana (F’ei cv Asupina). In addition, sequences of banana ferritin, an iron storage protein, have been isolated from Cavendish banana. The aim of the research described in this thesis was to evaluate the function of these genes to assess their suitability for the biofortification of banana fruit. In addition, a range of banana-derived promoters were characterised to determine their suitability for controlling the expression of transgenes in banana fruit. Due to the time constraints involved with generating transgenic banana fruit, rice was used as the model crop to investigate the functionality of the banana-derived APsy2a and ferritin genes. Using Agrobacterium-mediated transformation, rice callus was transformed with APsy2a +/- the bacterial-derived carotene desaturase gene (CrtI) each under the control of the constitutive maize poly-ubiquitin promoter (ZmUbi) or seed-specific rice glutelin1 (Gt1) promoter. The maize phytoene synthase (ZmPsy1) gene was included as a control. On selective media, with the exception of ZmUbi-CrtI-transgenic callus, all antibiotic resistant callus displayed a yellow-orange colour from which the presence of â-carotene was demonstrated using Raman spectroscopy. Although the regeneration of plants from yellow-orange callus was difficult, 16 transgenic plants were obtained and characterised from callus transformed with ZmUbi-APys2a alone. At least 50% of the T1 seeds developed a yellow-orange coloured callus which was found to contain levels of â-carotene ranging from 4.6-fold to 72-fold higher than that in non-transgenic rice callus. Using the seed-specific Gt1 promoter, 38 transgenic rice plants were generated from APsy2a-CrtI-transformed callus while 32 plants were regenerated from ZmPsy1-CrtI-transformed callus. However, when analysed for presence of transgene by PCR, all transgenic plants contained the APsy2a, ZmPsy1 or CrtI transgene, with none of the plants found to be co-transformed. Using Raman spectroscopy, no â-carotene was detected in-situ in representative T1 seeds. To investigate the potential of the banana-derived ferritin gene (BanFer1) to enhance iron content, rice callus was transformed with constitutively expressed BanFer1 using the soybean ferritin gene (SoyFer) as a control. A total of 12 and 11 callus lines independently transformed with BanFer1 and SoyFer, respectively, were multiplied and transgene expression was verified by RT-PCR. Pearl’s Prussian blue staining for in-situ detection of ferric iron showed a stronger blue colour in rice callus transformed with BanFer1 compared to SoyFer. Using flame atomic absorption spectrometry, the highest mean amount of iron quantified in callus transformed with BanFer1 was 30-fold while that obtained using the SoyFer was 14-fold higher than the controls. In addition, ~78% of BanFer1-transgenic callus lines and ~27% of SoyFer-transgenic callus lines had significantly higher iron content than the non-transformed controls. Since the genes used for enhancing micronutrient content need to be expressed in banana fruit, the activity of a range of banana-derived, potentially fruit-active promoters in banana was investigated. Using uidA (GUS) as a reporter gene, the function of the Expansin1 (MaExp1), Expansin1 containing the rice actin intron (MaExp1a), Expansin4 (MaExp4), Extensin (MaExt), ACS (MaACS), ACO (MaACO), Metallothionein (MaMT2a) and phytoene synthase (APsy2a) promoters were transiently analysed in intact banana fruit using two transformation methods, particle bombardment and Agrobacterium-mediated infiltration (agro-infiltration). Although a considerable amount of variation in promoter activity was observed both within and between experiments, similar trends were obtained using both transformation methods. The MaExp1 and MaExp1a directed high levels of GUS expression in banana fruit which were comparable to those observed from the ZmUbi and Banana bunchy top virus-derived BT4 promoters that were included as positive controls. Lower levels of promoter activity were obtained in both methods using the MaACO and MaExt promoters while the MaExp4, MaACS, and APsy2a promoters directed the lowest GUS activity in banana fruit. An attempt was subsequently made to use agro-infiltration to assess the expression of pVA biosynthesis genes in banana fruit by infiltrating fruit with constructs in which the ZmUbi promoter controlled the expression of APsy2a +/- CrtI, and with the maize phytoene synthase gene (ZmPsy1) included as a control. Unfortunately, the large amount of variation and inconsistency observed within and between experiments precluded any meaningful conclusions to be drawn. The final component of this research was to assess the level of promoter activity and specificity in non-target tissue. These analyses were done on leaves obtained from glasshouse-grown banana plants stably transformed with MaExp1, MaACO, APsy2a, BT4 and ZmUbi promoters driving the expression of the GUS gene in addition to leaves from a selection of the same transgenic plants which were growing in a field trial in North Queensland. The results from both histochemical and fluorometric GUS assays showed that the MaExp1 and MaACO promoters directed very low GUS activities in leaves of stably transformed banana plants compared to the constitutive ZmUbi and BT4 promoters. In summary, the results from this research provide evidence that the banana phytoene synthase gene (APsy2a) and the banana ferritin gene (BanFer1) are functional, since the constitutive over-expression of each of these transgenes led to increased levels of pVA carotenoids (for APsy2a) and iron content (for BanFer1) in transgenic rice callus. Further work is now required to determine the functionality of these genes in stably-transformed banana fruit. This research also demonstrated that the MaExp1 and MaACO promoters are fruit-active but have low activity in non-target tissue (leaves), characteristics that make them potentially useful for the biofortification of banana fruit. Ultimately, however, analysis of fruit from field-grown transgenic plants will be required to fully evaluate the suitability of pVA biosynthesis genes and the fruit-active promoters for fruit biofortification.

CDKN2A is not the principal target of deletions on the short arm of chromosome 9 in neuroendocrine (Merkel cell) carcinoma of the skin

The majority of small-cell lung cancers (SCLCs) express p16 but not pRb. Given our previous study showing loss of pRb in Merkel cell carcinoma (MCC)/neuroendocrine carcinoma of the skin and the clinicopathological similarities between SCLC and MCC, we wished to determine if this was also the case in MCC. Twenty-nine MCC specimens from 23 patients were examined for deletions at 10 loci on 9p and 1 on 9q. No loss of heterozygosity (LOH) was seen in 9 patients including 2 for which tumour and cell line DNAs were examined. Four patients had LOH for all informative loci on 9p. Ten tumours showed more limited regions of loss on 9p, and from these 2 common regions of deletion were determined. Half of all informative cases had LOH at D9S168, the most telomeric marker examined, and 3 specimens showed loss of only D9S168. A second region (IFNA-D9S126) showed LOH in 10 (44%) cases, and case MCC26 showed LOH for only D9S126, implicating genes centromeric of the CDKN2A locus. No mutations in the coding regions of p16 were seen in 7 cell lines tested, and reactivity to anti-p16 antibody was seen in all 11 tumour specimens examined and in 6 of 7 cell lines from 6 patients. Furthermore, all cell lines examined reacted with anti-p14(ARF) antibody. These results suggest that neither transcript of the CDKN2A locus is the target of deletions on 9p in MCC and imply the existence of tumour-suppressor genes mapping both centromeric and telomeric of this locus.

FGFR2 as a molecular target in endometrial cancer

Although molecularly targeted therapies have been effective in some cancer types, no targeted therapy is approved for use in endometrial cancer. The recent identification of activating mutations in fibroblast growth factor receptor 2 (FGFR2) in endometrial tumors has generated a new avenue for the development of targeted therapeutic agents. The majority of the mutations identified are identical to germline mutations in FGFR2 and FGFR3 that cause craniosynostosis and hypochondroplasia syndromes and result in both ligand-independent and ligand-dependent receptor activation. Mutations that predominantly occur in the endometrioid subtype of endometrial cancer, are mutually exclusive with KRAS mutation, but occur in the presence of PTEN abrogation. In vitro studies have shown that endometrial cancer cell lines with activating FGFR2 mutations are selectively sensitive to a pan-FGFR inhibitor, PD173074. Several agents with activity against FGFRs are currently in clinical trials. Investigation of these agents in endometrial cancer patients with activating FGFR2 mutations is warranted.

Detection versus false alarm characterisation of a vision-based airborne dim-target collision detection system

This paper presents a preliminary flight test based detection range versus false alarm performance characterisation of a morphological-hidden Markov model filtering approach to vision-based airborne dim-target collision detection. On the basis of compelling in-flight collision scenario data, we calculate system operating characteristic (SOC) curves that concisely illustrate the detection range versus false alarm rate performance design trade-offs. These preliminary SOC curves provide a more complete dim-target detection performance description than previous studies (due to the experimental difficulties involved, previous studies have been limited to very short flight data sample sets and hence have not been able to quantify false alarm behaviour). The preliminary investigation here is based on data collected from 4 controlled collision encounters and supporting non-target flight data. This study suggests head-on detection ranges of approximately 2.22 km under blue sky background conditions (1.26 km in cluttered background conditions), whilst experiencing false alarms at a rate less than 1.7 false alarms/hour (ie. less than once every 36 minutes). Further data collection is currently in progress.

Regulating greenhouse gas emissions from coal mining activities in the context of climate change

Current approaches to the regulation of coal mining activities in Australia have facilitated the extraction of substantial amounts of coal and coal seam gas. The regulation of coal mining activities must now achieve the reduction or mitigation of greenhouse gas emissions in order to address the challenge of climate change and achieve ecologically sustainable development. Several legislative mechanisms currently exist which appear to offer the means to bring about the reduction or mitigation of greenhouse gas emissions from coal mining activities, yet Australia’s emissions from coal mining continue to rise. This article critiques these existing legislative mechanisms and presents recommendations for reform.

Simple classification of walking activities using commodity smart phones

People interact with mobile computing devices everywhere, while sitting, walking, running or even driving. Adapting the interface to suit these contexts is important, thus this paper proposes a simple human activity classification system. Our approach uses a vector magnitude recognition technique to detect and classify when a person is stationary (or not walking), casually walking, or jogging, without any prior training. The user study has confirmed the accuracy.

Pollutant concentrations within households in Lao PDR and association with housing characteristics and occupants' activities

The paper presents the results of a study conducted to investigate indoor air quality within residential dwellings in Lao PDR. Results from PM 10, CO, and NO2 measurements inside 167 dwellings in Lao PDR over a five month period (December 2005-April 2006) are discussed as a function of household characteristics and occupant activities. Extremely high PM10 and NO2 concentrations (12 h mean PM10 concentrations 1275 ± 98 μg m-3 and 1183 ± 99 μg m-3 in Vientiane and Bolikhamxay provinces, respectively; 12 h mean NO2 concentrations 1210 ± 94 μg m-3 and 561 ± 45 μg m-3 in Vientiane and Bolikhamxay, respectively) were measured within the dwellings. Correlations, ANOVA analysis (univariate and multivariate), and linear regression results suggest a substantial contribution from cookingandsmoking.The PM10 concentrations were significantly higher in houses without a chimney compared to houses in which cooking occurred on a stove with a chimney. However, no significant differences in pollutantconcentrations were observed as a function of cooking location. Furthermore, PM10 and NO2 concentrations were higher in houses in which smoking occurred, suggestive of a relationship between increased indoor concentrations and smoking (0.05 < p < 0.10). Resuspension of dust from soil floors was another significant source of PM10 inside the house (634 μg m-3, p < 0.05).

Defining the role of phytoene synthase in carotenoid accumulation of high provitamin A bananas

Vitamin A deficiency (VAD) is a serious problem in developing countries, affecting approximately 127 million children of preschool age and 7.2 million pregnant women each year. However, this deficiency is readily treated and prevented through adequate nutrition. This can potentially be achieved through genetically engineered biofortification of staple food crops to enhance provitamin A (pVA) carotenoid content. Bananas are the fourth most important food crop with an annual production of 100 million tonnes and are widely consumed in areas affected by VAD. However, the fruit pVA content of most widely consumed banana cultivars is low (~ 0.2 to 0.5 ìg/g dry weight). This includes cultivars such as the East African highland banana (EAHB), the staple crop in countries such as Uganda, where annual banana consumption is approximately 250 kg per person. This fact, in addition to the agronomic properties of staple banana cultivars such as vegetative reproduction and continuous cropping, make bananas an ideal target for pVA enhancement through genetic engineering. Interestingly, there are banana varieties known with high fruit pVA content (up to 27.8 ìg/g dry weight), although they are not widely consumed due to factors such as cultural preference and availability. The genes involved in carotenoid accumulation during banana fruit ripening have not been well studied and an understanding of the molecular basis for the differential capacity of bananas to accumulate carotenoids may impact on the effective production of genetically engineered high pVA bananas. The production of phytoene by the enzyme phytoene synthase (PSY) has been shown to be an important rate limiting determinant of pVA accumulation in crop systems such as maize and rice. Manipulation of this gene in rice has been used successfully to produce Golden Rice, which exhibits higher seed endosperm pVA levels than wild type plants. Therefore, it was hypothesised that differences between high and low pVA accumulating bananas could be due either to differences in PSY enzyme activity or factors regulating the expression of the psy gene. Therefore, the aim of this thesis was to investigate the role of PSY in accumulation of pVA in banana fruit of representative high (Asupina) and low (Cavendish) pVA banana cultivars by comparing the nucleic acid and encoded amino acid sequences of the banana psy genes, in vivo enzyme activity of PSY in rice callus and expression of PSY through analysis of promoter activity and mRNA levels. Initially, partial sequences of the psy coding region from five banana cultivars were obtained using reverse transcriptase (RT)-PCR with degenerate primers designed to conserved amino acids in the coding region of available psy sequences from other plants. Based on phylogenetic analysis and comparison to maize psy sequences, it was found that in banana, psy occurs as a gene family of at least three members (psy1, psy2a and psy2b). Subsequent analysis of the complete coding regions of these genes from Asupina and Cavendish suggested that they were all capable of producing functional proteins due to high conservation in the catalytic domain. However, inability to obtain the complete mRNA sequences of Cavendish psy2a, and isolation of two non-functional Cavendish psy2a coding region variants, suggested that psy2a expression may be impaired in Cavendish. Sequence analysis indicated that these Cavendish psy2a coding region variants may have resulted from alternate splicing. Evidence of alternate splicing was also observed in one Asupina psy1 coding region variant, which was predicted to produce a functional PSY1 isoform. The complete mRNA sequence of the psy2b coding regions could not be isolated from either cultivar. Interestingly, psy1 was cloned predominantly from leaf while psy2 was obtained preferentially from fruit, suggesting some level of tissue-specific expression. The Asupina and Cavendish psy1 and psy2a coding regions were subsequently expressed in rice callus and the activity of the enzymes compared in vivo through visual observation and quantitative measurement of carotenoid accumulation. The maize B73 psy1 coding region was included as a positive control. After several weeks on selection, regenerating calli showed a range of colours from white to dark orange representing various levels of carotenoid accumulation. These results confirmed that the banana psy coding regions were all capable of producing functional enzymes. No statistically significant differences in levels of activity were observed between banana PSYs, suggesting that differences in PSY activity were not responsible for differences in the fruit pVA content of Asupina and Cavendish. The psy1 and psy2a promoter sequences were isolated from Asupina and Cavendish gDNA using a PCR-based genome walking strategy. Interestingly, three Cavendish psy2a promoter clones of different sizes, representing possible allelic variants, were identified while only single promoter sequences were obtained for the other Asupina and Cavendish psy genes. Bioinformatic analysis of these sequences identified motifs that were previously characterised in the Arabidopsis psy promoter. Notably, an ATCTA motif associated with basal expression in Arabidopsis was identified in all promoters with the exception of two of the Cavendish psy2a promoter clones (Cpsy2apr2 and Cpsy2apr3). G1 and G2 motifs, linked to light-regulated responses in Arabidopsis, appeared to be differentially distributed between psy1 and psy2a promoters. In the untranscribed regulatory regions, the G1 motifs were found only in psy1 promoters, while the G2 motifs were found only in psy2a. Interestingly, both ATCTA and G2 motifs were identified in the 5’ UTRs of Asupina and Cavendish psy1. Consistent with other monocot promoters, introns were present in the Asupina and Cavendish psy1 5’ UTRs, while none were observed in the psy2a 5’ UTRs. Promoters were cloned into expression constructs, driving the â-glucuronidase (GUS) reporter gene. Transient expression of the Asupina and Cavendish psy1 and psy2a promoters in both Cavendish embryogenic cells and Cavendish fruit demonstrated that all promoters were active, except Cpsy2apr2 and Cpsy2apr3. The functional Cavendish psy2a promoter (Cpsy2apr1) appeared to have activity similar to the Asupina psy2a promoter. The activities of the Asupina and Cavendish psy1 promoters were similar to each other, and comparable to those of the functional psy2a promoters. Semi-quantitative PCR analysis of Asupina and Cavendish psy1 and psy2a transcripts showed that psy2a levels were high in green fruit and decreased during ripening, reinforcing the hypothesis that fruit pVA levels were largely dependent on levels of psy2a expression. Additionally, semi-quantitative PCR using intron-spanning primers indicated that high levels of unprocessed psy2a and psy2b mRNA were present in the ripe fruit of Cavendish but not in Asupina. This raised the possibility that differences in intron processing may influence pVA accumulation in Asupina and Cavendish. In this study the role of PSY in banana pVA accumulation was analysed at a number of different levels. Both mRNA accumulation and promoter activity of psy genes studied were very similar between Asupina and Cavendish. However, in several experiments there was evidence of cryptic or alternate splicing that differed in Cavendish compared to Asupina, although these differences were not conclusively linked to the differences in fruit pVA accumulation between Asupina and Cavendish. Therefore, other carotenoid biosynthetic genes or regulatory mechanisms may be involved in determining pVA levels in these cultivars. This study has contributed to an increased understanding of the role of PSY in the production of pVA carotenoids in banana fruit, corroborating the importance of this enzyme in regulating carotenoid production. Ultimately, this work may serve to inform future research into pVA accumulation in important crop varieties such as the EAHB and the discovery of avenues to improve such crops through genetic modification.

The effect of housing characteristics and occupant activities on the respiratory health of women and children in Lao PDR

The paper presents the results of a study conducted into the relationship between dwelling characteristics and occupant activities with the respiratory health of resident women and children in Lao People’s Democratic Republic (PDR). Lao is one of the least developed countries in south-east Asia with poor life expectancies and mortality rates. The study, commissioned by the World Health Organisation, included questionnaires delivered to residents of 356 dwellings in nine districts in Lao PDR over a five month period (December 2005-April 2006), with the aim of identifying the association between respiratory health and indoor air pollution, in particular exposures related to indoor biomass burning. Adjusted odds ratios were calculated for each health outcome separately using binary logistic regression. After adjusting for age, a wide range of symptoms of respiratory illness in women and children aged 1-4 years were positively associated with a range of indoor exposures related to indoor cooking, including exposure to a fire and location of the cooking place. Among women, “dust always inside the house” and smoking were also identified as strong risk factors for respiratory illness. Other strong risk factors for children, after adjusting for age and gender, included dust and drying clothes inside. This analysis confirms the role of indoor air pollution in the burden of disease among women and children in Lao PDR.

Facet-controlled Self-assembly of ZnO Nanocrystals by Non-Hydrolytic Aminolysis and their Photodegradation Activities

We report a method for controlling the exposed facets and hence the dimensionality and shape of ZnO nanocrystals using a non-hydrolytic aminolysis synthesis route. The effects of changes to reaction conditions on ZnO formation were investigated and possible self-assembly mechanisms proposed. The crystal facet growth and hence morphologies of the ZnO nanocrystals were controlled by varying reaction temperature and the reactant ratio. Four distinct ZnO nanocrystal types were produced (nanocones, nanobullets, nanorods and nanoplates). The relative photocatalytic activities of the exposed facets of these ZnO nanostructures were also examined, which showed the activities obviously depended on the reactivity of exposed crystal facets in the order: {1011}>>{0001}, {1010}.

Proteasome activities decrease during dexamethasone-induced apoptosis of thymocytes

The induction of apoptosis in thymocytes by the glucocorticoid dexamethasone was used as a model system to investigate whether there are changes in 20 S and 26 S proteasome activities during apoptosis. We observed that thymocytes contain high concentrations of proteasomes and that following treatment with dexamethasone, cell extracts showed a decrease in proteasome chymotrypsin-like activity which correlated with the degree of apoptosis observed. The decrease in chymotrypsin-like activity of 20 S and 26S proteasomes was still apparent after these complexes had been partially puri®ed from apoptotic thymocyte extracts and was therefore not due to competition resulting from a general increase in protein turnover. The trypsin-like and peptidylglutamylpeptide hydrolase activities of proteasome complexes were also observed to decrease during apoptosis, but these decreases were reversed by the inhibition of apoptosis by the caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp(OMe)-¯uoromethylketone. However, the chymotrypsin-like activity of proteasomes decreased further in the presence of the apoptosis inhibitor. Val-Ala-Asp-¯uoromethylketone was found to inhibit the chymotrypsin- and trypsin-like activity of 26 S proteasomes in .itro. The decrease in proteasome activities in apoptosis did not appear to be due to a decrease in the concentration of total cellular proteasomes. Thus, the early decreases in 20 S and 26 S proteasome activities during apoptosis appear to be due to a down-regulation of their proteolytic activities and not to a decrease in their protein concentration. These data suggest that proteasomes may be responsible, in thymocytes, for the turnover of a protein that functions as a positive regulator of apoptosis.

Catalytic properties of 26 S and 20 S proteasomes and radiolabeling of MB1, LMP7, and C7 subunits associated with trypsin-like and chymotrypsin-like activities*

20 and 26 S proteasomes were isolated from rat liver. The procedure developed for the 26 S proteasome resulted in greatly improved yields compared with previously published methods. A comparison of the kinetic properties of 20 and 26 S proteasomes showed significant differences in the kinetic characteristics with certain substrates and differences in the effects of a protein substrate on peptidase activity. Observed differences in the kinetics of peptidylglutamyl peptide hydrolase activity suggest that the 26 S complex cannot undergo the conformational changes of 20 S proteasomes at high concentrations of the substrate benzyloxycarbonyl (Z) -Leu-Leu-Glu-b-naphthylamide. Various inhibitors that differentially affect the trypsin-like and chymotrypsin-like activities have been identified. Ala-Ala-Phe-chloromethyl (CH2Cl) inhibits chymotrypsin-like activity assayed with succinyl (Suc) -Leu-Leu-Val-Tyr-AMC, but surprisingly not hydrolysis of Ala-Ala-Phe-7-amido-4-methylcoumarin (AMC). Tyr-Gly-Arg-CH2Cl inhibits Suc-Leu-Leu-Val-Tyr-AMC hydrolysis as well as trypsinlike activity measured with t-butoxycarbonyl (Boc) -Leu-Ser-Thr-Arg-AMC, while Z-Phe-Gly-Tyr-diazomethyl (CHN2) was found to inhibit only the two chymotrypsin- like activities. Radiolabeled forms of peptidyl chloromethane and peptidyl diazomethane inhibitors, [3H]acetyl-Ala-Ala-Phe-CH2Cl, [3H]acetyland radioiodinated Tyr-Gly-Arg-CH2Cl, and Z-Phe-Gly- Tyr-(125I-CHN2), have been used to identify catalytic components associated with each of the three peptidase activities. In each case, incorporation of the label could be blocked by prior treatment of the proteasomes with known active site-directed inhibitors, calpain inhibitor 1 or 3,4-dichloroisocoumarin. Subunits of labeled proteasomes were separated either by reverse phase-HPLC and SDS-polyacrylamide gel electrophoresis or by twodimensional polyacrylamide gel electrophoresis followed by autoradiography/fluorography and immunoblotting with subunit-specific antibodies. In each case, label was found to be incorporated into subunits C7, MB1, and LMP7 but in different relative amounts depending on the inhibitor used, consistent with the observed effects on the different peptidase activities. The results strongly suggest a relationship between trypsin-like activity and chymotrypsin-like activity. They also help to relate the different subunits of the complex to the assayed multicatalytic endopeptidase activities