BIOCHEMICAL CHARACTERIZATION AND GENETIC MAPPING OF WILD TYPE AND MUTANT GLYCERALDEHYDE-3-PHOSPHATE DEHYDROGENASE ALLELES IN CHINESE HAMSTER OVARY CELLS

A UV-induced mutation of the enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPD) was characterized in the CHO clone A24. The asymmetric 4-banded zymogram and an in vitro GAPD activity equal to that of wild type cells were not consistent with models of a mutant heterozygote producing equal amounts of wild type and either catalytically active or inactive mutant subunits that interacted randomly. Cumulative evidence indicated that the site of the mutation was the GAPD structural locus expressed in CHO wild type cells, and that the mutant allele coded for a subunit that differed from the wild type subunit in stability and kinetics. The evidence included the appearance of a fifth band, the putative mutant homotetramer, after addition of the substrate glyceraldehyde-3-phosphate (GAP) to the gel matrix; dilution experiments indicating stability differences between the subunits; experiments with subsaturating levels of GAP indicating differences in affinity for the substrate; GAPD zymograms of A24 x mouse hybrids that were consistent with the presence of two distinct A24 subunits; independent segregation of A24 wild type and mutant electrophoretic bands from the hybrids, which was inconsistent with models of mutation of a locus involved in posttranslational modification; the mapping of both wild type and mutant forms of GAPD to chromosome 8; and the failure to detect any evidence of posttranslational modification (of other A24 isozymes, or through mixing of homogenates of A24 and mouse).^ The extent of skewing of the zymogram toward the wild type band, and the unreduced in vitro activity were inconsistent with models based solely on differences in activity of the two subunits. Comparison of wild type homotetramer bands in wild type cells and A24 suggested the latter had a preponderance of wild type subunits over mutant subunits, and had more GAPD tetramers than did CHO controls.^ Two CHO linkages, GAPD-triose phosphate isomerase, and acid phosphatase 2-adenosine deaminase were reported provisionally, and several others were confirmed. ^

FUNCTIONAL AND BIOCHEMICAL CHARACTERIZATION OF ADHESION DEFECTIVE CHINESE HAMSTER OVARY CELLS

Cell adhesion is an intricate process involving adhesion promoting ligands such as laminin and fibronectin, surface receptors for these ligands and a complex interplay of metabolic and cytoskeletal events (Geiger, BBA 737:305, 1983). Although considerable effort has been directed towards studying adhesion molecules such as fibronectin (Fn), very little is known about the mechanisms regulating the complex process of adhesion.^ I chose to use a CHO adhesion variant clone called AD('v)F11 as a tool to study the various steps which may be involved in adhesion. AD('v)F11 cells unlike wild type (WT), do not adhere to Fn-coated substrata, but will adhere to substrata coated with other extracellular components (Harper and Juliano, J Cell Biol. 91:647, 1981). I have found that although AD('v)F11 cells can bind Fn-coated latex beads to the same extent as WT cells, AD('v)F11 cells also differed from WT cells in that they did not aggregate in the presence of Fn-beads nor internalize Fn-beads. The defect in bead induced cell aggregation and internalization seem to be specific to Fn since lectin coated beads could aggregate AD('v)F11 cells as well as WT cells, and AD('v)F11 cells can also readily internalize lectins. These observations suggest that the defect associated with AD('v)F11 cells is distal to the initial binding to Fn to its cell surface receptor. To further investigate the biochemical defect associated with AD('v)F11 cells, a panel of compounds were examined for their ability to correct the non-adhesive phenotype of AD('v)F11 cells. Among the compounds tested, only those known to increase intracellular cAMP levels were found to be effective in correcting the adhesion defect of F11CA11 cells, a subclone of AD('v)F11 cells.^ Since cAMP effects in eukaryotic cells are mediated through phosphorylation events by the cAMP-dependent protein kinase (cAdPK) system, the phosphorylation pattern and cAdPK system of the F11CA11 cells were analyzed. Comparison between the phosphorylation pattern of intact untreated F11CA11 and WT cells, revealed the presence of a 50 kd phosphoprotein(s) in WT cells but not in F11CA11 cells. Results presented in this dissertation strongly indicate that the adhesion defect in F11CA11 is associated to an altered type I cAdPK that can be corrected by raising intracellular cAMP levels. (Abstract shortened with permission of author.) ^

Development and evaluation of a cervical cancer screening program for Chinese women

Despite of the proven efficacy of the Pap test, Asian populations still have low Pap screening compliance. The purpose of this dissertation was to investigate factors that influencing women's decision to obtain a Pap test, and to describe the development and evaluation of a cervical cancer educational program promoting the Pap screening behavior among women in Taiwan. ^ The first study examined factors associated with Pap screening compliance. Psychometric properties of measurement instruments were also assessed. The scale reliabilities were as the follows: Cronbach alpha 0.70 for knowledge scale, 0.88 for pros scale, 0.68 for cons scale, and 0.72 for perceived norms scale. Results from multiple logistic regression analysis, after adjusted for marital status, showed women who compliant to Pap screening guidelines had significantly higher knowledge, higher perceived benefits (pros), lower perceived barriers (cons), and higher perceived norms to receive a Pap test. ^ The second study described the development of a program called “Love yourself before you take care of your family”, designed to increase Pap screening behavior among women in Taiwan. The development of this program was guided by Intervention Mapping (IM), an innovative process of intervention design. The program used methods such as information transmission, modeling, persuasion, and facilitation. Strategies included direct mail campaigns, role model stories with women's testimonials, and phone intervention. ^ The third study examined the effectiveness of a randomized trial of the carefully-designed intervention (N = 424). Participants were female family members of inpatients admitted to one of the major teaching hospitals in Taiwan during August and September 1999. Women in the intervention group reported a higher rate of receiving a Pap test than women in the control group (50% versus 32%) after a three-month intervention (p = 0.002). Women in the intervention group showed increased knowledge (p = .016), perceived pros (p = 0.008), and susceptibility (p = .011) between baseline and follow-up. They also showed higher perceived pros of Pap tests than women in control group at follow-up (p = .031). This result suggested that program development based on theories and evidences could maximize the intervention impact for a specific target population. ^

Association between length of stay in the U.S. and diabetes risk factors among Chinese Americans in Houston, Texas in 2004-2005

Studies have suggested that acculturation is related to diabetes prevalence and risk factors among immigrant groups in the United States (U.S.), however scant data are available to investigate this relationship among Asian Americans and Asian American subgroups. The objective of this cross-sectional study was to examine the association between length of stay in the U.S. and type 2 diabetes prevalence and its risk factors among Chinese Americans in Houston, Texas. Data were obtained from the 2004-2005 Asian-American Health Needs Assessment in Houston, Texas (N=409 Chinese Americans) for secondary analysis in this study. Diabetes prevalence and risk factors (overweight/obesity and access to medical care) were based on self-report. Descriptive statistics summarized demographic characteristics, diabetes prevalence, and reasons for not seeing a doctor. Logistic regression, using an incremental modeling approach, was used to measure the association between length of stay and diabetes prevalence and related risk factors, while adjusting for the potential confounding factors of age, gender, education level, and income level. Although the prevalence of type 2 diabetes was highest among those living in the U.S. for more than 20 years, there was no significant association between length of stay in the U.S. and diabetes prevalence among these Chinese Americans after adjustment for confounding factors. No association was found between length of stay in the U.S. and overweight/obese status among this population either, after adjusting for confounding factors, too. On the other hand, a longer length of stay was significantly associated with increased health insurance coverage in both unadjusted and adjusted models. The findings of this study suggest that length of stay in the U.S. alone may not be an indicator for diabetes risk among Chinese Americans. Future research should consider alternative models to measure acculturation (e.g., models that reflect acculturation as a multi-dimensional, not uni-dimensional process), which may more accurately depict its effect on diabetes prevalence and related risk factors.^

Quality of Life in Chinese elderly people with depression

Background Depression is the most prevalent functional mental disorder of later life. It is estimated that about 5% of the elderly population of Hong Kong are suffering from depression. Aim To investigate the self-rated quality of life of community-dwelling elderly people diagnosed with depression, and to examine the relationships between quality of life and mental, physical health, functional status and social support. Methods and results A cross-sectional descriptive survey was conducted in psychiatric outpatient clinics. A convenience sample of 80 Chinese elderly people with a diagnosis of depressive disorder was recruited. Perception of quality of life was measured by the Hong Kong Chinese World Health Organization Quality of Life Scale-Brief Version. Participants' mental status, functional abilities, physical health condition, and social support status were assessed. Sixty-one (76.3%) participants were female. They were least satisfied with meaningfulness of life, life enjoyment, concentration and thinking, energy and work capacity. Functional abilities had a positive association with participants' perceived quality of life, level of depression and number of physical health conditions had a negative association. Participants had low ratings of quality of life when compared with healthy persons and persons with chronic physical problems. Findings are discussed in light of the socio-cultural environment in Hong Kong. Conclusion Comprehensive treatment and better control of depression, including different modes of medical and psychosocial intervention, could help to improve participants' perception of quality of life. A longitudinal study with a larger sample with various levels of depression and socio-demographic characteristics is recommended. Copyright © 2006 John Wiley & Sons, Ltd.

Factors influencing Hong Kong Chinese patients' decision-making in seeking early treatment for acute myocardial infarction

The purpose of this study was to identify, through in-depth interview, factors that influenced 27 Hong Kong Chinese patients' decision-making in seeking early treatment for acute myocardial infarction (AMI). The median delay time from the onset of symptoms to arrival at the hospital was 15.6 hours for men and 53.7 hours for women. Three major categories emerged from the data: (a) becoming aware of the threat, (b) maintaining a sense of normality, and (c) struggling to mobilize resources. A variety of decisions were made by patients from the onset of chest Pain to seeking help. These decisions were heavily influenced by healthcare factors (access to emergency medical service (EMS) and treatment), personal factors (cognitive interpretations of symptoms), sociocultural factors (family situation, cultural beliefs, and practices), and coping strategies. (c) 2006 Wiley Periodicals, Inc.

Using a mixed design study to develop a breast screening intervention among Chinese women in the UK

Breast cancer is the most common cancer among Chinese women living in the UK. However the literature suggests that Chinese women are less likely to attend breast screening than white British women. No studies have been conducted to explore reasons for low attendance among this specific population. The purpose of this thesis was to understand the psycho-social factors related to breast cancer prevention and screening among Chinese women in the UK, and then to inform a breast screening intervention design. Three studies were conducted. The first was a systematic review of interventions to increase breast screening among Chinese women living in Western countries. The second and third studies used focus groups to explore Chinese women’s beliefs about breast cancer prevention and screening practices among older and younger generations. Finally, Intervention Mapping was used to synthesise the findings of the focus groups with those of the systematic review to design an empirical and theoretical evidence based breast screening intervention directed at Chinese women who are non-adherent to the NHS Breast Screening Programme. The qualitative findings revealed that older participants held a more holistic view of health maintenance, and had less knowledge about breast cancer and its causes than younger participants. They showed positive attitudes to breast screening and most had responded to receiving a mammography invitation. Language was a key barrier to older participants using medical care and obtaining health-related information. Younger participants expressed high dissatisfaction with health care in UK and showed a strong ‘neo-fatalistic’ view of breast cancer prevention, believing the main cause of breast cancer to be genetic predisposition. The synthesis of findings suggest that healthcare providers need to take Chinese cultural and language concerns, but also the differences between generations, into account when designing and implementing breast screening services and educational programmes which target Chinese women.

Novel NR5A1 Missense Mutation in Premature Ovarian Failure: Detection in Han Chinese Indicates Causation in Different Ethnic Groups

Background The etiology of most premature ovarian failure (POF) cases is usually elusive. Although genetic causes clearly exist and a likely susceptible region of 8q22.3 has been discovered, no predominant explanation exists for POF. More recently, evidences have indicated that mutations in NR5A1 gene could be causative for POF. We therefore screened for mutations in the NR5A1 gene in a large cohort of Chinese women with non-syndromic POF. Methods Mutation screening of NR5A1 gene was performed in 400 Han Chinese women with well-defined 46,XX idiopathic non-syndromic POF and 400 controls. Subsequently, functional characterization of the novel mutation identified was evaluated in vitro. Results A novel heterozygous missense mutation [c.13T>G (p.Tyr5Asp)] in NR5A1 was identified in 1 of 384 patients (0.26%). This mutation impaired transcriptional activation on Amh, Inhibin-a, Cyp11a1and Cyp19a1 gene, as shown by transactivation assays. However, no dominant negative effect was observed, nor was there impact on protein expression and nuclear localization. Conclusions This novel mutation p.Tyr5Asp, in a novel non-domain region, is presumed to result in haploinsufficiency. Irrespectively, perturbation in NR5A1 is not a common explanation for POF in Chinese.

The effects of acute administration of Chinese aphrodisiacs sold in Blantyre City on sperm characteristics and fertility profile in guinea pigs

Background Chinese aphrodisiacs have become popular remedy for sexual dysfunction and improvement of libido in men in Malawi. However, selling of these drugs seems not to be well regulated. Probably the aphrodisiacs that are currently on the market have unknown efficacy, potency and safety profiles. The aim of this study was to assess the efficacy of imported Chinese aphrodisiacs using guinea pigs as a model. Materials and Methods Two types of drugs were purchased from vendors in Blantyre City. Tonic tea, which was purported to improve erectile function and libido, and sperm multiplier tablets which were claimed to increase the sperm count. The tonic tea was prepared by soaking one tea bag in 100ml boiling water. The tea was cooled and administered to eight male experimental animals in varying doses. Each animal was introduced into a separate cage with a female guinea pig. Sexual behaviour such as mounting, sniffing behind the female were observed and recorded. Each sperm multiplier tablet was dissolved in distilled water and administered to the experimental animals in the morning and evening for seven days. At the end of the treatment, the experimental and control animals were sacrificed, their semen collected and analysed sperm motility, concentration and morphology. Results For the tonic tea, there were no statistical differences between the experimental and the control animals in terms of the number of mountings and sniffing behind the female. The sperm multiplier drug showed statistically significant differences between the experimental and the control animals in terms of the sperm motility (78.24 ± 1.35 vs. 86.54 ± 1.88, p< 0.05), and concentration (54.28 ± 1.24 vs. 67.59 ± 2.12, p<0.05). Conclusion The tonic tea did not show any efficacy in improving erection and libido. The sperm multiplier tablets, purported to increase sperm production, significantly increased the sperm motility, sperm concentration in the treated animals.

Effects Of Sterilization Methods On The Physical, Chemical, And Biological Properties Of Silk Fibroin Membranes.

Silk fibroin has been widely explored for many biomedical applications, due to its biocompatibility and biodegradability. Sterilization is a fundamental step in biomaterials processing and it must not jeopardize the functionality of medical devices. The aim of this study was to analyze the influence of different sterilization methods in the physical, chemical, and biological characteristics of dense and porous silk fibroin membranes. Silk fibroin membranes were treated by several procedures: immersion in 70% ethanol solution, ultraviolet radiation, autoclave, ethylene oxide, and gamma radiation, and were analyzed by scanning electron microscopy, Fourier-transformed infrared spectroscopy (FTIR), X-ray diffraction, tensile strength and in vitro cytotoxicity to Chinese hamster ovary cells. The results indicated that the sterilization methods did not cause perceivable morphological changes in the membranes and the membranes were not toxic to cells. The sterilization methods that used organic solvent or an increased humidity and/or temperature (70% ethanol, autoclave, and ethylene oxide) increased the silk II content in the membranes: the dense membranes became more brittle, while the porous membranes showed increased strength at break. Membranes that underwent sterilization by UV and gamma radiation presented properties similar to the nonsterilized membranes, mainly for tensile strength and FTIR results.

Motivating Medical Students To Learn Basic Science Concepts Using Chronic Myeloid Leukemia As An Integration Theme.

To report on the use of chronic myeloid leukemia as a theme of basic clinical integration for first year medical students to motivate and enable in-depth understanding of the basic sciences of the future physician. During the past thirteen years we have reviewed and updated the curriculum of the medical school of the Universidade Estadual de Campinas. The main objective of the new curriculum is to teach the students how to learn to learn. Since then, a case of chronic myeloid leukemia has been introduced to first year medical students and discussed in horizontal integration with all themes taught during a molecular and cell biology course. Cell structure and components, protein, chromosomes, gene organization, proliferation, cell cycle, apoptosis, signaling and so on are all themes approached during this course. At the end of every topic approached, the students prepare in advance the corresponding topic of clinical cases chosen randomly during the class, which are then presented by them. During the final class, a paper regarding mutations in the abl gene that cause resistance to tyrosine kinase inhibitors is discussed. After each class, three tests are solved in an interactive evaluation. The course has been successful since its beginning, 13 years ago. Great motivation of those who participated in the course was observed. There were less than 20% absences in the classes. At least three (and as many as nine) students every year were interested in starting research training in the field of hematology. At the end of each class, an interactive evaluation was performed and more than 70% of the answers were correct in each evaluation. Moreover, for the final evaluation, the students summarized, in a written report, the molecular and therapeutic basis of chronic myeloid leukemia, with scores ranging from 0 to 10. Considering all 13 years, a median of 78% of the class scored above 5 (min 74%-max 85%), and a median of 67% scored above 7. Chronic myeloid leukemia is an excellent example of a disease that can be used for clinical basic integration as this disorder involves well known protein, cytogenetic and cell function abnormalities, has well-defined diagnostic strategies and a target oriented therapy.