Novel organizational features, captured cellular genes, and strain variability within the genome of KSHV/HHV8.

Strong serologic and molecular probe correlations indicate that the newly discovered gamma herpesvirus KSHV or HHV8 is the likely etiologic agent of all forms of Kaposi's sarcoma as well as BCBL/PEL and MCD in patients with acquired immunodeficiency syndrome (AIDS). Two large segments of HHV8 DNA from an AIDS-associated BCBL tumor covering genomic positions 0-52 kilobase [kb] and 108-140 kb have been cloned, mapped, and partially sequenced. Our studies have focused on novel viral proteins encoded within a 13-kb divergent locus (DL-B) by nine captured homologues of cellular genes, including vIL-6, vDHFR, vTS, vBcl-2, three C-C beta chemokines (vMIP-1A, vMIP-1B, and vBCK), and two LAP/PHD subclass zinc finger proteins (IE1A and IE1B). The HHV-8 vIL-6, vDHFR, vTS, and vBcl-2 proteins have all been shown to be active in a variety of appropriate functional assays, and transcripts from vIL-6, vMIP-1B, vIE1-A, vIE1-B, and vDHFR genes are all expressed as abundant single messenger RNA species after butyrate or phorbol ester (TPA) induction of the lytic cycle in HHV8-positive BCBL cell lines. All of these genes lie within a divergent transcriptional domain that contains a single central enhancer and associated untranslated leader region plus seven distinct proximal promoters, some of which are negatively regulated through AP-1 and ZRE motifs by the EBV ZTA transactivator. This region also encompasses a predicted complex oriLyt domain of 1050 bp that is duplicated in inverted orientation adjacent to the T0.7 latency RNA in another large divergent locus (DL-E). We have previously described three distinct subtypes of the HHV8 genome that differ by 1.0%-1.5% at the nucleotide level within the ORF26 and ORF75 genes. Certain strains or clades appear to have preferential geographic distributions, but it is not known as yet whether there are any specific disease associations. Interestingly, the A, B, and C subtypes of HHV-8 also proved to differ dramatically in coding content at both the extreme left and right ends of the unique segment of the genome as well as in the positions of the junctions with the terminal repeats. On the left-hand side, the receptor-like ORF-K1 protein is highly variable with A-strain subtypes displaying 15% amino acid differences from C strains and up to 30% differences from B strains. On the right-hand side, two unrelated alternative types of the putative multiple membrane spanning ORF-K15 protein are found.

Evaluation and mapping of the DNA binding and oligomerization domains of the IE2 regulatory protein of human cytomegalovirus using yeast one and two hybrid interaction assays.

The 86-kDa IE2 nuclear phosphoprotein encoded by the human cytomegalovirus (HCMV) major immediate-early (MIE) gene behaves as both a non-specific transactivator of viral and cellular gene expression and as a specific DNA-binding protein targeted to the cis-repression sequence (CRS) at the cap site of its own promoter/enhancer region. Although the IE2 protein produced in bacteria has been shown to bind to the 14-bp palindromic CRS motif and IE2 synthesized in vitro forms stable dimers in solution through the conserved C-terminus of the protein, there is no direct evidence as yet that the intracellular mammalian forms of IE2 do so. Here, we show that the intact HCMV IE2 protein both binds to CRS DNA and dimerizes in yeast cells. In a one-hybrid assay system, a GAL4/IE2 fusion protein expressed in yeast cells activated target HIS3 expression only when CRS sites were located upstream of the GAL1 minimal promoter, but failed to do so on mutant CRS sites, demonstrating a requirement for sequence-specific DNA-binding by IE2. Examination of a series of deletion and triple amino acid point mutations in the C-terminal half of IE2 mapped the domains required for DNA-binding in yeast to the entire region between codons 313 and 579, whereas in the previous in-vitro study with truncated bacterial GST fusion proteins, it was mapped to between codons 346 and 579. Transient co-transfection assays with deleted IE2 effector genes in Vero cells showed that the extra segment of IE2 between codons 313 and 346 is also required for both autoregulation and transactivation activity in mammalian cells. In a two-hybrid assay to study IE2 self-interations, we generated both GAL4 DNA-binding (DB) and activation domain (A)/IE2 fusion proteins and showed that IE2 could also dimerize or oligomerize through the C-terminus of the protein in yeast cells. Domains required for this interaction were all mapped to within the region between codons 388 and 542, which is coincident with the domain mapped previously for dimerization by co-translation and immunoprecipitation in vitro. Comparison of the domains of the IE2 protein required for CRS binding and dimerization in yeast suggests that these activities correlate precisely with requirements for the negative autoregulation function of the IE2 protein in mammalian cells.

Expression of human granulocyte-macrophage colony-stimulating factor gene in insect cells by a baculovirus vector.

A plasmid pAc373GM-CSF was constructed and co-transfected into Spodoptera frugiperda (Sf9) cells with wild-type Autographa californica nuclear polyhedrosis virus (AcNPV) DNA. The recombinant virus vAc373GM-CSF was identified and purified by several rounds of plaque hybridization. By assaying the culture medium, we demonstrated recombinant virus infected Sf9 cells expressing hGM-CSF. Recombinant hGM-CSFs with apparent molecular masses of 14.5, 15.5 and 16.5 kDa were detected by the Western blot method. All 3 forms have biological activity of hGM-CSF. Following N-glycanase treatment, a single band of 14.5-15.5 kDa appeared in SDS-PAGE. Western blot analysis of expression in Sf9 cell treated with tunicamycin revealed only the presence of the 14.5 kDa species. Thus, the signal sequence of recombinant hGM-CSF could be recognized and cleaved by infected insect cell and the resultant molecule secreted into the media.

The WAF1-mediated p53 growth-suppressor pathway is intact in the coronary arteries of heart transplant recipients.

It has been suggested that the interaction of cytomegalovirus (CMV) with the p53 tumor suppressor gene product plays a role in the development of coronary artery restenosis after angioplasty. CMV nucleic acids have been observed in the coronary arteries of allografted hearts, suggesting a possible role for the interaction of CMV with p53 in the development of accelerated graft arteriosclerosis in transplant recipients. Formalin-fixed, paraffin-embedded sections of coronary arteries from 19 transplanted hearts were immunostained for the p53 gene product using Target Unmasking Fluid (TUF)-mediated immunohistochemistry and the anti-p53 antibodies CM1 and DO7. Fresh-frozen sections of coronary arteries were also available from six of the 19 hearts, and these fresh-frozen sections were immunostained for the p53 gene product with the DO7 antibody and for WAF1 using the anti-WAF1 antibody EA10. Focal and weak staining for p53 was observed in smooth muscle and endothelial cells in two of 19 vessels, whereas the remaining 17 did not stain. CMV nucleic acids were previously shown in six of 13 of these hearts by in situ hybridization. The fresh-frozen sections of coronary arteries also did not stain for p53, but the smooth muscle cells in these vessels did stain intensely for WAF1. These results suggest three possibilities: (1) CMV-p53 interactions are not important in the development of accelerated graft arteriosclerosis; or (2) there is an interaction, but it is transient and not detectable at the time points examined in this study; or (3) there is an interaction, but binding of CMV to p53 leads to accelerated degradation of p53, as occurs with HPV-E6. The expression of WAF1 further suggests that the WAF1-mediated antiproliferative signal is intact in these vessels.

Detection of the Human Cytomegalovirus 2.0-kb Immediate Early Gene 1 Transcripts in Permissive and Nonpermissive Infections by RNA in situ Hybridization.

The immediate early gene 1 (IE1) is the first gene to be expressed following the entry of the human cytomegalovirus (HCMV) into the cell and it does not require prior protein synthesis for its expression. Therefore, the IE1 gene is a potential candidate for the development of probes to detect HCMV in various states of infection. Using strand-specific (32)P- or digoxigenin-labeled riboprobes derived from an exon-specific subgenomic fragment of the HCMV Towne IE1 gene, we performed Northern blot analysis and RNA in situ hybridization on HCMV-infected human (permissive cells) and mouse (nonpermissive cells) fibroblasts and on 10 formalin-fixed paraffin-embedded sections of human tissue. By Northern blot analysis and by in situ hybridization, expression of the 2.0-kb IE1 gene was found in permissive as well as in nonpermissive infections. Specific nuclear and cytoplasmic hybridization was found at 5, 10, 24 and 72 h after infection in human fibroblasts. In comparison, hybridization was first detected at 10 h after infection in mouse fibroblasts. Hybridization with the IE1 probe was detected in cells with and without cytopathic changes in the formalin-fixed paraffin-embedded HCMV-infected human tissues. Hybridization patterns of the IE1 riboprobe were compared to those of the HCMV 2.7-kb major early beta-riboprobe which we have previously described [Am J Pathol 141:1247-1254;1992]. Although both riboprobes hybridize to their respective target sequences in the consecutive tissue sections, the patterns of hybridization are different. On occasion, sections of HCMV-infected human tissue showing no specific hybridization for the 2.7-kb riboprobe will show specific in situ hybridization when using the IE1 riboprobe. Our results suggest that RNA in situ hybridization with a probe directed at the IE1 transcripts is an effective method of detecting early and late stages of both permissive and nonpermissive HCMV infections. Copyright 1997 S. Karger AG, Basel

Factors affecting human cytomegalovirus gene expression in human monocyte cell lines.

To understand the mechanisms for establishing and reactivating monocytes and macrophages from latency by human cytomegalovirus (HCMV), human monocyte cell lines were infected and HCMV gene expression was investigated. Indirect immunofluorescence assay (IFA) with monoclonal antibody to HCMV major immediate early (MIE) IE1 or IE2 proteins revealed that HCMV MIE genes were expressed at low levels in relatively more differentiated THP-1 cells with TPA treatment after virus infection (posttreatment). Less differentiated cells such as U937 or HL60 did not support MIE gene expression even after TPA treatment. If THP-1 cells were pretreated before virus infection with TPA and became differentiated at the time of HCMV infection, MIE gene expression increased by 5-6 fold. Therefore, the relative degree of monocyte cell differentiation appears to be an important factor for regulating HCMV gene expression. Further IFA studies using monoclonal antibodies specific for IE1 or IE2 proteins indicate that the sequence and general pattern of IE1 and IE2 gene expression in THP-1 cells treated with TPA were similar to those in permissive human fibroblast cells with some delay in time. Formation of the replication compartment detected with monoclonal antibody to HCMV polymerase accessory protein UL44 in THP-1 cells suggests a fully productive replication process of HCMV in these cells. Monocytes are known to be induced to differentiate by hydrocortisone (HC), tumor necrosis factor (TNF)-alpha or interferon (IFN)-gamma. HC, which is known to stimulate HCMV replication in permissive human fibroblast (HF) cells, enhanced HCMV gene expression by 2-3 fold in TPA-pre or posttreated THP-1 cells, but TNF-alpha or IFN-gamma had little effect. Nitric oxide (NO) is released by immune cells in the defense against foreign stimuli and was shown to inhibit HCMV gene expression in HF cells. Increasing NO by nitroprusside significantly reduced HCMV gene expression in THP-1 cells. Therefore, it appears that the expression of HCMV immediate early genes in THP-1 cells treated with TPA closely resembles those in permissive HF cells.

The motion control of a statically stable biped robot on an uneven floor.

This work studies the motion control of a statically stable biped robot having seven degrees of freedom. Statically stable walking of the biped robot is realized by maintaining the center-of-gravity inside the convex region of the supporting foot and/or feet during both single-support and double-support phases. The main points of this work are framing the stability in an easy and correct way, the design of a bipedal statically stable walker, and walking on sloping surfaces and stairs.

[Coping behaviors of the elderly with positive physical examination results in Kaohsiung City].

The objectives of this study were as follows: first, to investigate the coping behaviors of the elderly facing the stress of disease after free physical examination; Second, to identify personal and disease characteristics, and the social support that influences coping behaviors in the elderly. The study sample consisted of 661 elderly patients who were found to have abnormal findings during a free physical examination provided by 11 regional health stations in Kaohsiung City. Data were collected with questionnaires through home interview. Four hundred and eighty-four subjects completed the questionnaires. Data were analyzed using SAS computer program. The major findings were as follows: 1.Problem-oriented coping behaviors were the more frequently used. The most frequently used coping behaviors were "accepting the situation as it is", "reling on myself to solve problems", "hoping that things would get better", "seeking professional help", and "letting things follow their natural cause". 2. The relative variables for problem-oriented coping were marital status, religion, medical insurance, educational level, disease characteristics, and social support. Sex, marital status, religion, medical insurance, perceived disease severity, characteristics of health problem, and newly found abnormality at this free physical examination were the relative variables for affective-oriented coping. 3. The best predictive variables for using problem-oriented coping behaviors were medical insurance, self-esteem support from family and friends, tangible support from health professionals, emotional support from health professionals, and whether it was the first time the patients knew their health problems. Findings from this study had implications for development of nursing care plans for elderly clients, as well as for collaborative team exploration of coping behaviors in the elderly.

[Assessment of the health-promoting lifestyle profile on reliability and validity].

The purpose of this study is to test the reliability and validity of a translated lifestyle scale, Health-Promoting Lifestyle Profile (HPLP), for college students. Methods such as Cronbach's Alpha, test-retest, content validity, item analysis and factor analysis were utilized. Two hundred and fifty-two college students in Kaohsiung City were sampled as subjects and data were collected by interviews. On the basis of statistical data, 6 items were deleted from the original scale. The revised scale revealed good internal consistency (alpha = .93) and test-retest reliability (r = .84). Six factors, which were identified by means of factor analysis, well corresponded with the concept of original scale. These factors included self-actualization, health responsibility, stress management, interpersonal support, nutrition and exercise, which accounted for 52.8% of variance of the scores. In conclusion, this study has proved that the revised HPLP is an adequate scale to be utilized on future research for Taiwanese young adults.

[The participation of physical activity and its associated factors in the elderly].

The purpose of this study was to explore the participation of physical activity and its related factors in the elderly in the community. Five hundred elderly ( > or = 65 yrs) living in the San-Min district of Kaohsiung city was taken as study subjects. A structured questionnaire was used to gather data including the following items: (1) the participation of physical activity (2) modifying factors: demographic data, biological characteristics, previous experience in exercise and exercise knowledge (3) cognitive-perceptual factors: perceived health status, perceived self-efficacy, perceived benefits and perceived barriers to exercise (4) cues to action. The results showed 19.2% of the elderly never exercised, 15.9% had exercised previously but not now, 20.4% exercised intermittently, 1% exercised only on holidays and 43.8% exercised regularly. Within the elderly who exercised regularly, 89.2% exercised more than three times a week and at least 20 minutes each time, walking was the most popular exercise pattern. The participation of physical activity had significant relationship with modifying factors, cognitive-perceptual factors and cues to action. After logistic stepwise multiple regression, those who totally self-care, without a history of exercise injury during youth, male, higher perceived self-efficacy score, lower perceived barriers score and those with higher cues to action score tended to exercise regularly.

[The study of healthy life-style risk factors and health status for cardiovascular diseases for employees in Kaohsiung area].

To understand the healthy life style, health status and risk factors of employees, institutional employees in Kaohsiung area were studied by using questionnaire investigation and blood specimen examination. One-thousand-four hundred-fifty subjects were used, but 1147 questionnaires were completed. The complete rate was 79.1%. The reliability and validity of the healthy life style scale achieved an acceptable level. The results are as follows: 1. The mean total score of healthy life-style scale was 63.9. Four healthy behavior scored lowest: "consulted health professional about health information", "monitored self of blood pressure and blood sugar", did moderate exercise at least three times per week", "took leisure activities". 2. The severity of risk factor was as follows: (1) 32.2% of employees had abnormal value for blood cholesterol (> or = 200 mg/dl); (2) 22.9% of employees had abnormal blood pressure value; (3) 22.6% of employees were overweight or obese; (4) 16.7% of employees often smoked; (5) 13.6% of employees had abnormal values for blood triglyceride. (6) 2.1% of employees had abnormal blood sugar values. 3. 10.8% of subjects were perceieved to have a worsing health status; 14.5% of the subjects had chronic disease with physicians diagnosis and most of their (71%) were hypertersion. 4. The findings of demographic data showed that people who were male, aged over forty, had education below junior high school and those of blue collar class should be screened and monitored, for the risk factors at regular intervals. Similarly, People who are male, age below forty and blue-collar class should be encouraged to develop the healthy life-styles.

[Utilizing the PRECEDE model to predict health examination behavior of the elderly].

Health examination of the elderly in Taiwan, the reported rates of health examination of the elderly were always below 30%. In order to explore the predictors related to the behavior of health examination of the elderly, the PRECEDE model was adopted for this study. Both the 1200 participants and spare samples were recruited from eleven districts of Kaohsiung city. 1193 participants had been completed by home interview. Sample size was based on the distribution of the elderly in the whole city and the stratified sampling method was applied. There was no significant difference after comparing the rates of gender, age, and examination between sample and population. The results showed that seven factors could be used to predict the behavior of health examination of the elderly. In the individual predisposing factors, "numbers of chronic diseases", "knowledge of health examination", "previous health examination", and "health promotion behavior" were related to the behavior of health examination of the elderly. In reinforcing factors, "attitude of family members for health examination", and in enabling factors "the guide and the convenience of health-examined services" also showed significantly affecting examination behavior. Therefore, referring to the improvement to be the examination rate, we can intervene or do something through academic, practical and administrative approaches.

[Related factors contributing to the healthy lifestyle of urban employees through the PRECEDE model].

The purpose of this study was to assess the related factors to the healthy lifestyle of 1147 urban employees. The theoretical framework of this study was the PRECEDE model. The model included three factors: 1. predisposing factors cardiovascular disease knowledge, general self-efficacy, health related diet and exercise self-efficacy, attitude for maintaining healthy life-style, health value, health status and illness history of family); 2. enabling factors (health resource availability, health resource utility); and 3. reinforcing factors (social support, feelings of colleagues health practice); The three factors were measured by 12 scales, through which the reliability and validity were assessed. According to the stepwise multiple regression analysis, diet and exercise self-efficacy, attitude for maintaining healthy life-style, marital status, cardiovascular disease knowledge, health status, occupational level, social support and feel of colleague health practice were the significant factors which accounted for 38.2% of variance. Predisposing factors and reinforcing factors were the important factors of healthy lifestyle on urban employees.

[Preliminary study of drug use by the elderly].

Out of 425 cases aged 65 and above, two hundred and sixty six cases were visited and surveyed with a structured questionnaire. The visitation rate was 62.4%. The purposes of this study were to: 1. Investigate the symptoms of drug-use among the elderly. 2. Reveal the percentage and the duration of drug-use among the elderly. 3. Understand the major reasons and methods of drug-use among the elderly. The results of this study included: 1. Drug-use by the elderly was very popular and persistent. 2. Thirty percent of our subjects used nonprescription drugs. 3. The major symptoms of drug-use were eye-indisposed, hypertension, notalgia and muscle pain & neuralgia. Our suggestions were: 1. It is very important to investigate the practice of drug-use among the elderly, so that health education for high risk people can be promoted. 2. Prevention of hypertension and notalgia while young is better than a cure while old.

[The utilization of health care by the elderly with chronic health problems in Kaohsiung City].

The purpose of this study was (1) to ascertain whether or not the elderly utilized the health care system and did follow-up after the initial free health check-up and screening; (2) to investigate the types of chronic health problems the elderly who utilize health care had and their reasons for using the health care system; and (3) to explore related factors that influence the utilization of health care by the elderly. In this study, 484 elderly individuals were interviewed by home visits. All names were from the 11 public health clinics in Kaohsiung City, and all subjects had been diagnosed with one of the following disorders: heart disease, diabetes mellitus, hypertension, liver and biliary disorder, urinary disorder, or lung disease. Data were statistically analyzed by means of descriptive and analytical methods, %, t-test, Chi-Square test and odds ratio. The major findings of this study were as follows: (1) the utilization rate by chronically ill elderly was 80.2%. (2) elderly with multiple utilization of the health care system comprised 33.8%. (3) the variables influencing the utilization of the health care system included: perceived susceptibility, fixed physician, medical insurance, doctor's attitude, social support, community health nurse follow-up, the number of diseases, perceived severity of the illness, and attitude of health.

[Role and function of registered professional nurses in the health station: a Delphi study].

This research was motivated by the need for a specific role definition for Registered Professional Nurses in public health stations that will enable them to fulfil their full potential. An adapted Delphi technique was used to reveal areas of consensus among the views of experts from the practicing nursing profession, government and academia regarding the specific roles and functions of registered professional nurses, and to rank such roles and functions in order of importance. Three questionnaires were sent to 150 experts from the above sectors, of whom 99 completed all three questionnaires: 36 (36.36%) from nursing practice, 34 (34.34%) from government and 29 (29.29%) from academia. Respondents believed that the role of the Registered Professional Nurse should be extended from the traditional "case-oriented" role to a "community health-oriented" role, and that the depth and scope of care should be enhanced. Priority roles identified in the area of "service" were those of "community care provider" and "family health manager", and in the area of "administration", that of "health and hygiene planner"; the most important personal qualities required were identified as "abundant knowledge, skills and practical experience in public health and nursing". It is believed that this study is conducive to establishing a consistent view of the role and functions of the Registered Professional Nurse between the above sectors, and can also serve as a useful guide for curriculum design for Registered Professional Nurses' pre-vocational and in-service training.

The human cytomegalovirus 86K immediate early (IE) 2 protein requires the basic region of the TATA-box binding protein (TBP) for binding, and interacts with TBP and transcription factor TFIIB via regions of IE2 required for transcriptional regulation.

The 86K immediate early (IE) 2 protein of human cytomegalovirus trans-activates a number of homologous and heterologous promoters, including the cellular promoter for the 70K heat-shock protein (hsp70), and the human immunodeficiency virus long terminal repeat. We have previously shown that IE2 trans-activates these two promoters in a TATA-dependent manner, and that IE2 is able to form a direct contact with TATA-box binding protein (TBP) in vitro. We now show that IE2 binds to the basic repeat region of TBP. In addition IE2 can contact a second general transcription factor, TFIIB. We have mapped the TBP- and TFIIB-binding regions within IE2 and show that these regions overlap, and also lie within parts of the protein previously identified as being required for the trans-activation and autoregulation functions of IE2.

Identification and mapping of dimerization and DNA-binding domains in the C terminus of the IE2 regulatory protein of human cytomegalovirus.

The 80-kDa IE2 nuclear phosphoprotein encoded by the human cytomegalovirus (HCMV) major immediate-early (MIE) gene behaves both as a nonspecific transactivator of heterologous reporter genes and as a specific repressor of its own promoter-enhancer region. To begin to examine the biochemical properties of the IE2 protein, we prepared panels of N-terminal and C-terminal truncation mutants by in vitro translation procedures. In cross-linking experiments, the C-terminal half of IE2 (which is sufficient for down-regulation) formed dimers but N-terminal segments did not do so. Cotranslated Oct2/IE2 fusion proteins containing the same IE2 C-terminal region from codons 266 to 579 also formed mixed-subunit DNA-bound oligomeric complexes in gel mobility shift assays. Furthermore, an IE2 domain bounded by codons 388 to 542 proved to immunoprecipitate as heterodimers with cotranslated subunits containing known epitopes for specific antibodies. Deletion up to codon 428 or truncation back to codon 504 prevented this interaction. In direct gel shift DNA-binding assays, a bacterial GST/IE2(346-579) fusion protein bound to a 30-mer oligonucleotide probe encompassing the major immediate-early gene negative cis-regulatory target DNA sequence but failed to bind to a single-base-pair insertion mutant probe (delta CRS). This specific DNA-binding activity was abolished by further deletion up to codon 388 on the N-terminal side or by truncation at codon 542 on the C-terminal side. Therefore, the minimal DNA-binding domain requires additional amino acid motifs on both sides of the dimerization domain. This segment of IE2 is functionally important for both transactivation and down-regulation and contains several highly conserved amino acid motifs that are shared amongst the equivalent HCMV, simian CMV, mouse CMV, rat CMV, and human herpesvirus 6 proteins from other betaherpesviruses.

[A survey of the relationships between children and their parents in chronic disease risk factors, health knowledge and lifestyles].

The purpose of this study is to understand the relationships for risk factors, health knowledge and lifestyles between children and their parents. 2806 primary schoolchildren in Kaohsiung City were measured for selected risk factors: serum cholesterol (CHOL), triglyceride (TG), systolic and diastolic blood pressure and height and weight for obese index (Rohrer's index). Children whose Rohrer's index > or = 150, or blood pressure, or CHOL, or TG level were above the 95th percentile distribution in the same age group were categorized as the risk group, the other children fell into the non-risk group. Using random sampling, 180 children were chosen from risk group and non-risk group respectively. A structured questionnaire about health knowledge and lifestyles were filled out by these children and their parents. In the risk group, the questionnaire was completed by 157 children, 103 fathers and 112 mothers. In the non-risk group, 102 children, 83 fathers and 82 mothers completed the questionnaire. The same risk factors examination of children was given to parents; 86 fathers and 144 mothers completed the examination from the risk group, while 56 fathers and 98 mothers completed the examination in the non-risk group. The results showed that: (1) there were significant positive correlations between children and their parents in CHOL, TG and obese index in risk group; children of mothers with higher CHOL and TG levels also showed higher CHOL and TG levels, (2) childrens' health knowledge was not correlated with parents' health knowledge, (3) childrens' lifestyles showed a significant positive correlation with mothers' lifestyles, (4) health knowledge has a significant positive correlation with childrens' and parents' lifestyles, (5) healthy lifestyles have a negative correlation with Rohrer's index in children and a negative correlation with TG level in fathers. The results suggest the presence of certain relationships for risk factors and lifestyles between children and their parents.

Binding of the human cytomegalovirus 80-kDa immediate-early protein (IE2) to minor groove A/T-rich sequences bounded by CG dinucleotides is regulated by protein oligomerization and phosphorylation.

The 80-kDa immediate-early regulatory protein IE2 of human cytomegalovirus (HCMV) functions as an essential positive transactivator of downstream viral promoters, but it also specifically down-regulates transcription from the major immediate-early promoter through a 14-bp DNA target motif known as the cis-repression signal (CRS) located at the transcription start site. The IE2 protein purified from bacteria as a fusion product of either staphylococcal Protein A/IE2(290-579) or glutathione-S-transferase (GST)/IE2(346-579) bound specifically to a [32P]-labeled CRS oligonucleotide probe in an in vitro electrophoretic mobility shift assay (EMSA). In contrast, no direct interaction with the CRS probes could be detected with IE2 wild-type protein in extracts from infected or transfected mammalian cells or when synthesized by in vitro translation. However, in vitro phosphorylation of GST/IE2(346-579) by incubation with either the catalytic subunit of protein kinase A (PKA) or a HeLa cell nuclear extract strongly inhibited its DNA-binding activity. This process required ATP hydrolysis and could be reversed by subsequent incubation with bacterial alkaline phosphatase. Importantly, dephosphorylation of the constitutively expressed native IE2 protein present in a nuclear extract from the U373(A45) cell line unmasked a specific CRS DNA-binding activity that could be supershifted with anti-IE2 monoclonal antibody (mAb). A series of high-molecular-weight hetero-oligomeric DNA-bound structures of intermediate mobility were formed in EMSA assays when a mixture of staphylococcal Protein A/IE2 and GST/IE2 was coincubated with the CRS probe. Coincubation with a DNA-binding negative but dimerization-competent GST/IE2 deletion mutant competitively inhibited DNA-binding by staphylococcal Protein A/IE2, whereas coincubation with a GST/IE2 deletion mutant that lacked the ability to both dimerize and bind to DNA failed to influence the mobility of the DNA-bound staphylococcal Protein A/IE2 protein. Therefore, IE2 appears to bind to DNA as a higher-order oligomer in which the presence of subunits with mutant DNA-binding domains interferes with the overall DNA-binding function. A series of point mutations introduced into each of nine conserved motifs throughout the DNA-binding and dimerization domain, all of which abolish the ability of the transfected intact IE2 protein to autoregulate the MIE promoter, also all lacked the ability to bind to CRS sequences as GST/IE2(346-379) fusion proteins. Detailed analysis of point mutations in the 14-bp CRS target DNA binding motif revealed that IE2 binds in a relatively sequence-independent manner to 10-bp-long A/T-rich DNA elements bounded on each side by CG dinucleotides. Moreover, the A/T-rich minor groove binding agent distamycin, but not the G/C-rich minor groove binding agent chromomycin-A3, actively competed with IE2 for binding to the CRS motif in a dose-dependent fashion. In conclusion, IE2 binds preferentially as multimerized dimers to A/T-rich sequences in the minor groove that are flanked on both sides by appropriately spaced CG dinucleotides, and inhibition of the DNA-binding or oligomerization activity by PKA phosphorylation probably accounts for the inactivity of the mammalian and in vitro translated forms of the protein.