bluuebunny/crossref_metadata_2025 huggingface dataset:
Single‐blind follow‐up study on the effectiveness of a symbiotic preparation in irritable bowel syndrome
Authors: TSUCHIYA J , BARRETO R , OKURA R , KAWAKITA S , FESCE E , MAROTTA F
Published: October 2004

OBJECTIVE:  Experimental and clinical studies have shown that a novel symbiotic (known as SCM‐III) exerts a beneficial effect on gut translocation and local and systemic inflammatory and microbial metabolic parameters. The present investigation was a preliminary trial on the effectiveness of SCM‐III for irritable bowel syndrome (IBS).

METHODS:  Sixty‐eight consecutive adult patients with IBS who were free from lactose malabsorption, abdominal surgery, overt psychiatric disorders and ongoing psychotropic drug therapy or ethanol abuse were studied prospectively and divided into 2 groups that were comparable for age, gender, body size, education and pattern of presenting symptoms. The 2 groups were blindly given for 12 weeks either SCM‐III 10 mL t.i.d or the same dosage of heat‐inactivated symbiotic.

RESULTS:  Treatment with SCM‐III was ‘effective’ or ‘very effective’ in more than 80% of the patients (P < 0.01 vs baseline values and control). Less than 5% reported ‘not effective’ as the final evaluation compared with over 40% of patients in the control group. After 6 weeks of treatment, a significant improvement of pain and bloating was reported in the treatment group compared with control and baseline values. There was also a benefit for bowel habits, mostly for patients with constipation or alternating bowel habits. No overt clinical or biochemical adverse side‐effects were recorded.

CONCLUSION:  Compared with baseline values and the control group, SCM‐III resulted in a significant increase in lactobacilla, eubacteria and bifidobacteria, which suggests that some selected IBS patients could benefit substantially from symbiotics, but the treatment may need to be given on a cyclic schedule because of the temporary modification of the fecal flora.
Clinical significance of functional constipation categorized by colonic transit time and pelvic floor electromyography
Authors: XIONG Guan Ying , ZHAO Zhi Quan
Published: October 2004

OBJECTIVE:  Functional constipation (FC) is a common gastrointestinal disorder, the incidence of which is increasing each year with the changes in life style and diet, and the age of patients is becoming younger. Correct categorization will guide the treatment of FC and in the present study colonic transit time (CTT) and pelvic floor electromyography (PFE) were evaluated for their clinical significance in categorizing FC.

METHODS:  Thirty‐two cases of FC diagnosed by Rome II criteria with a duration of 6 months to 30 years were enrolled. Radio‐opaque markers were used to detect CTT, the transit index (TI) and colonic evacuation rate; PFE was used to observe whether there was any paradoxical movement between the pelvic floor and abdominal muscles.

RESULTS:  Colonic transit time was recorded in 32 patients: 20 had outlet obstruction constipation and 12 had slow transit type. In the outlet obstruction type, PFE showed varying degrees of paradoxical movement between the muscles, whereas in the slow transit type, only 4 cases showed it.

CONCLUSION:  Functional constipation can be categorized by CTT and PFE, which are simple, painless and feasible to perform.
Mechanism of hepatocytes apoptosis induced by the proapoptosis protein Bid
Authors: BAI Li , CAO Chuan Ping , MAO Gao Ping
Published: October 2004

OBJECTIVE:  To investigate the mechanism of hepatocyte apoptosis induced by the proapoptosis protein Bid.

METHODS:  Mouse primary hepatocytes were isolated from wild‐type and Bid‐deficient mice and treated with tumor necrosis factor‐alpha (TNF‐α) or anti‐Fas antibody to induce cell apoptosis. Immunofluorescence staining of Bax was performed to recognize Bax translocation and its conformational change. The wild‐type mice or wild‐type mice transfected with the adenovirus carried DN‐FADD (Dominant Negative–Fas Associated Death Domain) and the Bid‐deficient mice were injected with anti‐Fas antibody 2 h before being killed. Caspase 3 and 8 activities were measured. Bands of Bid cleavage and Bax conformational change were detected by Western blot.

RESULTS:  Death receptors, including TNF‐α and anti‐Fas antibody, induced hepatocytes apoptosis, Bax translocation and conformational change through activation of Bid, which caused Bax to be inserted into the mitochondrial membrane of hepatocytes. The translocation and insertion of Bax were blocked when Bid was knocked out or blocked, and hepatocyte apoptosis was delayed or inhibited.

CONCLUSION:  Hepatocyte apoptosis induced by death receptors is regulated by Bid and the translocation and insertion of Bax are also dependent on Bid.
Peutz–Jeghers syndrome: case reports and update on diagnosis and treatment
Authors: DONG Ke , LI Bo
Published: October 2004

OBJECTIVE:  To analyze the etiology and diagnostic methods of Peutz–Jeghers syndrome (PJS) and thus establish a treatment strategy.

METHODS:  Clinical data from six patients with PJS were evaluated from the aspect of familial history, carcinogenesis and recurrence of polyps.

RESULTS:  The fathers of four and the mother of one of the six patients had PJS. The grandfather of three of the six patients had PJS. There was a history of cancer in three of the five families. Case 4 underwent two laparotomies for intussusceptions caused by recurrent polyps of the small intestine. Case 5 also had recurrent small intestinal polyps and required a laparotomy after 1 year of initial treatment. Polyps in cases 1 and 4 showed adenomatous changes and those in case 2 were associated with gastric cancer.

CONCLUSIONS:  Patients with PJS have a strong family history of cancer and a high incidence of recurrence of small intestinal polyps. Malignant changes of polyps may follow the hamartoma–adenoma–carcinoma sequence. Careful follow‐up is mandatory for gastrointestinal tract symptoms, and other solid organs that are susceptible to malignant change.
Selection bias, comparative advantage and heterogeneous returns to education: evidence from China in 2000
Authors: Heckman James J. , Li Xuesong
Published: October 2004

Abstract.  This paper uses Chinese micro data and new semi‐parametric methods to estimate the current return to college education allowing for heterogeneous returns and for self‐selection into schooling based on them. OLS and IV methods do not properly account for this sorting. Our estimates suggest that, for a randomly selected young person from an urban area, college attendance leads to a 43% increase in lifetime earnings (nearly 11% annually) in 2000, compared with just 36% (nearly 9% annually) for those who do not attend. Our evidence suggests that the return to education has increased substantially in China since the early 1990s.
Differential rewards to, and contributions of, education in urban China's segmented labor markets
Authors: Maurer‐Fazio Margaret , Dinh Ngan
Published: October 2004

Abstract.  Using worker data from a 1999–2000 urban enterprise survey, we examine the effects of education on the current earnings of continuously employed urban workers, migrants and laid off but subsequently re‐employed workers. We also decompose the earnings differentials between each of these groups of workers and then assess the contribution of education to explanations of the differentials. The empirical results demonstrate that returns to education increase with marketization and competition in the workplace. We also find educational attainment to be an important explanator of the earnings differentials between institutionally differentiated groups of workers in China's urban labor markets.
Reporting errors, ability heterogeneity, and returns to schooling in China
Authors: Li Haizheng , Luo Yi
Published: October 2004

Abstract.  This study is aimed at providing a more accurate estimate of the returns to schooling in China by controlling for unobserved ability heterogeneity and measurement errors. We identify a unique instrument to correct for the omitted ability bias. We find that the attenuation bias caused by measurement error dominates the omitted ability bias in the OLS estimation. Based on the GMM estimation, for young workers in China, the return to schooling is 15.0% overall and 16.9% for women.
Wage‐related labour standards and FDI in China: some survey findings from Guangdong province
Authors: Liu Minquan , Xu Luodan , Liu Liu
Published: October 2004

Abstract.  The paper presents findings from a survey of foreign invested enterprises (FIEs) in Guangdong, China, on the relationship between foreign direct investment (FDI) and wage‐related labour standards (regular wages, and compliance with official overtime and minimum wages), which show that wage‐related standards are statistically higher in FIEs whose home countries’ standards are higher, after controlling for other influences. However, a cost‐reduction FIE is more likely to be associated with inferior standards. The results call for a finer examination of the relationship between FDI and labour standards beyond the simple ‘race to the bottom’ thesis.
Studies on the use of chhana and paneer whey in the preparation of puras (pancakes)
Authors: JINDAL A R , SHOREE M , SHUKLA F C , SINGH B
Published: October 2004

Puras (pancakes) are widely accepted traditional Indian foods. Studies were conducted on the use of chhana and paneer whey in the preparation of sweet and salty puras. Six samples of chhana and paneer whey were substituted in sweet and salty puras at different whey to water ratios (0 : 100, 20 : 80, 40 : 60, 60 : 40, 80 : 20, 100 : 0). The effects of chhana and paneer whey on sensory evaluation showed that sweet puras containing mixed milk chhana whey and salty puras containing cow's milk chhana whey scored highest with respect to appearance, body and texture, flavour and taste, and overall acceptability. In general, commercial samples scored less, which may be due to poor handling and maintenance of whey by commercial manufacturers. Whey substitutions also improved fat, total solids, protein and ash of both sweet and salty puras. It is interesting to note that none of sweet and salty pura samples was below the limits acceptable to the panellists. It can be thus concluded that chhana and paneer whey can be used successfully in the preparation of puras for value addition.
Effect of different manufacturing parameters on the characteristics of Graviera Kritis cheese
Authors: MOATSOU GOLFO , MOSCHOPOULOU EKATERINI , ANIFANTAKIS EMMANOUEL
Published: October 2004

The objective of the present study was to improve the characteristics of Graviera Kritis cheese. The influence of lactic acid and propionic acid starters was studied and then the effect of washing and salting the curd combined with the use of starters was investigated. Although the traditional technology without starters resulted in the highest organoleptic characteristics, it was shown that the use of starters could improve texture characteristics of the cheeses if it is combined with a curd wash to control acidity development. Lower pH values and higher contents of low molecular weight nitrogenous substances were consistent with texture defects. Moreover, the ripening at higher temperatures increased proteolysis levels and decreased cheese quality.
The technology of low‐fat cheese manufacture
Authors: BANKS JEAN M
Published: October 2004

Growth in the low‐fat cheese market has been slower than would be anticipated on the basis of increased consumer awareness of dietary fat intake. Consumer dissatisfaction with the quality of first‐generation low‐fat products has highlighted the need for improved technology. Significant advances in understanding the biochemical and physicochemical characteristics of low‐fat variants in the past decade have led to novel technological developments. Approaches that have the potential to improve the flavour, texture and functionality of reduced‐ and low‐fat cheese are reviewed here. Topics include the control of processing variables, the selection of appropriate starter and adjunct bacteria, and the use of fat mimetics to improve texture. Factors influencing flavour and texture development in low‐fat variants are also considered.
Biobehavioral Correlates of Relocation in the Frail Elderly: Salivary Cortisol, Affect, and Cognitive Function
Authors: Hodgson Nancy , Freedman Vicki A. , Granger Douglas A. , Erno Amy
Published: October 2004

Objectives: To examine affect and physiological stress in frail older adults in response to a voluntary nursing home relocation.

Design: Randomized, controlled trial.

Setting: Long‐term care facility located within the greater Philadelphia, Pennsylvania, community.

Participants: Seventy‐seven nursing home residents, aged 65 and over.

Intervention: Experimental group residents were relocated to a newly built nursing home facility with a cluster design in the fall of 2001; control group residents were moved after study completion in the spring of 2002.

Measurements: Mini‐Mental State Examination scores, Observed Affect Rating Scale scores, salivary cortisol, blood pressure, and pulse obtained 1 week before moving and 1 week and 4 weeks after moving.

Results: Relocated nursing home residents demonstrated significant differences in salivary cortisol and mood from a randomly selected group of residents that had not yet moved. Relocation resulted in significantly higher cortisol levels 1 week after the move (P=.005), followed by a significant decline in afternoon cortisol at 4 weeks after the move (P=.03). Moreover, relocated residents had significantly lower depression and anxiety symptoms and pulse rates than residents who had not yet moved.

Conclusion: These findings have important implications for planning medical and social services for relocated elderly. Efforts should be made to prepare individuals for the initial stressors associated with relocation, but it also appears that the stress imposed by relocation is time limited and may begin to ease as early as 4 weeks postmove.
Urinary Incontinence and Psychological Distress in Community‐Dwelling Older African Americans and Whites
Authors: Bogner Hillary R.
Published: October 2004

Objectives: To compare the association between urinary incontinence (UI) and psychological distress in older African Americans and whites.

Design: A population‐based longitudinal survey.

Setting: Continuing participants in a study of community‐dwelling adults who were initially living in East Baltimore in 1981.

Participants: African Americans and whites aged 50 and older at follow‐up interviews performed between 1993 and 1996 for whom complete data were available (n=747).

Measurements: Participants were classified as incontinent if any uncontrolled urine loss within the 12 months before the interview was reported. Psychological distress was assessed using the General Health Questionnaire (GHQ).

Results: African Americans with UI were more likely to experience psychological distress as measured using the GHQ than were African Americans without UI (unadjusted odds ratio=4.22, 95% confidence interval=1.72–10.39). In multivariate models that controlled for age, sex, education, functional status, cognitive status, and chronic medical conditions, this association remained statistically significant. The association between UI and psychological distress did not achieve statistical significance in whites.

Conclusion: The effect of UI on emotional well‐being may be greater for African Americans than for whites.
Patient Relocation in the 6 Months After Hip Fracture: Risk Factors for Fragmented Care
Authors: Boockvar Kenneth S. , Litke Ann , Penrod Joan D. , Halm Ethan A. , Morrison R. Sean , Silberzweig Stacey B. , Magaziner Jay , Koval Kenneth , Siu Albert L.
Published: October 2004

Objectives: To describe the incidence and patterns of patient relocation after hip fracture, identify factors associated with relocation, and examine effect of relocation on outcomes.

Design: Prospective cohort study.

Setting: Four hospitals in the New York metropolitan area.

Participants: A total of 562 patients hospitalized for hip fracture discharged alive in 1997 to 1998.

Measurements: Patient characteristics and hospital course were ascertained using patient or surrogate interview, research nurse assessment, and medical record review. Patient location was ascertained at five time points using patient or surrogate interview, and hospital readmissions were identified using New York state and hospital admission databases. Mobility was measured using patient or surrogate report using the Functional Independence Measure.

Results: During 6 months of follow‐up, the mean number of relocations per patient±standard deviation was 3.5±1.5 (range 2–10). Forty‐one percent of relocations were between home and hospital, 36% between rehabilitation or nursing facility and hospital, 17% between rehabilitation or nursing facility and home, and 4% between two rehabilitation/nursing facilities. In a Poisson regression model that controlled for patient characteristics, hospital course, and length of follow‐up, factors associated with relocation (P<.05) were absence of dementia, in‐hospital delirium, one or more new impairments at hospital discharge, hospital discharge other than to home, and not living at home alone prefracture. Relocation was not significantly associated with immobility or mortality at 6 months (odds ratio=1.14, 95% confidence interval=0.97–1.35).

Conclusion: Subgroups of patients with elevated risk of relocation after hip fracture may be target groups for intensive care coordination and care planning interventions.
Inflammatory and Thrombotic Blood Markers and Walking‐Related Disability in Men and Women with and Without Peripheral Arterial Disease
Authors: McDermott Mary M. , Guralnik Jack M. , Greenland Philip , Green David , Liu Kiang , Ridker Paul M. , Chan Cheeling , Criqui Michael H. , Ferrucci Luigi , Taylor Lloyd M. , Pearce William H. , Schneider Joseph R. , Oskin Sarah I.
Published: October 2004

Objectives: To determine whether higher circulating levels of thrombotic and inflammatory markers are associated with greater disability.

Design: Cross‐sectional.

Setting: Academic medical center.

Participants: A total of 346 men and women with peripheral arterial disease (PAD) and 203 without PAD.

Measurements: Disability measures were the Walking Impairment Questionnaire (WIQ) distance, speed, and stair‐climbing scores and the 36‐item Short‐Form (SF‐36) physical functioning score. The SF‐36 and WIQ are scored on a 0 to 100 scale (100=best).

Results: In persons with PAD, higher D‐dimer levels were associated with lower WIQ speed scores (P<.001), lower stair‐climbing scores (P<.04), and poorer SF‐36 physical functioning scores (P<.01), adjusting for known and potential confounders. In participants without PAD, higher D‐dimer levels were associated with lower WIQ distance scores (P<.03), lower speed scores (P<.05), and poorer SF‐36 physical functioning scores (P<.02). Higher high‐sensitivity C‐reactive protein (hsCRP) levels were associated with lower WIQ distance (P<.02) and speed scores (P<.001) in persons without PAD. Most of these associations were attenuated after additional adjustment for objectively measured functional limitations.

Conclusion: Higher circulating D‐dimer and hsCRP levels are associated with greater disability in walking and physical functioning in individuals with and without PAD. Physiological changes that result in walking disability may mediate these associations.
The Coming Epidemic of Obesity in Elderly Americans
Authors: Arterburn David E. , Crane Paul K. , Sullivan Sean D.
Published: October 2004

Objectives: To estimate the prevalence of obesity in elderly Americans in 2010 and to discuss the health and economic implications of these estimates.

Design: Three methods of generating future point‐prevalence estimates using data from consecutive cross‐sectional studies.

Setting: All regions of the United States.

Participants: Estimates were based on five nationally representative surveys of the adult population of the United States, conducted from 1960 to 2000, and population estimates from the U.S. Census Bureau.

Measurements: Changes in obesity (body mass index (BMI) ≥30 kg/m2) and normal weight (BMI <25 kg/m2) prevalence for men and women by 10‐year U.S. birth cohorts were examined. The prevalence of obesity and normal weight in the elderly in 2010 was estimated under three different scenarios of obesity prevalence change.

Results: It was estimated that the prevalence of obesity in adults aged 60 and older will increase from 32.0% in 2000 to 37.4% in 2010 (range 33.6–39.6%). The number of obese adults aged 60 and older will increase from 14.6 to 20.9 million (range 18.8–22.2 million). Similarly, it was estimated that the prevalence of normal weight among adults aged 60 and older will decrease from 30.6% in 2000 to 26.7% in 2010 (range 31.0–24.7%).

Conclusion: The prevalence of obesity in elderly Americans will likely continue to increase, challenging healthcare delivery and financing systems in the United States.
Inappropriate Medication Use and Health Outcomes in the Elderly
Authors: Fu Alex Z. , Liu Gordon G. , Christensen Dale B.
Published: October 2004

Inappropriate medication use is a major problem for the elderly. Although increasing attention has been paid to inappropriate prescription medication use, most previous research has been limited to the investigation of prevalence and trends. Few studies provide the empirical evidence for the adverse effect of inappropriate medication use on health outcomes at the national level. This study is the first attempt to assess the relationship between inappropriate prescription use and health status for the elderly in the United States.

Based on the 1996 Medical Expenditure Panel Survey, inappropriate medication use in a national representative elderly population was first identified using Beers criteria. A survey type of ordered probit model was then estimated to quantify the effect of inappropriate drug use on patient self‐perceived health status measured using a five‐point scale (poor, fair, good, very good, and excellent). After controlling for a set of possible confounding factors, it was found that individuals using inappropriate medications in Round 1 were more likely than those not using inappropriate medications to report poorer health status in Round 2. Other risk factors for poor health status include a higher number of prescriptions, being black, having low education, and having one or more chronic diseases.

This study provides strong evidence of a significant adverse effect of inappropriate medication use on patient health status. These findings lend partial support to the use of Beers criteria in assessing the quality of prescribing and the appropriateness of medication use in the elderly population.
Androgen action on human skin – from basic research to clinical significance
Authors: Zouboulis Christos C. , Degitz Klaus
Published: October 2004

Abstract:  Androgens affect several functions of the human skin, such as sebaceous gland growth and differentiation, hair growth, epidermal barrier homeostasis and wound healing. Their effects are mediated by binding to nuclear androgen receptors. Androgen activation and deactivation are mainly intracellular events. They differ from cell type to cell type and between cells at different locations. The major circulating androgens, dehydroepiandrosterone sulfate and androstenedione, are predominantly produced in the adrenal glands, and testosterone and 5α‐dihydrotestosterone are mainly synthesized in the gonads. Testosterone in women and 5α‐dihydrotestosterone in both genders are also synthesized in the skin. Skin cells express all androgen metabolizing enzymes required for the independent cutaneous synthesis of androgens and the development of hyperandrogenism‐associated conditions and diseases, such as seborrhea, acne, hirsutism and androgenetic alopecia. The major thrust of drug design for the treatment of androgen‐associated disorders has been directed against several levels of androgen function and metabolism. Partial effectiveness has only been achieved either by androgen depletion, inhibition of androgen metabolism or blockade of the androgen receptor.
Vitamin D and skin: new aspects for dermatology
Authors: Lehmann Bodo , Querings Kerstin , Reichrath Jörg
Published: October 2004

Abstract:  It has been shown that epidermal keratinocytes have the capacity for the UVB‐induced photochemical conversion of 7‐dehydrocholesterol to vitamin D3, and also for the enzymatically controlled hydroxylation of the photolysis product. This metabolic loop results in the formation of the biologically active final product 1α,25‐dihydroxyvitamin D3 (1α,25(OH)2D3, calcitriol). The epidermal synthesis of calcitriol is of fundamental relevance because calcitriol regulates important cellular functions in keratinocytes and immunocompetent cells. Because of their anti‐proliferative and prodifferentiating effects, calcitriol and other vitamin D analogs are highly efficient in the treatment of psoriasis vulgaris. In addition, the known therapeutic effect of UVB light therapy in the treatment of psoriasis may, at least in part, be mediated via UVB‐induced synthesis of calcitriol. Increasing evidence now indicates that cutaneous vitamin D synthesis is of great importance for the prevention of a broad variety of diseases, including various malignancies. It has been postulated that cancer mortality could be reduced via careful UV exposure or, more safely, via oral substitution with vitamin D. These new findings must be taken into account when establishing new sun protection guidelines for the prevention of skin cancer. In addition, better understanding of the metabolism of vitamin D in the skin has opened up new perspectives for the therapeutic application of vitamin D analogs, e.g. in inflammatory skin diseases.
Novel aspects in cutaneous biology of acetylcholine synthesis and acetylcholine receptors
Authors: Kurzen Hjalmar , Schallreuter Karin U.
Published: October 2004

Abstract:  Extraneuronal acetylcholine (ACh) has been demonstrated to influence a plethora of cutaneous cell functions in an autocrine, paracrine and endocrine fashion. Through the differentiation‐specific expression of its different nicotinic (nACh‐R) and muscarinic (mACh‐R) receptors, ACh acts upon keratinocyte proliferation and migration, terminal differentiation and barrier formation, sweat and sebum secretion as well as microcirculation and angiogenesis. Only very recently it has been recognized that acetylcholinesterase, but not cholineacetyltransferase, activity is regulated by hydrogen peroxide. Considering that the outer layer of the human skin can be a target for UV‐generated H2O2 in the millimolar range, this mechanism needs to be taken into account for the regulation of ACh homeostasis in skin biology. Consequently, ACh can accumulate, as shown, for example, in the depigmentation process in vitiligo. There is a highly regulated distribution of ACh‐R in human epidermis and adnexal structures, supporting previously observed effects of cholinergic compounds on keratinocyte biology. Most significantly, the regulated expression of ACh‐R in sebaceous glands advocates a role for ACh in sebum production and as a promoter of sebocyte differentiation, thus offering an explanation for skin diseases associated with altered sebum production after chronic nicotine exposure. So far, ACh‐induced sweat production has been thought to be under the exclusive control of mACh‐R. However, recently, the presence of both different nACh‐R and mACh‐R in myoepithelial and acinar cells of eccrine sweat glands has been documented, indicating a more complex regulation of sweat production and expulsion.
Neutral endopeptidase and angiotensin‐converting enzyme – key enzymes terminating the action of neuroendocrine mediators
Authors: Scholzen Thomas E. , Luger Thomas A.
Published: October 2004

Abstract:  Zinc‐metalloproteases, such as neutral endopeptidase (NEP) and angiotensin‐converting enzyme (ACE), effectively control the bioavailability of peptide mediators released from sensory nerves, immune and skin cells during the cutaneous response to endogenous or exogenous noxious stimuli. Functional inactivation of NEP or ACE by transient inhibition or permanent genomic deletion results in a relative abundance of substance P (SP) and bradykinin (BK); this augments murine allergic contact dermatitis (ACD) by affecting ACD sensitization and elicitation, which involves neurokinin 1 receptors (NK1), BK receptors (B2) and an intact cutaneous sensory nervous system. Present evidence suggests that increased SP via NK1 is capable of boosting important functions of SP‐ and NK1‐expressing dendritic cells (DCs) and T cells (TCs) in an auto‐ or paracrine manner, which promotes ACD antigen sensitization. Moreover, skin inflammation or wounding in vivo, as well as treatment of epidermal and dermal cells by UV light and inflammatory mediators in vitro, regulates NEP and ACE expression and activity. Likewise, NEP and ACE are capable of processing neuroendocrine hormones, such as adrenocorticotropin and α‐melanocyte‐stimulating hormone. Thus, present data indicate that ACE and NEP, via proteolytic cleavage of peptide mediators and growth factors, represent important control factors for the inflammatory response in skin disorders such as psoriasis or allergic inflammation, but may also be capable of affecting pigmentation, cell survival, wound healing and tissue regeneration.
Construction of a Herpes Simplex Virus Type 1 Mutant with Only a Three-Nucleotide Change in the Branchpoint Region of the Latency-Associated Transcript (LAT) and the Stability of Its Two-Kilobase LAT Intron
Authors: Ng Alan K. , Block Timothy M. , Aiamkitsumrit Benjamas , Wang Mengjun , Clementi Emily , Wu Ting-Ting , Taylor John M. , Su Ying-Hsiu
Published: October 2004

Previous studies using a eukaryotic expression system indicated that the unusual stability of the latency-associated transcript (LAT) intron was due to its nonconsensus branchpoint sequence (T.-T Wu, Y.-H. Su, T. M. Block, and J. M. Taylor, Virology, 243:140-149, 1998). The present study investigated the role of the branchpoint sequence in the stability of the intron expressed from the herpes simplex virus type 1 (HSV-1) genome and the role of LAT intron stability in the HSV-1 life cycle. A branchpoint mutant called Sy2 and the corresponding rescued viruses, SyRA and SyRB, were constructed. To preserve the coding sequence of the immediate early gene icp0, which overlaps with the branchpoint region of the 2-kb LAT, a 3-nucleotide mutation into the branchpoint region of the 2-kb LAT was introduced, resulting in a branchpoint that is 85% identical to the consensus intron branchpoint sequence of eukaryotic cells. As anticipated, there was a 90- to 96-fold reduction in 2-kb LAT accumulation following productive infection in tissue culture and latent infection in mice with Sy2, as determined by Northern blot analysis. These results clearly suggest that the accumulation of the 2-kb intron in tissue culture and in vivo is, at least in part, due to the nonconsensus branchpoint sequence of the LAT intron. Interestingly, a failure to accumulate LAT was associated with greater progeny production of Sy2 at a low multiplicity of infection (0.01) in tissue culture, but not in mice. However, the ability of mutant Sy2 to reactivate from trigeminal ganglia (TG) derived from latently infected mice was indistinguishable from that of wild-type virus, as assayed in the mouse TG explant reactivation system.
Nonrandom Dimerization of Murine Leukemia Virus Genomic RNAs
Authors: Flynn Jessica A. , An Wenfeng , King Steven R. , Telesnitsky Alice
Published: October 2004

Retroviral genomes consist of two unspliced RNAs linked noncovalently in a dimer. Although these two RNAs are generally identical, two different RNAs can be copackaged when virions are produced by coinfected cells. It has been assumed, but not tested, that copackaging results from random RNA associations in the cytoplasm to yield encapsidated RNA homodimers and heterodimers in Hardy-Weinberg proportions. Here, virion RNA homo- and heterodimerization were examined for Moloney murine leukemia virus (MLV) using nondenaturing Northern blotting and a novel RNA dimer capture assay. The results demonstrated that coexpressed MLV RNAs preferentially self-associated, even when RNAs were identical in known packaging and dimerization sequences or when they differed overall by less than 0.1%. In contrast, HIV-1 RNAs formed homo- and heterodimers in random proportions. We speculate that these species-specific differences in RNA dimer partner selection may at least partially explain the higher frequency of genetic recombination observed for human immunodeficiency virus type 1 than for MLV.
The L2 Minor Capsid Protein of Human Papillomavirus Type 16 Interacts with a Network of Nuclear Import Receptors
Authors: Darshan Medha S. , Lucchi John , Harding Emily , Moroianu Junona
Published: October 2004

The L2 minor capsid proteins enter the nucleus twice during viral infection: in the initial phase after virion disassembly and in the productive phase when, together with the L1 major capsid proteins, they assemble the replicated viral DNA into virions. In this study we investigated the interactions between the L2 protein of high-risk human papillomavirus type 16 (HPV16) and nuclear import receptors. We discovered that HPV16 L2 interacts directly with both Kapβ
2
and Kapβ
3
. Moreover, binding of Ran-GTP to either Kapβ
2
or Kapβ
3
inhibits its interaction with L2, suggesting that the Kapβ/L2 complex is import competent. In addition, we found that L2 forms a complex with the Kapα
2
β
1
heterodimer via interaction with the Kapα
2
adapter. In agreement with the binding data, nuclear import of L2 in digitonin-permeabilized cells could be mediated by either Kapα
2
β
1
heterodimers, Kapβ
2
, or Kapβ
3
. Mapping studies revealed that HPV16 L2 contains two nuclear localization signals (NLSs), in the N terminus (nNLS) and C terminus (cNLS), that could mediate its nuclear import. Together the data suggest that HPV16 L2 interacts via its NLSs with a network of karyopherins and can enter the nucleus via several import pathways mediated by Kapα
2
β
1
heterodimers, Kapβ
2
, and Kapβ
3
.
Analysis of Adaptive Mutations in Kunjin Virus Replicon RNA Reveals a Novel Role for the Flavivirus Nonstructural Protein NS2A in Inhibition of Beta Interferon Promoter-Driven Transcription
Authors: Liu Wen Jun , Chen Hua Bo , Wang Xiang Ju , Huang Hester , Khromykh Alexander A.
Published: October 2004

The establishment of persistent noncytopathic replication by replicon RNAs of a number of positive-strand RNA viruses usually leads to generation of adaptive mutations in nonstructural genes. Some of these adaptive mutations (e.g., in hepatitis C virus) increase the ability of RNA replication to resist the antiviral action of alpha/beta interferon (IFN-α/β); others (e.g., in Sindbis virus) may also lead to more efficient IFN production. Using puromycin-selectable Kunjin virus (KUN) replicon RNA, we identified two adaptive mutations in the NS2A gene (producing Ala30-to-Pro and Asn101-to-Asp mutations in the gene product; for simplicity, these will be referred to hereafter as Ala30-to-Pro and Asn101-to-Asp mutations) that, when introduced individually or together into the original wild-type (wt) replicon RNA, resulted in ∼15- to 50-fold more efficient establishment of persistent replication in hamster (BHK21) and human (HEK293 and HEp-2) cell lines. Transfection with a reporter plasmid carrying the luciferase gene under the control of the IFN-β promoter resulted in ∼6- to 7-fold-higher luciferase expression in HEp-2 cells stably expressing KUN replicon RNA with an Ala30-to-Pro mutation in the NS2A gene compared to that observed in HEp-2 cells stably expressing KUN replicon RNA with the wt NS2A gene. Moreover, cotransfection of plasmids expressing individual wt or Ala30-to-Pro-mutated NS2A genes with the IFN-β promoter reporter plasmid, followed by infection with Semliki Forest virus to activate IFN-β promoter-driven transcription, showed ∼7-fold inhibition of luciferase expression by the wt but not by the Ala30-to-Pro-mutated NS2A protein. The results show for the first time a role for the flavivirus nonstructural protein NS2A in inhibition of IFN-β promoter-driven transcription and identify a single-amino-acid mutation in NS2A that dramatically reduces this inhibitory activity. The findings determine a new function for NS2A in virus-host interactions, extend the range of KUN replicon vectors for noncytopathic gene expression, and identify NS2A as a new target for attenuation in the development of live flavivirus vaccines.
Cellular Immune Responses in Seronegative Sexual Contacts of Acute Hepatitis C Patients
Authors: Kamal Sanaa M. , Amin Ashraf , Madwar Mohamed , Graham Camilla S. , He Qi , Al Tawil Ahmed , Rasenack Jens , Nakano Tatsunori , Robertson Betty , Ismail Alaa , Koziel Margaret James
Published: October 2004

Acute hepatitis C virus (HCV) is typically defined as new viremia and antibody seroconversion. Rates and immunologic correlates of hepatitis C clearance have therefore been based on clearance of viremia only in individuals who initially had an antibody response. We sought to characterize the immunological correlates of clearance in patients with acute hepatitis C and their sexual contacts. We prospectively determined CD4+and CD8+cytotoxic T-lymphocyte responses in index patients with acute HCV and their sexual contacts who developed acute infection, either with or without spontaneous clearance, as well as those contacts who never developed viremia. Responses were measured using proliferation and ELISpot assays for CD4+and CD8+responses. We demonstrate in this prospective study that cellular immune responses can develop in exposed but persistently aviremic and antibody-negative individuals as well as those individuals with spontaneous clearance of acute HCV. These findings lend further credence to the importance of cellular immune responses in recovery from HCV and suggest that low exposure to HCV may lead to development of HCV-specific immune responses without ongoing HCV replication. This finding has important implications for HCV vaccine and therapeutic development.
Dynamic Chromatin Boundaries Delineate a Latency Control Region of Epstein-Barr Virus
Authors: Chau Charles M. , Lieberman Paul M.
Published: October 2004

The oncogenic potential of latent Epstein-Barr virus (EBV) can be regulated by epigenetic factors controlling LMP1 and EBNA2 gene transcription. The EBV latency control region (LCR) constitutes ∼12 kb of viral sequence spanning the divergent promoters of LMP1 and EBNA2 and encompasses the EBV latent replication origin OriP and RNA polymerase III-transcribed EBV-encoded RNA genes. We have used the chromatin immunoprecipitation assay to examine the chromatin architecture of the LCR in different types of EBV latency programs. We have found that histone H3 K4 methylation (H3mK4) was enriched throughout a large domain that extended from internal repeat 1 (IR1) to the terminal repeat in type III latency where EBNA2 and LMP1 genes are expressed. In type I latency where EBNA2 and LMP1 genes are transcriptionally silent, the H3mK4 domain contracts and does not enter the EBNA2 or LMP1 promoters. In contrast, histone H3 K9 methylation (H3mK9), associated with silent heterochromatin, was enriched in the EBNA2 and LMP1 upstream control regions in type I but not type III cells. MTA [5′-deoxy-5′(methylthio)adenosine], a pharmacological inhibitor of protein methylation, globally reduced histone H3mK4 and inhibited EBNA2 transcription in type III cells. 5′-Azacytidine, an inhibitor of DNA methylation that derepresses EBNA2 transcription in type I latency, caused H3mK4 expansion and a corresponding loss of H3mK9 at IR1. The chromatin boundary protein and transcription repressor CCCTC-binding factor was enriched at the EBNA2 transcription control region in type I but not type III cells. We also present evidence that OriP binding factors EBNA1 and ORC2 can interact with sequences outside of OriP including a region within IR1 that may influence EBNA2 transcription status. These results indicate that types I and III latency programs have distinct histone methylation patterns in the LCR and suggest that chromatin architecture coordinates gene expression of LMP1 and EBNA2.
CpG Oligodeoxynucleotides Block Human Immunodeficiency Virus Type 1 Replication in Human Lymphoid Tissue Infected Ex Vivo
Authors: Schlaepfer Erika , Audigé Annette , von Beust Barbara , Manolova Vania , Weber Markus , Joller Helene , Bachmann Martin F. , Kundig Thomas M. , Speck Roberto F.
Published: October 2004

Oligodeoxynucleotides (ODNs) with immunomodulatory motifs control a number of microbial infections in animal models, presumably by acting through toll-like receptor 9 (TLR9) to induce a number of cytokines (e.g., alpha interferon and tumor necrosis factor alpha). The immunomodulatory motif consists of unmethylated sequences of cytosine and guanosine (CpG motif). ODNs without CpG motifs do not trigger TLR9. We hypothesized that triggering of TLR9 generates a cellular environment unfavorable for human immunodeficiency virus (HIV) replication. We tested this hypothesis in human lymphocyte cultures and found that phosphorothioate-modified ODN CpG2006 (type B ODNs) inhibited HIV replication nearly completely and prevented the loss of CD4+T cells. ODNs CpG2216 and CpG10 (type A ODNs) were less effective. CpG2006 blocked HIV replication in purified CD4+T cells and T-cell lines; CpG10 was ineffective in this setting, indicating that type A ODNs may inhibit HIV replication in CD4+T-cell lines indirectly through a separate cell subset. However, control ODNs without CpG motifs also showed anti-HIV effects, indicating that these effects are nonspecific and not due to TLR9 triggering. The mechanism of action is not clear. CpG2006 and its control ODN blocked syncytium formation in a cell fusion-based assay, but CpG10, CpG2216, and their control ODNs did not. The latter types interfered with the HIV replication cycle during disassembly or reverse transcription. In contrast, CpG2006 and CpG2216 specifically induced cytokines critical to initiation of the innate immune response. In summary, the nonspecific anti-HIV activity of CpG ODNs, their ability to stimulate HIV replication in latently infected cells, potentially resulting in their elimination, and their documented ability to link the innate and adaptive immune responses make them attractive candidates for further study as anti-HIV drugs.
Secretion of a TNFR:Fc Fusion Protein following Pulmonary Administration of Pseudotyped Adeno-Associated Virus Vectors
Authors: Sandalon Ziv , Bruckheimer Elizabeth M. , Lustig Kurt H. , Rogers Linda C. , Peluso Richard W. , Burstein Haim
Published: October 2004

This study evaluated and compared delivery of the tumor necrosis factor alpha receptor (TNFR)-immunoglobulin G1 (IgG1) Fc fusion (TNFR:Fc) gene to the lung by single and repeat administrations of multiple pseudotyped adeno-associated virus (AAV) vectors as a means for achieving systemic distribution of the soluble TNFR:Fc protein. A single endotracheal administration of AAV[2/5]cytomegalovirus (CMV)-TNFR:Fc vector (containing the AAV2 inverted terminal repeats and AAV5 capsid) to the rat lung resulted in long-term, high levels of serum TNFR:Fc protein that gradually declined over a period of 8 months. Endotracheal delivery of AAV[2/1]CMV-TNFR:Fc resulted in serum TNFR:Fc protein levels that were detectable for at least 4 months but were 10-fold lower than that of the AAV[2/5] vector. In contrast, secretion of the TNFR:Fc protein following pulmonary delivery of AAV[2/2]CMV-TNFR:Fc vector was very inefficient, and the protein was detected in the blood only when an airway epithelial cell-specific promoter, CC10, was substituted for the CMV enhancer/promoter to control transgene expression. In the context of AAV[2/5], the CC10 promoter was as efficient as CMV enhancer/promoter in generating similar levels of systemic TNFR:Fc protein, suggesting that this protein is secreted primarily from the airway epithelium. In mice, comparable long-term secretion of TNFR:Fc protein was demonstrated after AAV[2/2] and AAV[2/5] delivery, although the kinetics of transduction appeared to be different. All pseudotyped AAV vectors elicited serum anti-AAV capsid-neutralizing antibody responses, but these did not prevent lung transduction and efficient secretion of TNFR:Fc protein to the circulation following readministration with AAV[2/5]. These results highlight the potential utility of AAV vectors containing serotype 5 capsid to deliver and redeliver genes of secreted proteins to the lung to achieve long-term systemic protein expression.
Expression of Human Immunodeficiency Virus Type 1 Gag Modulates Ligand-Induced Downregulation of EGF Receptor
Authors: Valiathan Rajeshwari R. , Resh Marilyn D.
Published: October 2004

Many enveloped viruses use the ESCRT proteins of the cellular vacuolar protein sorting pathway for efficient egress from the cell. Recruitment of the ESCRT proteins by human immunodeficiency virus type 1 (HIV-1) Gag is required for HIV-1 particle budding and egress. ESCRT proteins normally function at endosomal membranes, where they facilitate the downregulation of mitogen-activated receptors such as EGF receptor (EGFR) through multivesicular body biogenesis. It is not known whether the Gag-mediated recruitment of ESCRT proteins functionally depletes the pool of these molecules that is available for the downregulation of EGFR. Here we show that the expression of HIV-1 Gag decreases the rate of EGFR downregulation, as assessed by decreases in the rates of
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I-EGF and EGFR degradation. The effect of Gag was dependent on the presence of the TSG101 binding motif (PTAP) within the Gag C-terminal p6 domain. Cells expressing HIV-1 Gag retained more EGFR in late endosomes. This effect occurred when Gag was expressed alone from a heterologous promoter and when Gag expression was driven by the HIV-1 long terminal repeat within pHXB2ΔBalD25S, a noninfectious lentiviral vector. Gag-expressing cells exhibited higher levels of activated mitogen-activated protein kinase for longer times after EGF addition than did cells that did not express HIV-1 Gag. These results indicate that HIV-1 Gag can impinge upon the functioning of the cellular vacuolar protein sorting pathway and reveal yet another facet of the intricate effects of HIV-1 infection on host cell physiology.
Inhibition of Interferon-Mediated Antiviral Activity by Murine Gammaherpesvirus 68 Latency-Associated M2 Protein
Authors: Liang Xiaozhen , Shin Young C. , Means Robert E. , Jung Jae U.
Published: October 2004

Upon viral infection, the major defense mounted by the host immune system is the activation of the interferon (IFN)-mediated antiviral pathway. In order to complete their life cycle, viruses that are obligatory intracellular parasites must modulate the host IFN-mediated immune response. Murine gammaherpesvirus 68 (γHV68) infects a wide range of cell types and establishes latent infections in mice. Here we demonstrate that the γHV68 latency-associated M2 protein has a cell-type-dependent localization pattern: M2 is present in the cytoplasm and plasma membrane in lymphocytes, whereas it is present primarily in the nucleus in epithelial and fibroblast cells. A mutational analysis indicated that the internal positively charged amino acids of M2 are required for its nuclear localization in fibroblasts. Purification of the M2 complex showed that M2 specifically interacts with the cellular p32 acidic protein through its central positively charged region and that this interaction recruits the cellular p32 protein to the nucleus in fibroblasts. Regardless of its localization, M2 expression effectively induced the downregulation of STAT1 and/or STAT2 in both A20 B lymphocytes and NIH 3T3 fibroblasts, resulting in the inhibition of IFN-α/β- and IFN-γ-mediated transcriptional activation. Finally, the M2 interaction with the p32 protein appeared to contribute to its ability to inhibit IFN-mediated transcriptional activation. These results indicate that γHV68 harbors a latency-associated M2 gene that antagonizes IFN-mediated host innate immunity and thus could play an important role in the establishment and maintenance of viral latency in infected animals.