Prospective enzymes for omega-3 PUFA biosynthesis found in endoparasitic classes within the phylum Platyhelminthes.

The free-living infectious stages of macroparasites, specifically, the cercariae of trematodes (flatworms), are likely to be significant (albeit underappreciated) vectors of nutritionally important polyunsaturated fatty acids (PUFA) to consumers within aquatic food webs, and other macroparasites could serve similar roles. In the context of de novo omega-3 (n-3) PUFA biosynthesis, it was thought that most animals lack the fatty acid (FA) desaturase enzymes that convert stearic acid (18:0) into ɑ-linolenic acid (ALA; 18:3n-3), the main FA precursor for n-3 long-chain PUFA. Recently, novel sequences of these enzymes were recovered from 80 species from six invertebrate phyla, with experimental confirmation of gene function in five phyla. Given this wide distribution, and the unusual attributes of flatworm genomes, we conducted an additional search for genes for de novo n-3 PUFA in the phylum Platyhelminthes. Searches with experimentally confirmed sequences from Rotifera recovered nine relevant FA desaturase sequences from eight species in four genera in the two exclusively endoparasite classes (Trematoda and Cestoda). These results could indicate adaptations of these particular parasite species, or may reflect the uneven taxonomic coverage of sequence databases. Although additional genomic data and, particularly, experimental study of gene functionality are important future validation steps, our results indicate endoparasitic platyhelminths may have enzymes for de novo n-3 PUFA biosynthesis, thereby contributing to global PUFA production, but also representing a potential target for clinical antihelmintic applications.

Phosphoinositide involvement in phagocytosis and phagosome maturation.

Cells of the innate immune system engulf invading microorganisms into plasma membrane-derived vacuoles called phagosomes. Newly formed phagosomes gradually acquire microbicidal properties by a maturation process which involves sequential and coordinated rounds of fusion with endomembranes and concomitant fission. Some pathogens interfere with this maturation sequence and thereby evade killing by the immune cells, managing to survive intracellularly as parasites. Phosphoinositides seem to be intimately involved in the processes of phagosome formation and maturation, and initial observations suggest that the ability of some microorganisms to survive intracellularly is associated with alterations in phosphoinositide metabolism. This chapter presents a brief overview of phosphoinositides in cells of the immune system, their metabolism in the context of phagocytosis and phagosome maturation and their possible derangements during infectious pathogenosis.

Phagosome maturation: a few bugs in the system.

Cells of the innate immune system ingest and destroy invading microorganisms by initially engulfing them into a specialized vacuole, known as the phagosome. The membrane of the forming phagosome is similar to the plasmalemma and its contents resemble the extracellular milieu. As such, the nascent phagosome is not competent to kill and eliminate the ingested microorganisms. However, shortly after sealing, the phagosome undergoes a series of rapid and extensive changes in its composition, the result of a sophisticated sequence of membrane fusion and fission reactions. Understanding the molecular basis of these events is of particular importance, since they are often the target of disruption by intracellular parasites such as Mycobacterium, Salmonella and Legionella. The objective of this review is to summarize the current knowledge of the molecular mechanisms underlying phagosomal maturation and its subversion by parasitic microorganisms.

Distinct roles of class I and class III phosphatidylinositol 3-kinases in phagosome formation and maturation.

Phagosomes acquire their microbicidal properties by fusion with lysosomes. Products of phosphatidylinositol 3-kinase (PI 3-kinase) are required for phagosome formation, but their role in maturation is unknown. Using chimeric fluorescent proteins encoding tandem FYVE domains, we found that phosphatidylinositol 3-phosphate (PI[3]P) accumulates greatly but transiently on the phagosomal membrane. Unlike the 3'-phosphoinositides generated by class I PI 3-kinases which are evident in the nascent phagosomal cup, PI(3)P is only detectable after the phagosome has sealed. The class III PI 3-kinase VPS34 was found to be responsible for PI(3)P synthesis and essential for phagolysosome formation. In contrast, selective ablation of class I PI 3-kinase revealed that optimal phagocytosis, but not maturation, requires this type of enzyme. These results highlight the differential functional role of the two families of kinases, and raise the possibility that PI(3)P production by VPS34 may be targeted during the maturation arrest induced by some intracellular parasites.

The genomic structure of SYCP3, a meiosis-specific gene encoding a protein of the chromosome core.

SYCP3 localizes to the lateral elements of the synaptonemal complex and is essential for male meiosis. The genomic structure of SYCP3 consists of nine exons spanning approximately 14 kb. In mouse and rat, but not in hamster, the putative translation start of SYCP3 is present in the first exon. The putative promoter of SYCP3 was also cloned and shown to drive transcription of a reporter gene in somatic cells.

Indirect role for COPI in the completion of FCgamma receptor-mediated phagocytosis.

Recent evidence suggests that extension of pseudopods during phagocytosis requires localized insertion of endomembrane vesicles. The nature of these vesicles and the processes mediating their release and insertion are unknown. COPI plays an essential role in the budding and traffic of membrane vesicles in intracellular compartments. We therefore assessed whether COPI is also involved in phagosome formation. We used ldlF cells, a mutant line derived from Chinese hamster ovary cells that express a temperature-sensitive form of epsilonCOP. To confer phagocytic ability to ldlF cells, they were stably transfected with Fc receptors type IIA (FcgammaRIIA). In the presence of functional COPI, FcgammaRIIA-transfected ldlF cells effectively internalized opsonized particles. In contrast, phagocytosis was virtually eliminated after incubation at the restrictive temperature. Similar results were obtained impairing COPI function in macrophages using brefeldin A. Notably, loss of COPI function preceded complete inhibition of phagocytosis, suggesting that COPI is indirectly required for phagocytosis. Despite their inability to internalize particles, COPI-deficient cells nevertheless expressed normal levels of FcgammaRIIA, and signal transduction appeared unimpeded. The opsonized particles adhered normally to COPI-deficient cells and were often found on actin-rich pedestals, but they were not internalized due to the inability of the cells to extend pseudopods. The failure to extend pseudopods was attributed to the inability of COPI-deficient cells to mobilize endomembrane vesicles, including a VAMP3-containing compartment, in response to the phagocytic stimulus.

Role of COPI in phagosome maturation.

Phagosomes mature by sequentially fusing with endosomes and lysosomes. Vesicle budding is presumed to occur concomitantly, mediating the retrieval of plasmalemmal components and the regulation of phagosomal size. We analyzed whether fission of vesicles from phagosomes requires COPI, a multimeric complex known to be involved in budding from the Golgi and endosomes. The role of COPI was studied using ldlF cells, that harbor a temperature-sensitive mutation in epsilon-COP, a subunit of the coatomer complex. These cells were made phagocytic toward IgG-opsonized particles by heterologous expression of human FcgammaRIIA receptors. Following incubation at the restrictive temperature, epsilon-COP was degraded in these cells and their Golgi complex dispersed. Nevertheless, phagocytosis persisted for hours in cells devoid of epsilon-COP. Retrieval of transferrin receptors from phagosomes became inefficient in the absence of epsilon-COP, while clearance of the FcgammaRIIA receptors was unaffected. This indicates that fission of vesicles from the phagosomal membrane involves at least two mechanisms, one of which requires intact COPI. Traffic of fluid-phase markers and aggregated IgG-receptor complexes along the endocytic pathway was abnormal in epsilon-COP-deficient cells. In contrast, phagosome fusion with endosomes and lysosomes was unimpaired. Moreover, the resulting phagolysosomes were highly acidic. Similar results were obtained in RAW264.7 macrophages treated with brefeldin A, which precludes COPI assembly by interfering with the activation of adenosine ribosylation factor. These data indicate that neither phagosome formation nor maturation are absolutely dependent on COPI. Our findings imply that phagosomal maturation differs from endosomal progression, which appears to be more dependent on COPI-mediated formation of carrier vesicles.

Localized biphasic changes in phosphatidylinositol-4,5-bisphosphate at sites of phagocytosis.

Phagocytosis requires localized and transient remodeling of actin filaments. Phosphoinositide signaling is believed to play an important role in cytoskeletal organization, but it is unclear whether lipids, which can diffuse along the membrane, can mediate the focal actin assembly required for phagocytosis. We used imaging of fluorescent chimeras of pleckstrin homology and C1 domains in live macrophages to monitor the distribution of phosphatidylinositol-4,5-bisphosphate (4,5-PIP(2)) and diacylglycerol, respectively, during phagocytosis. Our results reveal a sequence of exquisitely localized, coordinated steps in phospholipid metabolism: a focal, rapid accumulation of 4,5-PIP(2) accompanied by recruitment of type Ialpha phosphatidylinositol phosphate kinase to the phagosomal cup, followed by disappearance of the phosphoinositide as the phagosome seals. Loss of 4,5-PIP(2) correlated with mobilization of phospholipase Cgamma (PLCgamma) and with the localized formation of diacylglycerol. The presence of 4, 5-PIP(2) and active PLCgamma at the phagosome was shown to be essential for effective particle ingestion. The temporal sequence of phosphoinositide metabolism suggests that accumulation of 4,5-PIP(2) is involved in the initial recruitment of actin to the phagocytic cup, while its degradation contributes to the subsequent cytoskeletal remodeling.

Phagosomal maturation, acidification, and inhibition of bacterial growth in nonphagocytic cells transfected with FcgammaRIIA receptors.

Phagocytosis and killing of microbial pathogens by professional phagocytes is an essential component of the innate immune response. Recently, heterologous transfection of individual receptors into nonmyeloid cells has been used successfully to elucidate the early steps that signal phagosome formation. It is unclear, however, whether the vacuoles formed by such transfected cells are bona fide phagosomes, capable of fusion with endomembranes, of luminal acidification, and of controlling the growth of microorganisms. The aim of the current study was to determine whether COS-1 and Chinese hamster ovary cells, rendered phagocytic by expression of human FcgammaRIIA receptors, express the cellular machinery required to support phagosomal maturation. Immunolocalization studies demonstrated that early endosomes, as well as late endosomes and/or lysosomes, fuse sequentially with phagosomes in the transfectants. Microfluorescence ratio imaging of particles labeled with pH-sensitive dyes revealed that maturation of the phagosome was accompanied by luminal acidification. The drop in pH, which attained levels comparable to those reported in professional phagocytes, was prevented by inhibitors of vacuolar-type H(+)-ATPases. Optimal phagosomal acidification required elevation of cytosolic [Ca(2+)], suggesting that it results from fusion of endomembranes bearing proton pumps. Moreover, the transfected cells effectively internalized live bacteria. Opsonization was essential for bacterial internalization, implying that it occurred by FcgammaRIIA-mediated phagocytosis, as opposed to invasion. Uptake into phagolysosomes was associated with inhibition of bacterial growth, due at least in part to the low intraphagosomal pH. These studies indicate that the biochemical events that follow receptor-mediated particle internalization in cells transfected with FcgammaRIIA receptors closely resemble the process of phagosomal maturation in neutrophils and macrophages. FcgammaRIIA-transfected cells can, therefore, be used as a model for the study of additional aspects of phagocyte biology.

Patients with alcohol problems in primary care: understanding their resistance and motivating change.

How can physicians motivate patients with alcohol problems when they resist advice to change? A framework for understanding patient resistance is described to help physicians use this motivational approach more effectively with patients.

Understanding and preventing substance abuse by adolescents: a guide for primary care clinicians.

Psychoactive drug use by teens is a common occurrence. This article examines the influences that promote and deter experimentation with and hazardous use of psychoactive substances. Clinical guidance is offered on how to assess and intervene with teens and their parents at various developmental phases and levels of involvement with drugs. Understanding how youth make decisions to change their behavior can assist a clinician in helping a teenager avoid these problems.

Family involvement in routine health care: a survey of patients' behaviors and preferences.

BACKGROUND: The purpose of this study was to assess the behavior and preferences of patients regarding family involvement in their routine health care visits. METHODS: A self-administered questionnaire was given to a convenience sample of patients visiting a family medicine center for an appointment. RESULTS: Thirty-nine percent of patients came to the physician's office with a family member or friend. Married patients and those with higher emotional involvement scores were significantly more likely to come to the office with someone. Two thirds of accompanied patients reported that this person came into the examination room with them. One third of the accompanied patients, however, thought that their physician was unaware that someone had accompanied them to the office. The majority (55%) of patients indicated that they would prefer to have a friend or family member in the examination room with them for some of their visits. No patient indicated that they never wanted a family member or friend to come into the examination room. CONCLUSIONS: Patients prefer direct family involvement in their health care more often than what occurs in practice. Physicians can easily address this discrepancy by asking patients whether and in what way they would like others to be involved in their health care.

Secondary prevention of excessive alcohol use: assessing the prospects of implementation.

BACKGROUND: Alcohol risk and harm reduction is a public health approach that goes beyond specialized treatments for alcoholism. The greatest potential for reducing alcohol risk and harm in a population depends on the extent to which health care practitioners use secondary prevention programmes. OBJECTIVE: We aim to assess the factors that affect the prospects of disseminating comprehensive, secondary prevention programmes into mainstream practice. METHOD: A decision balance was used to assess the prospects of practitioners implementing comprehensive programmes systematically. The stages-of-change model provides perspectives about behaviour change with regard to patients, practitioners and practice settings. RESULTS AND CONCLUSIONS: Programme implementation is extremely unlikely given the current organization of health care settings. To maintain the use of such programmes, we need to change the "unit of leverage" in the system: from the clinical encounter--that is, practitioners working with individual patients in a case-finding manner--to an organizational level--that is, the appropriate use of managerial and information systems supporting health care settings to identify at-risk patients systematically as they enter primary care and hospital settings. With appropriate infrastructure support, practitioners will be able to fulfil the potential for as well as maintain the use of comprehensive, secondary prevention programmes to reduce alcohol risk and harm in the population.

Motivating change in health behavior. Implications for health promotion and disease prevention.

Motivational approaches that build on the concepts of risk and harm reduction are new developments in the application of behavioral sciences to health promotion and disease prevention. This article develops a generic approach for helping practitioners motivate patients to change unhealthy behaviors.

The role of the Society of Teachers of Family Medicine in health care reform: a membership survey.

BACKGROUND AND OBJECTIVES: We conducted a survey of STFM members to: 1) measure perceived knowledge of and support for four health care reform proposals, 2) rate the members' priorities about specific legislative activities relevant to STFM, health care reform, and STFM general activities, and 3) assess interest in STFM developing a monograph on health care reform. METHODS: A self-administered questionnaire was mailed to a 15% random sample (n = 470) of STFM members. Researchers were blinded to the identity of respondents. RESULTS: Three hundred seven members returned completed surveys (response rate = 65%). Members were largely divided in their support between the pay or play and the single payer plans, with 39% (95% confidence interval (CI) 33%-44%) preferring the former and 34% (95% CI = 31%-37%) preferring the latter. Employer mandate and tax credit plans were rated less favorably. Overall, members rated their perceived knowledge about these plans as fair to good. In terms of rating their priorities about STFM activities, members gave the highest ratings to STFM legislative activities specific to the needs of academic family medicine (eg, faculty development, reimbursement for clinical and teaching activities, and research). These specific legislative activities were rated higher than all of the general categories of STFM activities. CONCLUSION: STFM members want STFM to advocate for specific legislation pertinent to the development of academic family medicine. Although most members support comprehensive health care reform, no single plan is preferred by a majority of members.

Dealing with substance misuse, abuse, and dependency.

Using this model, clinicians can enhance skills for screening, assessing, and aiding at-risk and problem drinkers. The six-step model incorporates the transtheoretic model of behavior change and uses motivational interviewing strategies and the concept of brief, early interventions. Primary care physicians can apply this model for patients in their offices and in hospital settings where they provide continuity of care. Furthermore, physicians can also use this model to intervene successfully at both the secondary and tertiary levels of prevention. In essence, this model uses a variety of strategies to aid at-risk and problem drinkers. These strategies can help patients and families overcome their ignorance about the role that alcohol plays in their lives and to motivate them toward a healthier lifestyle. Physicians can select strategies that range from simple advice to motivational counseling. Depending on the presenting problems and the likelihood and severity of an alcohol problem, the physician can select strategies described in this model to develop an individualized approach to motivate at-risk and problem drinkers to move through the phases of behavioral change: precontemplation, contemplation, preparation, action, and maintenance. Such an approach can help patients take responsibility for changing their drinking habits.

Home assessment of adherence to long-term medication in the elderly.

BACKGROUND: Drug nonadherence to long-term medication is a common and poorly understood problem in the elderly. A study was conducted to assess whether elderly patients would accept a research assistant conducting pill counts in their homes, and to examine how nonadherence was associated with patient variables. METHODS: A letter and a telephone follow-up call were used to identify eligible patients (aged 65 years and over, with two or more chronic diseases). RESULTS: A total of 98 eligible patients were identified. Fifty-nine agreed to participate in the study. Of the 59 participants, 54.7% were nonadherent to their medication regimen. Nonadherence was defined as an overall mean level of compliance of less than 80%. Drug regimen nonadherence was associated with the inability to read medication labels (P less than .01), but not with impaired visual acuity, the number of prescribed medications, the type of medication container lid, depression, cognitive impairment, perceived health status, or the cost of medications. Frequency of drug administration affected patient adherence to the medication regimen. Mean adherence of patients to prescriptions for drugs to be taken once or twice daily was 72%, whereas drugs to be taken three or four times daily had a mean adherence rate of 54% (P less than .01). CONCLUSIONS: Using the simple pill count method on home visits, rates of nonadherence to long-term medication were comparable to those found in studies using electronic pill-counting devices. Larger studies are needed to clarify how other variables, in addition to patient inability to read medication labels, are associated with noncompliance with medication regimens for chronic diseases in elderly patients.