Use of serum-free hormonally defined media to evaluate the effects of growth factors and inhibitors on proliferation of estrogen-responsive mammary and pituitary tumor cells in culture

Summary Serum-free defined media have been developed for assay of the mitogenic effects of growth factors on human MCF-7, human T-47D, and mouse COMMA-D mammary cells as well as for identification of mitogens and inhibitors of GH₄C₁ rat pituitary tumor cell growth. These lines were shown to grow in vivo in response to a variety of hormones including estrogens and thyroid hormones. With mammary cells, complete hormonally and nutritionally defined media were established that supported continuous passage at 50 to 90% of the serum stimulated rate. The strategy used to measure mitogens for mammary cells was to identify nutritional conditions where the growth rate was reduced greatly without impairing the response to picomolar to nanomolar concentration of growth factors. The effects of polypeptide growth factors and tissue extracts were estimated by their addition to basal medium and measuring cell number increases or labeled thymidine incorporation into DNA. In a variation of this methodology, the MTW9/PL2 rat mammary cells were used to identify secreted autocrine growth factors; nutritionally defined conditions were sought for growth of these rat cells in the complete absence of exogenous growth factors. The factors secreted into the medium were detected by bioassays with COMMA-D or MCF-7 mammary cell lines. The effects of growth factors-inhibitors on pituitary cells were measured by a related method; the GH₄C₁ cells were grown at less than optimal rates in a defined medium designated TRM-1. Addition of mitogens to TRM-1 stimulated pituitary cell growth whereas addition of inhibitors caused reduced levels of growth. The methods described in this report offer new means of assaying growth factors-inhibitors for a range of mammary and pituitary tumor cells.     

Interpersonal stressors and resources as predictors of parental adaptation following pediatric traumatic injury

The authors examined the relationship of preinjury interpersonal resources and stressors to parental adaptation following pediatric traumatic brain injury (TBI) and orthopedic injury. Parents of children with severe TBI (n = 53), moderate TBI (n = 56), and orthopedic injuries (n = 80) were assessed soon after injury, 6 and 12 months after the initial evaluation, and at an extended follow-up with a mean of 4 years postinjury. General linear model analyses provide support for both main and moderating effects of stressors and resources on parental adjustment. Support from friends and spouse was associated with less psychological distress, whereas family and spouse stressors were associated with greater distress. The results also reveal a marked decline in injury-related stress over follow-up for families in the severe TBI group who reported a combination of high stressors and high resources. The decline suggests that interpersonal resources attenuated long-term family burden because of severe TBI. The findings are discussed in terms of their implications for intervention following TBI.     

Influences of chorion type on measurements of the corpus callosum in adult monozygotic male twins?

MRI imaging was used to estimate volumes of corpus callosum structure in 45 pairs of identical (monozygotic, MZ) twins from the National Heart, Lung, and Blood Institute (NHLBI) twin study. Age range of the study subjects was from 68–78 years. Finger, palm, and footprint data (dermatoglyphics) collected at previous examinations of the NHLBI twin study were available for 39 pairs. The dermatoglyphics were scored for an index to retrospectively assess chorion type in MZ twin‐pairs. The results indicated an association between variability in various structures of the corpus callosum with some of these dermatoglyphic traits, suggesting greater structural variation within pairs with dichorionic placentas. In contrast, total intracranial volume, which has similar heritability estimates as a result of shared genetic effects with the corpus callosum, was unrelated to the dermatoglyphic traits. The results provide indirect evidence that the intrauterine environment may influence twin‐pair similarity of corpus callosum measures in adults. Am. J. Hum. Biol. 14:338–346, 2002. © 2002 Wiley‐Liss, Inc.     

Detection of Cattle Naturally Infected with Anaplasma marginale in a Region of Endemicity by Nested PCR and a Competitive Enzyme-Linked Immunosorbent Assay Using Recombinant Major Surface Protein 5

A competitive enzyme-linked immunosorbent assay using recombinant major surface protein 5 (rMSP5-cELISA) of Anaplasma marginale was validated in a naturally infected cattle herd in an area of eastern Oregon where A. marginale is endemic. The true positive and negative A. marginale infection status of 235 randomly selected cattle was determined by using a nested PCR (nPCR) coupled with msp5 sequence analysis and hybridization. Judgment of the reliability of the nPCR and hybridization for detection of persistent infections was based on three observations. First, the nPCR was able to detect as few as 30 infected erythrocytes per ml. Second, the nPCR was able to consistently detect low levels of rickettsemia in seven carrier cattle experimentally infected with A. marginale. Third, msp5 sequence analysis showed >95% identity among 30 nPCR amplicons from cattle naturally infected with field strains of A. marginale. The nPCR and hybridization identified 151 infected and 84 uninfected cattle among the 235 animals tested. With a cutoff point of 28%, the rMSP5-cELISA showed a sensitivity of 96% and a specificity of 95%. These results indicate that the rMSP5-cELISA can sensitively and specifically detect cattle with naturally acquired persistent A. marginale infections and suggest that it is an excellent assay for epidemiological studies, eradication programs, and regulation of international cattle movement.     

Preferences for self-care or professional advice for minor illness: a discrete choice experiment

AIM: To determine the relative importance of factors that influence decision making in the management of minor illness, and how people trade between these factors. DESIGN OF STUDY: Discrete choice experiment.Setting:Scottish electoral roll. METHOD: Six hundred and fifty-two responders of a previous national survey were invited to complete a discrete choice experiment questionnaire. This was used to measure relative preferences for managing symptoms of minor illness often associated with analgesic use. Three attributes were identified as important to participants: type of management, availability, and cost of managing symptoms. Trade-offs between these attributes were examined. RESULTS: A 57% response rate was achieved (51% valid response rate). People preferred to manage symptoms by self-care and were willing to pay almost pounds 23 to do so. Community pharmacy was the preferred source of advice. Responders preferred less waiting time and paying less money when managing symptoms, and were willing to trade between factors. A less preferred type of management became more attractive when waiting times and cost were reduced. CONCLUSION: Findings suggest that self-care is the preferred method of managing symptoms of minor illness. When developing services to support self-care, policy makers should invest in services that reduce waiting times and incur least cost to users.     

Genes for glycosylphosphatidylinositol toxin biosynthesis in Plasmodium falciparum

About 2.5 million people die of Plasmodium falciparum malaria every year. Fatalities are associated with systemic and organ-specific inflammation initiated by a parasite toxin. Recent studies show that glycosylphosphatidylinositol (GPI) functions as the dominant parasite toxin in the context of infection. GPIs also serve as membrane anchors for several of the most important surface antigens of parasite invasive stages. GPI anchoring is a complex posttranslational modification produced through the coordinated action of a multicomponent biosynthetic pathway. Here we present eight new genes of P. falciparum selected for encoding homologs of proteins essential for GPI synthesis: PIG-A, PIG-B, PIG-M, PIG-O, GPI1, GPI8, GAA-1, and DPM1. We describe the experimentally verified mRNA and predicted amino acid sequences and in situ localization of the gene products to the parasite endoplasmic reticulum. Moreover, we show preliminary evidence for the PIG-L and PIG-C genes. The biosynthetic pathway of the malaria parasite GPI offers potential targets for drug development and may be useful for studying parasite cell biology and the molecular basis for the pathophysiology of parasitic diseases.     

Changes in human muscle transverse relaxation following short-term creatine supplementation

The rapid increase in body mass that often occurs following creatine (Cr) supplementation is believed to be due to intracellular water retention. The purpose of this study was to determine whether Cr consumption alters the magnetic resonance (MR) transverse relaxation (T(2)) distribution of skeletal muscle. Transverse relaxation can be used to model water compartments within a cell or tissue. In this double-blind study, subjects were asked to supplement their normal diet with creatine monohydrate (20 g day(-1) for 5 days) mixed with a grape drink (Creatine group, n = 7), or the grape drink alone (Placebo group, n = 8). Phosphorous MR spectroscopy was used to determine the effectiveness of the supplementation protocol. Subjects that responded to the Cr supplementation (i.e. showed a > 5 % increase in the ratio of the levels of phosphocreatine (PCr) and ATP) were placed in the Creatine group. Both proton MR imaging and spectroscopy were used to acquire T(2) data, at 1.89 T, from the flexor digitorum profundus muscle of each subject before and after supplementation. Following the supplementation period, the Creatine group showed a gain in body mass (1.2 +/- 0.8 kg, P < 0.05, mean +/- S.D.), and an increase in PCr/ATP ratio (23.8 +/- 16.4 %, P < 0.001). Neither group showed any changes in intracellular pH or T(2) calculated from MR images. However, the spectroscopy data revealed at least three components (> 5 ms) at approximately 20, 40 and 125 ms in both groups. Only in the Creatine group was there an increase in the apparent proton concentration of the two shorter components combined (+5.0 +/- 4.7 %, P < 0.05). According to the cellular water compartment model, the changes observed in the shorter T(2) components are consistent with an increase in intracellular water.     

Babesia bovis merozoite surface antigen 1 and rhoptry-associated protein 1 are expressed in sporozoites, and specific antibodies inhibit sporozoite attachment to erythrocytes

We examined Babesia bovis sporozoites for the expression of two molecules, merozoite surface antigen 1 (MSA-1) and rhoptry-associated protein 1 (RAP-1), that are postulated to be involved in the invasion of host erythrocytes. Both MSA-1 and RAP-1 were transcribed and expressed in infectious sporozoites. Importantly, monospecific MSA-1 and RAP-1 antisera each inhibited sporozoite invasion of erythrocytes in vitro. This is the first identification of antigens expressed in Babesia sp. sporozoites and establishes that, at least in part, sporozoites and merozoites share common targets of antibody mediated inhibition of erythrocyte invasion.