Differential roles of the Leloir pathway enzymes and metabolites in defining galactose sensitivity in yeast

The metabolism of galactose via enzymes of the Leloir pathway: galactokinase, galactose-1-P uridylyltransferase, and UDP galactose-4'-epimerase, is a process that has been conserved from Escherichia coli through humans. Impairment of this pathway in patients results in the disease galactosemia. Despite decades of study, the underlying pathophysiology in galactosemia remains unknown. Here we have defined the functional and metabolic implications of impaired galactose metabolism in yeast, by asking two questions: (1) What is the impact of loss of each of the three Leloir enzymes on the ability of cells to metabolize galactose, and on their sensitivity to galactose, and (2) what is the relationship between gal-1P and galactose-sensitivity in yeast? Our results demonstrate that only transferase-null cells are able to deplete their medium of galactose; deletion of kinase or epimerase halts this process. In contrast, only kinase-null cultures grow well in glycerol/ethanol medium despite the addition of galactose; both transferase and epimerase-null yeast arrest growth under these conditions. Indeed, epimerase-null yeast arrest growth at galactose concentrations 10-fold lower than do their transferase-null counterparts. Secondary deletion of kinase relieves growth arrest in both strains. Finally, rather than a continuous relationship between gal-1P and growth arrest, we observed a threshold level of gal-1P (approximately 10 nmol/mg cell DM) above which both transferase-null and epimerase-null cultures could not grow. These results both confirm and significantly extend prior knowledge of galactose metabolism in yeast, and set the stage for future studies into the mediators and mechanism of Leloir-impaired galactose sensitivity in eukaryotes.     

Update: Health Insurance and Utilization of Care Among Rural Adolescents

CONTEXT: Adolescence is critical for the development of adult health habits. Disparities between rural and urban adolescents and between minority and white youth can have life-long consequences. PURPOSE: To compare health insurance coverage and ambulatory care contacts between rural minority adolescents and white and urban adolescents. METHODS: Cross-sectional design using data from the 1999-2000 National Health Interview Survey, a nationally representative sample of US households. Analysis was restricted to white, black, and Hispanic children aged 12 through 17 (8,503 observations). Outcome measures included health insurance, ambulatory visit within past year, usual source of care (USOC), and well visit within past year. Independent variables included race, residence, demographics, facilitating/enabling characteristics, and need. RESULTS: Across races, rural adolescents were as likely to have insurance (86.8% vs 87.7%) but less likely to report a preventive visit (60.1% vs 65.5%) than urban children; residence did not affect the likelihood of a visit or a USOC. Minority rural adolescents were less likely than whites to be insured, report a visit, or have a USOC. Most race-based differences were not significant in multivariate analysis holding constant living situation, caretaker education, income, and insurance. Low caretaker English fluency, limited almost exclusively to Hispanics, was an impediment to all outcomes. CONCLUSIONS: Most barriers to care among rural and minority youth are attributable to factors originating outside the health care system, such as language, living situation, caretaker education, and income. A combination of outreach activities and programs to enhance rural schools and economic opportunities will be needed to improve coverage and utilization among adolescents.     

Comparative preclinical and clinical pharmacokinetics of a cremophor-free, nanoparticle albumin-bound paclitaxel (ABI-007) and paclitaxel formulated in Cremophor (Taxol).

PURPOSE: To compare the preclinical and clinical pharmacokinetic properties of paclitaxel formulated as a Cremophor-free, albumin-bound nanoparticle (ABI-007) and formulated in Cremophor-ethanol (Taxol). EXPERIMENTAL DESIGN: ABI-007 and Taxol were given i.v. to Harlan Sprague-Dawley male rats to determine pharmacokinetic and drug disposition. Paclitaxel pharmacokinetic properties also were assessed in 27 patients with advanced solid tumors who were randomly assigned to treatment with ABI-007 (260 mg/m(2), 30 minutes; n = 14) or Taxol (175 mg/m(2), 3 hours; n = 13), with cycles repeated every 3 weeks. RESULTS: The volume of distribution at steady state and clearance for paclitaxel formulated as Cremophor-free nanoparticle ABI-007 were significantly greater than those for paclitaxel formulated with Cremophor (Taxol) in rats. Fecal excretion was the main elimination pathway with both formulations. Consistent with the preclinical data, paclitaxel clearance and volume of distribution were significantly higher for ABI-007 than for Taxol in humans [21.13 versus 14.76 L/h/m(2) (P = 0.048) and 663.8 versus 433.4 L/m(2) (P = 0.040), respectively]. CONCLUSIONS: Paclitaxel formulated as ABI-007 differs from paclitaxel formulated as Taxol, with a higher plasma clearance and a larger volume of distribution. This finding is consistent with the absence of paclitaxel-sequestering Cremophor micelles after administration of ABI-007. This unique property of ABI-007 could be important for its therapeutic effectiveness.     

Cardiovascular Effects of Medical Marijuana: A Systematic Review

Utilization of marijuana as a medicinal agent is becoming increasingly popular, and so far, 25 states have legalized it for medical purposes. However, there is emerging evidence that marijuana use can result in cardiovascular side effects, such as rhythm abnormalities, syncope/dizziness, and myocardial infarction, among others. Further, there are currently no stringent national standards or approval processes, like Food and Drug Administration (FDA) evaluation, in place to assess medical marijuana products. This review includes the largest up-to-date pooled population of patients with exposure to marijuana and reported cardiovascular effects. Although purported as benign by many seeking to advance the use of marijuana as an adjunctive medical therapy across the country, marijuana is associated with its own set of cardiovascular risks and deserves further definitive study and the same level of scrutiny we apply in research of all other types of medications. When used as a medicinal agent, marijuana should be regarded accordingly, and both clinical providers and patients must be aware of potential adverse effects associated with its use for early recognition and management.     

Damage patterns observed in mtDNA control region MPS data for a range of template concentrations and when using different amplification approaches

International Journal of Legal Medicine - Massively parallel sequencing (MPS) of mitochondrial (mt) DNA allows practitioners the ability to fully resolve heteroplasmic sites. In forensic DNA...     

The Communication Supports Inventory-Children & Youth (CSI-CY), a new instrument based on the ICF-CY

Purpose: Two studies are presented that evaluated the Communication Supports Inventory-Children & Youth (CSI-CY), an instrument designed to facilitate the development of communication-related educational goals for students with complex communication needs (CCN). The CSI-CY incorporates a code set based on the ICF-CY. The studies were designed to determine the effect of using the CSI-CY on IEP goals for students with CCN and to evaluate consumer satisfaction.

Method: In Study 1, sixty-one educators and speech–language pathologists were randomly assigned to either (a) provide a student’s current IEP (control group) or (b) complete the CSI-CY prior to preparing a student’s next IEP and to submit the new IEP (experimental group). Study 2 was a field test to generate consumer satisfaction data.

Results: Study 1 showed that IEP goals submitted by participants in the experimental group referenced CSI-CY-related content significantly more frequently than did those submitted by control participants. Study 2 revealed high satisfaction with the instrument.

Conclusions: The code set basis of the CSI-CY extends the common language of the ICF-CY to practical educational use for children with CCN across diagnostic groups. The CSI-CY is well regarded as an instrument to inform the content of communication goals related to CCN.

• Implications for Rehabilitation

• The CSI-CY will guide rehabilitation professionals to develop goals for children with complex communication impairments.

• The CSI-CY is a new instrument that is based on the ICF-CY for documentation of communication goals.

     

Incorporation of human complement C8 into the membrane attack complex is mediated by a binding site located within the C8beta MACPF domain

Human C8 is one of five complement components (C5b, C6, C7, C8, C9) that interact to form the membrane attack complex (MAC). C8 is an oligomeric protein composed of a disulfide-linked C8alpha-gamma heterodimer and a noncovalently associated C8beta chain. C8alpha and C8beta are homologous; both contain N- and C-terminal modules and an intervening approximately 40 kDa segment referred to as the membrane attack complex/perforin (MACPF) domain. C8beta participates in at least two binding interactions. It has a high affinity binding site for C8alpha, which facilitates its interaction with C8alpha-gamma. C8beta also mediates incorporation of C8 into the MAC by binding to C5b-7, an intermediate in the MAC assembly pathway. Little is known about the location or properties of the respective binding sites on C8beta. In this study, the MACPF domain of C8beta (betaMACPF) was expressed in Escherichia coli and its role in binding C8alpha and C5b-7 examined. Recombinant betaMACPF was shown to bind C8alpha-gamma in solution and form a noncovalent complex (betaMACPF*C8alpha-gamma) that exhibited C8 hemolytic activity. betaMACPF was also capable of binding independently to erythrocytes carrying C5b-7. Subsequent addition of C8alpha-gamma and C9 to these cells produced a hemolytically active MAC. The ability to produce a soluble, recombinant betaMACPF that retains the binding functions of C8beta suggests this segment of C8beta is an independently folded domain. Furthermore, results indicate the principal binding sites for C8alpha and C5b-7 are located within this domain, and that C8beta binding specificity is not determined by the N- and C-terminal modules.     

Effect of Rhamnolipids on Permeability Across Caco-2 Cell Monolayers

Purpose

This report describes the effect of rhamnolipids (RLs), an amphiphilic biosurfactant produced by the bacterium Pseudomonas aeruginosa, on the integrity and permeability across Caco-2 cell monolayers.

Methods

We measured the trans-epithelial electrical resistance (TEER) and permeability of [¹⁴C]mannitol across Caco-2 cell monolayers upon incubation with 0.01–5.0% v/v RLs as a function of incubation time (30, 60, 90, and 120 min). We also studied the recovery of RL-treated Caco-2 cell monolayers upon incubation with Kaempferol, which is a natural flavonoid that promotes the assembly of the tight junctions.

Results

TEER of Caco-2 cell monolayers incubated with 0.01–5.0% v/v RLs solution dropped to 80–28% of that of untreated cells. Decline in TEER was associated with an increase in [¹⁴C]mannitol permeability as a function of RLs concentration and incubation time with Caco-2 cells. Incubation of RLs-treated Caco-2 cell monolayers with normal culture medium for 48 h did not restore barrier integrity. Whereas, incubation of a RLs-treated Caco-2 cells with culture medium containing Kaempferol for 24 h restored barrier function indicated by the higher TEER and lower [¹⁴C]mannitol permeability values.

Conclusions

These results show the ability of RLs to modulate the integrity and permeability of Caco-2 cell monolayers in a concentration- and time-dependent fashion, which suggest their potential to function as a non-toxic permeation enhancer.     

American College of Surgeons/Association for Surgical Education medical student simulation-based surgical skills curriculum needs assessment

Background

Simulation can enhance learning effectiveness, efficiency, and patient safety and is engaging for learners.

Methods

A survey was conducted of surgical clerkship directors nationally and medical students at 5 medical schools to rank and stratify simulation-based educational topics. Students applying to surgery were compared with others using Wilcoxon's rank-sum tests.

Results

Seventy-three of 163 clerkship directors (45%) and 231 of 872 students (26.5%) completed the survey. Of students, 28.6% were applying for surgical residency training. Clerkship directors and students generally agreed on the importance and timing of specific educational topics. Clerkship directors tended to rank basic skills, such as examination skills, higher than medical students. Students ranked procedural skills, such as lumbar puncture, more highly than clerkship directors.

Conclusions

Surgery clerkship directors and 4th-year medical students agree substantially about the content of a simulation-based curriculum, although 4th-year medical students recommended that some topics be taught earlier than the clerkship directors recommended. Students planning to apply to surgical residencies did not differ significantly in their scoring from students pursuing nonsurgical specialties.