Molecular mechanism of temperature sensing by the circadian clock of Neurospora crassa

Expression levels and ratios of the long (l) and short (s) isoforms of the Neurospora circadian clock protein FREQUENCY (FRQ) are crucial for temperature compensation of circadian rhythms. We show that the ratio of l-FRQ versus s-FRQ is regulated by thermosensitive splicing of intron 6 of frq, a process removing the translation initiation site of l-FRQ. Thermosensitivity is due to inefficient recognition of nonconsensus splice sites at elevated temperature. The temperature-dependent accumulation of FRQ relative to bulk protein is controlled at the level of translation. The 5'-UTR of frq RNA contains six upstream open reading frames (uORFs) that are in nonconsensus context for translation initiation. Thermosensitive trapping of scanning ribosomes at the uORFs leads to reduced translation of the main ORF and allows adjustment of FRQ levels according to ambient temperature.     

Lipopolysaccharide-binding proteins of Limulus amebocyte lysate.

Limulus amebocyte lysate, obtained from horseshoe crab (Limulus polyphemus) blood cells, contains a coagulation system which is activated by bacterial lipopolysaccharide (LPS). A chromatographic fraction of Limulus lysate, containing the endotoxin-sensitive factor(s) which initiates the coagulation cascade, was studied. We utilized a photoreactive, cleavable, radiolabeled derivative of Salmonella minnesota LPS, LPS-(p-azidosalicylamido)-1,3'-dithiopropionamide (LPS-ASD), to identify LPS-binding proteins. The lysate fraction was incubated with LPS-ASD, and LPS-binding proteins were identified by autoradiography of sodium dodecyl sulfate-polyacrylamide gels. An 82-kDa protein, a major protein component of this fraction from Limulus lysate, was identified as a LPS-binding protein in a majority of lysates. Incubation of whole Limulus lysate with antiserum to this protein resulted in enhanced sensitivity of the lysate to LPS, suggesting that this 82-kDa protein is a negative regulator of coagulation. A minor 50-kDa protein component of lysate also was identified as a LPS-binding protein and is a candidate for the LPS-sensitive coagulation protein in L. polyphemus.     

Risk factors for wheezing in a subtropical environment: role of respiratory viruses and allergen sensitization

BACKGROUND: Risk factors for acute wheezing among children in subtropical areas are largely unknown. OBJECTIVE: To investigate the role of viral infections, allergen sensitization, and exposure to indoor allergens as risk factors for acute wheezing in children 0 to 12 years old. METHODS: One hundred thirty-two children 0 to 12 years of age who sought emergency department care for wheezing and 65 children with no history of wheezing were enrolled in this case-control study. Detection of respiratory syncytial virus antigen, rhinovirus and coronavirus RNA, adenovirus, influenza, and parainfluenza antigens was performed in nasal washes. Total IgE and specific IgE to mites, cockroach, cat, and dog were measured with the CAP system. Major allergens from mites, cockroach, cat, and dog were quantified in dust samples by ELISA. Univariate and multivariate analyses were performed by logistic regression. RESULTS: In children under 2 years of age, infection with respiratory viruses and family history of allergy were independently associated with wheezing (odds ratio, 15.5 and 4.2; P = .0001 and P = .008, respectively). Among children 2 to 12 years old, sensitization to inhalant allergens was the major risk factor for wheezing (odds ratio, 2.7; P = .03). High-level allergen exposure, exposure to tobacco smoke, and lack of breast-feeding showed no association with wheezing. CONCLUSIONS: Some risk factors for wheezing previously identified in temperate climates were present in a subtropical area, including respiratory syncytial virus infection in infants and allergy in children older than 2 years. Rhinovirus was not associated with wheezing and did not appear to be a trigger for asthma exacerbations.     

The use of multistate life-table models for improving population health

We demonstrate how incidence, prevalence, remission, mortality(IPRM) models may be constructed on population life-tables,how the incidence of a condition may be calculated, and howthe consequences of demographic changes and public health interventionsmay be predicted. We illustrate the methodology by applyingit to the epidemiology of diabetes, physical inactivity andobesity in New Zealand.     

Video-rate nonlinear microscopy of neuronal membrane dynamics with genetically encoded probes

Biological membranes decorated with suitable contrast agents give rise to nonlinear optical signals such as two-photon fluorescence and harmonic up-conversion when illuminated with ultra-short, high-intensity pulses of infrared laser light. Microscopic images based on these nonlinear contrasts were acquired at video or higher frame rates by scanning a focused illuminating spot rapidly across neural tissues. The scan engine relied on an acousto-optic deflector (AOD) to produce a fast horizontal raster and on corrective prisms to offset the AOD-induced dispersion of the ultra-short excitation light pulses in space and time. Two membrane-bound derivatives of the green fluorescent protein (GFP) were tested as nonlinear contrast agents. Synapto-pHluorin, a pH-sensitive GFP variant fused to a synaptic vesicle membrane protein, provided a time-resolved fluorescent read-out of neurotransmitter release at genetically specified synaptic terminals in the intact brain. Arrays of dually lipidated GFP molecules at the plasma membrane generated intense two-photon fluorescence but no detectable second-harmonic power. Comparison with second-harmonic generation by membranes stained with a synthetic styryl dye suggested that the genetically encoded chromophore arrangement lacked the orientational anisotropy and/or dipole density required for efficient coherent scattering of the incident optical field.     

Analgesia after inguinal herniorrhaphy with laparoscopic inspection of the peritoneum in children. Caudal block versus ilioinguinal/iliohypogastric block

The authors prospectively evaluated the efficacy of caudal epidural block versus local infiltration combined with ilioinguinal/iliohypogastric block for analgesia after inguinal herniorrhaphy with laparoscopic inspection of the peritoneum. During standardized anesthetic care, 24 children were randomized to Group I (caudal epidural block with 1.2 mL/kg of 0.25% bupivacaine) or to Group II (local infiltration with an ilioinguinal/iliohypogastric block). Postoperative pain scores were significantly lower at all four evaluation points in Group I than in Group II. Patients in Group I had a significantly decreased requirement for supplemental intravenous fentanyl. Intra-operative requirements for isoflurane were decreased in Group I. The expired concentration of isoflurane was 0.4 +/- 0.1 (mean +/- SEM) in Group I and 1.5 +/- 0.3 in Group II. Time to extubation was 3.8 +/- 0.5 minutes in Group I and 8.2 +/- 1.1 minutes in Group II. The time from arrival in the postanesthesia care unit until discharge home was 113 +/- 3 minutes in Group I and 152 +/- 11 minutes in Group II. Caudal epidural block was more effective than local infiltration in controlling pain after herniorrhaphy with laparoscopy in children and resulted in earlier discharge home.