Cytoplasmic CUG RNA Foci Are Insufficient to Elicit Key DM1 Features

The genetic basis of myotonic dystrophy type I (DM1) is the expansion of a CTG tract located in the 3′ untranslated region of DMPK. Expression of mutant RNAs encoding expanded CUG repeats plays a central role in the development of cardiac disease in DM1. Expanded CUG tracts form both nuclear and cytoplasmic aggregates, yet the relative significance of such aggregates in eliciting DM1 pathology is unclear. To test the pathophysiology of CUG repeat encoding RNAs, we developed and analyzed mice with cardiac-specific expression of a beta-galactosidase cassette in which a (CTG)₄₀₀ repeat tract was positioned 3′ of the termination codon and 5′ of the bovine growth hormone polyadenylation signal. In these animals CUG aggregates form exclusively in the cytoplasm of cardiac cells. A key pathological consequence of expanded CUG repeat RNA expression in DM1 is aberrant RNA splicing. Abnormal splicing results from the functional inactivation of MBNL1, which is hypothesized to occur due to MBNL1 sequestration in CUG foci or from elevated levels of CUG-BP1. We therefore tested the ability of cytoplasmic CUG foci to elicit these changes. Aggregation of CUG RNAs within the cytoplasm results both in Mbnl1 sequestration and in approximately a two fold increase in both nuclear and cytoplasmic Cug-bp1 levels. Significantly, despite these changes RNA splice defects were not observed and functional analysis revealed only subtle cardiac dysfunction, characterized by conduction defects that primarily manifest under anesthesia. Using a human myoblast culture system we show that this transgene, when expressed at similar levels to a second transgene, which encodes expanded CTG tracts and facilitates both nuclear focus formation and aberrant splicing, does not elicit aberrant splicing. Thus the lack of toxicity of cytoplasmic CUG foci does not appear to be a consequence of low expression levels. Our results therefore demonstrate that the cellular location of CUG RNA aggregates is an important variable that influences toxicity and support the hypothesis that small molecules that increase the rate of transport of the mutant DMPK RNA from the nucleus into the cytoplasm may significantly improve DM1 pathology.     

Moxifloxacin and azithromycin but not amoxicillin protect human respiratory epithelial cells against streptococcus pneumoniae in vitro when administered up to 6 hours after challenge

We determined the protective effect of moxifloxacin, azithromycin, and amoxicillin against Streptococcus pneumoniae infection of respiratory cells. Moxifloxacin and azithromycin effectively killed intracellular S. pneumoniae strains and protected respiratory epithelial cells significantly even when given 6 h after S. pneumoniae challenge. Amoxicillin was less effective.     

Early diagnosis of septic arthritis in immunocompromised patients

Objectives

Septic arthritis results in rapid joint destruction if not properly diagnosed and treated. A work up for septic arthritis includes a peripheral white blood cell count, inflammatory markers, and a joint aspiration. In the general population, the interpretation of these labs has been well-defined by prior studies. To this point, no study has determined how immunosuppressive states affect this work up.

An osteopenic nonfracture syndrome with features of mild osteogenesis imperfecta associated with the substitution of a cysteine for glycine at triple helix position 43 in the pro alpha 1(I) chain of type I collagen.

Mutations affecting the pro alpha 1(I) or pro alpha 2(I) collagen genes have been identified in each of the major clinical types of osteogenesis imperfecta. This study reports the presence of a heritable connective tissue disorder in a family with an osteopenic syndrome which has features of mild osteogenesis imperfecta but was considered idiopathic osteoporosis in the proband. At age 38, while still premenopausal, she was found to have osteopenia, short stature, hypermobile joints, mild hyperelastic skin, mild scoliosis, and blue sclerae. There was no history of vertebral or appendicular fracture. Hip and vertebral bone mineral density measurements were consistent with marked fracture risk. Delayed reduction SDS-PAGE of pepsin-digested collagens from dermal fibroblast cultures demonstrated an anomalous band migrating between alpha 1(I) and alpha 1(III). This band merged with the normal alpha-chains upon prereduction, indicating an unexpected cysteine residue. Cyanogen bromide peptide mapping suggested that the mutation was in the smaller NH2-terminal peptides. cDNA was reverse transcribed from mRNA and amplified by the polymerase chain reaction. A basepair mismatch between proband and control alpha 1(I) cDNA hybrids was detected by chemical cleavage with hydroxylamine:piperidine. The cysteine substitution was thus localized to alpha 1(I) exon 9 within the cyanogen bromide 4 peptide. Nucleotide sequence analysis localized a G----T point mutation in the first position of helical codon 43, replacing the expected glycine (GGT) residue with a cysteine (TGT). The prevalence of similar NH2-terminal mutations in subjects with this phenotype which clinically overlaps idiopathic osteoporosis remains to be determined.     

Effects of early and late intravenous norepinephrine infusion on cerebral perfusion,microcirculation, brain-tissue oxygenation,and edema formation in brain-injured rats

OBJECTIVES: Reduction of cerebral perfusion during the early phase after traumatic brain injury is followed by a later phase of normal to increased perfusion. Thus, pharmacologically elevating mean arterial blood pressure with the aim of improving cerebral perfusion may exert different time-dependent effects on cortical perfusion, microcirculation, tissue oxygenation and brain edema formation after traumatic brain injury. DESIGN: Randomized, placebo-controlled trial. SETTING: Experimental laboratory at a university hospital. SUBJECTS: A total of 37 male Sprague-Dawley rats subjected to a focal cortical contusion. INTERVENTIONS: At 4 or 24 hrs after focal traumatic brain injury, mean arterial blood pressure was increased to 120 mm Hg for 90 mins by infusing norepinephrine. In rats receiving physiologic saline, mean arterial blood pressure remained unchanged. In the first series, pericontusional cortical perfusion was measured using the laser Doppler flowmetry scanning technique before injury and before, during, and after the infusion period. In a second series, intracranial and cerebral perfusion pressure and intraparenchymal perfusion and tissue oxygen measured within the contused and pericontusional cortex were recorded continuously before, during, and after norepinephrine infusion. Changes in cortical microcirculation were investigated by orthogonal polarization spectral imaging. At the end of each experiment, hemispheric swelling and water content were determined gravimetrically. MEASUREMENTS AND MAIN RESULTS: At 4 and 24 hrs after traumatic brain injury, intravenous norepinephrine significantly increased pericontusional cortical perfusion, which was also reflected by an increase in diameters and flow velocities of pericontusional arterioles and venules. Cerebral perfusion pressure and intraparenchymal perfusion and tissue oxygen were significantly increased during norepinephrine infusion at 4 and 24 hrs. Hemispheric swelling and water content showed no difference between the groups. CONCLUSIONS: After cortical impact injury, early and late intravenous norepinephrine infusion pressure-dependently increased cerebral perfusion and tissue oxygenation without aggravating or reducing brain edema formation. Future studies are warranted to determine long-term changes of short and prolonged norepinephrine-induced increases in mean arterial blood pressure and cerebral perfusion pressure.     

New dip-and-read test for determining leukocytes in urine

A new reagent strip for the determination of leukocytes in urine (LEUKOSTIX; Ames) is described. The test is based on the esterase activity in leukocytes as a marker. Upon contact between the reagent matrix and a urine containing leukocytes, an amino acid ester is hydrolyzed by the esterase to its corresponding alcohol. The free alcohol then couples with a diazonium salt to produce a purple azo dye. The relative concentration of leukocytes in the urine is obtained by visually comparing the strip reaction with a color chart. Performance of the strip was evaluated in a clinical study involving eight different sites and 867 urine specimens. The comparison method was sediment microscopy; specimens containing five cells or more per high-power field were considered to be positive. Sensitivity was 76.3%, specificity 80.8%. Performance was comparable with that of the CHEMSTRIP LN (Boehringer-Mannheim Diagnostics, Inc.) leukocyte test, which we evaluated concurrently.     

Reporter gene technology to assess activity of antimycobacterial agents in macrophages.

Reporter strains of Mycobacterium tuberculosis and Mycobacterium bovis BCG endogenously expressing firefly luciferase were used in bioluminescence assays to evaluate the activities of isoniazid and rifampin against mycobacteria sequestered in human macrophages. This methodology allowed the efficacy of antibiotics against intracellular mycobacteria to be assessed without the labor-intensive procedures and protracted incubation requirements associated with conventional CFU determinations.