Hyaluronidase increases electrogene transfer efficiency in skeletal muscle

Electrogene transfer (EGT) of plasmid DNA into skeletal muscle is a promising strategy for the treatment of muscle disorders and for the systemic secretion of therapeutic proteins. We report here that preinjecting hyaluronidase (HYAse) significantly increases the gene transfer efficiency of muscle EGT. Three constructs encoding mouse erythropoietin (pCMV/mEPO), secreted alkaline phosphatase (pCMV/SeAP), and luciferase (pGGluc) were electroinjected intramuscularly in BALB/c mice and rabbits with and without HYAse pretreatment. Preinjection 1 or 4 hr before EGT increased EPO gene expression by about 5-fold in mice and maintained higher gene expression than plasmid EGT alone. A similar increment in gene expression was observed on pretreatment with HYAse and electroinjection of pCMV/mEPO into rabbit tibialis muscle. The increment of gene expression in rabbits reached 17-fold on injection of plasmid pCMV/SeAP and 24-fold with plasmid pGGluc. Injection of a plasmid encoding beta-galactosidase (pCMV/beta gal/NLS) and subsequent staining with 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside indicated that HYAse increased the tissue area involved in gene expression. No irreversible tissue damage was observed on histological analysis of treated muscles. HYAse is used in a variety of clinical applications, and thus the combination of HYAse pretreatment and muscle EGT may constitute an efficient gene transfer method to achieve therapeutic levels of gene expression.     

Relative contribution of ecto-ATPase and ecto-ATPDase pathways to the biphasic effect of ATP on acetylcholine release from myenteric motoneurons

Background and purpose:

The relative contribution of distinct ecto-nucleotidases to the modulation of purinergic signalling may depend on differential tissue distribution and substrate preference.

Experimental approach:

Extracellular ATP catabolism (assessed by high-performance liquid chromatography) and its influence on [³H]acetylcholine ([³H]ACh) release were investigated in the myenteric plexus of rat ileum in vitro.

Key results:

ATP was primarily metabolized via ecto-ATPDase (adenosine 5′-triphosphate diphosphohydrolase) into AMP, which was then dephosphorylated into adenosine by ecto-5′-nucleotidase. Alternative conversion of ATP into ADP by ecto-ATPase (adenosine 5′-triphosphatase) was more relevant at high ATP concentrations. ATP transiently increased basal [³H]ACh outflow in a 2′,3′-O-(2,4,6-trinitrophenyl)adenosine-5′-triphosphate (TNP-ATP)-dependent, tetrodotoxin-independent manner. ATP and ATPγS (adenosine 5′-[γ-thio]triphosphate), but not α,β-methyleneATP, decreased [³H]ACh release induced by electrical stimulation. ADP and ADPβS (adenosine 5′[β-thio]diphosphate) only decreased evoked [³H]ACh release. Inhibition by ADPβS was prevented by MRS 2179 (2′-deoxy-N⁶-methyl adenosine 3′,5′-diphosphate diammonium salt, a selective P2Y₁ antagonist); blockade of ADP inhibition required co-application of MRS 2179 plus adenosine deaminase (which inactivates endogenous adenosine). Blockade of adenosine A₁ receptors with 1,3-dipropyl-8-cyclopentyl xanthine enhanced ADPβS inhibition, indicating that P2Y₁ stimulation is cut short by tonic adenosine A₁ receptor activation. MRS 2179 facilitated evoked [³H]ACh release, an effect reversed by the ecto-ATPase inhibitor, {"type":"entrez-protein","attrs":{"text":"ARL67156","term_id":"1186396857","term_text":"ARL67156"}}ARL67156, which delayed ATP conversion into ADP without affecting adenosine levels.

Conclusions and implications:

ATP transiently facilitated [³H]ACh release from non-stimulated nerve terminals via prejunctional P2X (probably P2X₂) receptors. Hydrolysis of ATP directly into AMP by ecto-ATPDase and subsequent formation of adenosine by ecto-5′-nucleotidase reduced [³H]ACh release via inhibitory adenosine A₁ receptors. Stimulation of inhibitory P2Y₁ receptors by ADP generated alternatively via ecto-ATPase might be relevant in restraining ACh exocytosis when ATP saturates ecto-ATPDase activity.     

Effect of Establishing Guidelines on Appropriate Urinary Catheter Placement

Objectives: Avoiding placement of unnecessary urinary catheters (UCs) in the emergency department (ED) affects UC utilization during hospitalization. The authors sought to evaluate the effect of establishing institutional guidelines for appropriate UC placement coupled with emergency physician (EP) education on UC utilization. Methods: Urinary catheter utilization was measured before and after the establishment of guidelines and EP education. Data collected included the presence of a UC on ED arrival, placement of a UC in the ED, documentation of a physician order for UC placement, reasons for placement, and compliance with the guidelines. Chi‐square analyses were used to study the association between pre‐ and postintervention time periods and catheter use. Results: A total of 377 (15%) patients had UCs; only 151 (47%) UCs initially placed in the ED had a physician order documented. UC placement was appropriately indicated in 75.5% of patients with a documented physician order, but in only 52% of cases without a documented physician order (p < 0.001). The physician intervention was associated with an overall reduction in UC utilization from 16.4% to 13% (p = 0.018). Physicians ordered 40% fewer UCs postintervention compared to preintervention. Preintervention, a physician order for UC placement was found indicated in 72.6% patients, compared to 82.2% patients with UC placed postintervention (p = 0.21). Conclusions: Establishing guidelines for UC placement and physician education in the ED were associated with a marked reduction in utilization. However, addressing appropriate UC utilization may require evaluating other factors such as nursing influence on utilization. ACADEMIC EMERGENCY MEDICINE 2010; 17:337–340 © 2010 by the Society for Academic Emergency Medicine     

Efficacy of Crocus sativus L. on reduction of cadmium‐induced toxicity on spermatogenesis in adult rats

Cadmium is a toxic heavy metal element, which probably cause infertility by impairment in spermatogenesis. The present work aimed (i) to study the toxic effect of cadmium on spermatogenesis in rat, as well as (ii) the protective effect of Crocus sativus L. on cadmium‐intoxicated rats. Cadmium chloride was administered intraperitoneally during 16 days at intervals of 48 h between subsequent treatments. Crocus sativus L. was pre‐treated in both of control and cadmium‐injected rats. Animals were sacrificed on day 17 after the first treatment. The left cauda epididymis was removed and immediately immersed into Hank's balanced salt solution for the evaluation of sperm count and viability, and left testis was fixed in 10% formalin for histological evaluation. Following contamination with cadmium, a decrease was observed in the number and viability of cauda epididymis sperm, which were increased by Crocus sativus L. pre‐treatment (P < 0.05). In addition, cadmium decreased both cell proliferation and Johnsen Scores in the seminiferous tubules, which were reversed by Crocus sativus pre‐treatment (P < 0.05). Furthermore, cadmium‐induced decrease in the amount of free serum testosterone as well as an increase in lipid peroxidation activity in the testicular tissue was reversed by Crocus sativus L. (P < 0.05). These findings may support the concept that Crocus sativus L. can improve the cadmium toxicity on spermatogenesis.     

Sexual behavior,communication, and chlamydial infections among college women

The sexual practices, partner communication patterns, and prevalence of chlamydial infection were determined in a sample of college women. Specific inclusion criteria were used to screen 146 participants. The presence of a mucopurulent cervical discharge was the only criterion significantly associated with infection. Only 12% of the sample used condoms alone or in combination with another birth control method, and only 31% of the women discussed with their partner their own or their partner's sexual history. Thirty‐eight percent of the sample reported a history of one to four episodes of a sexually transmitted disease, and 21% of the women stated they had had two or more sexual partners within the past 6 months. Health providers, counselors, and women themselves must address this health issue with more assertive behavior and communication skills.