Systemic delivery of enkephalin peptide through eyes

Leu-enkephalin is one of the important peptides which could become useful drugs in the clinics because of its analgesic action and its availability in mass quantity through biotechnology production. It is found in this study that enkephalin can be effectively absorbed systemically through eyes without using a surfactant as absorption enhancer. Enkephalin at 0.125% (31.25 micrograms/25 microliter) reached a plateau of blood concentration at 11.5 ng/ml in 3-4 hrs and stayed high for 8-9 hrs. In contrast, the blood concentration of enkephalin declined rapidly after i.v. administration with a T1/2 of less than 30 min and reached the lowest point at 22 ng/ml in 5 hrs. With higher concentrations at 1% (.25 mg/25 microliter) and 5% (1.25 mg/25 microliter) similar absorption kinetics was observed except that they reached higher plateau of blood concentration at 72 ng/ml and 233 ng/ml, respectively.     

Biochemical characterization of crystallins from frog lenses

Lens crystallins were isolated from the homogenate of frog (Rana catesbeiana) eye lenses by gel permeation chromatography and characterized by gel electrophoresis, amino acid analysis and circular dichroism. Four well-defined fractions corresponding to alpha/beta-, beta-, frog 39.5 kDa and gamma-crystallins comprising the relative weight percentages in the total soluble cytoplasmic proteins of 18%, 15%, 14% and 48% respectively were obtained. The native molecular masses for each purified fraction were determined to be 432, 207, 40 and 23 kDa, respectively. The polypeptide compositions as determined by SDS-gel electrophoresis revealed the typical subunit structures of mammalian crystallins with the exception of 39.5 kDa monomeric crystallin, which has not been shown in other classes of vertebrate lenses. The spectra of circular dichroism indicate a predominant beta-sheet structure in all four fractions, which also bears a resemblance to the secondary structure of mammalian crystallins. Comparison of the amino acid compositions of frog crystallins with those of mammalian and fish crystallins suggests that gamma-crystallin from the frog is more closely related to that of porcine than fish crystallins, and the frog 39.5 kDa, frog beta- and lamprey 48 kDa crystallins are probably mutually interrelated.     

Plasma-transforming growth factor-alpha expression in residents of an arseniasis area in Taiwan

Abstract

Epidemiological studies have demonstrated an association between long-term exposure to inorganic arsenic and the related adverse effects such as cancers, skin lesions, and vascular diseases. Although several hypotheses have been proposed for the mechanism of arsenic-induced pathogenesis, it remains imperfectly understood. Recent studies have suggested that alterations in growth signal transduction pathways, particularly involving transforming growth factor-alpha (TGF-alpha), may be important. Immunoassays were used to determine the plasma levels of TGF-alpha and epidermal growth factor receptor (EGFR), which is the receptor for TGF-alpha, in residents of an arseniasis area of Taiwan in relation to their estimated cumulative arsenic exposure from drinking water. No relationship between arsenic exposure and EGFR was found. However, among the high cumulative exposure group (>6 ppm-years), levels of plasma TGF-alpha (25.5±38.2 pg ml^?1) and the proportion of individuals with TGF-alpha over-expression (29.4%) were significantly higher (p<0.05) than normal, healthy unexposed controls (8.1±5.6 pg ml^?1, 8.6%, respectively). There was a significant linear trend between cumulative arsenic exposure and the prevalence of plasma TGF-alpha over-expression after adjusting for age and sex (p=0.019). The results suggest that plasma TGF-alpha expression may be a useful biomarker when detecting adverse effects on arsenic exposed population.     

Isolation and physical characterization of bovine lens crystallins.

The soluble proteins from bovine lens homogenate were separated on Sepharose CL-6B (2 X 200 cm) in 0.05 M tris-NaHSO3 pH 8.2 buffer containing 20 mM EDTA. Five sharp and defined fractions (HM alpha, alpha, beta H, beta L, gamma) were obtained. Each crystallin fraction was further purified by rechromatography on the same column. Each protein fraction was pure as judged by ultracentrifugation and SDS-gel electrophoresis. The molecular weights of the five fractions were 3.04 x 10(6), 5.83 x 10(5), 1.58 x 10(5) , 4.59 x 10(4), 2.14 x 10(4) as determined from sedimentation coefficient and intrinsic viscosity data by Scheraga-Mandelkern equation, which was in close agreement with that obtained by gel filtration. The polypeptide composition of crystallins as determined by SDS-gel electrophoresis revealed one band for high molecular weight alpha (HM alpha) and alpha, three for beta H, two for beta L and one for gamma. The gross CD patterns of crystallins were about the same in the peptide region (200 nm similar to or approximately 250 nm) with a minimum centered at about 217 nm, indicative of a beta-sheet structure in all crystallins. The [theta] values at 217 nm ranged from --1700 to --3700 degrees cm2 per decimole. The CD spectra of these crystallins in the aromatic region (250 nm similar to or approximately 300 nm) were different, reflecting the different contributions of aromatic amino acids to the tertiary structure of crystallins.     

Sequence comparison of gamma-crystallins from the reptilian and other vertebrate species

Lens crystallins were isolated from homogenates of reptilian eye lenses (Caiman crocodylus apaporiensis) by gel-permeation chromatography and characterized by gel electrophoresis, and amino acid and N-terminal sequence analyses. Four fractions corresponding to alpha-, delta/epsilon/beta-, beta- and gamma-crystallins were identified on the basis of their electrophoretic patterns as revealed by SDS gel electrophoresis. Comparison of the amino acid contents of reptilian crystallins with those of mammals suggests that each orthologous class of crystallins from the evolutionarily distant species still exhibits similarity in their amino acid compositions and probably sequence homology as well. All fractions except that of gamma-crystallin were found to be N-terminally blocked. N-terminal sequence analysis of the purified gamma-crystallin subfractions showed extensive homology between the reptilian gamma-crystallin polypeptides themselves and also those from other vertebrate species, suggesting the existence of a multigene family and their close relatedness to gamma-crystallins of other vertebrates.     

Toll-Like Receptor 9 Alternatively Spliced Isoform Negatively Regulates TLR9 Signaling in Teleost Fish

Toll-like receptor 9 (TLR9) recognizes and binds unmethylated CpG motifs in DNA, which are found in the genomes of bacteria and DNA viruses. In fish, Tlr9 is highly diverse, with the number of introns ranging from 0 to 4. A fish Tlr9 gene containing two introns has been reported to express two alternatively spliced isoforms, namely gTLR9A (full-length) and gTLR9B (with a truncated Cʹ-terminal signal transducing domain), whose regulation and function remain unclear. Here, we report a unique regulatory mechanism of gTLR9 signaling in orange-spotted grouper (Epinephelus coioides), whose gTlr9 sequence also contains two introns. We demonstrated that the grouper gTlr9 gene indeed has the capacity to produce two gTLR9 isoforms via alternative RNA splicing. We found that gTLR9B could function as a negative regulator to suppress gTLR9 signaling as demonstrated by the suppression of downstream gene expression. Following stimulation with CpG oligodeoxynucleotide (ODN), gTLR9A and gTLR9B were observed to translocate into endosomes and co-localize with ODN and the adaptor protein gMyD88. Both gTLR9A and gTLR9B could interact with gMyD88; however, gTLR9B could not interact with downstream IRAK4 and TRAF6. Further analysis of the expression profile of gTlr9A and gTlr9B upon immune-stimulation revealed that the two isoforms were differentially regulated in a time-dependent manner. Overall, these data suggest that fish TLR9B functions as a negative regulator, and that its temporal expression is mediated by alternative RNA splicing. This has not been observed in mammalian TLR9s and might have been acquired relatively recently in the evolution of fish.     

The Role of Principal and Secondary Diagnoses of Hospitalized Eye Trauma: A Nationwide Cohort in Taiwan, 1996-2010

Purpose

To estimate the rate of hospitalized eye trauma in Taiwan and investigate the role between principal and secondary diagnoses of such trauma.

Methods

Nationwide fixed cohort study of 1,000,000 beneficiaries from the Taiwan Longitudinal Health Database was used and 4819 patients who were hospitalized for eye trauma during 1996-2010 were analyzed.

Results

During 1996-2010, the incidence rates of hospitalized eye trauma (per 100 000 person-years) were 35.0 (95% confidence interval (CI), 34.0 to 36.0) for total diagnosis, 9.8 (95% CI, 9.3 to 10.3) for a principal diagnosis, and 25.3 (95% CI, 24.4 to 26.1) for a secondary diagnosis. The sex risk ratio was 3.1 for a principal diagnosis and 2.1 for a secondary diagnosis. The main causes of eye trauma were traffic accident, work accident, assault (among males <60 years of age), and falls (among elderly men and women). The proportion admitted to an ophthalmic department among those with a principal diagnosis of eye trauma (64.8%) was significantly higher than among those with a secondary diagnosis (2.3%) (p<.0001). Patients with a principal diagnosis of eye trauma had shorter hospital stays (7.1±10.2 days) and lower fatality (0.07%) than those with a secondary diagnosis of eye trauma (10.0±31.6 days and 0.3%, respectively).

Conclusion

Data only from ophthalmic admissions tends to underestimate the true incidence rate of hospitalized eye trauma. Patients with a principal diagnosis of eye trauma had less severe injuries than did those with a secondary diagnosis.     

Serum Ferritin Is Inversely Correlated with Testosterone in Boys and Young Male Adolescents: A Cross-Sectional Study in Taiwan

Objective

The transition from childhood to teenaged years is associated with increased testosterone and a decreased iron status. It is not clear whether higher testosterone levels cause the decreased iron status, and to what extent, obesity-related inflammation influences the iron-testosterone relationship. The aim of the present study was to examine relationships of testosterone, iron status, and anti-/proinflammatory cytokines in relation to nutritional status in boys and young adolescent Taiwanese males.

Methods

In total, 137 boys aged 7~13 yr were included. Parameters for obesity, the iron status, testosterone, and inflammatory markers were evaluated.

Results

Overweight and obese (ow/obese) boys had higher mean serum testosterone, interleukin (IL)-1β, and nitric oxide (NO) levels compared to their normal-weight counterparts (all p<0.05). Mean serum ferritin was slightly higher in ow/obese boys compared to normal-weight boys, but this did not reach statistical significance. A multiple linear regression showed that serum ferritin (β = -0.7470, p = 0.003) was inversely correlated with testosterone, while serum IL-10 (β = 0.3475, p = 0.009) was positively associated with testosterone after adjusting for covariates. When normal-weight boys were separately assessed from ow/obesity boys, the association between testosterone and serum ferritin became stronger (β = -0.9628, p<0.0001), but the association between testosterone and IL-10 became non-significant (β = 0.1140, p = 0.4065) after adjusting for covariates. In ow/obese boys, only IL-10 was weakly associated with serum testosterone (β = 0.6444, p = 0.051) after adjusting for age.

Conclusions

Testosterone and serum ferritin are intrinsically interrelated but this relationship is weaker in ow/obese boys after adjusting for age.