Differentiation-associated modulation of heparan sulfate structure and function in CaCo-2 colon carcinoma cells

Heparan sulfate species expressed by different cell and tissue types differ in their structural and functional properties. Limited information is available on differences in regulation of heparan sulfate biosynthesis within a single tissue or cell population under different conditions. We have approached this question by studying the effect of cell differentiation on the biosynthesis and function of heparan sulfate in human colon carcinoma cells (CaCo-2). These cells undergo spontaneous differentiation in culture when grown on semipermeable supports; the differentiated cells show phenotypic similarity to small intestine enterocytes. Metabolically labeled heparan sulfate was isolated from the apical and basolateral media from cultures of differentiated and undifferentiated cells. Compositional analysis of disaccharides, derived from the contiguous N-sulfated regions of heparan sulfate, indicated a greater proportion of 2-O- sulfated iduronic acid units and a smaller amount of 6-O-sulfated glucosamine units in differentiated than in undifferentiated cells. By contrast, the overall degree of sulfation, the chain length and the size distribution of the N-acetylated regions were similar regardless the differentiation status of the cells. The structural changes were found to affect the binding of heparan sulfate to the long isoform of platelet-derived growth factor A chain but not to fibroblast growth factor 2. These findings show that heparan sulfate structures change during cell differentiation and that heparan sulfate-growth factor interactions may be affected by such changes.      

The Cutting Balloon--a new technology?

The Cutting Balloon consists of a standard balloon dilatation catheter with four microtome-sharp blades that incise the plaque and minimize arterial wall trauma. It was used in 31 patients; nine had calcified arteries, ten had non-compliant lesions, three had in-stent restenosis and nine had aorto-ostial lesions. Seventeen lesions were predilated, 28 were post-dilated and 18 required stent implantation. The procedure was very effective in aorto-ostial lesions, non-compliant lesions that were not responsive to high-pressure balloon dilatation, and was partially successful in calcified arteries. It has a very specific niche in selected lesions.     

Evaluation of the prevalence and economic burden of adverse drug reactions presenting to the medical emergency department of a tertiary referral centre: a prospective study

Background  

Adverse drug reactions (ADRs) are now recognized as an important cause of hospital admissions, with a proportion ranging from 0.9–7.9%. They also constitute a significant economic burden. We thus aimed at determining the prevalence and the economic burden of ADRs presenting to Medical Emergency Department (ED) of a tertiary referral center in India     

Evaluation of APOE polymorphisms and the risk for age-related macular degeneration in a Southeastern Brazilian population

This study aimed to evaluate the role of APOE polymorphisms (rs429358 and rs7412) in the risk of age-related macular degeneration in a sample of the Southeastern Brazilian population. Seven hundred and five unrelated individuals were analyzed, 334 with age-related macular degeneration (case group), and 371 without the disease (control group). In the case group, patients were further stratified according to disease phenotypes, divided into dry and wet age-related macular degeneration, and non-advanced and advanced age-related macular degeneration. APOE polymorphisms (rs429358 and rs7412) were evaluated through polymerase chain reaction and direct sequencing. In the comparison of cases vs. controls, none of the associations reached statistical significance, considering the Bonferroni-adjusted P-value, although there was a suggestive protection for the E3/E4 genotype (OR = 0.626; P-value = 0.037) and E4 carriers (OR = 0.6515; P-value = 0.047). Statistically significant protection for both the E3/E4 genotype and E4 carriers was observed in the comparisons: advanced age-related macular degeneration vs. controls (OR = 0.3665, P-value = 0.491 × 10⁻³ and OR = 0.4031, P-value = 0.814 × 10⁻³, respectively), advanced age-related macular degeneration vs. non-advanced age-related macular degeneration (OR = 0.2529, P-value = 0.659 × 10⁻⁴ and OR = 0.2692, P-value = 0.631 × 10⁻⁴, respectively). In the comparison of wet age-related macular degeneration vs. control, protection was statistically significant only for E3/E4 (OR = 0.4052, P-value = 0.001). None of the comparisons demonstrated any significant association for E2 genotypes or E2 carriers in age-related macular degeneration risk in this study. Findings suggest a protective role of the E4 haplotype in the APOE gene in the risk for advanced and wet forms of age-related macular degeneration, in a sample of the Brazilian population. To our knowledge, this is the first Brazilian study to show the association between APOE polymorphisms and age-related macular degeneration.     

Pregnancies following long luteal phases in southern white rhinoceros (Ceratotherium simum simum)

All extant species in the Rhinocerotidae family are experiencing escalating threats in the wild, making self-sustaining captive populations essential genetic reservoirs for species survival. Assisted reproductive technologies (ARTs) will become increasingly important for achieving and maintaining ex situ population sustainability and genetic diversity. Previous reports have shown that a large proportion of captive southern white rhinoceros (SWR) females are irregularly cyclic or acyclic, and that cycling females display two different estrous cycle lengths of approximately 30 or 70 days. It has been suggested that the longer estrous cycle length is infertile or subfertile, as no term pregnancies have been observed following long cycles. Here we report the achievement of two pregnancies following long luteal phases, using ovulation induction and artificial insemination with either fresh or frozen-thawed semen. One female SWR conceived on the first insemination attempt and gave birth to a live offspring. A second female conceived twice in consecutive long cycles although the first embryo was resorbed by 33 days post-insemination. A pregnancy from this female's second insemination is ongoing with expected parturition in November 2019. Whether prolonged estrous cycles in SWR are subfertile or infertile in natural breeding situations remains unclear. However, our findings demonstrate that the application of ARTs following prolonged cycles can result the successful establishment of pregnancies in SWR. Therefore, with ARTs, female SWR otherwise considered nonreproductive due to long estrous cycles may still have the potential for representation and contribution to the ex situ population.     

Mutagenicity of the mycotoxin diacetoxyscirpenol on somatic and germ cells of mice

Genotoxicity of diacetoxyscirpenol (DAS) was studied on laboratory mice after intraperitoneal injection with single and repeated doses. DAS was administrated at three different dose levels (0.5, 0.75, and 1.0 mg/kg body weight). The study was conducted on both somatic and germ cells additional to the sperm morphology analysis. DAS treatment resulted in a significant reduction (P<0.01) in mitotic activity at all levels of doses tested, confirming that DAS is a potent protein and DNA synthesis inhibitor. At somatic cells (bone marrow) both structural and numerical chromosome abnormalities were observed. Single dose treatment showed significant abnormalities only with high dose treatment. In contrast, at repeated dose similar abnormalities were also observed with some significance but no systematic relation between the administrated dose and abnormalities ratio could be settled. In germ cells (testicles), structural and numerical abnormalities were also observed. In general, the frequencies of scored abnormalities at germ cells were lower than that the somatic cells. Sperm count test revealed a decrease in the number of released sperm after toxin treatment. Abnormalities of sperm shape (head and tail) were observed, confirming the positive correlation between cytogenetic damage and sperm abnormality. The results also proved that DAS is a very toxic mycotoxin, in addition to inducing chromosomal abnormalities, it causes a severe inhibition of DNA synthesis which subsequently affects the cell cycle and cell division. A good system for good harvesting practice and good food technology can lower the risk for the consumers.     

Expression and characterization of human apolipoprotein A-I in Chinese hamster ovary cells

We produced human apolipoprotein A-I (apoA-I) in Chinese hamster ovary (CHO) cells. The CHO cells were transfected with an expression plasmid which placed the human apoA-I gene under the direction of the human metallothionein II gene promoter. Isolation of a clonal cell line resulted in high level expression of apoA-I. Greater than 30% of total protein secreted by these CHO cells was apoA-I, which enabled us to purify apoA-I with a single step purification scheme. As a result, large quantities of apoA-I can be produced and isolated without having to rely on plasma sources. Structural characterization of the recombinant apoA-I showed it to be identical to authentic apoA-I from human serum high density lipoprotein. Furthermore, we demonstrated approximately equal to 90% of the apoA-I secreted by CHO cells is processed, mature protein. A portion of the secreted recombinant apoA-I was associated with lipid and floated at a density approximately equal to 1.10 g/ml. Additional analysis identified the presence of five isoforms of apoA-I in the CHO cell conditioned medium. Processing and post-translational modification of the recombinant apoA-I occurred in the CHO cell cultures in the absence of serum components. We conclude that the human apoA-I produced by CHO cells is identical to circulating, mature apoA-I in humans and that recombinant mammalian expression offers an opportunity to investigate apoA-I processing.     

Reduced cardiac myofibrillar Mg-ATPase activity without changes in myosin isozymes in patients with end-stage heart failure

In this study we tested the hypothesis that reduced myofibrillar ATPase activities in end-stage heart failure are associated with a redistribution of myosin isozymes. Cardiac myofibrils were isolated from left ventricular free wall from normal human hearts and hearts at end-stage heart failure caused by coronary artery diseases, cardiomyopathy or immunological rejection. The hearts had been excised in preparation for a heart transplant. Myofibrillar Ca^2–-dependent Mg-ATPase and myosin Ca^–- and K^–EDTA-ATPase activities were compared. Possible changes in myosin isozyme distribution in the diseased heart were investigated using polyacrylamide gel electrophoresis of native myosin in the presence of pyrophosphate. Significant reduction in myofibrillar Ca²⁺-dependent Mg-ATPase with no changes in the sensitivity of the myofibrils to Ca⁺ was observed in heart with coronary artery diseases (25.2 to 27.1% at pCa 5.83 to pCa 5.05), cardiomyopathy (21.1 to 25.5% at pCa 5.41 to pCa 5.05), and in the immunologically rejected heart (18.4 to 22.8% at pCa 5.41 to pCa 5.05). Significantly lower myosin Ca²⁺-ATPase was observed with coronary artery diseases only and myosin K-EDTA activities did not differ in diseased and normal hearts. Polyacrylamide gel electrophoresis of native myosin from the normal and three models of end-stage heart failure revealed two distinct bands in the human left ventricle and one diffuse band in the human right atria. No apparent differences in myosin isoenzyme pattern were observed between the normal and diseased hearts. Further evaluation is needed to clarify the ATPase nature of the two bands.