Homocysteine contribution to DNA damage in cystathionine β-synthase-deficient patients

High blood levels of homocysteine (Hcy) are found in patients affected by homocystinuria, a genetic disorder caused by deficiency of cystathionine β-synthase (CBS) activity, as well as in nutritional deficiencies (vitamin B₁₂ or folate) and in abnormal renal function. We previously demonstrated that lipid and protein oxidative damage is increased and the antioxidant defenses diminished in plasma of CBS-deficient patients, indicating that oxidative stress is involved in the pathophysiology of this disease. In the present work, we extended these investigations by evaluating DNA damage through the comet assay in peripheral leukocytes from CBS-deficient patients, as well as by analyzing of the in vitro effect of Hcy on DNA damage in white blood cells. We verified that DNA damage was significantly higher in the CBS-deficient patients under treatment based on a protein-restricted diet and pyridoxine, folic acid, betaine and vitamin B₁₂ supplementation, when compared to controls. Furthermore, the in vitro study showed a concentration-dependent effect of Hcy inducing DNA damage. Taken together, the present data indicate that DNA damage occurs in treated CBS-deficient patients, possibly due to high Hcy levels.     

X-Ray crystal and molecular structure of 1,2,3,5-tetra-O-acetyl-4-deoxy-4-C-[(S)-ethylphosphinyl]-α-d-ribofuranose,and conformations of aldopentofuranose analogs having phosphorus in the hemiacetal ring

X-Ray crystallographic analysis was performed on the compound to which had been assigned the structure 1,2,3,5-tetra-O-acetyl-4-deoxy-4-C-[(S)-ethylphosphinyl]-α-d-ribofuranose. The results showed that the compound has the proposed configuration, the five-membered ring is in the ³T₂ conformation with a tendency towards the E₂ form, the substituents on C-1, C-4, and P-5 are linked bisectionally, and the acetoxyl groups on C-2 and C-3 are respectively attached axially and equatorially. Based on the X-ray crystallographic and ¹H-n.m.r.-spectral data, favored conformations of P-in-ring analogs of aldopentofuranose peracetates in solution are discussed.     

Monensin interferes with the determination of the mesodermal cell line in embryos of Patella vulgata

Summary In embryos of the equally cleaving marine gastropod Patella vulgata, the mesodermal stem cell is determined during the interval between the fifth and sixth cleavage by means of cellular interactions between one of the four vegetally located macromeres with the overlying animal micromeres. Shortly before and during this interaction phase an extracellular matrix (ECM) is present between the interacting cells. In this study the glycosylation-perturbing ionophore monensin was used to investigate the possible morphogenetic significance of the ECM. Incubation of 32-cell-stage Patella embryos in 10^–6 M monensin results in radialized embryos in which none of the four macromeres interacts with the overlying animal micromeres. None of the macromeres is determined, therefore, to form mesoderm in such embryos. Trochophore larvae reared from these embryos retain their radial symmetry, as is indicated by the presence of four shell glands and four blastopore- or stomodeum-like invaginations in these larvae. The monensin-treated embryos probably secrete abnormal ECM that does not provide the proper conditions for the blastomeres to stretch and interact with the micromeres. Changes in intracellular ionic concentrations may also be involved.     

Induction of yeast-like cells in a strain of Fonsecaea pedrosoi cultured under very acidic conditions

Yeast-like cells with a conidiogenesis of the annellidic type were obtained by culturing a strain of Fonsecaea pedrosoi, the principal agent of chromomycosis, under very acidic conditions (_pH 2.5). These annellides resemble those of such pathogenic black yeasts as Exophiala jeanselmei.     

Effects of fixation on the preservation of peroxisomal structures for immunofluorescence studies using HepG2 cells as a model system

Summary The immunofluorescence technique has become an important tool for the investigation of peroxisomes in cell culture. We have used this method for the study of peroxisomes in the human hepatoblastoma cell line HepG2. A marked heterogeneity of peroxisomal forms was detected. Besides spherical (about 100 nm) and rod-shaped structures (about 300 nm) many elongated, undulating tubular forms (up to 5 m) were found. Further observations indicate that the appearance of the peroxisomal forms in immunofluorescence is dependent on the fixation procedure used. Whereas the fixation with methanol-acetone (–20°C) or ethanol results in a punctate pattern with spherical particles, the use of formaldehyde/Triton X-100 fixation shows well-preserved tubules and rods. These observations may be of special importance for studies on the biogenesis of peroxisomes.     

Evaluation of the Auxacolor system for the identification of clinical yeast isolates

In the last decade the number of systemic yeast infections has increased significantly. Although Candida albicans is the most frequently isolated yeast from clinical specimens, the emergence of non-albicans species has clearly been a recent concern. As a consequence, there is a greater need for rapid and accurate methods for yeast identification. The aim of this study was to evaluate the performance of the AUXACOLOR system (Sanofi Diagnostics Pasteur) for the identification of clinically relevant yeasts, as compared with the conventional method. Yeast isolates (n = 97) belonging to 12 species were identified by the commercial system and the classic method. Correct identifications were obtained by using AUXACOLOR system in 79.4% of the isolates tested. Misidentification occurred in 5.2% of the strains and 15.5% were not identified due to a failure in the manufacturer's data base. In order to improve its accuracy, there is a need for expanding the database or revamping the tests included in the system. This revised version was published online in June 2006 with corrections to the Cover Date.     

Comparison of two methods of synchronization of estrus in brown brocket deer (Mazama gouazoubira)

This study aimed to establish a protocol for synchronization of estrus in brown brocket deer (Mazama gouazoubira). Two groups of hinds (n = 3) were submitted to two different protocols: Treatment 1 received an intravaginal progesterone (CIDR^®) device for 8 days, followed by 265 μg injection of cloprostenol at the time of removal; and Treatment 2 received two injections of 265 μg of cloprostenol 11 days apart. After 30 days, each group of three hinds received the other treatment. Treatment efficacy was evaluated by reproductive behavior, fecal progestin and estrogen concentration and the observation of CL by laparoscopy 6 days after the end of estrus. All the hinds (100%) had estrous behavior upon the completion of treatment, but a significant difference occurred between the time of onset, 70.5 ± 5.0 h for Treatment 1 and 52.3 ± 5.6 h for Treatment 2. The mean estrus duration time (34.7 ± 4.50 and 37.0 ± 8.11 h), ovulation rates (5/6 and 4/6), mean CL size (4.85 ± 0.74 and 3.21 ± 0.19 mm) and mean fecal progestin concentration at 6 days after the end of estrus (865.53 ± 76.59 and 1073.35 ± 106.82 ng/g feces) were not significantly different between treatments. There was no difference in fecal estrogen concentrations throughout the treatment and the greatest values of the estrogen:progestin ratio coincided with estrous behavior. Although fertility was not evaluated directly, both treatments were effective in synchronizing estrus in the species M. gouazoubira, with the formation of functional corpora lutea.     

Single nucleotide polymorphisms in the open reading frame of the stearoyl-CoA desaturase gene and resulting genetic variants in Canadian Holstein and Jersey cows.

Stearoyl-CoA desaturase (SCD) catalyzes the synthesis of conjugated linoleic acid (CLA) and mono-unsaturated fatty acids (MUFA) from their saturated counterparts in the mammary gland and adipose tissue of ruminant animals. We hypothesize that single nucleotide polymorphisms (SNPs) in the SCD gene account for some of the differences in SCD activity, and consequently for some of the variations in CLA and MUFA content of milk fat between Holsteins and Jersey cows and within these two breeds. We analyzed the open reading frame of the SCD gene of 44 Holsteins and 48 Jerseys for SNPs by sequencing. Three SNPs: 702A --> G, 762T --> C and 878C --> T were identified in both breeds and a further SNP, 435G --> A, was unique to Holsteins. The SNPs characterized four different genetic variants in Holsteins: A (G(435)A(702)T(762)C(878)), A1 (A(435)A(702)T(762)C(878)), B (G(435)G(702)C(762)T(878)) and B1 (A(435)G(702)C(762)T(878)), with only variants A and B in Jerseys. SNP 878C --> T resulted in a non-synonymous codon change while the rest resulted in synonymous codon changes giving rise to two protein variants, A having alanine and B having valine. Allele A was the most prevalent in the two breeds. These differences may, therefore, contribute to existing variations in CLA and fat content between and within Canadian Holstein and Jersey cows.